A type I and type II chemical biology toolbox to overcome the hypoxic tumour microenvironment for photodynamic therapy.

Photodynamic therapy (PDT) is a minimally invasive therapeutic modality employed for the treatment of various types of cancers, localized infections, and other diseases. Upon illumination, the photo-excited photosensitizer generates singlet oxygen and other reactive species, thereby inducing cytotoxicity in the target cells. The hypoxic tumour microenvironment (TME), however, poses a limitation on the supply of oxygen in tumour tissues. Moreover, under such conditions, tumour metastasis and drug resistance frequently occur, further compromising the efficacy of PDT in combating tumours. Traditionally, type I photosensitizers with lower oxygen consumption demonstrate significant potential in overcoming hypoxic environments and play a crucial role in determining the therapeutic efficacy of PDT because type I photosensitizers can generate highly cytotoxic free radicals. In comparison, type II photosensitizers exhibit high oxygen dependence. The rate of reactive oxygen species (ROS) generation in the type II process is significantly higher than that in the type I process. Thus, the efficiency and selectivity of PDT depend on the properties of the photosensitizer. Here, the recent development and application of type I and type II photosensitizers, mainly in the past year, are summarized. The design methods, electronic structures, photophysical properties, lipophilic properties, electric charge, and other molecular characteristics of these photosensitizers are discussed in detail. These modifications alter the microstructure of photosensitizers and directly impact the results of PDT. The main content of this paper will have a positive promoting and inspiring effect on the future development of PDT.

Engagement of N6-methyladenisine methylation of Gng4 mRNA in astrocyte dysfunction regulated by CircHECW2.

The N6-methyladenosine (m6A) modification is the most prevalent modification of eukaryotic mRNAs and plays a crucial role in various physiological processes by regulating the stability or function of target mRNAs. Accumulating evidence has suggested that m6A methylation may be involved in the pathological process of major depressive disorder (MDD), a common neuropsychiatric disorder with an unclear aetiology. Here, we found that the levels of the circular RNA HECW2 (circHECW2) were significantly increased in the plasma of both MDD patients and the chronic unpredictable stress (CUS) mouse model. Notably, the downregulation of circHECW2 attenuated astrocyte dysfunction and depression-like behaviors induced by CUS. Furthermore, we demonstrated that the downregulation of circHECW2 increased the expression of the methylase WTAP, leading to an increase in Gng4 expression via m6A modifications. Our findings provide functional insight into the correlation between circHECW2 and m6A methylation, suggesting that circHECW2 may represent a potential target for MDD treatment.

Integrating spatial and single-cell transcriptomics to characterize the molecular and cellular architecture of the ischemic mouse brain.

Neuroinflammation is acknowledged as a pivotal pathological event after cerebral ischemia. However, there is limited knowledge of the molecular and spatial characteristics of nonneuronal cells, as well as of the interactions between cell types in the ischemic brain. Here, we used spatial transcriptomics to study the ischemic hemisphere in mice after stroke and sequenced the transcriptomes of 19,777 spots, allowing us to both visualize the transcriptional landscape within the tissue and identify gene expression profiles linked to specific histologic entities. Cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of ischemia-associated gene expression in the peri-infarct area of the ischemic hemisphere. Analysis of ligand-receptor interactions in cell communication revealed galectin-9 to cell-surface glycoprotein CD44 (LGALS9-CD44) as a critical signaling pathway after ischemic injury and identified microglia and macrophages as the main source of galectins after stroke. Extracellular vesicle-mediated Lgals9 delivery improved the long-term functional recovery in photothrombotic stroke mice. Knockdown of Cd44 partially reversed these therapeutic effects, inhibiting oligodendrocyte differentiation and remyelination. In summary, our study provides a detailed molecular and cellular characterization of the peri-infact area in a murine stroke model and revealed Lgals9 as potential treatment target that warrants further investigation.

Fluorogenic methodology for visualization of phase separation in chemical biology.

Phase separation is a common biological phenomenon in the liquid environment of organisms. Phase separation has been shown to be a key cause of many existing incurable diseases, such as the protein aggregates formed by phase separation of Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Parkinson's disease, etc. Tracking the occurrence of phase separation in vivo is critical to many disease detection methods and solving many treatment problems. Its physicochemical properties and visual detection methods have flourished in the last few years in chemical biology, among which the fluorogenic toolbox has great application potential compared to the traditional detection methods that cannot visualize the phase separation process intuitively, but just show some parameters indirectly. This paper reviews the mechanism and disease correlation proven in recent years for phase separation and analyzes the detection methods for phase separation, including functional microscope imaging techniques, turbidity monitoring, macromolecule congestion sensing, in silico analysis, etc. It is worth mentioning that the qualitative and quantitative analysis of aggregates formed by phase separation using in vitro parameters has successfully provided basic physical and chemical properties for phase separation aggregates, and is an important cornerstone for researchers to carry forward the past and break through the existing technical shackles to create new in vivo monitoring methods such as fluorescence methodology. Crucially, fluorescence methods for cell microenvironment imaging based on different mechanisms are discussed, such as AIE-based probes, TICT-based probes and FRET-based probes, etc.

Rational Modulation of BODIPY Photosensitizers to Design Metal-Organic Framework-Based NIR Nanocomposites for High-Efficiency Photodynamic Therapy in a Hypoxic Environment.

Photodynamic therapy (PDT) is a promising noninvasive treatment that has drawn great attention. However, the hypoxic environment in tumors seriously limits the therapeutic effect of oxygen-dependent chemicals and PDT. Herein, a versatile nanocomposite DF-BODIPY@ZIF-8 with oxygen-generating ability was developed based on zeolitic imidazolate framework-8 (ZIF-8) by loading the near-infrared photosensitizer DF-BODIPY to overcome hypoxia-induced drug resistance in cancer therapy. ZIF-8 can catalyze the decomposition of hydrogen peroxide in tumors and increase the dissolved oxygen concentration, resulting in a significant improvement in PDT efficacy. Additionally, we found that enhancing the electronegativity of substituents can effectively reduce the energy level difference (ΔEst) between the minimum singlet state (S1) and the lowest triplet state (T1), leading to the enhancement of the singlet oxygen quantum yield. In vitro experiments suggested that DF-BODIPY@ZIF-8 indeed had a higher singlet oxygen quantum yield and better tumor cell phototoxicity than free DF-BODIPY. In vivo experiments also demonstrated that DF-BODIPY@ZIF-8 could effectively eliminate 4T1 tumors under light irradiation. Thus, we conclude that increasing the electronegativity of substituents and introducing a ZIF-8 material can effectively improve the singlet oxygen quantum yield and overcome the hypoxia limitations for high-efficiency PDT.

A chemical biology toolbox to overcome the hypoxic tumor microenvironment for photodynamic therapy: a review.

Cancer is a disease that seriously threatens human health. Over the past few decades, researchers have continued to find ways to cure cancer. Currently, the most commonly used clinical techniques are surgery, chemotherapy, radiotherapy and so on. Among them, photodynamic therapy (PDT) has received extensive attention due to its better therapeutic effect and lower side effects. However, the inherent microenvironmental hypoxia of tumor tissue leads to unsatisfactory therapeutic effects. Therefore, researchers have conducted in-depth research on the hypoxia problem in PDT therapy. This review classified photodynamic therapy according to the response mechanism and summarized the strategies developed to overcome tumor hypoxia in recent years. Among them, research strategies can be divided into five types: type I PDT photosensitizers, introducing exogenous oxygen, O2 carriers using nanomaterials, generating endogenous oxygen by catalytic reactions, and combination with prodrugs that inhibit the consumption of endogenous oxygen. Finally, we also list some studies using combination therapy, such as microbes, photothermal therapy, etc. It can be guaranteed that the review can provide theoretical guidance for the development of anti-hypoxic PDT tools.

Extracellular vesicle-mediated delivery of circDYM alleviates CUS-induced depressive-like behaviours.

Major depressive disorder (MDD) is the most prevalent psychiatric disorder worldwide and severely limits psychosocial function and quality of life, but no effective medication is currently available. Circular RNAs (circRNAs) have been revealed to participate in the MDD pathological process. Targeted delivery of circRNAs without blood-brain barrier (BBB) restriction for remission of MDD represents a promising approach for antidepressant therapy. In this study, RVG-circDYM-extracellular vesicles (RVG-circDYM-EVs) were engineered to target and preferentially transfer circDYM to the brain, and the effect on the pathological process in a chronic unpredictable stress (CUS) mouse model of depression was investigated. The results showed that RVG-circDYM-EVs were successfully purified by ultracentrifugation from overexpressed circDYM HEK 293T cells, and the characterization of RVG-circDYM-EVs was successfully demonstrated in terms of size, morphology and specific markers. Beyond demonstrating proof-of-concept for an RNA drug delivery technology, we observed that systemic administration of RVG-circDYM-EVs efficiently delivered circDYM to the brain, and alleviated CUS-induced depressive-like behaviours, and we discovered that RVG-circDYM-EVs notably inhibited microglial activation, BBB leakiness and peripheral immune cells infiltration, and attenuated astrocyte disfunction induced by CUS. CircDYM can bind mechanistically to the transcription factor TAF1 (TATA-box binding protein associated factor 1), resulting in the decreased expression of its downstream target genes with consequently suppressed neuroinflammation. Taken together, our findings suggest that extracellular vesicle-mediated delivery of circDYM is effective for MDD treatment and promising for clinical applications.

N6-Methyladenosine Modification of Fatty Acid Amide Hydrolase Messenger RNA in Circular RNA STAG1-Regulated Astrocyte Dysfunction and Depressive-like Behaviors.

BACKGROUND: N6-methyladenosine (m6A) is the most abundant epigenetic modification in eukaryotic messenger RNAs and is essential for multiple RNA processing events in physiological and pathological processes. However, precisely how m6A methylation is involved in major depressive disorder (MDD) is not fully understood. METHODS: Circular RNA STAG1 (circSTAG1) was screened from the hippocampus of chronic unpredictable stress-treated mice using high-throughput RNA sequencing. Microinjection of circSTAG1 lentivirus into the mouse hippocampus was used to observe the role of circSTAG1 in depression. Sucrose preference, forced swim, and tail suspension tests were performed to evaluate the depressive-like behaviors of mice. Astrocyte dysfunction was examined by GFAP immunostaining and 3D reconstruction. Methylated RNA immunoprecipitation sequence analysis was used to identify downstream targets of circSTAG1/ALKBH5 (alkB homolog 5) axis. Cell Counting Kit-8 assay was performed to evaluate astrocyte viability in vitro. RESULTS: circSTAG1 was significantly decreased in the chronic unpredictable stress-treated mouse hippocampus and in peripheral blood of patients with MDD. Overexpression of circSTAG1 notably attenuated astrocyte dysfunction and depressive-like behaviors induced by chronic unpredictable stress. Further examination indicated that overexpressed circSTAG1 captured ALKBH5 and decreased the translocation of ALKBH5 into the nucleus, leading to increased m6A methylation of fatty acid amide hydrolase (FAAH) messenger RNA and degradation of FAAH in astrocytes with subsequent attenuation of depressive-like behaviors and astrocyte loss induced by corticosterone in vitro. CONCLUSIONS: Our findings dissect the functional link between circSTAG1 and m6A methylation in the context of MDD, providing evidence that circSTAG1 may be a novel therapeutic target for MDD.

Extracellular Vesicle-Mediated Delivery of Circular RNA SCMH1 Promotes Functional Recovery in Rodent and Nonhuman Primate Ischemic Stroke Models.

BACKGROUND: Stroke is a leading cause of adult disability that can severely compromise the quality of life of patients, yet no effective medication currently exists to accelerate rehabilitation. A variety of circular RNA (circRNA) molecules are known to function in ischemic brain injury. Lentivirus-based expression systems have been widely used in basic studies of circRNAs, but safety issues with such delivery systems have limited exploration of the potential therapeutic roles for circRNAs. METHODS: Circular RNA SCMH1 (circSCMH1) was screened from the plasma of patients with acute ischemic stroke by using circRNA microarrays. Engineered rabies virus glycoprotein-circSCMH1-extracellular vesicles were generated to selectively deliver circSCMH1 to the brain. Nissl staining was used to examine infarct size. Behavioral tasks were performed to evaluate motor functions in both rodent and nonhuman primate ischemic stroke models. Golgi staining and immunostaining were used to examine neuroplasticity and glial activation. Proteomic assays and RNA-sequencing data combined with transcriptional profiling were used to identify downstream targets of circSCMH1. RESULTS: CircSCMH1 levels were significantly decreased in the plasma of patients with acute ischemic stroke, offering significant power in predicting stroke outcomes. The decreased levels of circSCMH1 were further confirmed in the plasma and peri-infarct cortex of photothrombotic stroke mice. Beyond demonstrating proof-of-concept for an RNA drug delivery technology, we observed that circSCMH1 treatment improved functional recovery after stroke in both mice and monkeys, and we discovered that circSCMH1 enhanced the neuronal plasticity and inhibited glial activation and peripheral immune cell infiltration. CircSCMH1 binds mechanistically to the transcription factor MeCP2 (methyl-CpG binding protein 2), thereby releasing repression of MeCP2 target gene transcription. CONCLUSIONS: Rabies virus glycoprotein-circSCMH1-extracellular vesicles afford protection by promoting functional recovery in the rodent and the nonhuman primate ischemic stroke models. Our study presents a potentially widely applicable nucleotide drug delivery technology and demonstrates the basic mechanism of how circRNAs can be therapeutically exploited to improve poststroke outcomes.

Circulating Circular RNAs as Biomarkers for the Diagnosis and Prediction of Outcomes in Acute Ischemic Stroke.

Background and Purpose- Circular RNAs (CircRNAs) show promise as stroke biomarkers because of their participation in various pathophysiological processes associated with acute ischemic stroke (AIS) and stability in peripheral blood. Methods- A circRNA microarray was used to identify differentially expressed circulating circRNAs in a discovery cohort (3 versus 3). Validation (36 versus 36) and replication (200 versus 100) were performed in independent cohorts by quantitative polymerase chain reaction. Platelets, lymphocytes, and granulocytes were separated from blood to examine the origins of circRNAs. Results- There were 3 upregulated circRNAs in Chinese population-based AIS patients compared with healthy controls. The combination of 3 circRNAs resulted in an area under the curve of 0.875, corresponding to a specificity of 91% and a sensitivity of 71.5% in AIS diagnosis. Furthermore, the combination of change rate in 3 circRNAs within the first 7 days of treatment showed an area under the curve of 0.960 in predicting stroke outcome. There was significant increase in lymphocytes and granulocytes for circPDS5B (circular RNA PDS5B) and only in granulocytes for circCDC14A (circular RNA CDC14A) in AIS patients compared with healthy controls. Conclusions- Three circRNAs could serve as biomarkers for AIS diagnosis and prediction of stroke outcomes. The elevated levels of circPDS5B and circCDC14A after stroke might be because of increased levels in lymphocytes and granulocytes.

Baicalin improves intestinal microecology and abnormal metabolism induced by high-fat diet.

Short-chain fatty acids (SCFAs) are produced by the fermentation of dietary fiber by the gut microbiota and are beneficial to the health of the body. Insufficient SCFAs productions are associated with type 2 diabetes (T2D). We used a long-term high-fat diet to simulate the pathogenesis of T2D and studied the effects of baicalin on gut microbiota and metabolites in mice as well as its mechanism, providing a theoretical basis for the treatment of T2D. Baicalin groups were given 200 mg/kg/day, and control groups were given an equal volume of 0.5% sodium carboxymethyl cellulose solution for 15 weeks. 16S rRNA amplicon pyrosequences was performed to evaluate the gut microbiota composition, and gas chromatography was used to detect SCFAs in stool samples in the different experimental groups. The abundance of gut microbiota in the high-fat model group was altered, and was associated with a decreased production of SCFAs. The microbiota abundance of the baicalin group was closer to that of the control group, increasing the population of SCFA-producing bacteria spp and improving metabolic syndrome, including abnormal glucose and lipid metabolism caused by a high-fat diet. Baicalin may improve abnormalities in glycolipid metabolism by affecting the production of SCFAs.