Dual-color Fluorescence Research Articles

Molecular Engineering of Luminogens for High-Integrity Imaging of Hydrogen Polysulfides via Activatable Aggregation-Induced Dual-Color Fluorescence.

Understanding biological events associated with H2Sn rather than mediated by H2S is of great significance but remains to be solved due to a lack of high-integrity imaging tools. In this study, we report a chemoselective probe for H2Sn over H2S through the molecular engineering of luminogens. Based on our search for H2Sn-activatable probes with high selectivity, we fabricate water-soluble and biocompatible nanoprobes. Such a designed nanoprobe shows rare aggregation-induced dual-color fluorescence responses to H2Sn, lighting up bright emissions at 588 and 750 nm, respectively. By use of this activatable dual-color fluorescence, high-integrity identification of intracellular H2Sn was successfully realized. Thus, our approach to H2Sn-activated multicolor fluorescent probes could provide valuable insight into interrogating H2Sn-mediated biological events.

Dual-color nanospheres based on aggregation-induced emission and catalytic hairpin assembly for simultaneous imaging of acrylamide and miR-21 in living cells

Acrylamide (AA) is a heat-processed potent food carcinogen that is widely used in industry, posing a significant risk to human health. Therefore, it is necessary to investigate the toxic effects and mechanism of AA. miR-21 is a representative biomarker during AA-induced carcinogenesis. Here, dual-color aggregation-induced emission nanoparticles (AIENPs) were developed for the detection and simultaneous imaging of AA and miR-21. AIENPs were synthesized by combining aggregation-induced emission (AIE) dyes and a poly (styrene-co-maleic anhydride) (PSMA) amphiphilic polymer modified with hairpin DNA. Upon AA intervention and aptamer recognition, cDNA was dissociated, leading to miR-21 overexpression and initiating the catalytic hairpin assembly cycle. Consequently, fluorescence quenching was observed due to FRET between AIENPs and labeled quenchers. The relative fluorescence intensities of dual-color AIENPs displayed good linear relationships with logarithmic AA and miR-21 concentrations. Moreover, there was a gradual decrease in dual-color AIENP fluorescence as the HepG2 cell concentration of AA (0–500 μM) and stimulation time (0–12 h) increased, making it possible to simultaneously image AA and AA-induced miR-21. The findings of this work are valuable for revealing the cytotoxic mechanism of AA.

Cxcl12a plays an essential role in pharyngeal cartilage development.

Background: Neural crest cells constitute a distinct set of multipotent cells that undergo migration along predefined pathways, culmination in the differentiation into a plethora of cell types, including components of the pharyngeal cartilage. The neurocranium is composite structure derived from both cranial neural crest and mesoderm cells, whereas the pharyngeal skeletal elements-including the mandibular and branchial arches-are exclusively formed by craniofacial neural crest cells. Previous studies have elucidated the critical involvement of the chemokine signaling axis Cxcl12b/Cxcr4a in craniofacial development in zebrafish (Danio rerio). Nonetheless, the function contribution of Cxcl12a and Cxcr4b-the homologous counterparts of Cxcl12b and Cxcr4a-remain largely unexplored. Methods: In the present study, mutant lines for cxcl12a and cxcr4b were generated employing CRISPR/Cas9 system. Temporal and spatial expression patterns of specific genes were assessed using in situ hybridization and dual-color fluorescence in situ hybridization techniques. High-resolution confocal microscopy was utilized for in vivo imaging to detect the pharyngeal arch or pouch patterning. Additionally, cartilage formation within the craniofacial region was analyzed via Alcian blue staining, and the proliferation and apoptosis rates of craniofacial neural crest cells were quantified through BrdU incorporation and TUNEL staining. Results: Our data reveals that the deletion of the chemokine gene cxcl12a results in a marked diminution of pharyngeal cartilage elements, attributable to compromised proliferation of post-migratory craniofacial neural crest cells. Subsequent experiments confirmed that Cxcl12a and Cxcl12b exhibit a synergistic influence on pharyngeal arch and pouch formation. Conclusion: Collectively, the present investigation furnishes compelling empirical evidence supporting the indispensable role of Cxcl2a in craniofacial cartilage morphogenesis, albeit cxcr4b mutants exert a minimal impact on this biological process. We delineate that Cxcl12a is essential for chondrogenesis in zebrafish, primarily by promoting the proliferation of craniofacial neural crest cells. Furthermore, we proposed a conceptual framework wherein Cxcl12a and Cxcl12b function synergistically in orchestrating both the pharyngeal arch and pouch morphogenesis.

Exploring ESInterPT behaviors of water sensitive probes: 3HF derivative clusters

The fluorescent probe molecules 3-hydroxyflavone derivatives (PPC, EPC and MNC) have been synthesized and for the first time realized the dual color fluorescence imaging of lipid droplets (J. Am. Chem. Soc., 2021, 143, 3169–3179), and PPC and EPC demonstrate the fluorescent sensitivity to water. In this work, the excited-state intermolecular proton transfer (ESInterPT) process in the clusters of 3HF-D with water molecules ((3HF-D-(H2O)n, abbreviated as D-(H2O)n: n = 1, 2, 3, 1 + 2, 2 + 1; D = PPC, EPC, MNC) are investigated by means of the time-dependent density functional theory (TDDFT) method. Based on the calculated potential energy scheme, we found that the dynamics and thermodynamic of excited-state intermolecular proton transfer in D-(H2O)n are different and dependent on the number of water molecules, but all proton transfer processes follow the concerted mechanism. We further reveal the water sensitivity of PPC, EPC and MNC, and theoretically confirm the feasibility of PPC and EPC as water sensitive probes. Besides we found the water sensitivity is correlated with charge transfer degree. This work provides significant insight into fluorescent sensing water using 3HF derivatives which is useful for rational design of new organic fluorescent probes with excellent performance.

Effects of biophysical membrane properties on recognition of phosphatidylserine, or phosphatidylinositol 4-phosphate by lipid biosensors LactC2, or P4M

Lipid biosensors are molecular tools used both in vivo and in vitro applications, capable of selectively detecting specific types of lipids in biological membranes. However, despite their extensive use, there is a lack of systematic characterization of their binding properties in various membrane conditions. The purpose of this study was to investigate the impact of membrane properties, such as fluidity and membrane charge, on the sensitivity of two lipid biosensors, LactC2 and P4M, to their target lipids, phosphatidylserine (PS) or phosphatidylinositol 4-phosphate (PI4P), respectively. Dual-color fluorescence cross-correlation spectroscopy, employed in this study, provided a useful technique to investigate interactions of these recombinant fluorescent biosensors with liposomes of varying compositions. The results of the study demonstrate that the binding of the LactC2 biosensor to low levels of PS in the membrane is highly supported by the presence of anionic lipids or membrane fluidity. However, at high PS levels, the presence of anionic lipids does not further enhance binding of LactC2. In contrast, neither membrane charge, nor membrane fluidity significantly affect the binding affinity of P4M to PI4P. These findings provide valuable insights into the role of membrane properties on the binding properties of lipid biosensors.

Tuning the Dual-Color Fluorescence Emission of Nitrogen-Doped Carbon Dots by Changing Nitrogen Doping Amounts

Tuning the Dual-Color Fluorescence Emission of Nitrogen-Doped Carbon Dots by Changing Nitrogen Doping Amounts

Effects of Sufentanil Nanoparticles on Proliferation and Apoptosis of Human Lung Adenocarcinoma Cells

This research was aimed to investigate the effects of sufentanil nanoparticles (Suf-NPs) on the proliferation and apoptosis of human lung adenocarcinoma (LUAD) cells, and to provide theoretical support for future drug therapy. Based on the preparation method of solid lipid nanoparticles (SLN), stearic acid and lecithin were used as materials to prepare Suf-NPs. The physical and chemical properties of Suf-NPs were analyzed by scanning electron microscope and particle size analyzer. A549 human LUAD cell line was selected. According to different cell treatment methods, A549 cells were randomly divided into control (Con), sufentanil (Suf), blank SLN, and Suf-NPs groups. Con was cultured routinely, and Suf group was treated with 0.02 μg/mL sufentanil. Suf-NPs group was treated with Suf-NPs, and the concentration was the same as that in the Suf group. The SLN group was added with blank SLN suspension. CCK8 assay was adopted to test the proliferation inhibition rate (PIR) of A549 cells treated with SUF-NPs. Flow cytometry and dual-color fluorescence microscopy were adopted to detect the apoptosis rate. The expression levels of apoptosis-related proteins P53, Bax, and Bcl-2 were detected by Western blot. The results revealed that the average particle size of Suf-NPs was (113.86±2.03) nm, the zeta potential was (−28.44±1.52) mV, the encapsulation efficiency (PER) was (82.02±3.56) %, and the drug loading (DL) capacity was (50.31±6.49) %. As against Con, the PIR, apoptosis rate, P53, and Bax protein expression was significantly higher (P <0.001), and Bcl-2 protein was markedly inferior in Suf, SLN, and Suf-NPs groups (P <0.05). Compared with the Suf, the Suf-NPs group had markedly higher PIR, apoptosis rate, and expression of P53 and Bax proteins (P < 0.05), and markedly lower expression of Bcl-2 protein (P <0.05). Suf-NPs can markedly restrain A549 human LUAD cells, promote their apoptosis, and affect the expression of related genes and proteins. It provides a theoretical basis for the future development of Suf-NPs as a drug for the treatment of lung cancer (LC).

Compact and robust dual-color linearly polarized illumination source for three-photon fluorescence imaging

The miniaturized femtosecond laser in near infrared-II region is the core equipment of three-photon microscopy. In this paper, we design a compact and robust illumination source that emits dual-color linearly polarized light for three-photon microscopy. Based on an all-polarization-maintaining passive mode-locked fiber laser, we shift the center wavelength of the pulses to the 1.7[Formula: see text][Formula: see text]m band utilizing cascade Raman effect, thereby generate dual-wavelength pulses. To enhance clarity, the two wavelengths are separated through the graded-index multimode fiber. Then we obtain the dual-pulse sequences with 1639.4[Formula: see text]nm and 1683.7[Formula: see text]nm wavelengths, 920[Formula: see text]fs pulse duration, and 23.75[Formula: see text]MHz pulse repetition rate. The average power of the signal is 53.64[Formula: see text]mW, corresponding to a single pulse energy of 2.25[Formula: see text]nJ. This illumination source can be further amplified and compressed for three-photon fluorescence imaging, especially dual-color three-photon fluorescence imaging, making it an ideal option for biomedical applications.

Bioluminescence imaging on-chip platforms for non-invasive high-content bioimaging

Incorporating non-invasive biosensing features in organ-on-chip models is of paramount importance for a wider implementation of these advanced in vitro microfluidic platforms. Optical biosensors, based on Bioluminescence Imaging (BLI), enable continuous, non-invasive, and in-situ imaging of cells, tissues or miniaturized organs without the drawbacks of conventional fluorescence imaging. Here, we report the first-of-its-kind integration and optimization of BLI in microfluidic chips, for non-invasive imaging of multiple biological readouts. The cell line HEK293T-GFP was engineered to express NanoLuc® luciferase under the control of a constitutive promoter and were cultured on-chip in 3D, in standard ECM-like hydrogels, to assess optimal cell detection conditions. Using real-time in-vitro dual-color microscopy, Bioluminescence (BL) and fluorescence (FL) were detectable using distinct imaging setups. Detection of the bioluminescent signals were observed at single cell resolution on-chip 20 min post-addition of Furimazine substrate and under perfusion. All hydrogels enabled BLI with higher signal-to-noise ratios as compared to fluorescence. For instance, agarose gels showed a ∼5-fold greater BL signal over background after injection of the substrate as compared to the FL signal. The use of BLI with microfluidic chip technologies opens up the potential for simultaneous in situ detection with continuous monitoring of multicolor cell reporters. Moreover, this can be achieved in a non-invasive manner. BL has great promise as a highly desirable biosensor for studying organ-on-chip platforms.

Reconsideration of the ESIPT off mechanism for fluorescent probe MNC in aqueous solution

Fluorescent probes with excited state intramolecular proton transfer (ESIPT) properties play a significant role in the research of life science and material science. Guo et al. designed 3-hydroxy-2-(6-Methoxynaphthalen-2-yl)-4H-chromen-4-one (MNC) as a control to achieve the dual-color fluorescence imaging of lipid droplets and endoplasmic reticulum (ER). They deemed that the ESIPT process would be turned off in ER with high water content [J. Am. Chem. Soc. 2021, 143, 3169–3179]. However, contrary to the conventional ESIPT off case, the enol* state fluorescence intensity that should have been enhanced was severely quenched in water. Here, combined with ultrafast spectrum, steady-state fluorescence spectrum and potential energy surface, the mechanism of ESIPT process of MNC turned off in water is revised. Furthermore, the formation of aggregated states in water is responsible for the quenching of MNC fluorescence. This work is expected to provide broader ideas for the design of hydrophobic fluorescent probes.

A Fluorescence Cross-Correlation-Spectroscopy-Based Immunoassay for Rapid, Selective, and Accurate Protein Sizing in Human Plasma, Applied to the von Willebrand Factor.

Multimeric abnormalities in plasma von Willebrand factor (VWF) cause bleeding or clotting disorders. Electrophoretic analysis of multimers is used to detect such abnormalities but is qualitative, slow, and difficult to standardize. Fluorescence correlation spectroscopy (FCS) is a good alternative but is affected by low selectivity and concentration bias. Here, we report the development of a homogeneous immunoassay based on dual-color fluorescence cross-correlation spectroscopy (FCCS) that overcomes these challenges. By performing a mild denaturation treatment followed by reacting with polyclonal antibodies, the concentration bias was drastically reduced. The use of a dual antibody assay improved selectivity. Diffusion times of immunolabeled VWF were measured with FCCS and standardized relative to calibrator measurements. The assay measures size changes in VWF using 1 μL of plasma and less than 10 ng of antibody per measurement and was validated over a 16-fold range of VWF antigen concentration (VWF:Ag), with a sensitivity of VWF:Ag 0.8%. Concentration bias and imprecision were less than 10%. Measurements were unaffected by hemolytic, icteric, or lipemic interference. Strong correlations were obtained with reference densitometric readouts (0.97 for calibrators, 0.85 for clinical samples), and significant differences were found between normal (n = 10), type 2A (n = 5), and type 2B (n = 5) von Willebrand's disease and acquired thrombotic thrombocytopenic purpura (n = 10) samples (p < 0.01). This FCCS based immunoassay accurately and selectively determines changes in the multimeric status of plasma VWF and may be used as a simpler, faster, and a standardizable alternative for multimer analysis, following further clinical validation in larger cohorts.

Double cervices in a Gypsy Vanner mare

A 7-year, purebred Gypsy Vanner, maiden mare was presented with abnormal cervical tissue that was detected during prebreeding examination. External vulvar examination, transrectal and ultrasonographic examination of uterus, and internal vaginal vault and cervical examination (via speculum and endoscopy) were conducted. Karyotype analysis included sex chromosome study with dual-color fluorescence in situ hybridization and polymerase chain reaction for SRY gene. Mare had normal 64,XX karyotype with no evidence of mosaicism. Two distinct, patent, cervical ora separated by a frenulum, leading to a single uterine body, were observed. It was recommended to avoid breeding because of dystocia risk. To the authors’ knowledge, this is the first reported case of this nature in a mare.

Green synthesis of glyco-CuInS2 QDs with visible/NIR dual emission for 3D multicellular tumor spheroid and in vivo imaging

Glyco-quantum dots (glyco-QDs) have attracted significant interest in bioimaging applications, notably in cancer imaging, because they effectively combine the glycocluster effect with the exceptional optical properties of QDs. The key challenge now lies in how to eliminate the high heavy metal toxicity originating from traditional toxic Cd-based QDs for in vivo bioimaging. Herein, we report an eco-friendly pathway to prepare nontoxic Cd-free glyco-QDs in water by the “direct” reaction of thiol-ending monosaccharides with metal salts precursors. The formation of glyco-CuInS2 QDs could be explained by a nucleation-growth mechanism following the LaMer model. As-prepared four glyco-CuInS2 QDs were water-soluble, monodispersed, spherical in shape and exhibited size range of 3.0–4.0 nm. They exhibited well-separated dual emission in the visible region (500–590 nm) and near-infrared range (~ 827 nm), which may be attributable to visible excitonic emission and near-infrared surface defect emission. Meanwhile, the cell imaging displayed the reversibly distinct dual-color (green and red) fluorescence in tumor cells (HeLa, A549, MKN-45) and excellent membrane-targeting properties of glyco-CuInS2 QDs based on their good biorecognition ability. Importantly, these QDs succeed in penetrating uniformly into the interior (the necrotic zone) of 3D multicellular tumor spheroids (MCTS) due to their high negative charge (zeta potential values ranging from − 23.9 to − 30.1 mV), which overcame the problem of poor penetration depth of existing QDs in in vitro spheroid models. So, confocal analysis confirmed their excellent ability to penetrate and label tumors. Thus, the successful application in in vivo bioimaging of these glyco-QDs verified that this design strategy is an effective, low cost and simple procedure for developing green nanoparticles as cheap and promising fluorescent bioprobes.

Construction of an Endogenously Activated Signal Amplification Assay for In Situ Imaging of MicroRNA and Guided Precise Photodynamic Therapy for Cancer Cells.

The construction of a sensitive strategy for in situ visualizing and dynamic tracing intracellular microRNA is of great importance. Via the toehold-mediated strand displacement process, the catalytic hairpin assembly (CHA) could offer several hundreds-fold signal amplifications. However, the CHA may produce certain background interferences since microRNA may exist in normal cells. In this work, we constructed an endogenously and sequentially activated signal amplification strategy to provide the amplified dual-color fluorescence imaging of microRNA in living cancer cells, which was comprised of two successive reaction processes: the activation of the preprotective catalytic probe by the endogenous glutathione (GSH) and the subsequent catalytic hairpin assembly on the surface of the upconversion nanoprobe triggered by the specific microRNA. Since the concentration of GSH in cancer cells was much higher than that in normal cells and the extracellular environment, the activation of the designed nanoprobe could be controlled at the desirable site. With the merits of the endogenous initiation and selective activation, the designed nanoprobe could achieve the bioimaging of microRNA in living cancer cells with high precision and reliability. Furthermore, via the introduction of a photosensitizer molecule into the DNA strand, the designed nanoplatform could achieve the precise photodynamic therapy (PDT) for cancer cells and malignant tumors under the irradiation of the NIR laser. This work provided a new avenue to achieve the accurate imaging of intracellular microRNA and guided precise PDT, which would offer powerful hints to the early diagnosis and therapy of malignant tumors.

Ultrathin C3N4 nanosheets-based oxidase-like 2D fluorescence nanozyme for dual-mode detection of organophosphorus pesticides

Engineering efficient dual-mode portable sensor with built-in cross reference correction is of great significance for onsite reliable and precise detection of organophosphorus pesticides (OPs) and evading the false-positive outputs, especially in emergency case. Currently, most nanozyme-based sensors for OPs monitoring primarily replied on the peroxidase-like activity, which involved unstable and toxic H2O2. In this scenario, a hybrid oxidase-like 2D fluorescence nanozyme (PtPdNPs@g-C3N4) was yielded by in situ growing PtPdNPs in the ultrathin two-dimensional (2D) graphitic carbon nitride (g-C3N4) nanosheet. When acetylcholinesterase (AChE) hydrolyzed acetylthiocholine (ATCh) to thiocholine (TCh), it ablated O2−• from the dissolved O2 catalyzed by PtPdNPs@g-C3N4’s oxidase-like activity, hampering the oxidation of o-phenylenediamine (OPD) into 2,3-diaminophenothiazine (DAP). Consequently, with the increasing concentration of OPs which inhibited the blocking effect by inactivating AChE, the produced DAP caused an apparent color change and a dual-color ratiometric fluorescence change in the response system. Through integrating into a smartphone, a H2O2-free 2D nanozyme-based onsite colorimetric and fluorescence dual-mode visual imaging sensor for OPs was proposed with acceptable results in real samples, which holds vast promise for further development of commercial point-of-care testing platform in early warning and controlling of OPs pollution for safeguarding environmental health and food safety.

Deep learning-assisted smartphone-based portable and visual ratiometric fluorescence device integrated intelligent gel label for agro-food freshness detection

Here, a smartphone-assisted dual-color ratiometric fluorescence smart gel label-based visual sensing platform was constructed for real-time evaluation of the freshness of agro-food based on the biogenic amines responses. Green-emission fluorescence carbon dots (CDs) coupled with blue-emission fluorescence bimetallic metal–organic framework (Fe/Zr-MOF) obtained dual-color CDs@Fe/Zr-MOF fluorescence nanoprobe acts as the response units. With the increase of SP and HIS content, the green fluorescence of CDs was enhanced, while the blue fluorescence of Fe/Zr-MOF was quenched. Therefore, this dual-color probe achieved a clear fluorescence color response to biogenic amines. The nanoprobe possessed sensitive and color-responsive with the LODs of 0.17 μM for SP and 2.95 μM for HIS in a wide range of 0–937.5 µM, respectively. Besides, these fluorescent nanoprobes were immobilized on the hydrogel carrier, and the intelligent fluorescent hydrogel tag can be obtained after freeze-drying, which realizes the real-time qualitative monitoring of SP and HIS in pork and shrimp samples.

Targeted photothrombotic subcortical small vessel occlusion using in vivo real-time fiber bundle endomicroscopy in mice.

The development of an accurate subcortical small vessel occlusion model for pathophysiological studies of subcortical ischemic stroke is still insignificant. In this study, in vivo real-time fiber bundle endomicroscopy (FBEµ) was applied to develop subcortical photothrombotic small vessel occlusion model in mice with minimal invasiveness. Our FBFµ system made it possible to precisely target specific blood vessels in deep brain and simultaneously observe the clot formation and blood flow blockage inside the target blood vessel during photochemical reactions. A fiber bundle probe was directly inserted into the anterior pretectal nucleus of the thalamus in brain of live mice to induce a targeted occlusion in small vessels. Then, targeted photothrombosis was performed using a patterned laser, observing the process through the dual-color fluorescence imaging. On day one post occlusion, infarct lesions are measured using TTC staining and post hoc histology. The results show that FBEµ applied to targeted photothrombosis can successfully generate a subcortical small vessel occlusion model for lacunar stroke.

Highly sensitive and specific detection of silver ions using a dual-color fluorescence co-localization strategy.

Ag+ ions are widely used in various fields of human life due to their unique properties and they threaten the environment and human health. The traditional methods for Ag+ detection commonly suffer from disadvantages including limited sensitivity, expensive equipment and complicated operating steps. Herein, we developed a highly specific dual-color fluorescence co-localization (DFC) strategy based on the C-Ag+-C structure for Ag+ detection. In this strategy, Ag+ ions can be captured to form C-Ag+-C base pairs, and these ions enable single-stranded DNAs to form double strands. The DFC strategy can exclude nonspecific interaction sites and greatly improve the sensitivity and specificity. By DFC of the QDs and Cy5 linked to the DNA strands, highly sensitive Ag+ detection was achieved in the concentration range from 0.14 pM to 200 nM, with a limit of detection (LOD) of 0.14 pM. Moreover, this method has been applied for the detection of Ag+ ions in real environmental samples with satisfactory recoveries. We believe that the DFC strategy is promising for Ag+ detection.

Multiplex Digital Polymerase Chain Reaction on a Droplet Array SlipChip for Analysis of KRAS Mutations in Pancreatic Cancer.

Pancreatic cancer is a terminal disease with high mortality and very poor prognosis. A sensitive and quantitative analysis of KRAS mutations in pancreatic cancer provides a tool not only to understand the biological mechanisms of pancreatic cancer but also for diagnosis and treatment monitoring. Digital polymerase chain reaction (PCR) is a promising tool for KRAS mutation analysis, but current methods generally require a complex microfluidic handling system, which can be challenging to implement in routine research and point-of-care clinical diagnostics. Here, we present a droplet-array SlipChip (da-SlipChip) for the multiplex quantification of KRAS G12D, V, R, and C mutant genes with the wild-type (WT) gene background by dual color (FAM/ROX) fluorescence detection. This da-SlipChip is a high-density microwell array of 21,696 wells of 200 pL in 4 by 5424 microwell format with simple loading and slipping operation. It does not require the same precise alignment of microfeatures on the different plates that are acquired by the traditional digital PCR SlipChip. This device can provide accurate quantification of both mutant genes and the WT KRAS gene. We collected tumor tissue, paired normal pancreatic tissue, and other normal tissues from 18 pancreatic cancer patients and analyzed the mutation profiles of KRAS G12D, V, R, and C in these samples; the results from the multiplex digital PCR on da-SlipChip agree well with those of next-generation sequencing (NGS). This da-SlipChip moves digital PCR closer to the practical point-of-care applications not only for detecting KRAS mutations in pancreatic cancer but also for other applications that require precise nucleic acid quantification with high sensitivity.

Off-the-shelf GMP-grade UC-MSCs as therapeutic drugs for the amelioration of CCl4-induced acute-on-chronic liver failure in NOD-SCID mice.

Umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs) are advanced therapy medicinal products (ATMPs) and thus act as an alternative to liver transplantation for acute-on-chronic liver failure (ACLF). Therewith, we are aiming to evaluate the pharmacologyandpharmacokinetics of GMP-grade UC-MSCs products on carbon tetrachloride (CCl4)-induced ACLF mouse model and the concomitant therapeutic dose for intravenous administration. For the purpose, the GMP-grade UC-MSCs products were transplanted intravenously into the aforementioned CCl4-induced ACLF NOD-SCID mouse model, and the therapeutic effect was evaluated with the aid of serological, biochemical and histological assessments. Meanwhile, the correlationshipbetween the treatment groups and other characteristics were determined by conducting principal component analysis (PCA). To further verify the spatio-temporal pharmacokinetics of UC-MSCs products on ACLF treatment, we took advantage of the bioluminescence imaging (BLI) technology with the dual-color fluorescence reporter construct (pLV-Fluc-eGFP). The biological characteristics of UC-MSCs products were in conformity with the International Society of Cell Therapy (ISCT) criteriaand the GMP requirements. ACLF mice with high dose of UC-MSCs treatment revealed significantly alleviated pathological manifestations with a dramatically improved survival rate, the alleviation of liver injury with reduced hepatic enzyme, inflammatory infiltration and inflammatory cytokines. Notably, UC-MSCs in ACLF mice displayed preferable homing and delayed attenuation in the damaged liver tissue. Collectively, our data indicated the feasibility of UC-MSC-based cytotherapy for ACLF model administration. Our findings have provided new references for pharmacologyandpharmacokinetics assessments, which will provide overwhelming evidence for pre-clinical study in vivo.