Toxicity of antimony trioxide nanoparticles on human hematopoietic progenitor cells and comparison to cell lines

Nanoparticles (NPs) are materials with one dimension in the range of 1–100 nm. The toxicity of NPs remains widely unknown and still poses concerns, due to the peculiar characteristics of materials in the nano-size range. We analyze the toxicity of seven NPs (Fe₂O₃, Fe₃O₄, Sb₂O₃, Au, TiO₂, Co, and Ag) on primary cultures of human hematopoietic progenitor cells from the bone marrow of healthy donors with CFU assays, and show that antimony oxide (Sb₂O₃) NPs and cobalt (Co) NPs have a toxic effect, while the other NPs have no effect at the tested concentrations (5, 25 and 100 μg/ml). While Co NPs suspension is toxic to both erythroid and granulocytic–monocytic precursors, Sb₂O₃ NPs at 5 μg/ml are specifically toxic to erythroid colony development, suggesting a highly selective type of toxicity. With liquid culture assays we show that Sb₂O₃ NPs impair the proliferation of erythroid progenitors, while no toxic effect is observed when Sb₂O₃ NPs are added during erythroid differentiation. CFU assays and liquid culture assays on seven human cell lines of hematopoietic origin (K562, HL-60, CEM, CEM-R, Thp-1, Jurkat, and Molt-4) show that, contrary to what observed on primary cultures of bone marrow progenitors, Sb₂O₃ NPs have no toxic effect on proliferation of any of the cell lines, raising concerns about the use of immortalized cell lines for nanotoxicology tests.     

Biological evaluation of Fe3O4–poly(l-lactide)–poly(ethylene glycol)–poly(l-lactide) magnetic microspheres prepared in supercritical CO2

The biocompatibility of Fe₃O₄–poly(l-lactide)–poly(ethylene glycol)–poly(l-lactide) magnetic microspheres (Fe₃O₄–PLLA–PEG–PLLA MMPs) prepared in a process of suspension-enhanced dispersion by supercritical CO₂ (SpEDS) was evaluated at various levels: cellular, molecular, and integrated. At the cellular level, the investigations of cytotoxicity and intracellular reactive oxygen species (ROS) generation indicate that the polymer-coated MMPs (2.0 mg/mL) had a higher toxicity than uncoated Fe₃O₄ nanoparticles, which led to about 20% loss of cell viability and an increase (0.2 fold) in ROS generation; the differences were not statistically significant (p > 0.05). However, an opposite phenomenon was observed in tests of hemolysis, which showed that the MMPs displayed the weakest hemolytic activity, namely only about 6% at the highest concentration (20 mg/mL). This phenomenon reveals that polymer-coated MMPs created less toxicity in red blood cells than uncoated Fe₃O₄ nanoparticles. At the molecular level, the MMPs were shown to be less genotoxic than Fe₃O₄ nanoparticles by measuring the micronucleus (MN) frequency in CHO-K1 cells. Furthermore, the mRNA expression of pro-inflammatory cytokines demonstrates that polymer-coated MMPs elicited a less intense secretion of pro-inflammatory cytokines than uncoated Fe₃O₄ nanoparticles. Acute toxicity tests of MMPs show quite a low toxicity, with an LD₅₀ > 1575.00 mg/kg. The evidence of low toxicity presented in the results indicates that the Fe₃O₄–PLLA–PEG–PLLA MMPs from the SpEDS process have great potential for use in biomedical applications.     

纳米锰锌铁氧体颗粒对L-02细胞的氧化损伤作用

目的 探讨磁性锰锌铁氧体纳米颗粒(Mn_0.5Zn_0.5Fe₂O₄)对人肝细胞株L-02的毒性作用机制。方法 Mn_0.5Zn_0.5Fe₂O₄ 800 mg·L^-1作用L-02细胞48 h,透射电镜观察细胞形态及超微结构的变化。Mn_0.5Zn_0.5Fe₂O₄ 200, 400和800 mg·L^-1作用48 h后,检测L-02细胞内丙二醛(MDA)的含量、超氧化物歧化酶(SOD)和还原型谷胱甘肽(GSH)的活性;荧光染色观察凋亡细胞形态;流式细胞术检测细胞周期及凋亡;荧光定量PCR仪检测胱天蛋白酶3 mRNA表达。结果 Mn_0.5Zn_0.5Fe₂O₄ 800 mg·L^-1作用48 h后,纳米颗粒进入细胞内,细胞膜发生破损,细胞器消失,染色体异常聚集。与正常对照组比较,Mn_0.5Zn_0.5Fe₂O₄ 200~800 mg·L^-1使细胞内MDA含量逐渐升高,SOD与GSH活性逐渐降低(P<0.05)。Mn_0.5Zn_0.5Fe₂O₄可使细胞周期发生改变,G₀/G₁期细胞百分率有降低的趋势,S期和G₂/M期细胞百分率有升高的趋势。Hoechst33258显示明显的细胞凋亡形态。Mn_0.5Zn_0.5Fe₂O₄可引起L-02细胞发生剂量依赖性的细胞凋亡,Mn_0.5Zn_0.5Fe₂O₄ 800 mg·L^-1作用48 h后,细胞凋亡率达到30.3%,是对照组细胞凋亡率(2.4%)的12.6倍。胱天蛋白酶3 mRNA表达量先增加后降低,但都明显高于正常对照组(P<0.05)。结论 Mn_0.5Zn_0.5Fe₂O₄可破坏细胞膜完整性并进入细胞内,诱导细胞发生氧化应激,改变细胞周期,引发细胞凋亡,产生细胞毒性。     

Fe3O4 nanoparticle redox system modulation via cell‐cycle progression and gene expression in human mesenchymal stem cells

The use of engineered nanoparticles (NPs) across multiple fields and applications has rapidly increased over the last decade owing to their unusual properties. However, there is an increased need in understanding their toxicological effect on human health. Particularly, iron oxide (Fe₃O₄) have been used in various sectors, including biomedical, food, and agriculture, but the current understanding of their impact on human health is inadequate. In this investigation, we assessed the toxic effect of Fe₃O₄ NPs on human mesenchymal stem cells (hMSCs) adopting cell viability, cellular morphological changes, mitochondrial transmembrane potential, and cell‐cycle progression assessment methodologies. Furthermore, the expression of oxidative stress, cell death, and cell‐cycle regulatory genes was assessed using quantitative polymerase chain reaction. The Fe₃O₄ NPs induced cytotoxicity and nuclear morphological changes in hMSCs by dose and time exposure. Cell‐cycle analysis indicated that Fe₃O₄ NPs altered the cell‐cycle progression through a decrease in the proportion of cells in the G₀–G₁ phase. The hMSC mitochondrial membrane potential loss increased with an increase in the concentration of Fe₃O₄ NPs exposure. The observed expression levels of the CYP1A, TNF3, TNFSF10, E2F1, and CCNC genes were significantly upregulated in hMSCs in response to Fe₃O₄ NPs exposure. Our findings suggest that Fe₃O₄ NPs caused metabolic stress through altered cell cycle, oxidative stress, and cell death regulatory gene expression in hMSCs. The results of this investigation revealed that Fe₃O₄ NPs exhibited moderate toxicity on hMSCs and that Fe₃O₄ NPs may have biomedical applications at low concentrations. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 901–912, 2016.     

Facile Synthesis of Core–shell Magnetic Mesoporous Silica Nanoparticles for pH‐sensitive Anticancer Drug Delivery

The facile synthesis of core–shell magnetic mesoporous silica nanoparticles (Fe₃O₄@mSiO₂ NPs) was reported in aqueous phase using cetyltrimethylammonium bromide as a template under alcohol‐free conditions. Compared to the conventional synthesis method for core–shell Fe₃O₄@mSiO₂ NPs, the approach in this study is rapid (only 5‐min reaction time), cheap (without using organic agents), and environmentally friendly (one‐step synthesis in alcohol‐free medium). Doxorubicin (DOX)‐loaded Fe₃O₄@mSiO₂ NPs exert extraordinarily high specificity for liver cancer cells, which was due to the pH‐sensitive doxorubicin release, as well as higher endocytosis capacity in liver cancer cells rather than normal liver cells. The potential advantages of using such Fe₃O₄@mSiO₂ NPs as the vehicle of anticancer drugs were that the Fe₃O₄@mSiO₂ NPs exhibit good biocompatibility, high loading and protection of the guest molecules, selective killing effect, and efficient cellular uptake. The exciting pH‐dependent release properties of doxorubicin‐loaded Fe₃O₄@mSiO₂ NPs make their use a promising strategy for enhancing efficient therapy toward tumors, while reducing the cytotoxicity of doxorubicin to human normal neutral tissue or cells.     

Iron oxide nanoparticles mediated cytotoxicity via PI3K/AKT pathway: Role of quercetin

Recently Fe₂O₃ NPs (iron oxide nanoparticles) have been extensively used in medical imaging and in industry also. As a result, people are increasingly exposed day by day to those nanoparticles. The adverse effect of Fe₂O₃ NPs is not so significant at lower doses but at higher doses Fe₂O₃ NPs causes significant damage to cells. The present study investigates the cell signaling mechanism of Fe₂O₃ NPs induced oxidative stress and cytotoxicity in vitro using murine hepatocytes as the working model. In addition, the cytoprotective action of quercetin in this pathophysiology has also been investigated. Dose-dependent studies suggest that incubation of hepatocytes with 250 μg/ml Fe₂O₃ NPs for 4 h significantly decreased the cell viability and intra-cellular antioxidant ability. This study also showed that exposure to Fe₂O₃ NPs caused hepatocytes death via apoptotic pathway. Incubation of hepatocytes with quercetin (50 μmol/L) prior to 1 h of Fe₂O₃ NPs exposure protects the cells from the altering activities of antioxidant indices, cytotoxicity and apoptotic death. Results suggest that Fe₂O₃ NPs induced cellular damage and quercetin plays a protective role in Fe₂O₃ NPs induced cytotoxicity and apoptotic death.     

Superparamagnetic nanovector with anti-cancer properties: γFe2O3@Zoledronate

We elaborate a magnetic nanovector to vectorize Zoledronate, an anti-cancer interest molecule of the hydroxmethylenebisphosphonate's family. In fact, Zoledronate is a powerful adjuvant in the treatment of bone diseases such as osteoporosis and Paget's disease. But, recent studies have shown that in addition to anti-osteoclastic properties, it presents antitumour properties notably in the case of breast and prostate cancer. However, these properties cannot be exploited due to their very high affinity to divalent cations and their preferentially accumulation in bone. To overcome this problem, one strategy is the vectorization trough maghemite nanocrystal functionalization. The specific surface coating permits to consider γFe₂O₃@Zoledronate as a drug delivery vehicle for therapeutic activity. The anchoring to the nanoparticle's surface allowed to increase their hydrophobicity and also to change the therapeutic target, increasing the Zoledronate intestinal absorption instead of their accumulation in bone. We show that Zoledronate link the nanoparticle surface through phosphonate groups. The biological in vitro tests performed on breast cancer cell line, MDA-MB 231, showed that γFe₂O₃@Zoledronate have antiproliferative activity. In addition, the γFe₂O₃ core could be used as MRI contrast agent for a good therapeutic evaluation.     

In vitro evaluation of the toxicity and underlying molecular mechanisms of Janus Fe3O4‐TiO2 nanoparticles in human liver cells

Recent studies show that Janus Fe₃O₄‐TiO₂ nanoparticles (NPs) have potential applications as a multifunctional agent of magnetic resonance imaging (MRI) and photodynamic therapy (PDT) for the diagnosis and therapy of cancer. However, little work has been done on their biological effects. To evaluate the toxicity and underlying molecular mechanisms of Janus Fe₃O₄‐TiO₂ nanoparticles, an in vitro study using a human liver cell line HL‐7702 cells was conducted. For comparison, the Janus Fe₃O₄‐TiO₂ NPs parent material TiO₂ NPs was also evaluated. Results showed that both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs decreased cell viability and ATP levels when applied in treatment, but increased malonaldehyde (MDA) and reactive oxygen species (ROS) generation. Mitochondria JC‐1 staining assay showed that mitochondrial membrane permeability injury occurred in both NPs treated cells. Cell viability analysis showed that TiO₂ NPs induced slightly higher cytotoxicity than Fe₃O₄‐TiO₂ NPs in HL7702 cells. Western blotting indicated that both TiO₂ NPs and Fe₃O₄‐TiO₂ NPs could induce apoptosis, inflammation, and carcinogenesis related signal protein alterations. Comparatively, Fe₃O₄‐TiO₂ NPs induced higher signal protein expressions than TiO₂ NPs under a high treatment dose. However, under a low dose (6.25 μg/cm²), neither NPs had any significant toxicity on HL7702 cells. In addition, our results suggest both Fe₃O₄‐TiO₂ NPs and TiO₂ NPs could induce oxidative stress and have a potential carcinogenetic effect in vitro. Further studies are needed to elaborate the detailed mechanisms of toxicity induced by a high dose of Fe₃O₄‐TiO₂ NPs.     

Fe3O4 nanoparticles with daunorubicin induce apoptosis through caspase 8-PARP pathway and inhibit K562 leukemia cell-induced tumor growth in vivo

Nanomaterials can enhance the delivery and treatment efficiency of anticancer drugs, but the mechanisms of the tumor-reducing activity of ferrous-ferric oxide (Fe₃O₄) nanoparticles (NPs) with daunorubicin (DNR) have not been established. Here we investigate the synergistic effects of Fe₃O₄ NPs with DNR on the induction of apoptosis using K562 leukemia cells. Fe₃O₄ NPs increased the ability of DNR to induce apoptosis in both adriamycin-sensitive and adriamycin-resistant K562 cells through the caspase 8-poly(ADP-ribose) polymerase pathway. Fe₃O₄ NPs combined with DNR also effectively inhibited the tumor growth induced by the inoculation of K562 cells into nude mice. The increased cell apoptotic rate was closely correlated with the enhanced inhibition of tumor growth. Biodistribution studies in xenograft tumors indicated that Fe₃O₄ NPs could be potentially excreted from the body via the gastrointestinal system. In conclusion, our study suggests that Fe₃O₄ NPs combined with anticancer drugs could serve as a better alternative for targeted therapeutic approaches to cancer treatments.

From the Clinical Editor

In this paper, the synergistic effects on tumor growth of ferrous-ferric oxide nanoparticles with daunorubicin are investigated. The combined treatment was demonstrated to be superior in a leukemia cell line murine model in vivo.     

Specific targeting of cancer cells by multifunctional mitoxantrone-conjugated magnetic nanoparticles

Abstract

We report on the synthesis of bifunctional mitoxantrone (MTX)-grafted magnetic nanoparticles (MNPs) modified by dopamine-polyethylene glycol-folic acid (DPA-PEG-FA) for targeted imaging and therapy of cancer. MNPs (～7–10?nm) were synthesized using the thermal decomposition reaction of Fe(acac)₃. Bromoacetyl (BrAc) terminal polyethylene glycol dopamine (DPA-PEG-BrAc) was synthesized and treated with ethylene diamine to form bifunctional PEG moiety containing dopamine at one end and amino group at the other end (i.e. DPA-PEG-NH₂). It was then reacted with Fe₃O₄ nanoparticles (NPs) to form Fe₃O₄-DPA-PEG-NH₂ NPs. The activated folic acid (FA) was chemically coupled to Fe₃O₄-DPA-PEG-NH₂, forming Fe₃O₄-DPA-PEG-FA. MTX was then conjugated to Fe₃O₄-DPA-PEG-FA, forming Fe₃O₄-DPA-PEG-FA-MTX. Physicochemical characteristics of the engineered MNPs were determined. The particle size analysis and electron microscopy showed an average size of ～35?nm for Fe₃O₄-DPA-PEG-FA-MTX NPs with superparamagnetic behavior. FT-IR spectrophotometry analysis confirmed the conjugation of FA and MTX onto the MNPs. Fluorescence microscopy, cytotoxicity assay and flow cytometry analysis revealed that the engineered Fe₃O₄-DPA-PEG-FA-MTX NPs were able to specifically bind to and significantly inhibit the folate receptor (FR)-positive MCF-7 cells, but not the FR-negative A549 cells. Based upon these findings, we suggest the Fe₃O₄-DPA-PEG-FA-MTX NPs as an effective multifunctional-targeted nanomedicine toward simultaneous imaging and therapy of FR-positive cancers.