Bioactive Layered Double Hydroxides for Synergistic Sonodynamic/Cuproptosis Anticancer Therapy with Elicitation of the Immune Response.

Sonodynamic therapy (SDT) has promising application prospects in tumor therapy. However, SDT does not eradicate metastatic tumors. Herein, Cu-substituted ZnAl ternary layered double hydroxide nanosheets (ZCA NSs) were developed as both sonosensitizers and copper nanocarriers for synergistic SDT/cuproptosis cancer therapy. An optimized electronic structure more conducive to the sonodynamic process was obtained from ZCA NSs via the Jahn-Teller effect induced by the introduction of Cu2+, and the synthesized ZCA NSs regulated the intricate tumor microenvironment (TME) by depleting endogenous glutathione (GSH) to amplify oxidative stress for further enhanced SDT performance. Furthermore, cuproptosis was evoked by intracellular overload of Cu2+ and amplified by SDT, leading to irreversible proteotoxicity. In vitro results showed that such synergetic SDT/cuproptosis triggered immunogenic cell death (ICD) and promoted the maturation of dendritic cells (DCs). Furthermore, the as-synthesized ZCA NS-mediated SDT/cuproptosis thoroughly eradicated the in vivo solid tumors and simultaneously elicited antitumor immunity to suppress lung and liver metastasis. Overall, this work established a nanoplatform for synergistic SDT/cuproptosis with a satisfactory antitumor immunity.

Guanidinylated/PEGylated chitosan in the bioink promotes the formation of multi-layered keratinocytes in a human skin equivalent.

The biological differences of skin between rodent and human beings and the strong appeal to replace the experimental animals have led to the development of alternative models with structures similar to the real human skin. Keratinocytes cultured in vitro on conventional dermal scaffolds tend to form monolayer rather than multi-layer epithelial tissue architectures. How to construct human skin or epidermal equivalents with multi-layered keratinocytes similar to real human epidermis remains one of the greatest challenges. Herein, a human skin equivalent with multi-layered keratinocytes was constructed by 3D bioprinting fibroblasts and subsequent culturing epidermal keratinocytes. Biocompatible guanidinylated/PEGylated chitosan (GPCS) was used as the main component of bioink to 3D bioprint tissue-engineered dermis. The function of GPCS to promote HaCat cell proliferation and connection was confirmed at the genetic, cellular, and histological levels. Compared with the skin tissues with mono-layered keratinocytes engineered with collagen and gelatin, adding GPCS in the bioink generated tissue-engineered human skin equivalents with multi-layered keratinocytes. Such human skin equivalents could be alternative models for biomedical, toxicological, and pharmaceutical research.

3D-printable supramolecular hydrogels with shear-thinning property: fabricating strength tunable bioink via dual crosslinking.

3-dimensional (3D) bioprinting technology provides promising strategy in the fabrication of artificial tissues and organs. As the fundamental element in bioprinting process, preparation of bioink with ideal mechanical properties without sacrifice of biocompatibility is a great challenge. In this study, a supramolecular hydrogel-based bioink is prepared by polyethylene glycol (PEG) grafted chitosan, α-cyclodextrin (α-CD) and gelatin. It has a primary crosslinking structure through the aggregation of the pseudo-polyrotaxane-like side chains, which are formed from the host-guest interactions between α-CD and PEG side chain. Apparent viscosity measurement shows the shear-shinning property of this bioink, which might be due to the reversibility of the physical crosslinking. Moreover, with ß-glycerophosphate at different concentrations as the secondary crosslinking agent, the printed constructs demonstrate different Young's modulus (p < 0.001). They could also maintain the Young's modulus in cell culture condition for at least 21 days (p < 0.05). By co-culturing each component with fibroblasts, CCK-8 assay demonstrate cellular viability is higher than 80%. After bioprinting and culturing, immunofluorescence staining with quantification indicate the expression of Ki-67, Paxillin, and N-cadherin is higher in day 14 than those in day 3 (p < 0.05). Oil red O and Nissl body specific staining reflect strength tunable bioink may have impact on the cell fate of mesenchymal stem cells (p < 0.05). This work might provide new idea for advanced bioink in the application of re-establishing complicated tissues and organs.

Enzymatically degradable alginate/gelatin bioink promotes cellular behavior and degradation in vitro and in vivo.

Bioink is of paramount importance in the process of three-dimensional extrusive bioprinting technology. Alginate is extensively used in cell-laden extrusive bioprinters with the advantage of biocompatibility, gelling and crosslinking features; however, the bioinert properties of alginate made it hard to degrade in vivo, and restrict cellular adhesion, extension and migration. In this study, we incorporated two concentrations of alginate lyase (0.5 mU ml-1 and 5 mU ml-1) into alginate/gelatin bioink to improve its degradation properties and effects on cellular behavior. The enzymatically degradable bioink demonstrated lower stiffness and higher porosity. Cellular proliferation, adhesion and extension were facilitated in the degradable bioink without sacrifice of cell viability. Additionally, the property of degradation still worked in vivo, with cellular infiltration and retention being observed in the grafted bioprinted constructs. The results suggest that alginate lyase could be incorporated into alginate/gelatin bioink. Degradation properties and cellular behavior could be promoted both in vitro and in vivo, providing a new avenue for the upgrade and modification of alginate-based bioink for further applications.

Two distinct conformers of SAG investigated by solution NMR and variable-temperature experiments.

SAG (Smo agonist) is high specific agonist of Smo receptor, it is widely used as chemical probe to explore the therapeutic value of activating the Hh-signaling pathway. In the present work, the coexistence of two distinct conformers (A and B) of SAG are revealed by solution NMR, the 3D structural difference of the two conformers are elucidated by ROESY spectroscopy and MMFF94 program. It is discovered that both conformers are agonists of Smo receptor, and the minor conformer (conformer B) in D2O solution has higher affinity to Smo receptor by molecular docking. The result showed the detail about two distinct conformers of SAG are involved in activation of Smo receptor, and also provides information for designing more effective agonist of Smo by mimicking B conformer of SAG. By exchange dynamics investigation using variable-temperature NMR experiments, the ratio of two conformers has been shown to be drastically solvent dependent, so, the menstruum type is another important influence factor of SAG bioactivity, when using SAG as chemical probe.

Validation of a solid-phase extraction and ultra-performance liquid chromatographic tandem mass spectrometric method for the detection of 16 glucocorticoids in pig tissues.

A sensitive analytical method has been developed and validated for simultaneous determination of 16 glucocorticoid (fluorometholone, flumethasone, triamcinolone, aldosterone, clobetasol propionate, methylprednisolone, fluocinolone acetonide, hydrocortisone, prednisone, dexamethasone, beclomethasone, prednisolone, budesonide, triamcinolone acetonide, fludrocortisone acetate, and cortisone) residues in pig tissues (muscle, liver, and kidney). These biosamples were hydrolyzed with beta-glucuronidase/arylsulfatase enzyme and passed through a Supelclean ENVI-Carb graphitized carbon black solid-phase extraction cartridge, followed by further purification using aminopropyl cartridges. Analytes were separated on an ultra-performance liquid chromatography BEH C18 column followed by tandem mass spectrometry (MS) with an electrospray ion source. The MS data acquisition was performed in the negative multireaction monitoring mode by a time-scheduled multireaction monitoring program. The assay for the 16 glucocorticoids were linear over the range of 1-250 microg/L for pork, liver, and kidney, with correlation coefficient >0.99. Estimated detection limits for the target analytes ranged from 0.03 to 0.30 microg/kg, and limits of quantitation ranged from 0.10 to 1.00 microg/kg. Recoveries of the glucocorticoids (spiked at levels of 0.4 and 2.0 microg/kg) ranged from 81.0 to 112.3%, with relative standard deviations between 2.6 and 16.6%.

[Simultaneous determination of twelve glucocorticoids residues in milk by ultra performance liquid chromatography-electrospray tandem mass spectrometry].

A comprehensive analytical method based on ultra performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS) with negative mode has been developed for the simultaneous determination of twelve glucocorticoids residues in milk. The multi-reaction monitoring mode was employed for the determination. Milk samples were extracted by sonication in a methanol/acetate buffer (pH 5.20) solution, and then defatted with n-hexane. Sample concentration and purification were performed using Oasis HLB, Sep-pak silica and Sep-pak amino-propyl solid phase extraction cartridges. The separation was performed on a Waters ACQUITY UPLC BEH C18 column (100 mm x 1.0 mm i. d., 1.7 microm) with gradient elution using methanol and water (containing 0.1% formic acid) at a flow rate of 0.1 mL/min. Identification of the glucocorticoids was done using retention times and the distribution of diagnostic ion pairs. Quantification of the glucocorticoids was based on the peak areas of the parent ion and a fragment ion with a higher signal. The limits of detection (LOD) of the method were from 0.02 to 0.38 microg/kg and the limits of quantification (LOQ) ranged from 0.07 to 1.27 microg/kg. Average recoveries for the twelve glucocorticoids (spiked at the levels of 2 and 0.4 microg/kg) ranged from 69.3% to 94.3%, with relative standard deviations between 3.5% and 16.7%. Routine tests showed that the method is fast, sensitive and specific for the determination of glucocorticoids residues in milk.

Simultaneous determination of seventeen glucocorticoids residues in milk and eggs by ultra-performance liquid chromatography/electrospray tandem mass spectrometry.

A comprehensive analytical method has been developed and validated for the simultaneous determination of seventeen glucocorticoid residues in eggs and milk. The mass spectrometer parameters, the composition of the mobile phase and the sample preparation method were firstly optimized to obtain maximum sensitivity. The samples were deconjugated with beta-glucuronidase/arylsulfatase enzyme and concentrated using an Oasis HLB solid-phase extraction cartridge, followed by cleanup with a dual Sep-pak silica and aminopropyl cartridge. The analytes were quantified by ultra-performance liquid chromatography (using a C18 column)/electrospray ionization tandem mass spectrometry (UPLC/ESI-MS/MS) operating in the negative ion mode. The assay for the 17 glucocorticoids was linear over the range of 1-200 microg/L for milk and egg samples with a high correlation coefficient (>0.99). The limits of quantification (LOQs) for the target analytes were 0.04-1.27 microg/kg for the egg samples and 0.03-0.73 microg/kg for the milk samples. The average extraction recoveries of the glucocorticoids from eggs and milk at two concentration levels (spiked at 0.40 and 2.00 microg/kg) were 65.6-118.7% and 61.5-119.6%, respectively, with relative standard deviations between 1.8-17.0% and 2.4-18.4%, respectively. Because of its high sensitivity, good precision and specificity, the method was found to be suitable for trace analysis of synthetic and natural glucocorticoids in complex biosamples such as eggs and milk.