Iron Oxide Nanoparticles Inhibit Tumor Progression and Suppress Lung Metastases in Mouse Models of Breast Cancer.

Systemic exposure to starch-coated iron oxide nanoparticles (IONPs) can stimulate antitumor T cell responses, even when little IONP is retained within the tumor. Here, we demonstrate in mouse models of metastatic breast cancer that IONPs can alter the host immune landscape, leading to systemic immune-mediated disease suppression. We report that a single intravenous injection of IONPs can inhibit primary tumor growth, suppress metastases, and extend survival. Gene expression analysis revealed the activation of Toll-like receptor (TLR) pathways involving signaling via Toll/Interleukin-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF), a TLR pathway adaptor protein. Requisite participation of TRIF in suppressing tumor progression was demonstrated with histopathologic evidence of upregulated IFN-regulatory factor 3 (IRF3), a downstream protein, and confirmed in a TRIF knockout syngeneic mouse model of metastatic breast cancer. Neither starch-coated polystyrene nanoparticles lacking iron, nor iron-containing dextran-coated parenteral iron replacement agent, induced significant antitumor effects, suggesting a dependence on the type of IONP formulation. Analysis of multiple independent clinical databases supports a hypothesis that upregulation of TLR3 and IRF3 correlates with increased overall survival among breast cancer patients. Taken together, these data support a compelling rationale to re-examine IONP formulations as harboring anticancer immune (nano)adjuvant properties to generate a therapeutic benefit without requiring uptake by cancer cells.

In situ theranostic platform combining highly localized magnetic fluid hyperthermia, magnetic particle imaging, and thermometry in 3D.

Theranostic platforms, combining diagnostic and therapeutic approaches within one system, have garnered interest in augmenting invasive surgical, chemical, and ionizing interventions. Magnetic particle imaging (MPI) offers a quite recent alternative to established radiation-based diagnostic modalities with its versatile tracer material (superparamagnetic iron oxide nanoparticles, SPION). It also offers a bimodal theranostic framework that can combine tomographic imaging with therapeutic techniques using the very same SPION. Methods: We show the interleaved combination of MPI-based imaging, therapy (highly localized magnetic fluid hyperthermia (MFH)) and therapy safety control (MPI-based thermometry) within one theranostic platform in all three spatial dimensions using a commercial MPI system and a custom-made heating insert. The heating characteristics as well as theranostic applications of the platform were demonstrated by various phantom experiments using commercial SPION. Results: We have shown the feasibility of an MPI-MFH-based theranostic platform by demonstrating high spatial control of the therapeutic target, adequate MPI-based thermometry, and successful in situ interleaved MPI-MFH application. Conclusions: MPI-MFH-based theranostic platforms serve as valuable tools that enable the synergistic integration of diagnostic and therapeutic approaches. The transition into in vivo studies will be essential to further validate their potential, and it holds promising prospects for future advancements.

Intravenous Injection of PEI-Decorated Iron Oxide Nanoparticles Impacts NF-kappaB Protein Expression in Immunologically Stressed Mice.

Nanoparticle-based formulations are considered valuable tools for diagnostic and treatment purposes. The surface decoration of nanoparticles with polyethyleneimine (PEI) is often used to enhance their targeting and functional properties. Here, we aimed at addressing the long-term fate in vivo and the potential "off-target" effects of PEI decorated iron oxide nanoparticles (PEI-MNPs) in individuals with low-grade and persistent systemic inflammation. For this purpose, we synthesized PEI-MNPs (core-shell method, PEI coating under high pressure homogenization). Further on, we induced a low-grade and persistent inflammation in mice through regular subcutaneous injection of pathogen-associated molecular patterns (PAMPs, from zymosan). PEI-MNPs were injected intravenously. Up to 7 weeks thereafter, the blood parameters were determined via automated fluorescence flow cytometry, animals were euthanized, and the organs analyzed for iron contents (atomic absorption spectrometry) and for expression of NF-κB associated proteins (p65, IκBα, p105/50, p100/52, COX-2, Bcl-2, SDS-PAGE and Western blotting). We observed that the PEI-MNPs had a diameter of 136 nm and a zeta-potential 56.9 mV. After injection in mice, the blood parameters were modified and the iron levels were increased in different organs. Moreover, the liver of animals showed an increased protein expression of canonical NF-κB signaling pathway members early after PEI-MNP application, whereas at the later post-observation time, members of the non-canonical signaling pathway were prominent. We conclude that the synergistic effect between PEI-MNPs and the low-grade and persistent inflammatory state is mainly due to the hepatocytes sensing infection (PAMPs), to immune responses resulting from the intracellular metabolism of the uptaken PEI-MNPs, or to hepatocyte and immune cell communications. Therefore, we suggest a careful assessment of the safety and toxicity of PEI-MNP-based carriers for gene therapy, chemotherapy, and other medical applications not only in healthy individuals but also in those suffering from chronic inflammation.

CXCR5+PD-1++ CD4+ T cells colonize infant intestines early in life and promote B cell maturation.

Gastrointestinal infections are a major cause for serious clinical complications in infants. The induction of antibody responses by B cells is critical for protective immunity against infections and requires CXCR5+PD-1++ CD4+ T cells (TFH cells). We investigated the ontogeny of CXCR5+PD-1++ CD4+ T cells in human intestines. While CXCR5+PD-1++ CD4+ T cells were absent in fetal intestines, CXCR5+PD-1++ CD4+ T cells increased after birth and were abundant in infant intestines, resulting in significant higher numbers compared to adults. These findings were supported by scRNAseq analyses, showing increased frequencies of CD4+ T cells with a TFH gene signature in infant intestines compared to blood. Co-cultures of autologous infant intestinal CXCR5+PD-1+/-CD4+ T cells with B cells further demonstrated that infant intestinal TFH cells were able to effectively promote class switching and antibody production by B cells. Taken together, we demonstrate that functional TFH cells are numerous in infant intestines, making them a promising target for oral pediatric vaccine strategies.

Selective Magnetic Nanoheating: Combining Iron Oxide Nanoparticles for Multi-Hot-Spot Induction and Sequential Regulation.

The contactless heating capacity of magnetic nanoparticles (MNPs) has been exploited in fields such as hyperthermia cancer therapy, catalysis, and enzymatic thermal regulation. Herein, we propose an advanced technology to generate multiple local temperatures in a single-pot reactor by exploiting the unique nanoheating features of iron oxide MNPs exposed to alternating magnetic fields (AMFs). The heating power of the MNPs depends on their magnetic features but also on the intensity and frequency conditions of the AMF. Using a mixture of diluted colloids of MNPs we were able to generate a multi-hot-spot reactor in which each population of MNPs can be selectively activated by adjusting the AMF conditions. The maximum temperature reached at the surface of each MNP was registered using independent fluorescent thermometers that mimic the molecular link between enzymes and MNPs. This technology paves the path for the implementation of a selective regulation of multienzymatic reactions.

The differences of the impact of a lipid and protein corona on the colloidal stability, toxicity, and degradation behavior of iron oxide nanoparticles.

AIM: In this study, the influence of a serum albumin (SA) and human plasma (HP) derived protein- and lipid molecule corona on the toxicity and biodegradability of different iron oxide nanoparticles (IONP) was investigated. METHODS: IONP were synthesized and physicochemically characterized regarding size, charge, and colloidal stability. The adsorbed proteins were quantified and separated by gel electrophoresis. Adsorbed lipids were profiled by ultraperformance liquid chromatography-ESI-tandem mass spectrometry. The biocompatibility was investigated using isolated erythrocytes and a shell-less hen's egg model. The biodegradability was assessed by iron release studies in artificial body fluids. RESULTS: The adsorption patterns of proteins and lipids varied depending on the surface characteristics of the IONP like charge and hydrophobicity. The biomolecule corona modified IONP displayed favorable colloidal stability and toxicological profile compared to IONP without biomolecule coronas, reducing erythrocyte aggregation and hemolysis in vitro as well as the corresponding effects ex ovo/in vivo. The coronas decreased the degradation speed of all tested IONP compared to bare particles, but, whereas all IONP degraded at the same rate for the SA corona, substantial differences were evident for IONP with HP-derived corona depending on the lipid adsorption profile. CONCLUSION: In this study the impact of the proteins and lipids in the biomolecule corona on the entire IONP application cycle from the injection process to the degradation was demonstrated.

Systemically delivered antibody-labeled magnetic iron oxide nanoparticles are less toxic than plain nanoparticles when activated by alternating magnetic fields.

OBJECTIVE: Toxicity from off-target heating with magnetic hyperthermia (MHT) is generally assumed to be understood. MHT research focuses on development of more potent heating magnetic iron oxide nanoparticles (MIONs), yet our understanding of factors that define biodistribution following systemic delivery remains limited. Preclinical development relies on mouse models, thus understanding off-target heating with MHT in mice provides critical knowledge for clinical development. METHODS: Eight-week old female nude mice received a single tail vein injection of bionized nanoferrite (BNF) MIONs or a counterpart labeled with a polyclonal human antibody (BNF-IgG) at 1 mg, 3 mg or 5 mg Fe/mouse on day 1. On day 3, mice were exposed to an alternating magnetic field (AMF) having amplitude of 32, 48 or 64 kA/m at ∼145 kHz for 20 min. Twenty-four hours later, blood, livers and spleens were harvested and analyzed. RESULTS: Damage to livers was apparent by histology and serum liver enzymes following MHT with BNF or BNF-IgG at doses ≥3 mg Fe and AMF amplitudes ≥48 kA/m. Differences between effects with BNF vs. BNF-IgG at a dose of 3 mg Fe were noted in all measures, with less damage and increased survival occurring in mice injected with BNF-IgG. Necropsies revealed severe damage to duodenum and upper small intestines, likely the immediate cause of death at the highest MHT doses. CONCLUSION: Results demonstrate that the MION coating affects biodistribution, which in turn determines off-target effects. Developments to improve heating capabilities of MIONs may be clinically irrelevant without better control of biodistribution.

Simulation of the long-term fate of superparamagnetic iron oxide-based nanoparticles using simulated biological fluids.

Aim: To simulate the stability and degradation of superparamagnetic iron oxide nanoparticles (MNP) in vitro as part of their life cycle using complex simulated biological fluids. Materials & methods: A set of 13 MNP with different polymeric or inorganic shell materials was synthesized and characterized regarding stability and degradation of core and shell in simulated biological fluids. Results: All MNP formulations showed excellent stability during storage and in simulated body fluid. In endosomal/lysosomal media the degradation behavior depended on shell characteristics (e.g., charge, acid-base character) and temperature enabling the development of an accelerated stress test protocol. Conclusion: Kinetics of transformations depending on the MNP type could be established to define structure-activity relationships as prediction model for rational particle design.

Improving the reliability of the iron concentration quantification for iron oxide nanoparticle suspensions: a two-institutions study.

Most iron oxide nanoparticles applications, and in special biomedical applications, require the accurate determination of iron content as the determination of particle properties from measurements in dispersions is strongly dependent on it. Inductively coupled plasma (ICP) and spectrophotometry are two typical worldwide used analytical methods for iron concentration determination. In both techniques, precise determination of iron is not straightforward and nanoparticle digestion and dilution procedures are needed prior to analysis. The sample preparation protocol has been shown to be as important as the analytical method when accuracy is aimed as many puzzling reported results in magnetic, colloidal, and structural properties are simply attributable to inadequate dissolution procedures. Therefore, a standard sample preparation protocol is needed to ensure the adequate and complete iron oxide nanoparticle dissolution and to harmonize this procedure. In this work, an interlaboratory evaluation of an optimized iron oxide nanoparticle digestion/dilution protocol was carried out. The presented protocol is simple, inexpensive, and does not involve any special device (as microwave, ultrasound, or other high-priced digestion devices). Then, iron concentration was measured by ICP-OES (performed in ICMM/CSIC-Spain) and spectrophotometry (NanoPET-Germany) and the obtained concentration values were analyzed to determine the most probable error causes. Uncertainty values as low as 1.5% were achieved after the optimized method was applied. Moreover, this article provides a list of recommendations to significantly reduce uncertainty in both sample preparation and analysis procedures. Graphical abstract ᅟ.

The effects of intraparticle structure and interparticle interactions on the magnetic hysteresis loop of magnetic nanoparticles.

Technologically relevant magnetic nanoparticles for biomedicine are rarely noninteracting single-domain nanoparticles; instead, they are often interacting, with complex physical and magnetic structures. In this paper, we present both experimental and simulated magnetic hysteresis loops of a system of magnetic nanoparticles with significant interparticle interactions and a well-defined intraparticle structure which are used for magnetic nanoparticle hyperthermia cancer treatment. Experimental measurements were made at 11 K on suspensions of magnetic nanoparticles dispersed in H2O which have been frozen in a range of applied magnetic fields to tune the interparticle interactions. Micromagnetic simulations of hysteresis loops investigated the roles of particle orientation with respect to the field and of particle chaining in the shape of the hysteresis loops. In addition, we present an analysis of the magnetic anisotropy arising from the combination of magnetocrystalline and shape anisotropy, given the well-defined internal structure of the nanoparticles. We find that the shape of the experimental hysteresis loops can be explained by the internal magnetic structure, modified by the effects of interparticle interactions from chaining.

Rapid Analysis of Cell-Nanoparticle Interactions using Single-Cell Raman Trapping Microscopy.

Iron oxide nanoparticles have been used in preclinical studies to label stem cells for non-invasive tracking and homing. The search continues for novel particle candidates that are suitable for clinical applications. Since standard analyses to investigate cell-particle interactions and safety are labor-intensive, an efficient procedure is required to guide future particle development and to exclude adverse health effects. The application of combined Raman trapping microscopy with fluidic chips is reported for the analysis of single cells labeled with different types of aminated iron oxide particles. Multivariate data analysis revealed Raman signal differences that could be clearly assigned to cell-particle interactions and cytotoxicity, respectively. A validation dataset verified that more than 95 % of the spectra were correctly classified. Thus, our approach enables rapid discrimination of non-hazardous from cytotoxic nanoparticles as a prerequisite for safe clinical applications.

Stem cell labeling with iron oxide nanoparticles: impact of 3D culture on cell labeling maintenance.

AIM: We aimed to analyze the suitability of nanoparticles (M4E) for safe human mesenchymal stem cell (hMSC) labeling and determined cell labeling maintenance in 2D and 3D culture. MATERIALS & METHODS: We investigated cell-particle interaction and the particles' impact on cell viability, growth and proliferation. We analyzed cell labeling maintenance in 2D and 3D culture invasively and noninvasively. RESULTS: M4E do not affect cell viability, growth and proliferation and do not cause chromosomal aberrations. Cell labeling maintenance is up to five-times higher in 3D conditions compared with 2D culture. CONCLUSION: M4E allow safe hMSC labeling and noninvasive identification. Our hMSC-loaded, 3D tissue-engineered construct could serve as a graft for regenerative therapies, in which M4E-labeled hMSCs can migrate to their target.

Effect of coating thickness of iron oxide nanoparticles on their relaxivity in the MRI.

OBJECTIVES: Iron oxide nanoparticles have found prevalent applications in various fields including drug delivery, cell separation and as contrast agents. Super paramagnetic iron oxide (SPIO) nanoparticles allow researchers and clinicians to enhance the tissue contrast of an area of interest by increasing the relaxation rate of water. In this study, we evaluate the dependency of hydrodynamic size of iron oxide nanoparticles coated with Polyethylene glycol (PEG) on their relativities with 3 Tesla clinical MRI. MATERIALS AND METHODS: We used three groups of nanoparticles with nominal sizes 20, 50 and 100 nm with a core size of 8.86 nm, 8.69 nm and 10.4 nm that they were covered with PEG 300 and 600 Da. A clinical magnetic resonance scanner determines the T1 and T2 relaxation times for various concentrations of PEG-coated nanoparticles. RESULTS: The size measurement by photon correlation spectroscopy showed the hydrodynamic sizes of MNPs with nominal 20, 50 and 100 nm with 70, 82 and 116 nm for particles with PEG 600 coating and 74, 93 and 100 nm for particles with PEG 300 coating, respectively. We foud that the relaxivity decreased with increasing overall particle size (via coating thickness). Magnetic resonance imaging showed that by increasing the size of the nanoparticles, r2/r1 increases linearly. CONCLUSION: According to the data obtained from this study it can be concluded that increments in coating thickness have more influence on relaxivities compared to the changes in core size of magnetic nanoparticles.

High specificity targeting and detection of human neuroblastoma using multifunctional anti-GD2 iron-oxide nanoparticles.

AIM: To develop biocompatible, tumor-specific multifunctional iron-oxide nanoconstructs targeting neuroblastoma, an aggressive pediatric malignancy. MATERIALS & METHODS: Clinical-grade humanized monoclonal antibody (hu14.18K322A), designed to target GD2 antigen on neuroblastoma with reduced nonspecific immune interactions, was conjugated to hydroxyethyl starch-coated iron-oxide nanoparticles. Targeting capability in vitro and in vivo was assessed by immunofluorescence, electron microscopy, analytical spectrophotometry, histochemistry and magnetic resonance R2* relaxometry. RESULTS: The biocompatible nanoconstructs demonstrated high tumor specificity in vitro and in vivo, and low background uptake in a mouse flank xenograft model. Specific accumulation in tumors enabled particle visualization and quantification by magnetic resonance R2* mapping. CONCLUSION: Our findings support the further development toward clinical application of this anti-GD2 iron-oxide nanoconstruct as diagnostic and therapeutic scaffold for neuroblastoma and potentially other GD2-positive malignancies.

Comparative in vitro study on magnetic iron oxide nanoparticles for MRI tracking of adipose tissue-derived progenitor cells.

Magnetic resonance imaging (MRI) using measurement of the transverse relaxation time (R2*) is to be considered as a promising approach for cell tracking experiments to evaluate the fate of transplanted progenitor cells and develop successful cell therapies for tissue engineering. While the relationship between core composition of nanoparticles and their MRI properties is well studied, little is known about possible effects on progenitor cells. This in vitro study aims at comparing two magnetic iron oxide nanoparticle types, single vs. multi-core nanoparticles, regarding their physico-chemical characteristics, effects on cellular behavior of adipose tissue-derived stem cells (ASC) like differentiation and proliferation as well as their detection and quantification by means of MRI. Quantification of both nanoparticle types revealed a linear correlation between labeling concentration and R2* values. However, according to core composition, different levels of labeling concentrations were needed to achieve comparable R2* values. Cell viability was not altered for all labeling concentrations, whereas the proliferation rate increased with increasing labeling concentrations. Likewise, deposition of lipid droplets as well as matrix calcification revealed to be highly dose-dependent particularly regarding multi-core nanoparticle-labeled cells. Synthesis of cartilage matrix proteins and mRNA expression of collagen type II was also highly dependent on nanoparticle labeling. In general, the differentiation potential was decreased with increasing labeling concentrations. This in vitro study provides the proof of principle for further in vivo tracking experiments of progenitor cells using nanoparticles with different core compositions but also provides striking evidence that combined testing of biological and MRI properties is advisable as improved MRI properties of multi-core nanoparticles may result in altered cell functions.

Synthesis and functionalisation of magnetic nanoparticles for hyperthermia applications.

A summary of recent developments in the synthesis, stabilisation and coating of magnetic iron oxide nanoparticles for hyperthermia applications is presented. Methods for synthesis in aqueous, organic and microemulsion systems are reviewed together with the resulting heating rates of the nanoparticles. Different stabilisation mechanisms for iron oxide nanoparticles from aqueous and organic media are discussed as intermediates for further coating and functionalisation. Coating with silica and/or polysaccharides is mainly used for design of nanoparticles especially for targeted hyperthermia application. These coatings permit versatile functionalisation as a basis for conjugating biomolecules, e.g. antibodies or peptides. Various strategies to conjugate biomolecules on the particle surface are discussed, with emphasis on methods that preserve biofunctionality after immobilisation. The efficiency of established methods such as carbodiimide coupling and oriented conjugation strategies is compared with new developments such as the bioorthogonal approaches that are based on the cycloaddition of strain-promoted alkynes with azides or nitrones. For targeted hyperthermia applications the study of the formation of a protein corona around nanoparticles with site-specific biomolecules on the surface is essential to achieve improved circulation times in the blood and reduced non-specific uptake by non-targeted organs for a high specific accumulation in the target tissue.

Phosphorylation of the HuR ligand APRIL by casein kinase 2 regulates CD83 expression.

Fully mature DC and, to a lesser extent, activated T and B cells express CD83, a surface molecule that appears to fulfil an important role in efficient T-cell activation. Recently, it has been shown that CD83 mRNA is transported from the nucleus to the cytoplasm by an uncommon route, involving the cellular RNA-binding protein HuR and the nuclear export receptor CRM1. Moreover, the shuttle phosphoprotein APRIL (ANP32B) has been shown to be required for HuR-mediated nucleocytoplasmic translocation of the CD83 mRNA by acting as an adaptor that links HuR and CRM1. Here, we are able to report that casein kinase 2 (CK2) phosphorylates APRIL on residue threonine244 (Thr(244)) and demonstrate that the CK2-specific inhibitor 4,5,6,7-tetrabromo-2-azabenzimidazole abolishes CD83 expression in activated Jurkat T cells by interfering with the nucleocytoplasmic translocation of CD83 mRNA. Depletion and knockdown studies demonstrate that the CK2 alpha' subunit is necessary for this regulation, whereas the CK2 alpha subunit seems to be dispensable. Taken together, the data presented significantly extend our knowledge of the complex regulation of CD83 mRNA processing and provides a novel strategy to interfere with CD83 expression.

Analysis of nucleocytoplasmic trafficking of the HuR ligand APRIL and its influence on CD83 expression.

Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system and are able to sensitize even naïve T cells. Mature DC are characterized by expression of CD83, a surface molecule that is proposed to be involved in efficient T cell activation. It has been recently shown that CD83 mRNA is transported from the nucleus to the cytoplasm in a HuR- and CRM1-dependent manner. Therefore we here investigated the impact of two known protein ligands of HuR, pp32 and APRIL, on CD83 expression. Both pp32 (ANP32A) and APRIL (ANP32B) are shuttle proteins, and it has been reported earlier that these HuR ligands can act as adaptors that link HuR and the CRM1-specific nuclear export pathway. By employing RNA interference (RNAi) technology we demonstrate that pp32 is dispensable for CD83 expression, whereas APRIL contributes to the nuclear export and subsequent translation of CD83 mRNA. Furthermore, we have determined the nuclear import signal (NLS) as well as the nuclear export signal (NES) of human APRIL. Moreover, we analyzed the status of phosphorylation of endogenous APRIL and identified threonine 244 to be an as yet unrecognized phosphate acceptor. Finally, we were able to show that phosphorylation of this specific amino acid residue regulates the nuclear export of APRIL. In sum, we report here the signal sequences in APRIL that mediate its intracellular trafficking and provide evidence that this protein ligand of HuR is an important player in the post-transcriptional regulation of CD83 expression by affecting the nucleocytoplasmic translocation of CD83 mRNA.