RESUMO
We show how the macrocyclic host cucurbit[8]uril (CB[8]) and a fluorescent dye form a biosensing ensemble while its cavity simultaneously traps tryptophan, the upstream substrate of IDO1 enzymes, therefore providing a label-free method to monitor the activity of IDO1 in real time. Incubation of malignant HeLa and HepG2 cells overexpressing IDO1 with the associative biosensor resulted in its spontaneous uptake and a fluorescence switch-on response in situ, which can be traced to the displacement of tryptophan from CB[8] upon IDO1-catalyzed oxidation. The results, for the first time, establish a supramolecular sensing concept for the detection of intracellular enzymatic activity in live cells, thus allowing direct cell-based analysis and inhibitor screening compatible with commercial instruments including microplate reader, fluorescent microscopy, and flow cytometry.
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes/química , Indolamina-Pirrol 2,3,-Dioxigenase/análise , Técnicas Biossensoriais/instrumentação , Hidrocarbonetos Aromáticos com Pontes/química , Linhagem Celular Tumoral , Células HeLa , Células Hep G2 , Humanos , Imidazóis/química , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Oxirredução , Triptofano/químicaRESUMO
Aberrant lipid metabolism impacts intratumoral T cell-mediated immune response and tumor growth. Fatostatin functions as an inhibitor of sterol regulatory element binding protein (SREBP) activation. However, the complex effects of fatostatin on cholesterol metabolism in the tumor microenvironment (TME) and its influence on T cell anti-tumor immunity remain unclear. In this study, fatostatin effectively suppressed B16 melanoma, MC38 colon cancer, and Lewis lung cancer (LLC) transplanted tumor growth in immunocompetent mice by reducing SREBPs-mediated lipid metabolism, especially cholesterol levels. Mechanistically, fatostatin decreased intracellular cholesterol accumulation and inhibited X-box binding protein 1 (XBP1)-mediated endoplasmic reticulum (ER) stress, reducing Treg cells and alleviating CD8+ T cell exhaustion in the TME, exerting anti-tumor activity. Neverthelessï¼ this effect was impaired in immunodeficient nude mice, suggesting fatostatin's anti-tumor efficacy in transplanted tumors partly relies on T cell-mediated anti-tumor immunity. Our study highlights SREBP2-mediated cholesterol metabolism as a potential strategy for anti-tumor immunotherapy, and confirms fatostatin's promise in tumor immunotherapy.
Assuntos
Melanoma Experimental , Piridinas , Tiazóis , Animais , Camundongos , Camundongos Nus , Melanoma Experimental/tratamento farmacológico , Metabolismo dos Lipídeos , Colesterol/metabolismo , Microambiente TumoralRESUMO
RATIONALE: Inflammatory bowel diseases (IBDs) are still awaiting innovative treatments that can maximize the efficiency of site-specific drug release in the colon while enhancing intestinal homeostasis. METHODS: Herein, we present multilayer-coated mesoporous silica (MSs) which release payload drugs specifically in the colon tract in the presence of azoreductase produced by the gut microbiota, and simultaneously rejuvenate the tryptophan metabolism of the microbiome to induce activation of the aryl hydrocarbon receptor (AHR) for increased anti-inflammatory effects. The MSs were prepared by using cucurbit[8]uril (CB[8]) as a supramolecular "handcuff" to assemble chitosan/hyaluronic acid multilayers on the periphery of a mesoporous silica core. RESULTS: Strikingly, although MSs remained fairly stable in both acidic and neutral pH, they exhibited excellent responsiveness towards dithionite, an azo-reducing agent employed as a substitute to mimic the specific azoreductase environment in vitro. In comparison with the drug in its free form, hydrocortisone-loaded MSs showed optimized accumulation of therapeutics in the colonic mucosa with minimized premature release in the upper gastrointestinal tract in in vivo imaging and biodistribution studies. The enhanced therapeutic effects of MSs were confirmed in dextran sodium sulfate-induced colitis in mice with promoted colonic epithelial barrier integrity, elevated level of AHR agonists and modulated production of inflammatory cytokines. Furthermore, 16S rRNA analysis showed that the disrupted gut homeostasis of colitic mice was partly corrected by MSs. CONCLUSION: This novel drug delivery system using self-assembly of tryptophan-functionalized chitosan, which was precomplexed with CB[8], and azobenzene-functionalized hyaluronic acid on the surface of mesoporous silica nanoparticles provides a synergistic gut microbiota-targeting approach for IBD therapy.
Assuntos
Colite , Microbioma Gastrointestinal , Animais , Hidrocarbonetos Aromáticos com Pontes , Colite/tratamento farmacológico , Colo , Homeostase , Imidazóis , Camundongos , Camundongos Endogâmicos C57BL , RNA Ribossômico 16S , Dióxido de Silício , Distribuição TecidualRESUMO
Nanosystems responsive to tumor-specific enzymes are considered as a highly attractive approach to intracellular drug release for targeted cancer therapy. Mesoporous silica nanoparticles are capped with tryptophan-mediated cucurbit[8]uril complex with Fe3 O4 to minimize the premature drug leakage while being able to deliver the payload on demand at the target tissue. The supramolecular interaction between tryptophan and cucurbit[8]uril is disrupted in the presence of indoleamine 2,3-dioxygenase 1 (IDO1) enzyme (abundant in the tumor intracellular microenvironment), which catalyzes the metabolism of tryptophan into N-formylkynurenine, resulting in the disassembly of the "gate-keeper" of the nanocarriers and intracellular release of therapeutics exclusively in tumor cells. The drug release from the nanocarrier with high selectivity to overexpressed IDO1 enzyme induces significant cytotoxicity against HepG2 cells in vitro, as well as the superior antitumor effects in vivo. This robust supramolecular nanosystem with sophisticated structure and property provides a promising platform for intracellular drug release targeting the intrinsic microenvironmental enzyme inside the tumor cells.