ROS-triggered degradable iron-chelating nanogels: Safely improving iron elimination in vivo.
ABSTRACT: Iron-mediated generation of highly toxic Reactive Oxygen Species (ROS) plays a major role in the process leading to iron overload-related diseases. The long-term subcutaneous administration of Deferoxamine (DFO) is currently clinically-approved to improve patient symptoms and survival. However, non-specific toxicity and short circulation times of the drug in humans often leads to poor patient compliance. Herein, thioketal-based ROS-responsive polymeric nanogels containing DFO moieties (rNG-DFO) were designed to chelate iron and to degrade under oxidative stimuli into fragments <10?nm to enhance excretion of iron-bound chelates. Serum ferritin levels and iron concentrations in major organs of IO mice decreased following treatment with rNG-DFO, and fecal elimination of iron-bound chelates increased compared to free DFO. Furthermore, rNG-DFO decreased iron mediated oxidative stress levels in vitro and reduced iron-mediated inflammation in the liver of IO mice. The study confirms that ROS-responsive nanogels may serve as a promising alternative to DFO for safer and more efficient iron chelation therapy.
Project description:Iron overload can increase cellular oxidative stress levels due to formation of reactive oxygen species (ROS); untreated, it can be extremely destructive to organs and fatal to patients. Since elevated oxidative stress levels are inherent to the condition in such patients, oxidation-induced degradable nanogels for iron chelation were rationally designed by simultaneously polymerizing oxidation-sensitive host-guest crosslinkers between ?-cyclodextrin (?-CD) and ferrocene (Fc) and iron chelating moieties composed of deferoxamine (DFO) into the final gel scaffold in reverse emulsion reaction chambers. UV-Vis absorption and atomic absorption spectroscopy (AAS) was used to verify iron chelating capability of nanogels. These materials can degrade into smaller chelating fragments at rates proportional to the level of oxidative stress present. Conjugating DFO reduces the cytotoxicity of the chelator in the macrophage cells. Importantly, the nanogel can effectively reduce cellular ferritin expression in iron overloaded cells and regulate intracellular iron levels at the same time, which is important for maintaining a homeostatic level of this critical metal in cells.
Project description:Deferoxamine (DFO) to treat iron overload (IO) has been limited by toxicity issues and short circulation times and it would be desirable to prolong circulation to improve non-transferrin bound iron (NTBI) chelation. In addition, DFO is currently unable to efficiently target the large pool of iron in the liver and spleen. Nanogel-Deferoxamine conjugates (NG-DFO) can prove useful as a model to investigate the pharmacokinetic (PK) properties and biodistribution (BD) behavior of iron-chelating macromolecules and their overall effect on serum ferritin levels. NG-DFO reduced the cytotoxicity of DFO and significantly reduced cellular ferritin levels in IO macrophages in vitro. PK/BD studies in normal rats revealed that NG-DFO displayed prolonged circulation and preferential accumulation into the liver and spleen. IO mice treated with NG1-DFO presented significantly lower levels of serum ferritin compared to DFO. Total renal and fecal elimination data point to the need to balance prolonged circulation with controlled degradation to accelerate clearance of iron-chelating macromolecules.
Project description:Background:Ferroptosis is a form of iron-dependent non-apoptotic cell death, with characteristics of loss of the activity of the lipid repair enzyme, glutathione (GSH) peroxidase 4 (GPX4), and accumulation of lethal reactive lipid oxygen species. The mechanism of ferroptosis in myelodysplastic syndrome (MDS) is unclear. Methods:Cell viability assay, reactive oxygen species (ROS) assay, GSH assay, and GPX activity assay were performed to study the regulation of ferroptosis in MDS cells obtained from MDS patients, the iron overload model mice, and cell lines. Results:The growth-inhibitory effect of decitabine could be partially reversed by ferrostatin-1 and iron-chelating agent [desferrioxamine (DFO)] in MDS cell lines. Erastin could increase the cytotoxicity of decitabine on MDS cells. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in MDS cells upon treatment with decitabine, which could be reversed by ferrostatin-1. The concentration of hemoglobin in peripheral blood of iron overload mice was negatively correlated with intracellular Fe2+ level and ferritin concentration. Iron overload (IO) led to decreased viability of bone marrow mononuclear cells (BMMNCs), which was negatively correlated with intracellular Fe2+ level. Ferrostatin-1 partially reversed the decline of cell viability in IO groups. The level of GSH and the activity of GPX4 decreased, whereas the ROS level increased in BMMNCs of IO mice. DFO could increase the level of GSH. Ferrostatin-1 and DFO could increase the GPX4 activity of BMMNCs in IO mice. Ferrostatin-1 could significantly reverse the growth-inhibitory effect of decitabine in MDS patients. Decitabine could significantly increase the ROS level in MDS groups, which could be inhibited by ferrostatin-1 or promoted by erastin. Ferrostatin-1 could significantly reverse the inhibitory effect of decitabine on GSH levels in MDS patients. Erastin combined with decitabine could further reduce the GSH level. Erastin could further decrease the activity of GPX4 compared with the decitabine group. Conclusion:Ferroptosis may account for the main mechanisms of how decitabine induced death of MDS cells. Decitabine-induced ROS raise leads to ferroptosis in MDS cells by decreasing GSH level and GPX4 activity.
Project description:Iron-deficient anemia is a prevalent disease among humans. We searched for genes regulated by iron deficiency and its regulated mechanism. cDNA microarrays were performed using Hepa1c1c7 cells treated with 100 ?M desferrioxamine (DFO), an iron chelator. Early growth response 1 (Egr1) was upregulated with at least 20-fold increase within 4 h and lasted for 24 h, which was confirmed by qRT-PCR. This activation was not seen by ferric ammonium citrate (FAC). DFO increased the transcriptional activity of Egr1-luc (-604 to +160) and serum response element (SRE)-luc reporters by 2.7-folds. In addition, cycloheximide lowered DFO-induced Egr1 mRNA levels. The upregulation of Egr1 by DFO was accompanied by sustained ERK signals along with phosphorylation of Elk-1. The ERK inhibitor (PD98059) prevented the DFO-induced Egr1 mRNAs. Overexpression of Elk-1 mutant (pElk-1S383A) decreased Egr1 reporter activity. DFO lowered reactive oxygen species (ROS) production and increased caspase 3/7 activity and cell death. DFO-induced iron deficiency upregulates Egr1 in part through transcriptional activation via ERK and Elk-1 signals, which may be important in the regulation of cell death in hepatoma cells. Our study demonstrated that iron depletion controlled the expression of Egr1, which might contribute to decisions about cellular fate in response to iron deficiency.
Project description:A new heterobifunctional reactive oxygen species (ROS) responsive thioketal linker and its synthesis are described. This linker allows for developing new ROS-responsive agents with two distinct functionalities using universal bioconjugation methods. The reaction kinetics of the thioketal cleavage in the presence of ROS is also described.
Project description:The bone marrow microenvironment plays important roles in the progression of the myelodysplastic syndrome (MDS). The higher incidence of ASXL1 and TET2 gene mutations in our iron overload (IO) MDS patients suggests that IO may be involved in the pathogenesis of MDS. The effects of IO damaging bone marrow mesenchymal stromal cells (MSCs) from higher-risk MDS patients were investigated. In our study, IO decreased the quantity and weakened the abilities of proliferation and differentiation of MSCs, and it inhibited the gene expressions of VEGFA, CXCL12, and TGF-?1 in MSCs regulating hematopoiesis. The increased level of reactive oxygen species (ROS) in MSCs caused by IO might be inducing apoptosis by activating caspase3 signals and involving in MDS progression by activating ?-catenin signals. The damages of MSCs caused by IO could be partially reversed by an antioxidant or an iron chelator. Furthermore, the MSCs in IO MDS/AML patients had increased levels of ROS and apoptosis, and the expressions of caspase3 and ?-catenin were increased even further. In conclusion, IO affects gene stability in higher-risk MDS patients and impairs MSCs by inducing ROS-related apoptosis and activating the Wnt/?-catenin signaling pathway, which could be partially reversed by an antioxidant or an iron chelator.
Project description:Essentials Potential neurodevelopmental side effects of thrombopoietin mimetics need to be considered. The effects of eltrombopag (ELT) on neuronal iron status and dendrite development were assessed. ELT crosses the blood-brain barrier and causes iron deficiency in developing neurons. ELT blunts dendrite maturation, indicating a need for more safety studies before neonatal use.Background Thrombocytopenia is common in sick neonates. Thrombopoietin mimetics (e.g. eltrombopag [ELT]) might provide an alternative therapy for selected neonates with severe and prolonged thrombocytopenia, and for infants and young children with different varieties of thrombocytopenia. However, ELT chelates intracellular iron, which may adversely affect developing organs with high metabolic requirements. Iron deficiency (ID) is particularly deleterious during brain development, impairing neuronal myelination, dopamine signaling and dendritic maturation and ultimately impairing long-term neurological function (e.g. hippocampal-dependent learning and memory). Objective To determine whether ELT crosses the blood-brain barrier (BBB), causes neuronal ID and impairs hippocampal neuron dendrite maturation. Methods ELT transport across the BBB was assessed using primary bovine brain microvascular endothelial cells. Embryonic mouse primary hippocampal neuron cultures were treated with ELT or deferoxamine (DFO, an iron chelator) from 7 days in vitro (DIV) through 14 DIV and assessed for gene expression and neuronal dendrite complexity. Results ELT crossed the BBB in a time-dependent manner. 2 and 6 ?m ELT increased Tfr1 and Slc11a2 (iron-responsive genes involved in neuronal iron uptake) mRNA levels, indicating neuronal ID. 6 ?m ELT, but not 2 ?m ELT, decreased BdnfVI, Camk2a and Vamp1 mRNA levels, suggesting impaired neuronal development and synaptic function. Dendrite branch number and length were reduced in 6 ?m ELT-treated neurons, resulting in blunted dendritic arbor complexity that was similar to DFO-treated neurons. Conclusions Eltrombopag treatment during development may impair neuronal structure as a result of neuronal ID. Preclinical in vivo studies are warranted to assess ELT safety during periods of rapid brain development.
Project description:Tropolones are naturally occurring seven-membered non-benzenoid aromatic compounds that are of interest due to their cytotoxic properties. MO-OH-Nap is a novel ?-substituted tropolone that induces caspase cleavage and upregulates markers associated with the unfolded protein response (UPR) in multiple myeloma (MM) cells. Given previous reports that tropolones may function as iron chelators, we investigated the effects of MO-OH-Nap, as well as the known iron chelator deferoxamine (DFO), in MM cells in the presence or absence of supplemental iron. The ability of MO-OH-Nap to induce apoptosis and upregulate markers of the UPR could be completely prevented by co-incubation with either ferric chloride or ammonium ferrous sulfate. Iron also completely prevented the decrease in BrdU incorporation induced by either DFO or MO-OH-Nap. Ferrozine assays demonstrated that MO-OH-Nap directly chelates iron. Furthermore, MO-OH-Nap upregulates cell surface expression and mRNA levels of transferrin receptor. In vivo studies demonstrate increased Prussian blue staining in hepatosplenic macrophages in MO-OH-Nap-treated mice. These studies demonstrate that MO-OH-Nap-induced cytotoxic effects in MM cells are dependent on the tropolone's ability to alter cellular iron availability and establish new connections between iron homeostasis and the UPR in MM.
Project description:Many drug controlled release methods have been integrated in multifunctional nanoparticles, such as pH-, redox-, temperature-, enzyme-, and light-responsive release. However, few report is associated with the ROS responsive drug controlled release. Herein, a thioketal linker-based ROS responsive drug (camptothecin conjugated with thioketal linker, abbreviated as TL-CPT) was prepared and the thioketal linker could be cleaved by ROS(reactive oxygen species). To achieve cancer simultaneous optical imaging, photodynamic therapy and chemotherapy, the photosensitizer Chlorin e6(Ce6), TL-CPT and carboxyl-mPEG were loaded on the upconversion nanoparticles (UCNPs), which were named as Ce6-CPT-UCNPs. Under 980 nm laser irradiation, Ce6-CPT-UCNPs emitted a narrow emission band at 645-675 nm which was overlapped with Ce6 absorption peak. Ce6 absorbed the light to produce ROS, which was used for photodynamic therapy and to cleave the thioketal linker in Ce6-CPT-UCNPs to release camptothecin for chemotherapy. Meanwhile, Ce6 absorbed the light, was used for near-infrared fluorescence imaging. The in vivo biodistribution studies showed that the prepared nanoparticles had high orthotopic lung cancer targeting efficiency. The in vivo therapeutic results demonstrated that NCI-H460 lung cancers could be completely eliminated by combining chemo- and photodynamic therapy under 980 nm laser irradiation. The prepared multifunctional Ce6-CPT-UCNPs have great potential in applications such as cancer targeted fluorescent imaging, simultaneous ROS activated chemo- and photodynamic therapy in near future.
Project description:Iron overload (IO) has been reported to contribute to mesenchymal stromal cell (MSC) damage, but the precise mechanism has yet to be clearly elucidated. In this study, we found that IO increased cell apoptosis and lowered cell viability in MSCs, accompanied by extensive mitochondrial fragmentation and autophagy enhancement. All these effects were reactive oxygen species (ROS) dependent. In MSCs with IO, the ATP concentrations were significantly reduced due to high ROS levels and low electron respiratory chain complex (ETC) II/III activity. Reduced ATP phosphorylated AMP-activated protein kinase (AMPK). Activation of AMPK kinase complexes triggered mitochondrial fission. Moreover, gene knockout of AMPK via CRISPR/Cas9 reduced cell apoptosis, enhanced cell viability and attenuated mitochondrial fragmentation and autophagy caused by IO in MSCs. Further, AMPK-induced mitochondrial fragmentation of MSCs with IO was mediated via phosphorylation of mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for the GTPase dynamin-related protein 1 (Drp1). Gene knockdown of MFF reversed AMPK-induced mitochondrial fragmentation in MSCs with IO. In addition, MSCs from IO patients with myelodysplastic syndrome (MDS) showed increased cell apoptosis, decreased cell viability, higher ROS levels, lower ATP concentrations and increased mitochondrial fragmentation compared with MSCs from non-IO patients. In addition, iron chelation or antioxidant weakened the activity of the AMPK/MFF/Drp1 pathway in MDS-MSCs with IO from several patients, accompanied by attenuation of mitochondrial fragmentation and autophagy. Taken together, the AMPK/MFF/Drp1 pathway has an important role in the damage to MDS-MSCs caused by IO.