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The generation of temperature gradients on nanoparticles heated externally by a magnetic field is crucially important in magnetic hyperthermia therapy. But the intrinsic low heat i n g power of magnetic nanoparticles, at the conditions allowed for human use, is a limitation that restricts the general implementation of the technique. A promising alternative is local intracellular hyperthermia, whereby cell death (by apoptosis, necroptosis, or other mechanisms) is attained by small amounts of heat generated at thermosensitive intracellular sites. However , the few experiments conducted on the temperature determination of magnetic nanoparticles have found temperature increments that are much higher t h a n the theoretical predictions, thus supporting the local hyperthermia hypothesis. Reliable intracellular temperature measurements are needed to get an accurate picture and resolve the discrepancy. In this paper, we report the real-time variation of the local temperature on gamma-Fe2O3 magnetic nanoheaters using a Sm3+/Eu3+ ratiometric luminescent thermometer located on its surface during exposure to an external alternating magnetic field. We measure maximum temperature increments of 8 degrees C on the surface of the nanoheaters without any appreciable temperature increase on the cell membrane. Even with magnetic fields whose frequency and intensity are still well within health safety limits, these local temperature increments are sufficient to produce a small but noticeable cell death, which is enhanced considerably as the magnetic field intensity is increased to the maximum level tolerated for human use, consequently demonstrating the feasibility of local hyperthermia.

All products labeled as sterile are required to be free of microbial contamination throughout their shelf life (obligatory critical quality attribute). Container closure integrity (CCI) needs to be addressed with a holistic life cycle strategy comprising adequate primary packaging components selection and the assessment of critical unit operations and critical process parameters (CPPs) according to quality by design (QbD) principles. The helium leak method is currently the most sensitive CCI test method and preferably used for the initial container closure system (CCS) qualification and characterization studies. Currently, two different measuring principles are used in the pharmaceutical industry, and no data is available in the public domain for typical method performance parameters such as accuracy, precision, intermediate-precision, and limit of quantification of the method. Furthermore, the performance of different types and sizes of artificial leaks as well as certified helium leak standards have not yet been characterized across different test laboratories. In this multicompany study, we shared 17 artificially prepared leak samples using the most common types of artificial leaks in relevant nominal size ranges that are commercially available or can be easily prepared in a laboratory. Each participating company generated results according to their in-house methods, applying their established test parameters as the aim of the study was not to create a standard for helium leak measurements, but to compare real-world performance between different laboratories. Consequently, this study is not an interlaboratory study using the same test method across laboratories.

Biocompatibility restrictions have limited the use of magnetic nanoparticles for magnetic hyperthermia therapy to iron oxides, namely magnetite (Fe

Biocompatibility restrictions have limited the use of magnetic nanoparticles for magnetic hyperthermia therapy to iron oxides, namely magnetite (Fe3O4) and maghemite (γ-Fe2O3). However, there is yet another magnetic iron oxide phase that has not been considered so far, in spite of its unique magnetic properties: ε-Fe2O3. Indeed, whereas Fe3O4 and γ-Fe2O3 have a relatively low magnetic coercivity, ε-Fe2O3 exhibits a giant coercivity. In this report, the heating power of ε-Fe2O3 nanoparticles in comparison with γ-Fe2O3 nanoparticles of similar size (∼20 nm) was measured in a wide range of field frequencies and amplitudes, in uncoated and polymer-coated samples. It was found that ε-Fe2O3 nanoparticles primarily heat in the low-frequency regime (20–100 kHz) in media whose viscosity is similar to that of cell cytoplasm. In contrast, γ-Fe2O3 nanoparticles heat more effectively in the high frequency range (400–900 kHz). Cell culture experiments exhibited no toxicity in a wide range of nanoparticle concentrations and a high internalization rate. In conclusion, the performance of ε-Fe2O3 nanoparticles is slightly inferior to that of γ-Fe2O3 nanoparticles in human magnetic hyperthermia applications. However, these ε-Fe2O3 nanoparticles open the way for switchable magnetic heating owing to their distinct response to frequency., This work was supported by European Union's Horizon 2020 FET Open program [Grants no: 801305 and 829162] Spanish Ministry of Science Innovation and Universities [Grant no: PGC2018_095795_B_I00] and Diputación General de Aragón [E11/17R]. Authors would like to acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza. This work was developed within the scope of the projects CoolPoint P2020-PTDC-CTMNAN-4511-2014 and CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, financed by national funds through the FCT/MEC and co-financed by FEDER under the PT2020 Partnership Agreement., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

The metabolic activity of tumor cells is known to be higher as compared to that of normal cells, which has been previously exploited to deliver nanomedicines to highly metabolic tumor cells. Unfortunately, current strategies, which are mostly based on complex energy sources, such as sugars, showed insufficient accumulation at the target sites. We here report the coating of IONPs with two essential units of cellular metabolism: adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). ATP and NADPH were directly bound to the IONPs’ surface using a simple aqueous method. Both molecules were used as coatings, i.e. as stabilizing agents, but also simultaneously as targeting molecules to deliver IONPs to highly metabolic tumor cells. Indeed, we found that the uptake of ATP-IONPs and NADPH-IONPs is correlated with the metabolic activity of tumor cells, especially regarding their cellular ATP levels and NADPH consumption. We also measured one of the highest MRI r2 relaxivities for both ATP-IONPs and NADPH-IONPs. With the direct coating of IONPs with ATP and NADPH, we therefore provide an optimal platform to stabilize IONPs and at the same time promising properties for the targeting and detection of highly metabolic tumor cells.

Due to their high chemical affinity towards metal ions, chelating agents (CAs) have been used for decades for water purification, but also for protection against metal intoxication and in nanomedicine as linking molecules at the surface of nanoparticles. However, this strong chemical activity could also impact their colloidal behavior, which is essential for biomedical applications. Therefore, we coated iron oxide nanoparticles (IONPs) with four CAs, differing in their number of active chemical groups, with variations from 2 to 5 dents containing carboxylic groups: iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). We found large differences between the CA-coated IONPs depending on the CA nature, especially regarding their agglomeration state and colloidal behavior, but also consequently their cellular uptake. Surprisingly, although CAs have been widely used for biomedical applications, CA-coated IONPs, especially IDA- and EDTA-coated IONPs, showed non-negligible toxicity. Moreover, for their application as contrast agents for MRI, we found that CA-coated IONPs displayed high r2 relaxivities, which differed according to their agglomeration state. Overall, our study suggests that CAs, depending on their chemical nature, can induce agglomeration and toxicity, which could be harmful in a clinical setting.

Undecylenic acid (UA), known as antifungal agent, still cannot be used to efficiently modify commercial dental materials in such a way that this affects Candida. Actually, issues with Candida infections and fungal resistance compromise the use of Poly(methyl-methacrylate) (PMMA) as dental material. The challenge remains to turn PMMA into an antifugal material, which can ideally affect both sessile (attached) and planktonic (free-floating) Candida cells. We aimed to tackle this challenge by designing PMMA-UA composites with different UA concentrations (3–12%). We studied their physico-chemical properties, the antifungal effect on Candida and the cytotoxicity toward human cells. We found that UA changes the PMMA surface into a more hydrophilic one. Mainly, as-preparation composites with ≥6% UA reduced sessile Candida for >90%. After six days, the composites were still efficiently reducing the sessile Candida cells (for ~70% for composites with ≥6% UA). Similar results were recorded for planktonic Candida. Moreover, the inhibition zone increased along with the UA concentration. The antifungal effect of UA was also examined at the surface of an UA-loaded agar and the minimal inhibitory concentration (MIC90) was below the lowest-studied 0.0125% UA. Furthermore, the embedded filamentation test after 24 h and 48 h showed complete inhibition of the Candida growth at 0.4% UA.

The overexpression of the folate receptor in most cancers has been widely exploited to specifically deliver folic acid (FA) coupled nanomedicines to tumors. However, complex coupling chemistry is often used to bind FA to the nanoparticles. Furthermore, very little has been reported for the targeting of nanomedicines to lymph node metastases (LNMs) of prostate cancer. We here report the simple and aqueous coating of iron oxide nanoparticles (IONPs) with FA for theranostics of LNMs of prostate cancer. FA was directly bound to the IONPs’ surface without the use of any linker, simultaneously playing the role of the coating molecule and targeting agent. We measured for FA-IONPs a hydrodynamic diameter around 100 nm and a negative surface charge, what is needed to access and to be retained in the lymphatic system for the LNMs targeting. We also show that FA-IONPs are specifically uptaken by prostate cancer cells expressing the prostate specific membrane antigen, including LNMs cells. FA-IONPs also displayed both high relaxivity for MRI detection and high specific absorption rate needed for hyperthermia treatment of tumors. Our study provides a theranostic platform for targeting LNMs of prostate cancer with high potential for their detection by MRI and treatment by hyperthermia.

This article belongs to the Special Issue Magnetic Nanoparticles in Biological Applications., Aqueous synthesis without ligands of iron oxide nanoparticles (IONPs) with exceptional properties still remains an open issue, because of the challenge to control simultaneously numerous properties of the IONPs in these rigorous settings. To solve this, it is necessary to correlate the synthesis process with their properties, but this correlation is until now not well understood. Here, we study and correlate the structure, crystallinity, morphology, as well as magnetic, relaxometric and heating properties of IONPs obtained for different durations of the hydrothermal treatment that correspond to the different growth stages of IONPs upon initial co-precipitation in aqueous environment without ligands. We find that their properties were different for IONPs with comparable diameters. Specifically, by controlling the growth of IONPs from primary to secondary particles firstly by colloidal and then also by magnetic interactions, we control their crystallinity from monocrystalline to polycrystalline IONPs, respectively. Surface energy minimization in the aqueous environment along with low temperature treatment is used to favor nearly defect-free IONPs featuring superior properties, such as high saturation magnetization, magnetic volume, surface crystallinity, the transversal magnetic resonance imaging (MRI) relaxivity (up to r₂ = 1189 mM-1·s-1 and r₂/r₁ = 195) and specific absorption rate, SAR (up to 1225.1 W·gFe-1)., This work was financially supported by the MagnetoTeranostics project that was scientifically evaluated by the Swiss National Science Foundation (SNSF), financed by the Swiss Confederation and funded by Nano-Tera.ch (Magnetotheranostics project Nº 530 627) and by the Nanoneck2 project funded by Eurostars (Project E!9309 Nanoneck2). Financial support from the Spanish Ministry of Science and Innovation research grant MAT2014-54975-R are gratefully acknowledged.

Nanoparticles (NPs) in contact with a biological environment get covered by proteins and some are loosely bound and some are tightly bound. The latter form a hard protein corona (HPC) which is known to determine their biological behavior. Therefore, in order to study the biological behaviour of NPs one needs to start from the HPC. However, established methods and standards of HPC isolation are still not known. This is especially a challenge in the case of magnetic NPs which form a major branch of nanomedicine. Therefore, we developed a novel HPC isolation method, a multi-step centrifugation method (MSCM), for single-domain magnetic NPs. The MSCM was applied to iron oxide NPs in interaction with human blood and lymph serum with different dilutions in triplicate. The analysis of the composition of the obtained HPCs showed the reproducibility of the MSCM. This new method was also compared with the existing magnetic separation method (MagSep) and a study of the obtained HPC allowed us to establish the validity limits of MagSep and MSCM on only superparamagnetic NPs and on any single-domain magnetic NPs, respectively. Surprisingly, the HPCs obtained by these two isolation methods were quite different, up to 50%, suggesting that only these proteins, which are found in the HPCs of both isolation methods, are in fact real HPCs.

We developed a method to correct absorbance-based toxicity tests to remove the influence of the deposited dose of nanoparticles. As a model of absorbance-based toxicity tests, we chose the frequently used MTS test. The corrected cell viabilities obtained with the MTS test matched the results of the fluorescence-based PrestoBlue® test.

In biological environments, the surface of nanoparticles (NPs) are modified by protein corona (PC) that determines their biological behavior. Unfortunately, in vitro tests still give different PC than in vivo tests causing in vitro-in vivo discrepancy; hence, in vitro studies are not indicative for the NPs' behavior in vivo. Here is demonstrated that PC in vitro is strongly influenced by the type of extracellular fluid (ECF), blood or lymph, by their high and low flow conditions and transitions between ECFs, and a combination of these parameters. As a result, this in vitro study approaches fluidic and dynamic variations to which NPs are exposed in vivo: different ECF that NPs encounter first in different injection routes, different transitions in-between ECFs during circulation, and simultaneous change in the exposed flow in these transitions. The most-abundant proteins in PCs are found to be not the most abundant in ECFs, but those having high affinity for binding to the surface of NPs. Moreover, some proteins are differently abundant in PCs at different flows, which indicate force-promoted binding, catch bonds. These results suggest that future in vitro studies should consider more complex incubation conditions to improve the in vitro-in vivo consistency necessary for translational research.

Iron oxide nanoparticles (IONPs) were synthesized by a novel aqueous synthesis route which combines co-precipitation (CP) and hydrothermal (HT) treatment, termed CP + HT, and compared with IONPs obtained by the standard CP method. Properties of both types of IONPs, including their morphology, diameters, composition, structure and crystallinity, as well as magnetic properties and toxicity were studied and correlated with the synthesis route. Their potential application as mediators for hyperthermia treatment has been evaluated by the specific absorption rate (SAR). Studies showed that IONPs obtained by a novel CP + HT route have a more controlled morphology, structure and crystallinity, leading to better magnetic properties and SAR as compared to IONPs synthesized by CP. Reported IONPs are also not toxic as shown by two assays in two cell lines. These results suggest that our IONPs are suitable for biomedical applications, especially as mediators for the hyperthermia treatment., This work was financially supported by the MagnetoTeranostics project that was scientifically evaluated by the Swiss National Science Foundation (SNSF), financed by the Swiss Confederation and funded by Nano-Tera.ch (Magnetotheranostics project No 530 627) and by the Nanoneck2 project funded by Eurostars (Project E!9309 Nanoneck2). We thank to the Interdisciplinary Centre for Electron Microscopy (CIME), Central Environmental Laboratory (CEL) and the Molecular and Hybrid Materials Characterisation Center (MHMC) all at EPFL for access to their facilities and technical support. We are also grateful to Phil Pattison and Swiss-Norwegian beamline (ESRF, Grenoble) for the synchrotron radiation XRD experiments. This work was partially supported by the Spanish Ministry of Science and Innovation (MAT2014-54975-R). The authors would like to acknowledge the use of Servicio General de Apoyo a la Investigacion-SAI, Universidad de Zaragoza.

Iron oxide nanoparticles (IONPs) were prepared via aqueous synthesis which combines alkaline co-precipitation (CP) of ferric and ferrous precursors with mild hydrothermal (HT) treatment without cupping agents (CA). In this novel synthesis route, CP + HT, we found the optimal synthesis conditions to obtain IONPs without a second phase and with the size larger than in standard CP: the equal number of Fe(II) and Fe(III) ions are co-precipitated with 6 M ammonia and further HT treated in mild conditions (120 °C for 24 h) without CA. The IONPs obtained by novel CP + HT route had faceted rectangular morphology, a mean TEM diameter of 21.5 ± 6.3 nm, a hydrodynamic diameter of 30.2 ± 9.1 nm and a zeta potential at pH 4 of 48.2 ± 0.6 mV. After the subsequent oxidation step, the final product (IONPs) was studied by XRD, FTIR and XPS, which confirmed the desired structure of γ-Fe2O3. Importantly, this synthesis was especially planned for the preparation of IONPs for biomedical applications. Thus, our novel synthesis was designed to be compliant with the regulations of nano-safety: no special atmosphere, no complex multistep size separation, no organic solvents or solvent exchange, no CA and their washing and the use of low temperature in the final optimised conditions. In addition, this simple synthesis route combines the CP and HT methods, which are both proven to be scalable. Moreover, repeatability and reproducibility of the optimal CP + HT synthesis were confirmed on the lab-scale; more than 100 repetitions with different dishes, different operators and different batches of chemicals were performed.

Tumor vasculature is known to be poorly organized leading to increased leakage of molecules to the extravascular space. This process can potentially increase interstitial fluid pressure impairing intra-tumoral blood flow and oxygen supply and can affect drug uptake. Anti-angiogenic therapies are believed to reduce vascular permeability, potentially reducing interstitial fluid pressure and improving the extravasation of small molecule-based chemotherapeutics. Here we show that pretreatment of human ovarian carcinoma tumors with sub-optimal doses of the VEGFR targeting tyrosine kinase inhibitor axitinib, but not the EGFR targeting kinase inhibitor erlotinib, induces a transient period of increased tumor oxygenation. Doxorubicin administered within this window was found to enter the extravascular tumor space more rapidly compared to doxorubicin when applied alone or outside this time window. Treatment with the chemotherapeutics, doxorubicin and RAPTA-C, as well as applying photodynamic therapy during this period of elevated oxygenation led to enhanced tumor growth inhibition. Improvement of therapy was not observed when applied outside the window of increased oxygenation. Taken together, these findings further confirm the hypothesis of angiostasis-induced vascular normalization and also help to understand the interactions between anti-angiogenesis and other anti-cancer strategies.

Photodynamic therapy (PDT) is an effective clinical treatment for a number of different cancers. PDT can induce hypoxia and inflammation, pro-angiogenic side effects, which may counteract its angio-occlusive mechanism. The combination of PDT with anti-angiogenic drugs offers a possibility for improved anti-tumour outcome. We used two tumour models to test the effects of the clinically approved angiostatic tyrosine kinase inhibitors sunitinib, sorafenib and axitinib in combination with PDT, and compared these results with the effects of bevacizumab, the anti-VEGF antibody, for the improvement of PDT. Best results were obtained from the combination of PDT and low-dose axitinib or sorafenib. Molecular analysis by PCR revealed that PDT in combination with axitinib suppressed VEGFR-2 expression in tumour vasculature. Treatment with bevacizumab, although effective as monotherapy, did not improve PDT outcome. In order to test for tumour vessel normalization effects, axitinib was also applied prior to PDT. The absence of improved PDT outcome in these experiments, as well as the lack of increased oxygenation in axitinib-treated tumours, suggests that vascular normalization did not occur. The current data imply that there is a future for certain anti-angiogenic agents to further improve the efficacy of photodynamic anti-cancer therapy.

Nanoparticles with smaller diameters have larger surface areas. Unfortunately, this fact is often neglected in their biomedical characterization, e.g. in the evaluation of their toxicity with cell viability tests. We here point out the different scientific conclusions drawn in such tests when considering the traditional mass or concentration of nanoparticles, or their surface areas.