Intrinsic Reactive Oxygen Species Research Articles

Recent Advances in the Development of Targeted Activation Strategies for Organoboron Prodrugs

Elevated levels of reactive oxygen species (ROS) are a hallmark of varieties of diseases such as cancer, inflammation, and neurodegenerative disorders. Inspired by the discrepancy of ROS concentrations between pathological tissues and the normal counterparts, an increasing number of ROS‐responsive theranostic prodrugs are developed in past years, with particularly high proportions of organoboron‐based prodrugs that can respond to H2O2. Unfortunately, increasing studies have demonstrated that the intrinsic ROS (H2O2) levels in most pathological tissue are only slightly higher than normal tissues and are not adequate to activate organoboron prodrugs; in contrast, several organoboron compounds have been clinically approved in which boronic acid acts as electrophilic warhead. To this end, developing more robust and universal approaches for boronic acid‐prodrug activation becomes highly attractive. In this context, we discuss the recently reported activation strategies for boron‐caged prodrugs with a particular focus on their design principles and activation mechanisms. The perspectives on the future directions for this important research area are discussed as well.

CD36-mediated ferroptosis destabilizes CD4+ T cell homeostasis in acute Stanford type-A aortic dissection

Acute type A aortic dissection (ATAAD) is a lethal pathological process within the aorta with high mortality and morbidity. T lymphocytes are perturbed and implicated in the clinical outcome of ATAAD, but the exact characteristics of T cell phenotype and its underlying mechanisms in ATAAD remain poorly understood. Here we report that CD4+ T cells from ATAAD patients presented with a hypofunctional phenotype that was correlated with poor outcomes. Whole transcriptome profiles showed that ferroptosis and lipid binding pathways were enriched in CD4+ T cells. Inhibiting ferroptosis or reducing intrinsic reactive oxygen species limited CD4+ T cell dysfunction. Mechanistically, CD36 was elevated in CD4+ T cells, whose blockade effectively alleviated palmitic acid-induced ferroptosis and CD4+ T cell hypofunction. Therefore, targeting the CD36-ferroptosis pathway to restore the functions of CD4+ T cells is a promising therapeutic strategy to improve clinical outcomes in ATAAD patients.

Mitochondrial DNA is a sensitive surrogate and oxidative stress target in oral cancer cells.

Cellular oxidative stress mediated by intrinsic and/or extrinsic reactive oxygen species (ROS) is associated with disease pathogenesis. Oxidative DNA damage can naturally be substituted by mitochondrial DNA (mtDNA), leading to base lesion/strand break formation, copy number changes, and mutations. In this study, we devised a single test for the sensitive quantification of acute mtDNA damage, repair, and copy number changes using supercoiling-sensitive quantitative PCR (ss-qPCR) and examined how oxidative stress-related mtDNA damage responses occur in oral cancer cells. We observed that exogenous hydrogen peroxide (H2O2) induced dynamic mtDNA damage responses, as reflected by early structural DNA damage, followed by DNA repair if damage did not exceed a particular threshold. However, high oxidative stress levels induced persistent mtDNA damage and caused a 5-30-fold depletion in mtDNA copy numbers over late responses. This dramatic depletion was associated with significant growth arrest and apoptosis, suggesting persistent functional consequences. Moreover, oral cancer cells responded differentially to oxidative injury when compared with normal cells, and different ROS species triggered different biological consequences under stress conditions. In conclusion, we developed a new method for the sensitive detection of mtDNA damage and copy number changes, with exogenous H2O2 inducing dynamic mtDNA damage responses associated with functional changes in stressed cancer cells. Finally, our method can help characterize oxidative DNA damage in cancer and other human diseases.

Carbon Dots in the Pathological Microenvironment: ROS Producers or Scavengers?

Reactive oxygen species (ROS), as metabolic byproducts, play pivotal role in physiological and pathological processes. Recently, studies on the regulation of ROS levels for disease treatments have attracted extensive attention, mainly involving the ROS-induced toxicity therapy mediated by ROS producers and antioxidant therapy by ROS scavengers. Nanotechnology advancements have led to the development of numerous nanomaterials with ROS-modulating capabilities, among which carbon dots (CDs) standing out as noteworthy ROS-modulating nanomedicines own their distinctive physicochemical properties, high stability, and excellent biocompatibility. Despite progress in treating ROS-related diseases based on CDs, critical issues such as rational design principles for their regulation remain underexplored. The primary cause of these issues may stem from the intricate amalgamation of core structure, defects, and surface states, inherent to CDs, which poses challenges in establishing a consistent generalization. This review succinctly summarizes the recently progress of ROS-modulated approaches using CDs in disease treatment. Specifically, it investigates established therapeutic strategies based on CDs-regulated ROS, emphasizing the interplay between intrinsic structure and ROS generation or scavenging ability. The conclusion raises several unresolved key scientific issues and prominent technological bottlenecks, and explores future perspectives for the comprehensive development of CDs-based ROS-modulating therapy.

Elucidating the role of intrinsic adenosine A1 receptors in acute alcoholism using human-induced pluripotent stem cell-derived hepatocytes.

Acute alcoholic hepatitis (AAH) from binge drinking is a serious disease. It is associated with a high mortality rate, especially among young adults. Apoptosis is known to be a primary cause of liver damage, and it can be induced by either intrinsic signaling pathways or by reactive oxygen species (ROS). Adenosine A1 receptors (ADORA1) are known to be involved in ethanol metabolism; however, underlying mechanism is not well understood. For investigating how the intrinsic ADORA1 function in ethanol metabolism in normal human hepatocytes without interference by extrinsic molecules, primary hepatocytes pose a challenge, due to unavoidable contamination by other kinds of cells in the liver. Also, they are difficult to culture stably. As a novel alternative, hepatocytes derived from human-induced pluripotent stem cells were employed because they display similar function to primary hepatocytes and they can be stably cultured. The dynamics and integrity of signal transduction mechanisms were investigated by following chronological changes in gene expression. This shed light on how and when the ADORA1 function and on causal relationships between the pathways and clinical symptoms. The findings of the present study shows that ADORA1 are most activated soon after exposure to ethanol, and transfection of small interfering RNA targeting ADORA1-messenger-RNA (ADORA1-siRNA) into the hepatocytes significantly suppresses production of actin protein and ROS. It suggests that ADORA1 in the liver contribute to apoptosis in acute alcoholism through both intrinsic pathway and ROS activity. Also, actin that is abundant in the cells could be an appropriate biomarker evaluating hepatic function status.

Bioinspired BSA@polydopamine@Fe Nanoprobe with Self-Purification Capacity for Targeted Magnetic Resonance Imaging of Acute Kidney Injury.

Contrast-enhanced magnetic resonance imaging (CE-MRI) of acute kidney injury (AKI) is severely hindered by the poor targeting capacity and potential toxicity of current contrast agents. Herein, we propose one-step fabrication of a bovine serum albumin@polydopamine@Fe (BSA@PDA@Fe, BPFe) nanoprobe with self-purification capacity for targeted CE-MRI of AKI. BSA endows the BPFe nanoprobe with renal tubule-targeting ability, and PDA is capable of completely inhibiting the intrinsic metal-induced reactive oxygen species (ROS), which are always involved in Fe/Mn-based agents. The as-prepared nanoprobe owns a tiny size of 2.7 nm, excellent solubility, good T1 MRI ability, superior biocompatibility, and powerful antioxidant capacity. In vivo CE-MRI shows that the BPFe nanoprobe can accumulate in the renal cortex due to the reabsorption effect toward the serum albumin. In the AKI model, impaired renal reabsorption function can be effortlessly detected via the diminishment of renal cortical signal enhancement. More importantly, the administration of the BPFe nanoprobe would not aggravate renal damage of AKI due to the outstanding self-purification capacity. Besides, the BPFe nanoprobe is employed for CE-MR angiography to visualize fine vessel structures. This work provides an MRI contrast agent with good biosafety and targeting ability for CE-MRI of kidney diseases.

ROS-Scavenging Lignin-Based Tolerogenic Nanoparticle Vaccine for Treatment of Multiple Sclerosis.

Multiple sclerosis (MS) is a demyelinating autoimmune disease, in which the immune system attacks myelin. Although systemic immunosuppressive agents have been used to treat MS, long-term treatment with these drugs causes undesirable side effects such as altered glucose metabolism, insomnia, and hypertension. Herein, we propose a tolerogenic therapeutic vaccine to treat MS based on lignin nanoparticles (LNP) with intrinsic reactive oxygen species (ROS)-scavenging capacity derived from their phenolic moieties. The LNP loaded with autoantigens of MS allowed for inducing tolerogenic DCs with low-level expression of costimulatory molecules while presenting antigenic peptides. Intravenous injection of an LNP-based tolerogenic vaccine into an experimental autoimmune encephalomyelitis (EAE) model led to durable antigen-specific immune tolerance via inducing regulatory T cells (Tregs). Autoreactive T helper type 1 cells, T helper type 17 cells, and inflammatory antigen presentation cells (APCs) were suppressed in the central nervous system (CNS), ameliorating ongoing MS in early and late disease states. Additionally, the incorporation of dexamethasone into an LNP-based tolerogenic nanovaccine could further improve the recovery of EAE mice in the severe chronic stage. As lignin is the most abundant biomass and waste byproduct in the pulping industry, a lignin-based tolerogenic vaccine could be a novel, cost-effective, high-value vaccine platform with potent therapeutic efficiency in treating autoimmune diseases.

Mitochondrial dsRNA from B-Precursor Acute Lymphoblastic Leukemia Cells Induce Formation of Cancer Associated Fibroblast in the Bone Marrow Leading to Formation of a Chemoprotective Bone Marrow Niche

Precursor B-cell Acute Lymphoblastic Leukemia (B-ALL) is typically a highly chemosensitive malignancy with the majority of children being cured by chemotherapy alone. Adult ALL is often chemosensitive at initial treatment, but long term disease free survival is less common. It is widely accepted that outcome in ALL closely relates to underlying genetic lesion (Moorman, Hematologica 2016) but no specific mechanisms by which ALL genetics influence outcome have been identified. We have previously demonstrated that an ‘activated mesenchymal stromal cell (MSC) niche’ develops in response to reactive oxygen species (ROS) -inducing chemotherapy in which mitochondria are actively transferred along tunnelling nanotubes to ‘rescue’ B-ALL cells from chemotherapy (Burt, et al Blood 2019). According to recent evidence that chemotherapy resistant ALL sub-clones may exist at diagnosis (Dobson et al Cancer Discovery 2020), we hypothesised that B-ALL cells may themselves directly activate bone marrow stromal cells to generate cancer associated fibroblasts (CAF) from MSC. To test this hypothesis, we first used our previously described in vitro niche model wherein either the HS27a MSC cell line or healthy donor (HD) MSCs are co-cultured with either B-ALL cell lines or primary patient B-ALL cells. Three of five B-ALL cell lines and 3 of 4 primary B-ALL samples tested, induced activation of the MSCs, as characterised by cytomorphological changes identified by confocal microscopy, up-regulation of CAF-relevant gene expression and increased IL6, IL8 and CCL2 protein secretion. The ability of the B-ALL cell lines to induce CAF correlated directly with the intrinsic ROS levels of the cell as determined by CellROX assay. In vivo, the CAF-inducing ALL cell lines also activated murine MSCs when established as xenografts in NOD-SCID gamma (NSG) mice. Immunohistochemistry of NSG murine femora revealed a very significant expansion in nestin+ cells after exposure to CAF-induing ALL cells compared to non-CAF-inducing controls. We established that the ALL cells activated MSC without contact, both from exposure of MSC to ALL cells in transwell and from exposure to B-ALL cell conditioned media (CM). RNA sequencing of HS27a stromal cells exposed to CAF-inducing B-ALL cell CM compared with non-CAF inducing controls unexpectedly showed that the most highly upregulated pathways were those involved in RNA sensing. Using a smaller gene panel selected from among the RNA-sensing pathway genes, we confirmed that this was also the case for primary, HD MSCs upon exposure to primary ALL patient samples. As a result, we sought evidence for the role of mitochondrial dsRNA, which has been shown to activate the anti-viral signalling pathway (Dhir et al, Nature 2018). Using the J2 anti-dsRNA monoclonal antibody in both confocal and flow cytometric assays, we observed very significantly higher levels of dsRNA in MSC which had been exposed to CAF inducing B-ALL cell CM than in non-CAF inducing B-ALL or MSC alone controls. We confirmed by RT-PCR for mitochondrial genes on dsRNA immunoprecipitated from CAF inducing B-ALL cell CM that the dsRNA was of mitochondrial origin. Using ELISA, we quantified dsRNA from total cell RNA extracted from paired primary B-ALL patient samples at diagnosis and in remission. We found significantly higher levels of dsRNA at diagnosis than in remission, with it being undetectable in most remission samples. Moreover, we confirmed by flow cytometry the presence of dsRNA in 6 B-ALL patient derived xenografts which was absent in the control NSG mice without leukemia. We used DMSO to degrade dsRNA from CAF inducing B-ALL cell CM, as is often done in Sanger sequencing and confirmed the degradation by ELISA. We then quantified gene expression of a selection of stromal cell activation marker genes, as well as genes in the RNA sensing pathway after exposure to either DMSO-treated B-ALL cell CM or non-treated control. There was a 2 to 4-fold reduction in expression of IL6, IL8 as well as of RIG-I, MX2, IFIH1, MAVS and OAS1 expression in DMSO-treated condition compared to control. We confirmed that IL6, IL8 and CCL2 protein secretion was also significantly abrogated in the test but not the control condition. We propose a model in which ROS-induced mitochondrial dysfunction in ALL cells leads to release of dsRNA which in turn activates bone marrow stromal cells leading to protective niche formation, despite lack of prior exposure to cytotoxic agents.

Polyphenolic Carbon Quantum Dots with Intrinsic Reactive Oxygen Species Amplification for Two-Photon Bioimaging and In Vivo Tumor Therapy.

Recent studies indicate that mitochondrial dysfunctions and DNA damage have a critical influence on cell survival, which is considered one of the therapeutic targets for cancer therapy. In this study, we demonstrated a comparative study of the effect of polyphenolic carbon quantum dots (CQDs) on in vitro and in vivo antitumor efficacy. Dual emissive (green and yellow) shape specific polyphenolic CQDs (G-CQDs and Y-CQDs) were synthesized from easily available nontoxic precursors (phloroglucinol), and the antitumor property of the as-synthesized probe was investigated as compared to round-shaped blue emissive CQDs (B-CQDs) derived from well-reported precursor citric acid and urea. The B-CQDs had a nuclei-targeting property, and G-CQDs and Y-CQDs had mitochondria-targeting properties. We have found that the polyphenol containing CQDs (at a dose of 100 μg mL-1) specifically attack mitochondria by excess accumulation, altering the metabolism, inhibiting branching pattern, imbalanced Bax/Bcl-2 homeostasis, and ultimately generating oxidative stress levels, leading to oxidative stress-induced cell death in cancer cells in vitro. We show that G-CQDs are the main cause of oxidative stress in cancer cells because of their ability to produce sufficient •OH- and 1O2 radicals, evidenced by electron paramagnetic resonance spectroscopy and a terephthalic acid test. Moreover, the near-infrared absorption properties of the CQDs were exhibited in two-photon (TP) emission, which was utilized for TP cellular imaging of cancer cells without photobleaching. The in vivo antitumor test further discloses that intratumoral injection of G-CQDs can significantly augment the treatment efficacy of subcutaneous tumors without any adverse effects on BalB/c nude mice. We believe that shape-specific polyphenolic CQD-based nanotheranostic agents have a potential role in tumor therapy, thus proving an insight on treatment of malignant cancers.

Redox-active polyphenol nanoparticles deprive endogenous glutathione of electrons for ROS generation and tumor chemodynamic therapy

Chemodynamic therapy (CDT) based on generating reactive oxygen species (ROS) is promising for cancer treatment. However, the intrinsic H2O2 is deficient for CDT, and glutathione (GSH) eliminates ROS to protect tumor cells from ROS cytotoxicity. Herein, we propose a strategy to switch the electron flow direction of GSH for O2 reduction and ROS generation rather than ROS clearance by using P(DA-Fc) nanoparticles, which are polymerized from ferrocenecarboxylic acid (Fc) coupled dopamine. P(DA-Fc) NPs with phenol-quinone conversion ability mimic NOX enzyme to deprive electrons from GSH to reduce O2 for H2O2 generation; the following •OH release can be triggered by Fc. Semiquinone radicals in P(DA-Fc) are significantly enhanced after GSH treatment, further demonstrated with strong single-electron reduction ability by calculation. In vitro and in vivo experiments indicate that P(DA-Fc) can consume intrinsic GSH to produce endogenous ROS; ROS generation strongly depends on GSH/pH level and eventually causes tumor cell death. Our work makes the first attempt to reverse the function of GSH from ROS scavenger to ROS producer, explores new roles of PDA-based nanomaterials in CDT beyond photothermal reagents and drug carriers, and provides a new strategy to improve the efficiency of CDT. Statement of SignificanceP(DA-Fc) nanoparticles performing tumor microenvironment response capacity and tumor reductive power utilize ability were fabricated for CDT tumor suppression. After endocytosis by tumor cells, P(DA-Fc) deprived GSH of electrons for H2O2 and •OH release, mimicking the intrinsic ROS production conducted by NADPH, further inducing tumor cell necrosis and apoptosis. Our work makes the first attempt to reverse the function of GSH from ROS scavenger to producer, explores new functions of PDA-based nanomaterials in CDT beyond photothermal reagents and drug carriers, and provides a new strategy to improve CDT efficiency.

Quantitative probing of reactive oxygen species and their selective degradation on contaminants in peroxymonosulfate-based process enhanced by picolinic acid

The processes of Fe(III) activated peroxymonosulfate (PMS) in degrading contaminants have been extensively studied. Herein, a biodegradable chelating agent, picolinic acid (PICA), was introduced to the PMS/Fe(III) process to improve the reaction efficiency. The emphases of this study were placed on the quantification of steady-state concentrations of reactive oxygen species (ROS). Experiments presented that five types of ROS, including Fe(IV), SO4•−, HO•, 1O2 and O2•− coexisted in this system. Four typical probe compounds were used to quantify the steady-state concentration of ROS under different variables. The steady-state concentration of Fe(IV) ([Fe(IV)]ss) was 3–5 orders of magnitude higher than that of other ROS, followed by 1O2 and SO4•−, whereas HO• had the lowest concentration. The reaction between PMS and PICA was first explored in our study and results showed that 1O2 and O2•− would form in this reaction. Owing to the hybrid oxidation by multiple ROS, this system showed high oxidation capacity, and could effectively degrade a variety of pollutants. The contributions of ROS to the alleviation of pollutants varied depending on their concentrations and specific reactivity of substrates. Generally, organic contaminants with phenol structures were prone to react with Fe(IV). Overall, this study compared the steady-state concentrations of different ROS and revealed the intrinsic ROS formation mechanisms.

Iron-Gallic Acid Peptide Nanoparticles as a Versatile Platform for Cellular Delivery with Synergistic ROS Enhancement Effect.

Cytosolic delivery of peptides is of great interest owing to their biological functions, which could be utilized for therapeutic applications. However, their susceptibility to enzymatic degradation and multiple cellular barriers generally hinders their clinical application. Integration into nanoparticles, which can enhance the stability and membrane permeability of bioactive peptides, is a promising strategy to overcome extracellular and intracellular obstacles. Herein, we present a versatile platform for the cellular delivery of various cargo peptides by integration into metallo-peptidic coordination nanoparticles. Both termini of cargo peptides were conjugated with gallic acid (GA) to assemble GA-modified peptides into nanostructures upon coordination of Fe(III). Initial pre-complexation of Fe(III) by poly-(vinylpolypyrrolidon) (PVP) as a template favored the formation of nanoparticles, which are able to deliver the peptides into cells efficiently. Iron-gallic acid peptide nanoparticles (IGPNs) are stable in water and are supposed to generate reactive oxygen species (ROS) from endogenous H2O2 in cells via the Fenton reaction. The strategy was successfully applied to an exemplary set of peptide sequences varying in length (1-7 amino acids) and charge (negative, neutral, positive). To confirm the capability of transporting bioactive cargos into cells, pro-apoptotic peptides were integrated into IGPNs, which demonstrated potent killing of human cervix carcinoma HeLa and murine neuroblastoma N2a cells at a 10 µM peptide concentration via the complementary mechanisms of peptide-triggered apoptosis and Fe(III)-mediated ROS generation. This study demonstrates the establishment of IGPNs as a novel and versatile platform for the assembly of peptides into nanoparticles, which can be used for cellular delivery of bioactive peptides combined with intrinsic ROS generation.

Flavonoids activated signaling pathways of mitochondrial biogenesis

The signaling pathways regulated by mitochondria involve among others, the intrinsic apoptotic pathway, calcium homeostasis and reactive oxygen species signaling, and ATP synthesis. The aim of this review is to narrate the importance of flavanoids (e.g., flavones, flavones, flavonols, flavonols-3-ols, anthocyanidins and isoflavones) in mitochondrial biogenesis. Therefore, the induction of mitochondrial biogenesis can be stimulated by nutrition by stimulating pathways that are responsible for activating certain kinases such as AMPK, p38 MAPK and Sirtuin 1, thereby signaling the expression of PGC-1α the conductor of mitochondrial biogenesis.

A conductive multifunctional hydrogel dressing with the synergistic effect of ROS-scavenging and electroactivity for the treatment and sensing of chronic diabetic wounds

Chronic diabetic wound with persistent inflammatory responses is still a serious threat to human health and life. Ideal wound dressings can be applied not only for covering the injury area, but also for regulating the inflammation to accelerate the wound healing and long-term monitoring of wound condition. However, there remains a challenge to design a multifunctional wound dressing for simultaneous treatment and monitoring of wound. Herein, an ionic conductive hydrogel with intrinsic reactive oxygen species (ROS)-scavenging properties and good electroactivity was developed for achieving the synergetic treatment and monitoring of diabetic wounds. In this study, we modified dextran methacrylate with phenylboronic acid (PBA) to prepare a ROS-scavenging material (DMP). Then the hydrogel was constructed by phenylboronic ester bonds induced dynamic crosslinking network, photo-crosslinked DMP and choline-based ionic liquid as the second network, and the crystallized polyvinyl alcohol as the third network, realizing good ROS-scavenging performance, high electroactivity, durable mechanical properties, and favorable biocompatibility. In vivo results showed that the hydrogel combined with electrical stimulation (ES) demonstrated good performance in promoting re-epithelization, angiogenesis and collagen deposition in chronic diabetic wound treatment by alleviating inflammation. Notably, with desirable mechanical properties and conductivity, the hydrogel could also precisely monitor movements of human body and possible tensile and compressive stresses of the wound site, providing timely alerts of excessive mechanical stress applied to the wound tissue. Thus, this “all-in-one” hydrogel exhibits great potential in constructing the next generation flexible bioelectronics for wound treatment and monitoring. Statement of significanceChronic diabetic wounds characterized by overexpressed reactive oxygen species (ROS) are still a serious threat to human health and life. However, there remains a challenge to design a multifunctional wound dressing for simultaneous wound treatment and monitoring. Herein, a flexible conductive hydrogel dressing with intrinsic ROS-scavenging properties and electroactivity was developed for the combined treatment and monitoring of the wound. The antioxidant hydrogel combined with electrical stimulation synergistically accelerated chronic diabetic wound healing by regulating oxidative stress, alleviating inflammation, promoting re-epithelization, angiogenesis and collagen deposition. Notably, with desirable mechanical properties and conductivity, the hydrogel also presented great potential in monitoring possible stresses of the wound site. The “all-in-one” bioelectronics integrating the treatment and monitoring functions present great application potential for accelerating chronic wound healing.

Pt(IV) prodrug initiated microparticles from microfluidics for tumor chemo-, photothermal and photodynamic combination therapy.

Multimodal treatment modalities hold great potential for cancer therapy, thus current efforts are focusing on the development of more effective and practical synergistic therapeutic platforms. Herein, we present a novel trans, trans,trans-[Pt(N3)2(OH)2(py)2] (Pt(IV)) prodrug-initiated hydrogel microparticles (MICG-Pt) with indocyanine green (ICG) encapsulation by microfluidics for efficiently synergistic chemo-, photothermal (PTT) and photodynamic therapy (PDT). The employed Pt(IV) could not only serves as an initiator to generate azidyl radical (N3 •) for photo-polymerization of methacrylate gelatin (GelMA) matrix, but also be reduced to high cytotoxic platinum(II) (Pt(II)) species for tumor chemotherapy. The laden ICG with highly photothermal heating ability and intrinsic reactive oxygen species (ROS) productivity endows the MICG-Pt with effective PTT/PDT performances upon near-infrared (NIR) light irradiation. In addition, benefiting from the production of oxygen during the photo-activation process of Pt(IV), the PDT efficacy of ICG-laden MICG-Pt could be further enhanced. Based on these advantages, we have demonstrated that the MICG-Pt could significantly eliminate cancer cells in vitro, and remarkably suppressed the tumor growth in vivo via synergistic chemotherapy, PTT, and PDT. These results indicate that such Pt(IV)-initiated hydrogel microparticles are ideal candidates of multimodal treatment platforms, holding great prospects for cancer therapy.

Photoactivation of Boronic Acid Prodrugs via a Phenyl Radical Mechanism: Iridium(III) Anticancer Complex as an Example.

Boronic acid (or ester) is a well-known temporary masking group for developing anticancer prodrugs responsive to tumoral reactive oxygen species (ROS), but their clinic application is largely hampered by the low activation efficiency. Herein, we report a robust photoactivation approach that can spatiotemporally convert boronic acid-caged iridium(III) complex IrBA into bioactive IrNH2 under hypoxic tumor microenvironments. Mechanistic studies show that the phenyl boronic acid moiety in IrBA is in equilibrium with phenyl boronate anion that can be photo-oxidized to generate phenyl radical, a highly reactive species that is capable of rapidly capturing O2 at extremely low concentrations (down to 0.02%). As a result, while IrBA could hardly be activated by intrinsic ROS in cancer cells, upon light irradiation, the prodrug is efficiently converted into IrNH2 even in limited O2 supply, along with direct damage to mitochondrial DNA and potent antitumor activities in hypoxic 2D monolayer cells, 3D tumor spheroids, and mice bearing tumor xenografts. Of note, the photoactivation approach could be extended to intermolecular photocatalytic activation by external photosensitizers with red absorption and to activate prodrugs of clinic compounds, thus offering a general approach for activation of anticancer organoboron prodrugs.

L-Ascorbic Acid 2-Phosphate Attenuates Methylmercury-Induced Apoptosis by Inhibiting Reactive Oxygen Species Accumulation and DNA Damage in Human SH-SY5Y Cells

Methylmercury (MeHg) is a toxin that causes severe neuronal oxidative damage. As vitamin C is an antioxidant well-known to protect neurons from oxidative damage, our goal was to elucidate its protective mechanism against MeHg-induced oxidative stress in human neuroblastomas (SHSY5Y). We treated cells with MeHg, L-ascorbic acid 2-phosphate (AA2P), or both, and used MTT, flow cytometry, and Western blot analyses to assess cell damage. We found that MeHg significantly decreased the survival rate of SH-SY5Y cells in a time- and dose-dependent manner, increased apoptosis, downregulated PAR and PARP1 expression, and upregulated AIF, Cyto C, and cleaved Caspase-3 expression. A time course study showed that MeHg increased reactive oxygen species (ROS) accumulation; enhanced apoptosis; increased DNA damage; upregulated expression ofγH2A.X, KU70, 67 and 57 kDa AIF, CytoC, and cleaved Caspase-3; and downregulated expression of 116 kDa PARP1, PAR, BRAC1, and Rad51. Supplementation with AA2P significantly increased cell viability and decreased intrinsic ROS accumulation. It also reduced ROS accumulation in cells treated with MeHg and decreased MeHg-induced apoptosis. Furthermore, AA2P conversely regulated gene expression compared to MeHg. Collectively, we demonstrate that AA2P attenuates MeHg-induced apoptosis by alleviating ROS-mediated DNA damage and is a potential treatment for MeHg neurotoxicity.

Theranostic Activity of Ceria-Based Nanoparticles toward Parental and Metastatic Melanoma: 2D vs 3D Models.

The time interval between the diagnosis of tumor in a patient and the initiation of treatment plays a key role in determining the survival rates. Consequently, theranostics, which is a combination of diagnosis and treatment, can be expected to improve survival rates. Early detection and immediate treatment initiation are particularly important in the management of melanoma, where survival rates decrease considerably after metastasis. The present work reports for the first time the application of fluorescein isothiocyanate (FITC)-tagged epidermal growth factor receptor (EGFR)-functionalized ceria nanoparticles, which exhibit intrinsic reactive oxygen species (ROS)-mediated anticancer effects, for the EGFR-targeted diagnosis and treatment of melanoma. The theranostic activity was demonstrated using two-dimensional (2D) and three-dimensional (3D) models of parental and metastatic melanoma. Confocal imaging studies confirm the diagnostic activity of the system. The therapeutic efficiency was evaluated using cell viability studies and ROS measurements. The ROS elevation levels are compared across the 2D and 3D models. Significant enhancement in the generation of cellular ROS and absence in mitochondrial ROS are observed in the 2D models. In contrast, significant elevations in both ROS types are observed for the 3D models, which are significantly higher for the metastatic spheroids than the parental spheroids, thus indicating the suitability of this nanoformulation for the treatment of metastatic melanoma.

The Role of Mitochondrial RNA from B-Precursor Acute Lymphoblastic Leukaemia Cells in Generating a Chemoprotective Bone Marrow Niche

Treatment resistant cells in precursor B-cell Acute Lymphoblastic Leukaemia (B-ALL) likely reside in a protective bone marrow niche with nestin-expressing mesenchymal stromal cells (MSC) (Duan et al Cancer Cell 2014). Previous work from our laboratory demonstrated that an 'activated MSC' niche, in which MSC have acquired features of cancer associated fibroblasts (CAF), develops in response to reactive oxygen species (ROS)-inducing chemotherapy. Mitochondria are actively transferred along tunneling nanotubes from activated MSC to B-ALL cells and can 'rescue' the cells from chemotherapy (Burt, et al Blood 2019). However, work from other groups suggests that chemoresistant ALL sub-clones exist prior to chemotherapy exposure, carrying a gene expression signature enriched for stemness, mitochondrial metabolism, stress responses and chromatin remodelling (Dobson et al Cancer Discovery 2020). We hypothesised that B-ALL cells may themselves directly activate bone marrow stromal cells and induce niche-formation prior to treatment, leading to ab-inito chemoresistance. To test this hypothesis, we first used our previously-described, in vitro niche model wherein either the HS27a MSC cell line or healthy donor MSCs were co-cultured with either B-ALL cell lines or primary patient B-ALL cells. Three of the five B-ALL cell lines and three of the four primary B-ALL samples that were tested induced activation of the MSCs, as characterised by cytomorphological changes identified by confocal microscopy, up-regulation of CAF-relevant gene expression and increased IL6, IL8 and CCL2 protein secretion. The ability of the B-ALL cell lines to activate MSC correlated directly with the intrinsic ROS level of the cell as determined by CellROX assay. Mitochondrial transfer from the cell-activated MSC to B-ALL cells, as measured by mitotracker dye transfer, occurred exactly as we had seen when we had used chemotherapy to activate the stroma. In vivo, the MSC-activating ALL cell lines also activated murine MSCs when established as xenografts in NOD-SCID gamma (NSG) mice. Immunohistochemistry of NSG murine femora revealed a very significant expansion in nestin+ cells after exposure to niche-induing ALL cells compared to non-niche-inducing controls. We aimed to determine the mechanism behind the B-ALL induced MSC activation. We determined that it was contact-independent, arising from exposure of MSC to ALL cells in transwell but also from exposure to B-ALL cell conditioned media alone. RNA sequencing of MSC after treatment with conditioned medium from activating B-ALL cells compared with non-activating controls unexpectedly showed that most highly upregulated pathways in the MSC-activating scenario were those involved in RNA sensing. Thus, we sought evidence for the role of mitochondrial dsRNA, as it had been recently shown to activate the anti-viral signalling pathway (Dhir et al, Nature 2018). Using the J2 anti-double stranded (ds) RNA monoclonal antibody in both confocal and flow cytometric assays, we observed very significantly higher levels of dsRNA in MSC which had been exposed to stromal-activating B-ALL cell conditioned medium than in non-activating B-ALL or MSC alone controls (see panel for image). We used the small molecule VBIT4, a voltage-dependent anion channel oligomerization inhibitor that decreases mitochondrial nucleic acid release to block this phenomenon in B-ALL cells. We quantified expression of a selection of stromal cell activation marker genes as well as genes in the RNA sensing pathway after treatment with VBIT4-exposed B-ALL cell CM or non-treated control. We observed a 2 to 4-fold reduction in expression of IL6 and CCL2 after VBIT4 exposure, compared to control. In the stromal cell RNA sensing pathway, there was a 15-fold reduction in expression of RIG-I and a 2 to 4-fold reduction in the interferon pathway genes MX2, IFIH1, MAVS and OAS1. Experiments to determine whether knock down of components of the antiviral sensing pathway also abrogates stromal activation and further murine experiments are ongoing. We propose a model in which ROS-induced mitochondrial dysfunction leads to release of dsRNA which in turn activates bone marrow stromal cells leading to protective niche formation and chemoprotection of acute lymphoblastic leukaemia cells despite lack of prior exposure to cytotoxic agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DNA Origami as a Nanomedicine for Targeted Rheumatoid Arthritis Therapy through Reactive Oxygen Species and Nitric Oxide Scavenging.

Rheumatoid arthritis (RA) severely threatens human health by causing inflammation, swelling, and pain in the joints and resulting in persistent synovitis and irreversible joint disability. In the development of RA, pro-inflammatory M1 macrophages, which express high levels of reactive oxygen species (ROS) and nitric oxide (NO), induce synovial inflammation and bone erosion. Eliminating ROS and NO in the inflamed joints is a potential RA therapeutic approach, which can drive the transition of pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype. Taking advantage of the intrinsic ROS- and NO-scavenging capability of DNA molecules, herein, we report the development of folic acid-modified triangular DNA origami nanostructures (FA-tDONs) for targeted RA treatment. FA-tDONs could efficiently scavenge ROS and NO and actively target M1 macrophages, facilitating the M1-to-M2 transition and the recovery of associated cytokines and biomarkers to the normal level. The therapeutic efficacy of FA-tDONs was examined in the RA mouse model. As validated by appearance, histological, and serum examinations, FA-tDONs treatment effectively alleviated synovial infiltration and cartilage damage, attenuating disease progression. This study demonstrated the usage of DNA origami for RA treatment and suggested its potential in other antioxidant therapies.