Intrahippocampal Supramolecular Assemblies Directed Bioorthogonal Liberation of Neurotransmitters to Suppress Seizures in Freely Moving Mice.

The precise delivery of anti-seizure medications (ASM) to epileptic loci remains the major challenge to treat epilepsy without causing adverse drug reactions. The unprovoked nature of epileptic seizures raises the additional need to release ASMs in a spatiotemporal controlled manner. Targeting the oxidative stress in epileptic lesions, here the reactive oxygen species (ROS) induced in situ supramolecular assemblies that synergized bioorthogonal reactions to deliver inhibitory neurotransmitter (GABA) on-demand, are developed. Tetrazine-bearing assembly precursors undergo oxidation and selectively self-assemble under pathological conditions inside primary neurons and mice brains. Assemblies induce local accumulation of tetrazine in the hippocampus CA3 region, which allows the subsequent bioorthogonal release of inhibitory neurotransmitters. For induced acute seizures, the sustained release of GABA extends the suppression than the direct supply of GABA. In the model of permanent damage of CA3, bioorthogonal ligation on assemblies provides a reservoir of GABA that behaves prompt release upon 365 nm irradiation. Incorporated with the state-of-the-art microelectrode arrays, it is elucidated that the bioorthogonal release of GABA shifts the neuron spike waveforms to suppress seizures at the single-neuron precision. The strategy of in situ supramolecular assemblies-directed bioorthogonal prodrug activation shall be promising for the effective delivery of ASMs to treat epilepsy.

Reductase-Labile Peptidic Supramolecular Hydrogels Aided in Oral Delivery of Probiotics.

Oral delivery of probiotics has been a promising method for treatment of inflammatory bowel diseases (IBDs). However, probiotics always suffer from substantial loss of viability due to the harsh gastrointestinal conditions, especially the highly acidic environment in the stomach and bile salts in the intestine. In addition, to overcome the challenging conditions, an ideal delivery of probiotics requires the on-demand release of probiotics upon environmental response. Herein, a novel nitroreductase (NTR) labile peptidic hydrogel based on supramolecular self-assembly is demonstrated. The efficient encapsulation of typical probiotic Escherichia coli Nissle 1917 (EcN) into supramolecular assemblies yielded a probiotic-loaded hydrogel (EcN@Gel). Such a hydrogel adequately protected EcN to improve its viability against harsh acid and bile salt environments during oral delivery. The upregulated NTR in the intestinal tract triggered the disassembly of the hydrogel and accomplished the controlled release of EcN locally. In ulcerative colitis (UC)-bearing mice, EcN@Gel showed significantly enhanced therapeutic efficacy by downregulating proinflammatory cytokines and repairing the intestinal barrier. Moreover, EcN@Gel remolded the gut microbiome by increasing the diversity and abundance of indigenous probiotics, contributing to ameliorated therapies of IBDs. The NTR-labile hydrogel provided a promising platform for the on-demand delivery of probiotics into the intestinal tract.

Bioorthogonal Disassembly of Tetrazine Bearing Supramolecular Assemblies Inside Living Cells.

Supramolecular assemblies are an emerging class of nanomaterials for drug delivery systems (DDS), while their unintended retention in the biological milieu remains largely unsolved. To realize the prompt clearance of supramolecular assemblies, the bioorthogonal reaction to disassemble and clear the supramolecular assemblies within living cells is investigated here. A series of tetrazine-capped assembly precursors which can self-assemble into nanofibers and hydrogels upon enzymatic dephosphorylation are designed. Such an enzyme-instructed supramolecular assembly process can perform intracellularly. The time-dependent accumulation of assemblies elicits oxidative stress and induces cellular toxicity. Tetrazine-bearing assemblies react with trans-cyclooctene derivatives, which lead to the disruption of π-π stacking and induce disassembly. In this way, the intracellular self-assemblies disassemble and are deprived of potency. This bioorthogonal disassembly strategy leverages the biosafety aspect in developing nanomaterials for DDSs.

Enzymatic non-covalent synthesis of supramolecular assemblies as a general platform for bioorthogonal prodrugs activation to combat drug resistance.

Multi-drug resistance (MDR) is one of the leading causes of the anticancer failures. Besides the blockage of the MDR pathways, the development of more potent drugs is with urgent needs, but has been postponed mainly due to an imbalance between safety and efficacy. The recent development of the bioorthogonal prodrug activation strategy has shown immense potential to balance safety and efficacy, while recent studies only focused on few drug entities such as doxorubicin and monomethyl auristatin E, leaving the vast collection of toxins undetermined. Here we have enumerated typical molecular entities ranging from food and drug administration (FDA) approved drugs to a heated antibody drug conjugates (ADC) warhead and a trichothecene toxin to demonstrate that the bioorthogonal caging and specific activation could serve as a general design to increase the therapeutic index of bioactive molecules. These prodrugs can be efficiently activated on-demand by the bioorthogonal activators whose distribution was regulated by the cancer cell specific enzymatic non-covalent synthesis of supramolecular self-assemblies. The prodrug activation not only enhanced the synergistic therapeutic effect within a broad range of dose ratios but also allowed the convenient switching of drug identities to successfully combat MDR tumor in vivo. In general, this strategy might serve as a general platform, which can be readily applicable to enlarge the therapeutic window for various bioactive molecules. We envision that the spatiotemporal controlled bioorthogonal prodrug activation would facilitate the discovery of anticancer drugs.

Enzyme-instructed supramolecular assemblies promote intracellular boron accumulation for boron neutron capture therapy.

Selective accumulation of boron agents in cancer cells is of critical importance for BNCT. Here we involve enzyme-instructed supramolecular assembly (EISA) to facilitate the accumulation of a typical boron agent borylphenylalanine (BPA) in cancer cells. By covalently conjugating BPA to the phosphorylated assembly precursor, the boron-bearing precursors undergo phosphatase-catalyzed dephosphorylation to yield assembly molecules, which then self-assemble to form nanomaterials. Due to the up-regulated phosphatase activity of cancer cells, kinetic preference allows the EISA to accumulate boron in HeLa cells selectively. Interestingly, by attaching BPA on the backbone or side-chain of precursor, the boron-bearing isomers show different assembly propensity with time-dependent morphology change, which leads to the differentiated accumulation of boron inside cells. Overall, the optimized boron-bearing assembly precursor could significantly improve the boron accumulation compared with BPA in cancer cells. In this study, we have demonstrated a convenient method to introduce boron agents to cancer cells. We envision that the EISA-mediated accumulation of boron will be helpful in the design of boron agents to facilitate BNCT treatment.

Preparation of Nitrogen-Doped Mesoporous Carbon for the Efficient Removal of Bilirubin in Hemoperfusion.

The development of bilirubin adsorbent with high selectivity, brilliant adsorption ability, and biocompatibility is still a considerable challenge. In this study, a copolymer-templated nitrogen-doped mesoporous carbon (CTNC) has been prepared via a simple carbonization procedure of well-defined polyacrylonitrile-block-poly(n-butyl acrylate) (PAN-b-PBA) block copolymer precursors. The structure and morphology were characterized by transmission electron microsphere (TEM), adsorption-desorption isotherms, and X-ray photoelectron spectroscopy (XPS). Owing to its high specific area, hierarchical open-porous structure, and the introduction of nitrogen atoms in graphitic sp2 network, the adsorbent CTNC exhibited high removal efficiency toward bilirubin and bile acid from human plasma. The removal rate of bilirubin was more than 50.7% with a minimal loss of albumin. Meanwhile, exceeding 95% of bile acid was eliminated. The effect of albumin on the adsorption kinetic of bilirubin was identified. The result indicated that the adsorption rate of BSA-bonded bilirubin experienced a decline than that of free bilirubin, but the adsorption capacity was still up to 39.8 mg/g within 2 h. Moreover, the effect of porosity and nitrogen contents on bilirubin adsorption ability and blood compatibility were systematically investigated. The material with lower nitrogen content showed only a negligible hemolysis activity. Therefore, the nitrogen-doped mesoporous carbon developed in this work has potential application in blood purification for the efficient removal of bilirubin.

Isothermal kinase-triggered supramolecular assemblies as drug sensitizers.

Protein kinases, the main regulators of a vast map of cellular processes, are the most attractive targets in drug discovery. Despite a few successful examples of protein kinase inhibitors, the drug discovery strategy of downregulating protein kinase activity has been quite limited and often fails even in animal models. Here, we utilize protein kinase A (PKA) activity to design PKA-triggered supramolecular assemblies with anticancer activities. Grafting a suitable peptide to PNIPAM raises the critical temperature of the LCST polymer above body temperature. Interestingly, the corresponding phosphorylated polymer has a critical temperature below body temperature, making this peptide-appended PNIPAM a suitable polymer for the PKA-triggered supramolecular assembly process. PKA-triggered assembly occurs selectively in PKA-upregulated MCF-7 cells, which disturbs the cytoskeleton and sensitizes cancer cells against doxorubicin. The chemosensitization is also observed in vivo to identify effective tumor inhibitors with satisfactory biocompatibility. Overall, this phosphorylation-induced (in principle, PKA-catalyzed) supramolecular assembly opens up a promising chemotherapy strategy for combating kinase-upregulated cancer.

Development of hydrophilic interaction chromatography with quadruple time-of-flight mass spectrometry for heparin and low molecular weight heparin disaccharide analysis.

RATIONALE: Heparin and low molecular weight heparin (LMWH) are widely used as clinical anticoagulants. The determination of their composition and structural heterogeneity still challenges analysts. METHODS: Disaccharide compositional analysis, utilizing heparinase-catalyzed depolymerization, is one of the most important ways to evaluate the sequence, structural composition and quality of heparin and LMWH. Hydrophilic interaction chromatography coupled with quadruple time-of-flight mass spectrometry (HILIC/QTOFMS) has been developed to analyze the resulting digestion products. RESULTS: HILIC shows good resolution and excellent MS compatibility. Digestion products of heparin and LMWHs afforded up to 16 compounds that were separated using HILIC and analyzed semi-quantitatively. These included eight common disaccharides, two disaccharides derived from chain termini, three 3-O-sulfo-group-containing tetrasaccharides, along with three linkage region tetrasaccharides and their derivatives. Structures of these digestion products were confirmed by mass spectral analysis. The disaccharide compositions of a heparin, two batches of the LMWH, enoxaparin, and two batches of the LMWH, nadroparin, were compared. In addition to identifying disaccharides, 3-O-sulfo-group-containing tetrasaccharides, linkage region tetrasaccharides were observed having slightly different compositions and contents in these heparin products suggesting that they had been prepared using different starting materials or production processes. CONCLUSIONS: Thus, compositional analysis using HILIC/QTOFMS offers a unique insight into different heparin products.

UP-HILIC-MS/MS to Determine the Action Pattern of Penicillium sp. Dextranase.

Investigation of the action pattern of enzymes acting on carbohydrates is challenging, as both the substrate and the digestion products are complex mixtures. Dextran and its enzyme-derived oligosaccharides are widely used for many industrial applications. In this work, a new method relying on ultra-performance hydrophilic interaction liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UP-HILIC-Q/TOF-MS/MS) was developed to analyze a complex mixture of dextran oligosaccharide products to determine the action pattern of dextranase. No derivatization of oligosaccharides was required and the impact of the α- and ß-configurations of the native oligosaccharides on the chromatographic separation was eliminated. The 1→6, 1→3, 1→4 backbone linkages and the branch linkages of these oligosaccharides were all distinguished from diagnostic ions in their MS/MS spectra, including fragments corresponding to (0,2)A, (0,3)A, (0,4)A, B-H2O, (2,5)A, and (3,5)A. The sequences of the oligosaccharide products were similarly established. Thus, the complex oligosaccharide mixtures in dextran digestion products were profiled and identified using this method. The more enzyme-resistant structures in dextran were established using much less sample, labor, time, and uncertainty than in previous studies. This method provides an efficient, sensitive, and straightforward way to monitor the entire process of digestion, establish the action pattern of the dextranase from Penicillium sp., and to support the proper industrial application of dextranase.

Impact of hydrolysis conditions on the detection of mannuronic to guluronic acid ratio in alginate and its derivatives.

Alginate is a linear and acidic polysaccharide, composed of (1 → 4) linked ß-D-mannuronic acid (ManA) and α-L-guluronic acid (GulA). The ratio of ManA to GulA (M/G) is one of the most important factors for the application of alginate and its derivatives in various areas. In this work, a robust and accurate method was developed to analyze M/G using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The impact of hydrolysis conditions on the release patterns of ManA and GulA from alginate and its derivatives was investigated. The release patterns of ManA and GulA need to be considered separately to obtain an accurate M/G. Several hydrolysis conditions were established that released ManA and GulA completely and maintained these saccharide residues intact. The proper M/G of alginates from different sources and its derivatives could then be calculated by integration of the corresponding ManA and GulA peaks.

Syntheses of aromatic substituted hydrazino-thiazole derivatives to clarify structural characterization and antioxidant activity between 3-arylsydnonyl and aryl substituted hydrazino-thiazoles.

This work clarifies the structural characterization and antioxidant activity between aromatic and 3-arylsydnonyl substituted hydrazino-thiazoles by further synthesizing a series of aromatic ring-substituted hydrazino-thiazole derivatives 8a-h and 9a-h. Hydrazino-thiazole derivatives 8a-h and 9a-h were obtained by reacting aromatic or heterocyclic aromatic aldehyde thiosemicarbazones 7a-h with cyclization reagents ethyl 2-chloroacetoacetate (2a) and 2-bromoacetophenone (2b), respectively. The ORTEP drawings of compounds 8g, 8h and 9f provide strong evidence of the structure of aromatic thiazole derivatives 8a-h and 9a-h. Undoubtedly, the structure of compounds 3e-h and 4e-h synthesized by the reaction of 3-aryl-4-formylsydnone thiosemicarbazones 1e-h with cyclization reagents 2a and 2b in the previous work should have the thiazole moiety, and not the thiazoline moiety. Both the new thiazole derivatives 8a-h and 9a-h and the 3-arylsydnonyl-substituted derivatives 3e-h and 4e-h were investigated to determine their antioxidant activity by two tests that have been highly documented-the direct scavenging effect on a stable free 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and the inhibition of the 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical. Results of this study demonstrate that not only the thiazole ring and the aryl ring has the contribution to the antioxidant activities, the sydnone ring of 3-arylsydnonyl moiety also has its considerable contribution.