Plant exosomes fused with engineered mesenchymal stem cell-derived nanovesicles for synergistic therapy of autoimmune skin disorders.

Existing therapeutics for autoimmune diseases remain problematic due to low efficacy, severe side effects, and difficulties to reach target tissues. Herein, we design multifunctional fusion nanovesicles that can target lesions for the treatment of autoimmune skin diseases. The grapefruit-derived exosome-like nanovesicles (GEVs) with anti-inflammatory and antioxidant effects are first encapsulated with CX5461, an immunosuppressant with anti-proliferative properties to form GEV@CX5461. In order to enhance therapeutic efficiency and safety, GEV@CX5461 are then fused with CCR6+ nanovesicles derived from membranes of engineered gingiva-derived mesenchymal stem cells (GMSCs). The resulting FV@CX5461 not only maintain the bioactivity of GEVs, CX5461, and GMSC membranes but also home to inflamed tissues rich in chemokine CCL20 through the chemotaxis function of CCR6 on FVs. Moreover, FV@CX5461 reduce the secretion of inflammatory factors, calm down Th17 cell activation, and induce Treg cell infiltration. Finally, impressive therapeutic efficiency in both psoriasis and atopic dermatitis disease models is demonstrated using FV@CX5461 to reshape the unbalanced immune microenvironment. A nanotherapeutic drug delivery strategy is developed using fusion nanovesicles derived from plant and animal cells with high clinical potential.

Lab in hydrogel portable kit: On-site monitoring of oxalate.

Oxalate is commonly employed as adjuvant of pesticide agent, causing renal injury of human even in trace residues. Despite the great achievements of the existing point-of-care testing (POCT) technology, accurate on-site screening of oxalate remains a tricky issue. To this aim, we proposed a "lab in a tube" platform which integrated portable hydrogel kit with smartphone for real-time monitoring of oxalate to achieve quantitatively precise analysis. In this work, a stimuli-responsive hydrogel-based kit was constructed via embedding manganese dioxide (MnO2) nanosheets into sodium alginate hydrogel system. Based on the intrinsic oxidase-like activity, MnO2 nanosheets-based nanozyme triggered color reaction by introducing a common sensing probe 3,3',5,5'-tetramethylbenzidine. Meanwhile, the presence of oxalate would decompose MnO2 nanosheets, inducing the decrease of nanozyme activity, which resulted in the color response of portable kit. Coupling with ImageJ software, the image information of kit captured via smartphone could be transduced into the hue intensity, which provided a directly quantitative tool to detect oxalate with a detection limit of 8.0 µmol L-1. This portable smartphone biosensor was successfully applied for screening urine sample within 10 min for high-throughput analysis (twelve samples) without the need for any advanced analytical instruments. Based on the merits of simple operation, cost-efficiency, and good selectivity, the availability of the miniaturized biosensor platform for POCT will achieve the requirements of routine screening and disease prevention.

Ratio fluorescence analysis of T4 polynucleotide kinase activity based on the formation of a graphene quantum dot-copper nanocluster nanohybrid.

In this work, a ratio fluorescence method was developed for T4 polynucleotide kinase (PNK) activity analysis based on the formation of a dual-emitting graphene quantum dot-copper nanocluster (GQD-CuNC) nanohybrid. An amino capped single-strand DNA (ssDNA) was firstly used to modify GQDs (GQD-ssDNA) and then hybridize with its complementary DNA strand to form double-stranded DNA functionalized GQDs (GQD-dsDNA). The dsDNA of GQD-dsDNA can act as an effective template for the preparation of CuNCs with fluorescence emission at 594 nm. When the dsDNA of GQD-dsDNA was phosphorylated through T4 PNK and subsequently degraded via λ exonuclease (λ exo) to produce mononucleotides and GQD-ssDNA, the formation of fluorescence CuNCs in GQD-CuNCs was blocked due to the lack of an effective dsDNA substrate, during which the fluorescence of GQDs at 446 nm in the nanohybrid was mostly not influenced. Thus, with the CuNCs serving as the reporter and GQDs as the reference signal, T4 PNK activity can be monitored through the change in the fluorescence intensity ratio (F594/F446) in the range of 0.01-10 U mL-1 with a detection limit (LOD) of 0.0037 U mL-1. Furthermore, the practicality of this T4 PNK activity analysis strategy in a complex sample was tested in cell lysates.

Copper nanoclusters/polydopamine nanospheres based fluorescence aptasensor for protein kinase activity determination.

A fluorescence aptasensor was constructed for protein kinase (PKA) activity detection by utilizing copper nanoclusters (CuNCs) and polydopamine nanospheres (PDANS). Through the π-π stacking interactions between adenosine triphosphate (ATP) aptamer and PDANS, the ATP aptamer modified CuNCs (apt-CuNCs) were absorbed onto PDANS surface, thus the fluorescence of apt-CuNCs were quenched through fluorescence resonance energy transfer (FRET) from apt-CuNCs to PDANS. In the presence of ATP, ATP specifically bound to aptamer, causing the dissociation of apt-CuNCs from PDANS surface and restoring the fluorescence of apt-CuNCs. However, PKA translated ATP into adenosine diphosphate (ADP), and ADP had no competence to combine with ATP aptamer, thus, apt-CuNCs were released and absorbed onto the PDANS surface to cause the fluorescence quenching of apt-CuNCs again. Therefore, PKA activity was conveniently detected via the fluorescence signal change. Under the optimal conditions, PKA activity was detected in the range of 0.05-4.5 U mL-1 with a detection limit of 0.021 U mL-1. Furthermore, the feasibility of the aptasensor for kinase inhibitor screening was explored via assessment of kinase inhibitor H-89 as one model. This aptasensor was also performed for PKA activity determination in HepG2 cell lysates with satisfactory results.

Pharmacokinetic comparisons of six components from raw and vinegar-processed Daphne genkwa aqueous extracts following oral administration in rats by employing UHPLC-MS/MS approaches.

A sensitive UHPLC-MS/MS approach was developed and validated for the quantification of genkwanin, 3'-hydroxygenkwanin, apigenin, luteolin, yuanhuacine and genkwadaphnin in biological samples after oral administration of raw and vinegar-processed Daphne genkwa. Liquiritin and glycyrrhetinic acid were employed as internal standards. Six components were extracted by using protein precipitation with acetonitrile. Chromatographic separation was achieved on a Waters BEH C18 column (50 mm × 2.1 mm, 1.7 µm) by using a mobile phase composed of water (containing 0.1% formic acid) and acetonitrile. Mass spectrometric detection was conducted using multiple reaction monitoring (MRM). The intra- and inter-day precisions of the six analytes were below 4.87%, and the accuracies were within ±5.0%. The extraction recoveries of the six constituents were determined between 97.5% and 105.4% and the matrix effects ranged from 97.3% to 103.7%. All the samples showed satisfactory precision and accuracy after various stability tests. The established approach was successfully applied to the comparative pharmacokinetic study. Compared to the raw group, the parameters of Cmax and AUC0-t of genkwanin, 3'-hydroxygenkwanin, apigenin and luteolin elevated remarkably (p < 0.05) after oral delivery of vinegar-processed Daphne genkwa while the parameters of Cmax and AUC0-t of yuanhuacine and genkwadaphnin decreased significantly (p < 0.05). The results revealed that vinegar-processing could enhance bioavailability of genkwanin, 3'-hydroxygenkwanin, apigenin and luteolin but reduce the bioavailability of yuanhuacine and genkwadaphnin.

UHPLC-MS/MS quantification combined with chemometrics for the comparative analysis of different batches of raw and wine-processed Dipsacus asper.

A rapid and sensitive ultra-high performance liquid chromatography with tandem mass spectrometry approach was established for the simultaneous determination of 4-caffeoylquinic acid, loganic acid, chlorogenic acid, loganin, 3,5-dicaffeoylquinic acid, dipsacoside B, asperosaponin VI, and sweroside in raw and wine-processed Dipsacus asper. Chloramphenicol and glycyrrhetinic acid were employed as internal standards. The proposed approach was fully validated in terms of linearity, sensitivity, precision, repeatability as well as recovery. Intra- and interassay variability for all analytes were 2.8-4.9 and 1.7-4.8%, respectively. The standard addition method determined recovery rates for each analytes (96.8-104.6%). In addition, the developed approach was applied to 20 batches of raw and wine-processed samples of Dipsacus asper. Principle component analysis and partial least squares-discriminate analysis revealed a clear separation between the raw group and wine-processed group. After wine-processing, the contents of loganic acid, chlorogenic acid, dipsacoside B, and asperosaponin VI were upregulated, while the contents of 3,5-dicaffeoylquinic acid, 4-caffeoylquinic acid, loganin, and sweroside were downregulated. Our results demonstrated that ultra-high performance liquid chromatography with tandem mass spectrometry quantification combined with chemometrics is a viable method for quality evaluation of the raw Dipsacus asper and its wine-processed products.