Transferrin-Conjugated Nanostructured Lipid Carriers for Targeting Artemisone to Melanoma Cells.

We report a successful formulation of Artemisone (ATM) in transferrin (Tf)-conjugated nanostructured lipid carriers (NLCs), achieving nearly a five-times increase in cell toxicity. The escalating cost of new drug discoveries led to the repurposing of approved drugs for new indications. This study incorporated Artemisone, an antimalarial drug, into a nanostructured lipid carrier (NLC) and tested for possible anticancer effects. The aim was to develop NLCs, and transferrin-conjugated NLCs (NLC-Tf) encapsulating Artemisone to enhance its delivery and anticancer activity. NLC formulations were prepared using high-pressure homogenization followed by ultrasonication and were characterized by particle size, zeta potential, and PDI. The conjugation of (Tf) to (NLC) was confirmed using IR, and the anticancer activity was tested using MTS assay. All formulations were in the nanometer size range (140-167 nm) with different zeta potential values. IR spectroscopy confirmed the successful conjugation of transferrin to NLC. Upon testing the formulations on melanoma cell lines using MTS assay, there was a significant decrease in viability and an increase in the encapsulated ATM-Tf toxicity compared to positive control ATM. The NLCs presented a promising potential carrier for delivering ATM to melanoma cells, and further conjugation with Tf significantly improved the ATM cytotoxicity.

Transferrin Immobilized Graphene Oxide Nanocomposite for Targeted Cancer Chemodynamic Therapy via Increasing Intracellular Labile Fe2+ Concentration.

Recently, different alternative regulated cell death (RCD) pathways, viz., necroptosis, pyroptosis, ferroptosis, cuproptosis etc., have been explored as important targets for the development of cancer medications in recent years, as these can change the immunogenicity of the tumor microenvironment (TME) and will finally lead to the inhibition of cancer progression and metastasis. Here, we report the development of transferrin immobilized graphene oxide (Tfn@GOAPTES) nanocomposite as a therapeutic strategy toward cancer cell killing. The electrostatic immobilization of Tfn on the GOAPTES surface was confirmed by different spectroscopy and microscopy techniques. The Tfn immobilization was found to be ∼74 ± 4%, whereas the stability of the protein on the GO surface suggested a robust nature of the nanocomposite. The MTT assay suggested that Tfn@GOAPTES exhibited cytotoxicity toward HeLa cells via increased lipid peroxidation and DNA damage. Western blot studies resulted in decreased expression of acetylation on lysine 40 of α-tubulin and increased expression of LC3a/b for Tfn@GOAPTES treated HeLa cells, suggesting autophagy to be the main cause of the cell death mechanism. Overall, we predict that the present approach can be used as a therapeutic strategy for cancer cell killing via selective induction of a high concentration of intracellular iron.

Transferrin maintains the motility rate, ATP content, and DNA integrity of common carp spermatozoa during short-term storage.

Short-term sperm storage is a straightforward and cost-effective method of managing logistics in large scale fish hatchery operations but may result in decline in sperm quality. For effective artificial reproduction of fish, use of an appropriate additive to optimize sperm storage conditions is essential. In this study, it was investigated the effect of purified seminal plasma transferrin (Tf) at 10 µg/ml on relevant parameters in common carp Cyprinus carpio sperm during short-term storage. We compared sperm motility and curvilinear velocity, adenosine triphosphate (ATP) content and DNA fragmentation of fresh spermatozoa to that stored for 24, 48, 72, and 144 h with or without Tf. The percentage of motile cells and the curvilinear velocity of spermatozoa in stored samples for 72 h with transferrin supplementation were greater compared to samples with no added protein. The ATP content in samples without added transferrin was reduced (P < 0.05) after 72 h of storage, in contrast to the levels observed in transferrin-supplemented sperm. A time-dependent increase in DNA fragmentation was observed. Significantly lower DNA damage, expressed as percent tail DNA (10.99 ±â€¯1.28) and olive tail moment (0.54 ±â€¯0.12), was recorded in Tf-supplemented samples stored for 48 h compared to that with no Tf. Hence, it is concluded that the beneficial effects of transferrin on common carp sperm could serve as an additional tool for developing and enhancing short-term sperm preservation procedures commonly used in aquaculture.

Transferrin promotes chondrogenic differentiation in condylar growth through inducing autophagy via ULK1-ATG16L1 axis.

Skeletal mandibular hypoplasia (SMH) is one of the most common skeletal craniofacial deformities in orthodontics, which was often accompanied by impaired chondrogenesis and increasing apoptosis of condylar chondrocytes. Therefore, protecting chondrocytes from apoptosis and promoting chondrogenesis in condylar growth is vital for treatment of SMH patients. Transferrin (TF) was highly expressed in condylar cartilage of newborn mice and was gradually declined as the condyle ceased growing. Interestingly, serum level of TF in SMH patients was significantly lower than normal subjects. Hence, the aim of our study was to investigate the effect of TF on survival and differentiation of chondrocytes and condylar growth. First, we found that TF protected chondrogenic cell line ATDC5 cells from hypoxia-induced apoptosis and promoted proliferation and chondrogenic differentiation in vitro. Second, TF promoted chondrogenic differentiation and survival through activating autophagic flux. Inhibiting autophagic flux markedly blocked the effects of TF. Third, TF significantly activated ULK1-ATG16L1 axis. Silencing either transferrin receptor (TFRC), ULK1/2 or ATG16 significantly blocked the autophagic flux induced by TF, as well as its effect on anti-apoptosis and chondrogenic differentiation. Furthermore, we established an organoid culture model of mandible ex vivo and found that TF significantly promoted condylar growth. Taken together, our study unraveled a novel function of TF in condylar growth that TF protected chondrocytes from hypoxia-induced apoptosis and promoted chondrogenic differentiation through inducing autophagy via ULK1-ATG16L1 axis, which demonstrated that TF could be a novel growth factor of condylar growth and shed new light on developing treatment strategy of SMH patients.

Transferrin-Targeted Cascade Nanoplatform for Inhibiting Transcription Factor Nuclear Factor Erythroid 2-Related Factor 2 and Enhancing Ferroptosis Anticancer Therapy.

Ferroptosis, an iron-dependent cell death driven by the lethal levels of lipid peroxidation (LPO), becomes a promising anticancer strategy. However, the anticancer efficacy of ferroptosis is often hindered by the activation of nuclear factor erythrocyte 2-associated factor 2 (Nrf2), which is an indispensable regulator of the cellular antioxidant balance by preventing the accumulation of intracellular reactive oxygen species (ROS). Herein, we present a rational design of a Tf-targeted cascade nanoplatform TPM@AM based on mesoporous polydopamine (MPDA) co-encapsulating a ferroptosis inducer (artesunate, ART) and an Nrf2-specific inhibitor (ML385) to enhance intracellular ROS and therefore amplify ferrotherapy. Transferrin (Tf) can specifically recognize the transferrin receptor (TfR) on the surface of the cell membrane, which binds and transports iron into cells. When TPM@AM is endocytosed, the high-acid tumor microenvironment and laser irradiation trigger the collapse of MPDA to release ART and ML385. Furthermore, MPDA endows the nanoplatform with photothermal capability. The nanoplatform exhibits high efficiency for synergistic tumor suppression, representing a spatiotemporal controllable therapeutic strategy for precise synergistic cancer therapy.

Reversal of Cisplatin Resistance in Ovarian Cancer by the Multitargeted Nanodrug Delivery System Tf-Mn-MOF@Nira@CDDP.

Cisplatin (CDDP) is a widely used chemotherapeutic drug with proven efficacy for treating tumors. However, its use has been associated with severe side effects and eventually leads to drug resistance, thus limiting its clinical application in patients with ovarian cancer (OC). Herein, we aimed to investigate the success rate of reversing cisplatin resistance using a synthetic, multitargeted nanodrug delivery system comprising a Mn-based metal-organic framework (Mn-MOF) containing niraparib (Nira) and CDDP alongside transferrin (Tf) conjugated to the surface (Tf-Mn-MOF@Nira@CDDP; MNCT). Our results revealed that MNCT can target the tumor site, consume glutathione (GSH), which is highly expressed in drug-resistant cells, and then decompose to release the encapsulated Nira and CDDP. Nira and CDDP play a synergistic role in increasing DNA damage and apoptosis, exhibiting excellent antiproliferation, migration, and invasion activities. In addition, MNCT significantly inhibited tumor growth in tumor-bearing mice and exhibited excellent biocompatibility without side effects. Furthermore, it depleted GSH, downregulated multidrug-resistant transporter protein (MDR) expression, and upregulated tumor suppressor protein phosphatase and tensin homolog (PTEN) expression, consequently reducing DNA damage repair and reversing cisplatin resistance. These results indicate that multitargeted nanodrug delivery systems can provide a promising clinical approach to overcoming cisplatin resistance. This study provides an experimental basis for further investigation into multitargeted nanodrug delivery systems to reverse cisplatin resistance in patients with OC.

Ferroportin inhibitor vamifeport ameliorates ineffective erythropoiesis in a mouse model of ß-thalassemia with blood transfusions.

ß-thalassemia is an inherited anemia characterized by ineffective erythropoiesis. Blood transfusions are required for survival in transfusion-dependent ß-thalassemia and are also occasionally needed in patients with non-transfusion-dependent ß-thalassemia. Patients with transfusion-dependent b-thalassemia often have elevated transferrin saturation (TSAT) and non-transferrin-bound iron (NTBI) levels, which can lead to organ iron overload, oxidative stress, and vascular damage. Vamifeport is an oral ferroportin inhibitor that was previously shown to ameliorate anemia, ineffective erythropoiesis, and dysregulated iron homeostasis in the Hbbth3/+ mouse model of ß-thalassemia, under non-transfused conditions. Our study aimed to assess the effects of oral vamifeport on iron-related parameters (including plasma NTBI levels) and ineffective erythropoiesis following blood transfusions in Hbbth3/+ mice. A single dose of vamifeport prevented the transient transfusion-mediated NTBI increase in Hbbth3/+ mice. Compared with vehicle treatment, vamifeport significantly increased hemoglobin levels and red blood cell counts in transfused mice. Vamifeport treatment also significantly improved ineffective erythropoiesis in the spleens of Hbbth3/+ mice, with additive effects observed when treatment was combined with repeated transfusions. Vamifeport corrected leukocyte counts and significantly improved iron-related parameters (serum transferrin, TSAT and erythropoietin levels) versus vehicle treatment in Hbbth3/+ mice, irrespective of transfusion status. In summary, vamifeport prevented transfusion-mediated NTBI formation in Hbbth3/+ mice. When given alone or combined with blood transfusions, vamifeport also ameliorated anemia, ineffective erythropoiesis, and dysregulated iron homeostasis. Administering vamifeport together with repeated blood transfusions additively ameliorated anemia and ineffective erythropoiesis in this mouse model, providing preclinical proof-of-concept for the efficacy of combining vamifeport with blood transfusions in ß-thalassemia.

Transferrin-targeted iridium nanoagglomerates with multi-enzyme activities for cerebral ischemia-reperfusion injury therapy.

Cerebral ischemia-reperfusion injury (CIRI) is a complex pathological condition with high mortality. In particular, reperfusion can stimulate overproduction of reactive oxygen species (ROS) and activation of inflammation, causing severe secondary injuries to the brain. Despite tremendous efforts, it remains urgent to rationally design antioxidative agents with straightforward and efficient ROS scavenging capability. Herein, a potent antioxidative agent was explored based on iridium oxide nano-agglomerates (Tf-IrO2 NAs) via the facile transferrin (Tf)-templated biomineralization approach, and innovatively applied to treat CIRI. Containing some small-size IrO2 aggregates, these NAs possess intrinsic hydroxyl radicals (•OH)-scavenging ability and multifarious enzyme activities, such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx). Moreover, they also showed improved blood-brain barrier (BBB) penetration and enhanced accumulation in the ischemic brain via Tf receptor-mediated transcytosis. Therefore, Tf-IrO2 NAs achieved robust in vitro anti-inflammatory and cytoprotection effects against oxidative stress. Importantly, mice were effectively protected against CIRI by enhanced ROS scavenging activity in vivo, and the therapeutic mechanism was systematically verified. These findings broaden the idea of expanding Ir-based NAs as potent antioxidative agents to treat CIRI and other ROS-mediated diseases. STATEMENT OF SIGNIFICANCE: (1) The ROS-scavenging activities of IrO2 are demonstrated comprehensively, which enriched the family of nano-antioxidants. (2) The engineering Tf-IrO2 nano-agglomerates present unique multifarious enzyme activities and simultaneous transferrin targeting and BBB crossing ability for cerebral ischemia-reperfusion injury therapy. (3) This work may open an avenue to enable the use of IrO2 to alleviate ROS-mediated inflammatory and brain injury diseases.

Effects of the addition of insulin-transferrin-selenium (ITS) and/or metformin to the in vitro maturation of porcine oocytes on cytoplasmic maturation and embryo development.

CONTEXT: One of the main problems of porcine in vitro maturation (IVM) is incomplete cytoplasmatic maturation. Nuclear and cytoplasmic maturation will determine the future success of fertilisation and embryo development. Insulin-transferrin-selenium (ITS) has insulin-like and antioxidant effects, and metformin (M) is an insulin-sensitiser and antioxidant drug. AIMS: To assess the effects of adding ITS and/or M in porcine IVM media on cytoplasmic maturation and early embryo development. METHODS: Cumulus -oocyte complexes (COC) were IVM with M (10-4 M), ITS (0.1% v/v), M+ITS or no adding (Control). KEY RESULTS: ITS increased glucose consumption compared to Control and M (P <0.01), and M+ITS did not differ from ITS or Control. Redox balance: M, ITS and M+ITS increased glutathione (P <0.01) and decreased lipid peroxidation (P <0.005). The viability of cumulus cells by flow cytometry increased with M (P <0.005) and decreased with ITS (P <0.001); M+ITS did not differ from Control. After IVF, M increased penetration and decreased male pronucleus (P <0.05). Embryo development: cleavage increased with M (P <0.05), and blastocysts increased with ITS and M+ITS (P <0.05). The number of blastocyst cells increased with ITS (P <0.05). CONCLUSIONS: Adding ITS and M+ITS to porcine IVM media benefits embryo development to blastocysts, but ITS alone has better effects than M+ITS. IMPLICATIONS: ITS is an excellent tool to improve IVM and embryo development after IVF in pigs.

Holographically Activatable Nanoprobe via Glutathione/Albumin-Mediated Exponential Signal Amplification for High-Contrast Tumor Imaging.

Glutathione (GSH)-activatable probes hold great promise for in vivo cancer imaging, but are restricted by their dependence on non-selective intracellular GSH enrichment and uncontrollable background noise. Here, a holographically activatable nanoprobe caging manganese tetraoxide is shown for tumor-selective contrast enhancement in magnetic resonance imaging (MRI) through cooperative GSH/albumin-mediated cascade signal amplification in tumors and rapid elimination in normal tissues. Once targeting tumors, the endocytosed nanoprobe effectively senses the lysosomal microenvironment to undergo instantaneous decomposition into Mn2+ with threshold GSH concentration of ≈ 0.12 mm for brightening MRI signals, thus achieving high contrast tumor imaging and flexible monitoring of GSH-relevant cisplatin resistance during chemotherapy. Upon efficient up-regulation of extracellular GSH in tumor via exogenous injection, the relaxivity-silent interstitial nanoprobe remarkably evolves into Mn2+ that are further captured/retained and re-activated into ultrahigh-relaxivity-capable complex by stromal albumin in the tumor, and simultaneously allows the renal clearance of off-targeted nanoprobe in the form of Mn2+ via lymphatic vessels for suppressing background noise to distinguish tiny liver metastasis. These findings demonstrate the concept of holographic tumor activation via both tumor GSH/albumin-mediated cascade signal amplification and simultaneous background suppression for precise tumor malignancy detection, surveillance, and surgical guidance.

Targeted delivery and apoptosis induction activity of peptide-transferrin targeted mesoporous silica encapsulated resveratrol in MCF-7 cells.

OBJECTIVES: Resveratrol (Res) was a naturally occurring polyphenol compound. It has various beneficial effects, including anti-inflammatory, anti-oxidant and anti-cancer effects. However, the anti-cancer activity was hindered by its low targeting and drug release performance. Thus, we synthesized transferrin-cathepsin B cleavable peptide modified mesoporous silica nanoparticle encapsulated Res (Tf-Res-MSN). METHODS: Res was encapsulated in mesoporous silica nanoparticles (MSN), which was a kind of drug carrier complex. Tf was modified to recognize the cancer cells. Cathepsin B cleavable peptide (Pep) was used to combine Res-MSN complex and Tf to construct the final product. Pep was used as linker and trigger for Res release. KEY FINDINGS: The smart nanocarriers were increased the drug release performance of Res in human breast cancer (MCF-7) cells. The physicochemical properties of Tf-Res-MSN were assessed by zeta potential, UV-Prove, diffraction scanning calorimetry (DSC), nitrogen physisorption analysis and transmission electron microscope (TEM). MTT assay, AO and Annexin V-FITC/PI staining were performed to explore the anti-tumour activity of Tf-Res-MSN. The results showed that Tf-Res-MSN significantly decreased cell viability and increased cell apoptosis. The inhibition rate and apoptotic rate of Tf-Res-MSN in MCF-7 cells were 95.75% and 80.8%, respectively. CONCLUSION: Our study demonstrated that Tf-Res-MSN was a valuable technique with potential value in breast cancer applications.

Neuroprotective Effects of Transferrin in Experimental Glaucoma Models.

Iron is essential for retinal metabolism, but an excess of ferrous iron causes oxidative stress. In glaucomatous eyes, retinal ganglion cell (RGC) death has been associated with dysregulation of iron homeostasis. Transferrin (TF) is an endogenous iron transporter that controls ocular iron levels. Intraocular administration of TF is neuroprotective in various models of retinal degeneration, preventing iron overload and reducing iron-induced oxidative stress. Herein, we assessed the protective effects of TF on RGC survival, using ex vivo rat retinal explants exposed to iron, NMDA-induced excitotoxicity, or CoCl2-induced hypoxia, and an in vivo rat model of ocular hypertension (OHT). TF significantly preserved RGCs against FeSO4-induced toxicity, NMDA-induced excitotoxicity, and CoCl2-induced hypoxia. TF protected RGCs from apoptosis, ferroptosis, and necrosis. In OHT rats, TF reduced RGC loss by about 70% compared to vehicle-treated animals and preserved about 47% of the axons. Finally, increased iron staining was shown in the retina of a glaucoma patient's eye as compared to non-glaucomatous eyes. These results indicate that TF can interfere with different cell-death mechanisms involved in glaucoma pathogenesis and demonstrate the ability of TF to protect RGCs exposed to elevated IOP. Altogether, these results suggest that TF is a promising treatment against glaucoma neuropathy.

Egg Protein Transferrin-Derived Peptides Irw (Lle-Arg-Trp) and Iqw (Lle-Gln-Trp) Prevent Obesity Mouse Model Induced by a High-Fat Diet via Reducing Lipid Deposition and Reprogramming Gut Microbiota.

Egg-derived peptides play important roles in insulin secretion and sensitivity, oxidative stress, and inflammation, suggesting their possible involvement in obesity management. Hence, the aim of this study is to explore the alleviating effects of IRW (lle-Arg-Trp) and IQW (lle-Gln-Trp) on obesity via the mouse model induced by a high-fat diet. The entire experimental period lasted eight weeks. The results demonstrated that IQW prevented weight gain (6.52%), decreased the glucose, low-density lipoprotein (LDL), malonaldehyde, triglycerides, total cholesterol (TC), and leptin levels, and increased the concentration of adiponectin (p < 0.05, n = 8). Although IRW failed to prevent weight gain, it reduced the concentration of glucose, high-density lipoprotein (HDL), LDL, and leptin, and increased the concentration of adiponectin (p < 0.05, n = 8). Moreover, IRW and IQW increased glucose tolerance and insulin resistance based on the results of the intraperitoneal glucose test and insulin tolerance test (p < 0.05, n = 8). The quantitative polymerase chain reaction results revealed that IRW and IQW downregulated the mRNA expression of DGAT1 (Diacylglycerol O-Acyltransferase 1), DGAT2 (Diacylglycerol O-Acyltransferase 2), TNF-α, IL-6, and IL-1ß of liver tissue (p < 0.05, n = 8). The results of the 16S ribosomal RNA amplicon sequencing showed that IQW and IRW tended to reduce the relative abundance of Firmicutes and Parabacteroides, and that IRW enhanced the abundance of Bacteroides (p < 0.05, n = 8). Collectively, IRW and IQW supplementation could alleviate the progression of obesity due to the fact that the supplementation reduced lipid deposition, maintained energy balance, and reprogrammed gut microbiota.

Bio-polymeric transferrin-targeted temozolomide nanoparticles in gel for synergistic post-surgical GBM therapy.

Spatiotemporal targeting of anti-glioma drugs remains a pressing issue in glioblastoma (GBM) treatment. We challenge this issue by developing a minimally invasive in situ implantable hydrogel implant comprising transferrin-targeted temozolomide-miltefosine nanovesicles in the surgically resected GBM cavity (tumour bed). Injection of the "nanovesicle in hydrogel system" in orthotopic GBM-bearing mice improved drug penetration into the peri-cavitary region (∼4.5 mm in depth) with the potential to act as a bridge therapy in the immediate postoperative period, before the initiation of adjuvant radiotherapy. The controlled and sustained release of temozolomide over a month in the surgical cavity eradicated the microscopic GBM cells present within the tumour bed, thereby augmenting the efficacy of adjuvant therapy. The drug (temozolomide and miltefosine) combination was tolerable and efficiently inhibited tumour growth, causing significant prolongation of the survival of tumour-bearing mice compared to that with the free drug. Direct implantation at the target site in the brain resulted in spatiotemporal anti-glioma activity with minimal extracranial and systemic distribution. Nanovesicle in flexible hydrogel systems can be used as potential platforms for the post-surgical management of GBM before initiating adjuvant radiation therapy.

Effect of transferrin glycation induced by high glucose on HK-2 cells in vitro.

Background and objective: Glycation is a common post-transcriptional modification of proteins. Previous studies have shown that advanced glycation end modified transferrin (AGE-Tf) levels in diabetic rat kidney tissues were increased; however, its role in diabetic nephropathy remains unclear. In this study, differences in glycation degree and Tf sites induced by differing high glucose concentrations in vitro and the effect on total iron binding capacity (TIBC) were observed. Moreover, the effect of AGE-Tf on human renal tubular epithelial cells (HK-2) was investigated. Methods: In vitro Tf was incubated with increasing glucose concentrations (0 mM, 5.6 mM, 11.1 mM, 33.3 mM, 100 mM, 500 mM, and 1,000 mM) for AGE-Tf. Differences in AGE-Tf glycation degree and TIBC level were analyzed via colorimetric method. The AGE-Tf glycation sites were identified with LC-MS/MS. HK-2 cells were treated with AGE-Tf prepared with different glucose concentrations (33.3 mM and 500 mM) in vitro. The effects of AGE-Tf on HK-2 cell viability, proliferation, oxidative stress index, and Tf receptor expression levels were then observed. Results: With increasing glucose concentrations (100 mM, 500 mM, and 1,000 mM) in vitro, Tf glycation degree was significantly increased. The TIBC levels of AGE-Tf were decreased significantly with increasing glucose concentrations (33.3 mM, 100 mM, 500 mM, and 1,000 mM). Four glycated modification sites in Tf and 17 glycated modification sites were detected in AGE-Tf (500 mM) by LC-MS/MS. The structural types of AGEs were CML, G-H1, FL-1H2O, FL, and MG-H1. No significant differences were found in the survival rate of HK-2 cells among the AGE-Tf (500 mM), AGE-Tf (33.3 mM), and Tf groups (all p > 0.05). The apoptosis rate of HK-2 cells in the AGE-Tf (500 mM) group was significantly higher than that in the AGE-Tf (33.3 mM) group. Additionally, both of them were significantly higher than that in the Tf group (both p < 0.05). The MDA levels of HK-2 cells in the AGE-Tf (500 mM) and AGE-Tf (33.3 mM) groups were higher than that in the Tf group, but not significantly (both p > 0.05). The T-AOC level of HK-2 in the AGE-Tf (500 mM) group was significantly lower than that in the AGE-Tf (33.3 mM) and Tf groups (both p < 0.001). The GSH level of HK-2 cells in the AGE-Tf (500 mM) group was significantly lower than that in the Tf group (p < 0.05). The expression level of TfR in the AGE-Tf (500 mM) group was also significantly lower than that in the Tf group (p < 0.05). Conclusion: The degree and sites of Tf glycation were increased in vitro secondary to high-glucose exposure; however, the binding ability of Tf to iron decreased gradually. After HK-2 was stimulated by AGE-Tf in vitro, the apoptosis of cells was increased, antioxidant capacity was decreased, and TfR expression levels were downregulated.

Labile iron, ROS, and cell death are prominently induced by haemin, but not by non-transferrin-bound iron.

BACKGROUND: In transfusion-related iron overload, haem-derived iron accumulation in monocytes/macrophages is the initial event. When iron loading exceeds the ferritin storage capacity, iron is released into the plasma. When iron loading exceeds transferrin binding capacity, labile, non-transferrin-bound iron (NTBI) appears and causes organ injury. Haemin-induced cell death has already been investigated; however, whether NTBI induces cell death in monocytes/macrophages remains unclear. MATERIAL AND METHODS: Human monocytic THP-1 cells were treated with haemin or NTBI, particularly ferric ammonium citrate (FAC) or ferrous ammonium sulfate (FAS). The intracellular labile iron pool (LIP) was measured using an iron-sensitive fluorescent probe. Ferritin expression was measured by western blotting. RESULTS: LIP was elevated after haemin treatment but not after FAC or FAS treatment. Reactive oxygen species (ROS) generation and cell death induction were remarkable after haemin treatment but not after FAC or FAS treatment. Ferritin expression was not different between the FAC and haemin treatments. The combination of an iron chelator and a ferroptosis inhibitor significantly augmented the suppression of haemin cytotoxicity (p = 0.011). DISCUSSION: The difference in LIP suggests the different iron traffic mechanisms for haem-derived iron and NTBI. The Combination of iron chelators and antioxidants is beneficial for iron overload therapy.

Active targeting of orthotopic glioma using biomimetic liposomes co-loaded elemene and cabazitaxel modified by transferritin.

BACKGROUND: Effective treatment of glioma requires a nanocarrier that can cross the blood-brain barrier (BBB) to target the tumor lesion. In the current study, elemene (ELE) and cabazitaxel (CTX) liposomes were prepared by conjugating liposomes with transferrin (Tf) and embedding the cell membrane proteins of RG2 glioma cells into liposomes (active-targeting biomimetic liposomes, Tf-ELE/CTX@BLIP), which exhibited effective BBB infiltration to target glioma. RESULTS: The findings showed that Tf-ELE/CTX@BLIP was highly stable. The liposomes exhibited highly significant homologous targeting and immune evasion in vitro and a 5.83-fold intake rate compared with classical liposome (ELE/CTX@LIP). Bioluminescence imaging showed increased drug accumulation in the brain and increased tumor penetration of Tf-ELE/CTX@BLIP in orthotopic glioma model nude mice. Findings from in vivo studies indicated that the antitumor effect of the Tf-ELE/CTX@BLIP led to increased survival time and decreased tumor volume in mice. The average tumor fluorescence intensity after intravenous administration of Tf-ELE/CTX@BLIP was 65.2, 12.5, 22.1, 6.6, 2.6, 1.5 times less compared with that of the control, CTX solution, ELE solution, ELE/CTX@LIP, ELE/CTX@BLIP, Tf-ELE/CTX@LIP groups, respectively. Histopathological analysis showed that Tf-ELE/CTX@BLIP were less toxic compared with administration of the CTX solution. CONCLUSION: These findings indicate that the active-targeting biomimetic liposome, Tf-ELE/CTX@BLIP, is a promising nanoplatform for delivery of drugs to gliomas.

Myogenic Differentiation of iPS Cells Shows Different Efficiency in Simultaneous Comparison of Protocols.

Induced pluripotent stem (iPS) cells constitute a perfect tool to study human embryo development processes such as myogenesis, thanks to their ability to differentiate into three germ layers. Currently, many protocols to obtain myogenic cells have been described in the literature. They differ in many aspects, such as media components, including signaling modulators, feeder layer constituents, and duration of culture. In our study, we compared three different myogenic differentiation protocols to verify, side by side, their efficiency. Protocol I was based on embryonic bodies differentiation induction, ITS addition, and selection with adhesion to collagen I type. Protocol II was based on strong myogenic induction at the embryonic bodies step with BIO, forskolin, and bFGF, whereas cells in Protocol III were cultured in monolayers in three special media, leading to WNT activation and TGF-ß and BMP signaling inhibition. Myogenic induction was confirmed by the hierarchical expression of myogenic regulatory factors MYF5, MYOD, MYF6 and MYOG, as well as the expression of myotubes markers MYH3 and MYH2, in each protocol. Our results revealed that Protocol III is the most efficient in obtaining myogenic cells. Furthermore, our results indicated that CD56 is not a specific marker for the evaluation of myogenic differentiation.

Iron-loaded transferrin potentiates erythropoietin effects on erythroblast proliferation and survival: a novel role through transferrin receptors.

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) is a glycoprotein cytokine that plays a major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration of Epo in bone marrow is hypothesized to be suboptimal, and the survival of erythroid cells has been suggested to depend on Epo sensitivity. However, the key factors that control Epo sensitivity remain unknown. Two types of transferrin receptors (TfRs), TfR1 and TfR2, are known to play a role in iron uptake in erythroid cells. Here, we hypothesized that TfRs may additionally modulate Epo sensitivity during erythropoiesis by modulating Epo receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) erythroid cell and human erythroid progenitor cell models, we report that iron-loaded transferrin, that is, holo-transferrin (holo-Tf), synergizes with suboptimal Epo levels to improve erythroid cell survival, proliferation, and differentiation. This is accomplished via the major signaling pathways of erythropoiesis, which include signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, we found that this cooperation is improved by, but does not require, the internalization of TfR1. Interestingly, we observed that loss of TfR2 stabilizes EpoR levels and abolishes the beneficial effects of holo-Tf. Overall, these data reveal novel signaling properties of TfRs, which involve the regulation of erythropoiesis through EpoR signaling.

A dual-targeting ruthenium nanodrug that inhibits primary tumor growth and lung metastasis via the PARP/ATM pathway.

BACKGROUND: Many studies have found that ruthenium complexes possess unique biochemical characteristics and inhibit tumor growth or metastasis. RESULTS: Here, we report the novel dual-targeting ruthenium candidate 2b, which has both antitumor and antimetastatic properties and targets tumor sites through the enhanced permeability and retention (EPR) effect and transferrin/transferrin receptor (TF/TFR) interaction. The candidate 2b is composed of ruthenium-complexed carboline acid and four chloride ions. In vitro, 2b triggered DNA cleavage and thus blocked cell cycle progression and induced apoptosis via the PARP/ATM pathway. In vivo, 2b inhibited not only Lewis lung cancer (LLC) tumor growth but also lung metastasis. We detected apoptosis and decreased CD31 expression in tumor tissues, and ruthenium accumulated in the primary tumor tissue of C57BL/6 mice implanted with LLC cells. CONCLUSIONS: Thus, we conclude that 2b targets tumors, inhibits tumor growth and prevents lung metastasis.