HMOX1 regulates ferroptosis via mic14 and its impact on chemotherapy resistance in small-cell lung cancer.

This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed bioinformatics, qPCR, and Western Blot to assess HMOX1 levels in SCLC versus normal tissues and its prognostic relevance. CCK-8, flow cytometry, and thiobarbituric acid assays determined HMOX1's impact on SCLC chemosensitivity, ferroptosis markers, lipid peroxidation, and mic14's role in chemoresistance. In the GSE40275 and GSE60052 cohorts, HMOX1 expression was downregulated in SCLC tissues compared to normal tissues. Higher HMOX1 expression was associated with improved prognosis in the Sun Yat-sen University Cancer Hospital cohort and GSE60052 cohort. The RNA and protein levels of HMOX1 were reduced in drug-resistant SCLC cell lines compared to chemosensitive cell lines. Upregulation of HMOX1 increased chemosensitivity and reduced drug resistance in SCLC, while downregulation of HMOX1 decreased chemosensitivity and increased drug resistance. Upregulation of HMOX1 elevated the expression of ferroptosis-related proteins ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while decreasing the expression of GPX4 and xCT. Conversely, downregulation of HMOX1 decreased the expression of ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while increasing the expression of GPX4 and xCT. Upregulation of HMOX1 promoted cellular lipid peroxidation, whereas downregulation of HMOX1 inhibited cellular lipid peroxidation. Upregulation of HMOX1 reduced the RNA level of mic14, while downregulation of HMOX1 increased the RNA level of mic14. mic14 exhibited inhibitory effects on cellular lipid peroxidation in SCLC cells and contributed to reduced chemosensitivity and increased drug resistance in chemoresistant SCLC cell lines. HMOX1 plays a role in ferroptosis by regulating mic14 expression, thereby reversing chemoresistance in SCLC.

Inhibitory effects of temozolomide on glioma cells is sensitized by RSL3-induced ferroptosis but negatively correlated with expression of ferritin heavy chain 1 and ferritin light chain.

Invasive growth of glioblastoma makes residual tumor unremovable by surgery and leads to disease relapse. Temozolomide is widely used first-line chemotherapy drug to treat glioma patients, but development of temozolomide resistance is almost inevitable. Ferroptosis, an iron-dependent form of non-apoptotic cell death, is found to be related to temozolomide response of gliomas. However, whether inducing ferroptosis could affect invasive growth of glioblastoma cells and which ferroptosis-related regulators were involved in temozolomide resistance are still unclear. In this study, we treated glioblastoma cells with RSL3, a ferroptosis inducer, in vitro (cell lines) and in vivo (subcutaneous and orthotopic animal models). The treated glioblastoma cells with wild-type or mutant IDH1 were subjected to RNA sequencing for transcriptomic profiling. We then analyze data from our RNA sequencing and public TCGA glioma database to identify ferroptosis-related biomarkers for prediction of prognosis and temozolomide resistance in gliomas. Analysis of transcriptome data from RSL3-treated glioblastoma cells suggested that RSL3 could inhibit glioblastoma cell growth and suppress expression of genes involved in cell cycle. RSL3 effectively reduced mobility of glioblastoma cells through downregulation of critical genes involved in epithelial-mesenchymal transition. Moreover, RSL3 in combination with temozolomide showed suppressive efficacy on glioblastoma cell growth, providing a promising therapeutic strategy for glioblastoma treatment. Although temozolomide attenuated invasion of glioblastoma cells with mutant IDH1 more than those with wild-type IDH1, the combination of RSL3 and temozolomide similarly impaired invasive ability of glioblastoma cells in spite of IDH1 status. Finally, we noticed that both ferritin heavy chain 1 and ferritin light chain predicted unfavorable prognosis of glioma patients and were significantly correlated with mRNA levels of methylguanine methyltransferase as well as temozolomide resistance. Altogether, our study provided rationale for combination of RSL3 with temozolomide to suppress glioblastoma cells and revealed ferritin heavy chain 1 and ferritin light chain as biomarkers to predict prognosis and temozolomide resistance of glioma patients.

ERK-Peptide-Inhibitor-Modified Ferritin Enhanced the Therapeutic Effects of Paclitaxel in Cancer Cells and Spheroids.

Rational design of a drug delivery system with enhanced therapeutic potency is critical for efficient tumor chemotherapy. Many protein-based drug delivery platforms have been designed to deliver drugs to target sites and improve the therapeutic efficacy. In this study, paclitaxel (PTX) molecules were encapsulated within an apoferritin nanocage-based drug delivery system with the modification of an extracellular-signal-regulated kinase (ERK) peptide inhibitor at the C-terminus of ferritin (HERK). Apoferritin is an endogenous nano-sized spherical protein which has the ability to specially bind to a majority of tumor cells via interacting with transferrin receptor 1. The ERK peptide inhibitor is a peptide which can disrupt the interaction of MEK with ERK in the mitogen-activated protein kinase/ERK pathway. By combining the targeted delivery effect of ferritin and the inhibitory effect of the ERK peptide inhibitor, the newly fabricated ferritin carrier nanoparticle HERK could still be taken up by tumor cells, and it displayed higher cell cytotoxicity than the parent ferritin. After loading with PTX, HERK-PTX displayed a favorable anticancer effect in human breast cancer cells MDA-MB-231 and lung carcinoma cells A549. The remarkable inhibitory effect on MDA-MB-231 tumor spheroids was also identified. These results indicated that the constructed HERK nanocarrier is a promising multi-functional drug delivery vehicle to enhance the therapeutic effect of drugs in cancer therapy.

Potential Role of H-Ferritin in Mitigating Valvular Mineralization.

Objective- Calcific aortic valve disease is a prominent finding in elderly and in patients with chronic kidney disease. We investigated the potential role of iron metabolism in the pathogenesis of calcific aortic valve disease. Approach and Results- Cultured valvular interstitial cells of stenotic aortic valve with calcification from patients undergoing valve replacement exhibited significant susceptibility to mineralization/osteoblastic transdifferentiation in response to phosphate. This process was abrogated by iron via induction of H-ferritin as reflected by lowering ALP and osteocalcin secretion and preventing extracellular calcium deposition. Cellular phosphate uptake and accumulation of lysosomal phosphate were decreased. Accordingly, expression of phosphate transporters Pit1 and Pit2 were repressed. Translocation of ferritin into lysosomes occurred with high phosphate-binding capacity. Importantly, ferritin reduced nuclear accumulation of RUNX2 (Runt-related transcription factor 2), and as a reciprocal effect, it enhanced nuclear localization of transcription factor Sox9 (SRY [sex-determining region Y]-box 9). Pyrophosphate generation was also increased via upregulation of ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase-2). 3H-1, 2-dithiole-3-thione mimicked these beneficial effects in valvular interstitial cell via induction of H-ferritin. Ferroxidase activity of H-ferritin was essential for this function, as ceruloplasmin exhibited similar inhibitory functions. Histological analysis of stenotic aortic valve revealed high expression of H-ferritin without iron accumulation and its relative dominance over ALP in noncalcified regions. Increased expression of H-ferritin accompanied by elevation of TNF-α (tumor necrosis factor-α) and IL-1ß (interleukin-1ß) levels, inducers of H-ferritin, corroborates the essential role of ferritin/ferroxidase via attenuating inflammation in calcific aortic valve disease. Conclusions- Our results indicate that H-ferritin is a stratagem in mitigating valvular mineralization/osteoblastic differentiation. Utilization of 3H-1, 2-dithiole-3-thione to induce ferritin expression may prove a novel therapeutic potential in valvular mineralization.

Protein nanocages that penetrate airway mucus and tumor tissue.

Reports on drug delivery systems capable of overcoming multiple biological barriers are rare. We introduce a nanoparticle-based drug delivery technology capable of rapidly penetrating both lung tumor tissue and the mucus layer that protects airway tissues from nanoscale objects. Specifically, human ferritin heavy-chain nanocages (FTn) were functionalized with polyethylene glycol (PEG) in a unique manner that allows robust control over PEG location (nanoparticle surface only) and surface density. We varied PEG surface density and molecular weight to discover PEGylated FTn that rapidly penetrated both mucus barriers and tumor tissues in vitro and in vivo. Upon inhalation in mice, PEGylated FTn with optimized PEGylation rapidly penetrated the mucus gel layer and thus provided a uniform distribution throughout the airways. Subsequently, PEGylated FTn preferentially penetrated and distributed within orthotopic lung tumor tissue, and selectively entered cancer cells, in a transferrin receptor 1-dependent manner, which is up-regulated in most cancers. To test the potential therapeutic benefits, doxorubicin (DOX) was conjugated to PEGylated FTn via an acid-labile linker to facilitate intracellular release of DOX after cell entry. Inhalation of DOX-loaded PEGylated FTn led to 60% survival, compared with 10% survival in the group that inhaled DOX in solution at the maximally tolerated dose, in a murine model of malignant airway lung cancer. This approach may provide benefits as an adjuvant therapy combined with systemic chemo- or immunotherapy or as a stand-alone therapy for patients with tumors confined to the airways.

Apoferritin and Apoferritin-Capped Metal Nanoparticles Inhibit Arginine Kinase of Trypanosoma brucei.

The aim of this study was to explore the inhibitory potential of apoferritin or apoferritin-capped metal nanoparticles (silver, gold and platinum) against Trypanosomabrucei arginine kinase. The arginine kinase activity was determined in the presence and absence of apoferritin or apoferritin-capped metal nanoparticles. In addition, kinetic parameters and relative inhibition of enzyme activity were estimated. Apoferritin or apoferritin-capped metal nanoparticles' interaction with arginine kinase of T. brucei led to a >70% reduction in the enzyme activity. Further analysis to determine kinetic parameters suggests a mixed inhibition by apoferritin or apoferritin-nanoparticles, with a decrease in Vmax. Furthermore, the Km of the enzyme increased for both ATP and L-arginine substrates. Meantime, the inhibition constant (Ki) values for the apoferritin and apoferritin-nanoparticle interaction were in the submicromolar concentration ranging between 0.062 to 0.168 nM and 0.001 to 0.057 nM, respectively, for both substrates (i.e., L-arginine and ATP). Further kinetic analyses are warranted to aid the development of these nanoparticles as selective therapeutics. Also, more studies are required to elucidate the binding properties of these nanoparticles to arginine kinase of T. brucei.

Protein Nanocage Mediated Fibroblast-Activation Protein Targeted Photoimmunotherapy To Enhance Cytotoxic T Cell Infiltration and Tumor Control.

Carcinoma-associated fibroblasts (CAFs) are found in many types of cancer and play an important role in tumor growth and metastasis. Fibroblast-activation protein (FAP), which is overexpressed on the surface of CAFs, has been proposed as a universal tumor targeting antigen. However, recent studies show that FAP is also expressed on multipotent bone marrow stem cells. A systematic anti-FAP therapy may lead to severe side effects and even death. Hence, there is an urgent need of a therapy that can selectively kill CAFs without causing systemic toxicity. Herein we report a nanoparticle-based photoimmunotherapy (nano-PIT) approach that addresses the need. Specifically, we exploit ferritin, a compact nanoparticle protein cage, as a photosensitizer carrier, and we conjugate to the surface of ferritin a FAP-specific single chain variable fragment (scFv). With photoirradiation, the enabled nano-PIT efficiently eliminates CAFs in tumors but causes little damage to healthy tissues due to the localized nature of the treatment. Interestingly, while not directly killing cancer cells, the nano-PIT caused efficient tumor suppression in tumor-bearing immunocompetent mice. Further investigations found that the nano-PIT led to suppressed C-X-C motif chemokine ligand 12 (CXCL12) secretion and extracellular matrix (ECM) deposition, both of which are regulated by CAFs in untreated tumors and mediate T cell exclusion that prevents physical contact between T cells and cancer cells. By selective killing of CAFs, the nano-PIT reversed the effect, leading to significantly enhanced T cell infiltration, followed by efficient tumor suppression. Our study suggests a new and safe CAF-targeted therapy and a novel strategy to modulate tumor microenvironment (TME) for enhanced immunity against cancer.

H-Ferritin Enriches the Curcumin Uptake and Improves the Therapeutic Efficacy in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a highly aggressive, invasive, and metastatic tumor. Although it is reported to be sensitive to cytotoxic chemotherapeutics, frequent relapse and chemoresistance often result in treatment failure. In this study, we developed a biomimetic nanodrug consisting of a self-assembling variant (HFn) of human apoferritin loaded with curcumin. HFn nanocage improved the solubility, chemical stability, and bioavailability of curcumin, allowing us to reliably carry out several experiments in the attempt to establish the potential of this molecule as a therapeutic agent and elucidate the mechanism of action in TNBC. HFn biopolymer was designed to bind selectively to the TfR1 receptor overexpressed in TNBC cells. HFn-curcumin (CFn) proved to be more effective in viability assays compared to the drug alone using MDA-MB-468 and MDA-MB-231 cell lines, representative of basal and claudin-low TNBC subtypes, respectively. Cellular uptake of CFn was demonstrated by flow cytometry and label-free confocal Raman imaging. CFn could act as a chemosensitizer enhancing the cytotoxic effect of doxorubicin by interfering with the activity of multidrug resistance transporters. In addition, CFn exhibited different cell cycle effects on these two TNBC cell lines, blocking MDA-MB-231 in G0/G1 phase, whereas MDA-MB-468 accumulated in G2/M phase. CFn was able to inhibit the Akt phosphorylation, suggesting that the effect on the proliferation and cell cycle involved the alteration of PI3K/Akt pathway.

Comparative analysis of two ferritin subunits from blunt snout bream (Megalobrama amblycephala): Characterization, expression, iron depriving and bacteriostatic activity.

Iron is an essential microelement for almost all living organisms, while an excess of iron is toxic, thus maintenance of iron homeostasis is vital. As iron storage protein, ferritin plays an important role in iron metabolism. In the present study, we cloned and characterized the ferritin H subunit from Megalobrama amblycephala, termed as MamFerH. An iron-responsive element (IRE) was predicted in the 5' untranslated region (UTR) of MamFerH, while its bulge structural was different from that of the reported ferritin M subunit (MamFerM). The MamFerH and MamFerM genes exhibited similar expression patterns during early development with specifically high expression post hatching, whereas their tissue expression patterns were different. Specifically, MamFerM was highly expressed in the spleen, liver and kidney, while MamFerH was predominantly expressed in the blood and brain, indicating their different functions. In addition, the expression of the two genes was induced upon Aeromonas hydrophila infection at both transcriptional and translational levels, and MamFerH was more efficient. Immunohistochemistry and immunofluorescence analysis confirmed their significant changes at protein level and distribution in the liver post infection, indicating their participation in host immune response. Furthermore, bacteriostatic experiment revealed that recombinant MamFerH displayed more significant inhibitory effect on the growth of A. hydrophila.

H-ferritin-nanocaged doxorubicin nanoparticles specifically target and kill tumors with a single-dose injection.

An ideal nanocarrier for efficient drug delivery must be able to target specific cells and carry high doses of therapeutic drugs and should also exhibit optimized physicochemical properties and biocompatibility. However, it is a tremendous challenge to engineer all of the above characteristics into a single carrier particle. Here, we show that natural H-ferritin (HFn) nanocages can carry high doses of doxorubicin (Dox) for tumor-specific targeting and killing without any targeting ligand functionalization or property modulation. Dox-loaded HFn (HFn-Dox) specifically bound and subsequently internalized into tumor cells via interaction with overexpressed transferrin receptor 1 and released Dox in the lysosomes. In vivo in the mouse, HFn-Dox exhibited more than 10-fold higher intratumoral drug concentration than free Dox and significantly inhibited tumor growth after a single-dose injection. Importantly, HFn-Dox displayed an excellent safety profile that significantly reduced healthy organ drug exposure and improved the maximum tolerated dose by fourfold compared with free Dox. Moreover, because the HFn nanocarrier has well-defined morphology and does not need any ligand modification or property modulation it can be easily produced with high purity and yield, which are requirements for drugs used in clinical trials. Thus, these unique properties make the HFn nanocage an ideal vehicle for efficient anticancer drug delivery.

Excitotoxic potential of exogenous ferritin and apoferritin: changes in ambient level of glutamate and synaptic vesicle acidification in brain nerve terminals.

Ferritin, an iron storage protein, is present in the serum and cerebrospinal fluid, has receptors on the cell surface, able to penetrate the brain-blood barrier, can be secreted from the cells, and leaks from destroyed cell in insult and brain trauma. The effect of exogenous ferritin on the key characteristic of glutamatergic neurotransmission was assessed in rat brain nerve terminals (synaptosomes). Exogenous ferritin (80 µg/ml, iron content 0.7%) significantly increased the ambient level of L-[(14)C]glutamate (0.200±0.015 versus 0.368±0.016 nmol/mg of protein) and endogenous glutamate (fluorimetric glutamate dehydrogenase assay) in the nerve terminals. This increase was not a result of augmentation of tonic release because the velocity of tonic release of L-[(14)C]glutamate was not changed significantly in ferritin-treated synaptosomes as compared to the control. Ferritin caused a decrease in synaptic vesicle acidification that was shown using fluorescent dye acridine orange. Iron-dependence of the effects of ferritin was analyzed with apoferritin (0.0025% residual iron). Apoferritin weakly affected the proton electrochemical gradient of synaptic vesicles but increased the ambient level and decreased the initial velocity of uptake of L-[(14)C]glutamate by synaptosomes, nevertheless these effects were ~30% lesser than those caused by ferritin. Exogenous ferritin can provoke the development of excitotoxicity increasing the ambient level of glutamate and lowering synaptic vesicle acidification and glutamate uptake in the nerve terminals, however these effects are not completely iron-dependent. Thus, in the CNS exogenous ferritin can act as a modulator of glutamate homeostasis in iron-dependent and iron-independent manner.

Cyclosporine A-loaded apoferritin alleviates myocardial ischemia-reperfusion injury by simultaneously blocking ferroptosis and apoptosis of cardiomyocytes.

Myocardial ischemia-reperfusion injury (MI/RI) seriously restricts the therapeutic effect of reperfusion. It is demonstrated that ferroptosis and apoptosis of cardiomyocytes are widely involved in MI/RI. Therefore, simultaneous inhibition of ferroptosis and apoptosis of cardiomyocytes can be a promising strategy to treat MI/RI. Besides, transferrin receptor 1 (TfR1) is highly expressed in ischemic myocardium, and apoferritin (ApoFn) is a ligand of the transferrin receptor. In this study, CsA@ApoFn was prepared by wrapping cyclosporin A (CsA) with ApoFn and actively accumulated in ischemic cardiomyocytes through TfR1 mediated endoctosis in MI/RI mice. After entering cardiomyocytes, ApoFn in CsA@ApoFn inhibited ferroptosis of ischemic cardiomyocytes by increasing the protein expression of GPX4 and reducing the content of labile iron pool and lipid peroxides. At the same time, CsA in CsA@ApoFn attenuated the apoptosis of ischemic cardiomyocytes through recovering mitochondrial membrane potential and reducing the level of reactive oxygen species, which played a synergistic role with ApoFn in the treatment of MI/RI. In conclusion, CsA@ApoFn restored cardiac function of MI/RI mice by simultaneously blocking ferroptosis and apoptosis of cardiomyocytes. ApoFn itself not only served as a safe carrier to specifically deliver CsA to ischemic cardiomyocytes but also played a therapeutic role on MI/RI. CsA@ApoFn is proved as an effective drug delivery platform for the treatment of MI/RI. STATEMENT OF SIGNIFICANCE: Recent studies have shown that ferroptosis is an important mechanism of myocardial ischemia-reperfusion injury (MI/RI). Therefore, simultaneous inhibition of ferroptosis and apoptosis of cardiomyocytes can be a promising strategy to treat MI/RI. Apoferritin, as a delivery carrier, can actively target to ischemic myocardium through binding with highly expressed transferrin receptor on ischemic cardiomyocytes. At the same time, apoferritin plays a protective role on ischemic cardiomyocytes by inhibiting ferroptosis. This strategy of killing two birds with one stone significantly improves the therapeutic effect on MI/RI while does not need more pharmaceutical excipients, which has the prospect of clinical transformation.

Ferritin heavy chain participated in ameliorating 3-nitropropionic acid-induced oxidative stress and apoptosis of goose follicular granulosa cells.

Oxidative stress is the major culprits responsible for ovarian dysfunction by damaging granulosa cells (GCs). Ferritin heavy chain (FHC) may participate in the regulation of ovarian function by mediating GCs apoptosis. However, the specific regulatory function of FHC in follicular GCs remains unclear. Here, 3-nitropropionic acid (3-NPA) was utilized to establish an oxidative stress model of follicular GCs of Sichuan white geese. To explore the regulatory effects of FHC on oxidative stress and apoptosis of primary GCs in geese by interfering or overexpressing FHC gene. After transfection of siRNA-FHC to GCs for 60 h, the expressions of FHC gene and protein decreased significantly (P < 0.05). After FHC overexpression for 72 h, the expressions of FHC mRNA and protein upregulated considerably (P < 0.05). The activity of GCs was impaired after interfering with FHC and 3-NPA coincubated (P < 0.05). When overexpression of FHC combined with 3-NPA treatment, the activity of GCs was remarkably enhanced (P < 0.05). After interference FHC and 3-NPA treatment, NF-κB and NRF2 gene expression decreased (P < 0.05), the intracellular reactive oxygen species (ROS) level increased greatly (P < 0.05), BCL-2 expression reduced, BAX/BCL-2 ratio intensified (P < 0.05), the mitochondrial membrane potential decreased notably (P < 0.05), and the apoptosis rate of GCs aggravated (P < 0.05). While overexpression of FHC combined with 3-NPA treatment could promote BCL-2 protein expression and reduce BAX/BCL-2 ratio, indicating that FHC regulated the mitochondrial membrane potential and apoptosis of GCs by mediating the expression of BCL-2. Taken together, our research manifested that FHC alleviated the inhibitory effect of 3-NPA on the activity of GCs. FHC knockdown could suppress the expression of NRF2 and NF-κB genes, reduce BCL-2 expression and augment BAX/BCL-2 ratio, contributing to the accumulation of ROS and jeopardizing mitochondrial membrane potential, as well as exacerbating GCs apoptosis.

Rational design of engineered H-ferritin nanoparticles with improved siRNA delivery efficacy across an in vitro model of the mouse BBB.

Gene therapy holds tremendous potential for the treatment of incurable brain diseases including Alzheimer's disease (AD), stroke, glioma, and Parkinson's disease. The main challenge is the lack of effective gene delivery systems traversing the blood-brain barrier (BBB), due to the complex microvessels present in the brain which restrict substances from the circulating blood passing through. Recently, increasing efforts have been made to develop promising gene carriers for brain-related disease therapies. One such development is the self-assembled heavy chain ferritin (HFn) nanoparticles (NPs). HFn NPs have a unique hollow spherical structure that can encapsulate nucleic acid drugs (NADs) and specifically bind to cancer cells and BBB endothelial cells (BBB ECs) via interactions with the transferrin receptor 1 (TfR1) overexpressed on their surfaces, which increases uptake through the BBB. However, the gene-loading capacity of HFn is restricted by its limited interior volume and negatively charged inner surface; therefore, these drawbacks have prompted the demand for strategies to remould the structure of HFn. In this work, we analyzed the three-dimensional (3D) structure of HFn using Chimera software (v 1.14) and developed a class of internally cationic HFn variants (HFn+ NPs) through arginine mutation on the lumenal surface of HFn. These HFn+ NPs presented powerful electrostatic forces in their cavities, and exhibited higher gene encapsulation efficacy than naive HFn. The top-performing candidate, HFn2, effectively delivered siRNA to glioma cells after traversing the BBB and achieved the highest silencing efficacy among HFn+ NPs. Overall, our findings demonstrate that HFn+ NPs obtained by this genetic engineering method provide critical insights into the future development of nucleic acid delivery carriers with BBB-crossing ability.

Self-recovery study of fluoride-induced ferroptosis in the liver of zebrafish (Danio rerio).

Ferroptosis plays a key role in fluorosis in aquatic organisms, but whether it is involved in fluoride-induced liver damage remains unclear. Previous studies have indicated that fluoride toxicity has the reversible tendency, but the mechanism of self-recovery after fluorosis in aquatic animals has not been elucidated. In this study, adult zebrafish and embryos were exposed to 0, 20, 40, 80 mg/L of fluoride for 30, 60 and 90 d and 3, 4 and 5 d post-fertilization (dpf), respectively. After 90 d, adult zebrafish were transferred to clean water for self-recovery of 30 d. The results showed that fluoride induced the prominent histopathologial changes in liver of adults, and the developmental delay and dark liver area in larvae. Fluoride significantly increased the iron overload, while decreased the expression levels of transferrin (tf), transferrin receptor (tfr), ferroportin (fpn), membrane iron transporter (fpn), and ferritin heavy chain (fth) in adults and larvae. Fluoride also induced the oxidative stress in adults and larvae by increasing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), while decreasing the glutathione (GSH) content and the levels of glutathione peroxidase 4 (gpx4) and solute carrier family 7 member 11 (slc7a11). Self-recovery relieved fluoride-induced ferroptosis by reducing the histopathological damage and oxidative stress, reversing the expression levels of fth and slc7a11, Fe2+ metabolism and GSH synthesis. Lipid peroxidation and Fe2+ metabolism may be the key factor in alleviating effects of self-recovery on fluoride toxicity. Moreover, males are more sensitive than females. Our results provide a theoretical basis for studying the alleviating effects of self-recovery on fluoride toxicity and the underlying mechanism of its protective effect.

Ferritin overexpression for noninvasive magnetic resonance imaging-based tracking of stem cells transplanted into the heart.

An unmet need in cardiac cell therapy is a noninvasive imaging technique capable of tracking changes in graft size over time and monitoring cell dynamics such as replication and death, factors to which commonly used superparamagnetic nanoparticles are insensitive. Our goal was to explore if overexpression of ferritin, a nontoxic iron-binding protein, can be used for noninvasive magnetic resonance imaging (MRI) of cells transplanted into the infarcted heart. Mouse skeletal myoblasts (C2C12 cells) were engineered to overexpress ferritin. Ferritin overexpression did not interfere with cell viability, proliferation, or differentiation into multinucleated myotubes. Ferritin overexpression caused a 25% decrease in T2 relaxation time in vitro compared to wild-type cells. Transgenic grafts were detected in vivo 3 weeks after transplantation into infarcted hearts of syngeneic mice as areas of hypointensity caused by iron accumulation in overexpressed ferritin complexes. Graft size evaluation by MRI correlated tighly with histologic measurements (R2 = .8). Our studies demonstrated the feasibility of ferritin overexpression in mouse skeletal myoblasts and the successful detection of transgenic cells by MRI in vitro and in vivo after transplantation into the infarcted mouse heart. These experiments lay the groundwork for using the MRI gene reporter ferritin to track stem cells transplanted to the heart.

Ca2+ participating self-assembly of an apoferritin nanostructure for nucleic acid drug delivery.

One of the most encountered obstacles for utilizing nano-sized vehicles to implement the in vivo delivery of nucleic acid drugs (NADs) is the possible steric hindrance caused by their intrinsic size and charge. In this work, we added Ca2+ for the pH triggered self-assembly process of H-apoferritin (HFn), to neutralize negative charges and help siRNA condense during complexation and particle formation. As expected, the internalization efficiency of siRNA in HFn particle formation could be enhanced 1.65-fold, compared with that without incorporated Ca2+. Furthermore, the calcification that occurred within the cavity of HFn particles endows them with endosomal escape capability, which could explain their contribution to the demonstrated in vitro and in vivo gene silencing effect achieved by the internalized siRNA. Thus, this Ca2+ participating self-assembly process of a protein nanostructure would lead to advanced internalization efficiency for NAD therapy.

Apoferritin nanocages with Au nanoshell coating as drug carrier for multistimuli-responsive drug release.

Cancer is one of the major causes of mortality worldwide. Therefore, it is necessary to provide an effective method of tumor therapy. Herein, we designed a new type of composite particle, apoferritin (APO) encapsulated doxorubicin (DOX), and the surface of APO was modified with Au nanoshell. As a nanocarrier, APO can carry chemotherapy drug DOX (APODOX) and release drug under acidic and high temperature conditions to reduce side effects of anticancer drugs. After covering Au nanoshell (APODOX-Au), the photothermal effect can be produced because of the unique surface plasmon resonance properties of gold nanoshell. This nanoplatform also provides the multi-stimuli responsive drug release system, which can achieve drug release in different conditions and have great potential in biomedical applications. Our investigation has demonstrated that APODOX-Au has good stability, high dispersibility and biocompatibility in vitro. The strong near-infrared absorption and good photothermal effect make sure the quick response to environmental changes (pH, temperature) to achieve drug release. These findings indicate that these nanoparticles have a potential application value in cancer treatment.

Ellipticine-loaded apoferritin nanocarrier retains DNA adduct-based cytochrome P450-facilitated toxicity in neuroblastoma cells.

Although ellipticine (Elli) is an efficient anticancer agent, it exerts several adverse effects. One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable nanocarrier, allowing targeted delivery to tumour tissue whereas avoiding healthy cells. We constructed a nanocarrier from apoferritin (Apo) bearing ellipticine, ApoElli, and subsequently characterized. The nanocarrier exhibits a narrow size distribution suggesting its suitability for entrapping the hydrophobic ellipticine molecule. Ellipticine was released from ApoElli into the water environment under pH 6.5, but only less than 20% was released at pH 7.4. The interaction of ApoElli with microsomal membrane particles containing cytochrome P450 (CYP) biotransformation enzymes accelerated the release of ellipticine from this nanocarrier making it possible to be transferred into this membrane system even at pH 7.4 and facilitating CYP-mediated metabolism. Reactive metabolites were formed not only from free ellipticine, but also from ApoElli, and both generated covalent DNA adducts. ApoElli was toxic in UKF-NB-4 neuroblastoma cells, but showed significantly lower cytotoxicity in non-malignant fibroblast HDFn cells. Ellipticine either free or released from ApoElli was concentrated in the nuclei of neuroblastoma cells, concentrations of which being significantly higher in nuclei of UKF-NB-4 than in HDFn cells. In HDFn the higher amounts of ellipticine were sequestrated in lysosomes. The extent of ApoElli entering the nuclei in UKF-NB-4 cells was lower than that of free ellipticine and correlated with the formation of ellipticine-derived DNA adducts. Our study indicates that the ApoElli form of ellipticine seems to be a promising tool for neuroblastoma treatment.

Recombinant ferritin-H induces immunosuppression in European sea bass larvae (Dicentrarchus labrax) rather than immunostimulation and protection against a Vibrio anguillarum infection.

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture. In this study, we evaluated if the recombinant sea bass ferritin-H could stimulate the innate immune system of gnotobiotic European sea bass larvae resulting in protection against a V. anguillarum challenge. We also evaluated the effect of a V. anguillarum infection on the transcription of immune-related genes in gnotobiotic European sea bass larvae. Recombinant sea bass ferritin-H was produced, encapsulated in calcium alginate microparticles and orally delivered to sea bass larvae at seven days after hatching. Our results showed V. anguillarum caused an acute infection, resulting in high mortality. The infection significantly upregulated the expression of tlr3, tlr5, cas1, il1ß, tnfα, mif, il10, cc1, cxcl8 at 18, 24 and 36 h post infection, but not of the chemokine receptor genes cxcr4 and ccr9. There was no protective effect of ferritin-H. Remarkably, ferritin-H caused significantly higher transcript levels for cxcr4 and ccr9. Sea bass ferritin-H was more likely involved in immune-suppression and results point in the direction of a negative regulation of CXCR4 resulting in inhibition of cell proliferation, differentiation and migration which is detrimental to innate immunity and might explain the non-protective effect of ferritin-H in fish larvae.