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Raluca Maria FRATILA Orla Carron Roxanne HACHANI
Irene MORALES CASERO Mikhail V. Zyuzin Helena Gavilán
Andreas SERGIDES Ivan Stijepović Lilin Wang
Aziliz Hervault Raquel Rodrigues Urszula Klekotka
Orla Carron Débora Bonvin David Cabrera
Mikhail V. Zyuzin Catalina Bordeianu Margarita Kakava
José Mariano Georgios Kasparis Maria Theodosiou
Edyta Cędrowska Aidin Lak Nguyen TK Thanh
Aziliz HERVAULT Maria Elena MATERIA Kuan Boone Tan

Real-time monitoring of cellular effects of sublethal magnetic hyperthermia.

The research proposed for this COST STSM is part of my on-going two-years Marie Skłodowska-Curie (MSC) Project OUTstandINg, which is focused on the covalent immobilization of magnetic nanoparticles (MNPs) on living cell plasma membranes using bioorthogonal click chemistry with the aim to address two fundamental questions in the field of magnetic hyperthermia: 1) how the subcellular localization (on the plasma membrane or inside the cells) of MNPs affects their heating behaviour when compared to MNPs in solution 2) how MNPs immobilization and sub-lethal magnetic hyperthermia impact different subcellular signalling pathways and the biophysics of cell membranes. The aim of the STSM at UCL Healthcare Biomagnetics Laboratory was to explore the possibility of real-time monitoring of the effect of sublethal magnetic hyperthermia on living cells, using the in situ MHT/imaging system developed by the host group (C. Blanco-Andujar et al., Nanomedicine, 2016, 11, 121). The preliminary studies carried out during the STSM were focused on optimizing the experimental conditions in order to maintain cells healthy and functioning normally on the microscope stage for the whole duration of the experiment (usually up to 16 h). This is one of the main technical challenges for performing live-cell imaging experiments and is fundamental for our studies as the decrease of cellular viability non-associated with the magnetic hyperthermia experiment must be minimized. To this end, MCF-7 (human breast adenocarcinoma) cells were cultured on 35-mm cell culture dishes at 37 ºC in a humidified atmosphere containing 5% CO2. Different control experiments were carried out to establish the best conditions for long-term imaging experiments. These experiments included cell growth using different cell culture dishes and use of an anti-evaporation oil to minimize changes in osmolarity due to evaporation, which can be severe when using relatively low volumes of cell culture medium in a 37 ºC environment. 


High frequency hysteresis loop meter: learning the set-up and performing magnetometric measurements for hyperthermia

 During the Short-Stay Scientific Mission, a systematic study of different γ-Fe2O3 and Fe3O4 nanoparticles has been done in order to investigate the effect of nanoparticle properties like size, saturation magnetization, magnetic anisotropies, etc. on the heating performance, by means of a hand-made high frequency hysteresis loop meter and a calorimeter. This study goes deep into the knowledge of the behavior and properties of magnetic nanoparticles subjected to high frequency alternating fields.

 

The analysed samples were ɣ-Fe2O3 nanoparticles of 8 nm, and uncoated and APS coated 12 nm and 14 nm; all the samples were synthetized by coprecipitation method. Nanoparticles of Fe3O4 with a size of 33 nm and a hybrid system of magnetite and gold were also analysed. A more complex system composed by ZnMnFe2O4@Fe3O4 in water and in isooctane was also measured as well as 6 nm ɣ-Fe2O3 nanoparticles with two different coatings, not being able to obtain any results of the measures since they did not warm up. All the colloids were previously characterized by TEM, XRD, DLS and SQUID magnetometry. From the SQUID and calorimetric measurements it has been seen that the coercive field and the SAR increase with the nanoparticle size until around 33 nm (monodomain-multidomain limit), having this Fe3O4 sample the higher heating efficiency of all, due probably to Brown relaxation. The comparison between uncoated and APS coated 12 nm maghemite shows an increase of the heating efficiency with the coating. For 12 nm and 14 nm maghemite samples, no differences were observed until around 30 mT; at higher fields, the 14 nm NPs have a SAR 13 % higher than the former. In the case of hybrid system of magnetite and gold, the magnetite NPs have a higher heating efficiency at low field, but at higher fields is the hybrid system the one with better performance.

SYNTHESIS OF METAL NANOPARTICLES FOR MAGNETIC HYPERTHEMIA

FeCo alloys are soft magnetic materials exhibiting low magnetic anisotropy constant (K Fe50Co50 = 1.5 x 104 J/m3) and the highest saturation magnetisation (Ms) among any other metal or alloy (bulk Ms: 240 – 245 emu/g). In addition, Co is the only element that increases the Ms when alloyed with Fe (55-65% Fe)1. These remarkable properties of FeCo nanoparticles (NPs), in superparamagnetic regime, in conjunction with their high heating rate2 ensure high potential for biomedical applications, such as Magnetic Resonance Imaging (MRI), Magnetic Particle Imaging (MPI), Magnetic Drug Targeting (MDT) and especially Magnetic Fluid Hyperthermia (MFH) for cancer treatment. However, the synthesis of these NPs remains a challenging task due to their poor chemical stability.

Herein we report the synthesis of FeCo NPs by thermal decomposition of organometallic precursors. The synthesis is based on the co-decomposition of Fe(CO)5 and Co(n3-C8H13)(n4-C8H12) in the presence of various ligands (oleic acid – OA, palmitic acid – PA, hexadecylamine – HDA, hexadecylamine hydrochloride – HDA.HCl) under 3 bars of H2 at 150 oC. During this study, we aimed to elucidate the effect of ligands, ligands’ concentration and ratio on the size, morphology and magnetic properties of the as-prepared NPs. In the most cases, spherical core-shell NPs were obtained with an average diameter less than 25 nm (Fig. 1). Among other techniques, WAXS, SQUID and TEM have been used for the determination of the structural, compositional and magnetic characteristics of these NPs.

 

References

1. Clifford, D. M.; Castano, C. E.; Lu, A. J.; Carpenter, E. E., Synthesis of FeCo alloy magnetically aligned linear chains by the polyol process: structural and magnetic characterization. J. Mater. Chem. C 2015, 3 (42), 11029-11035.

2. Kappiyoor, R.; Liangruksa, M.; Ganguly, R.; Puri, I. K., The effects of magnetic nanoparticle properties on magnetic fluid hyperthermia. Journal of Applied Physics 2010, 108 (9), 094702.


Study of the in vitro enhanced synergism of magnetic hyperthermia combined with doxorubicin delivery in the treatment of cancer using the bioluminescence imaging method

Magnetic nanocarriers have attracted increasing attention for multimodal cancer therapy due to the possibility to deliver heat and drugs locally and the possibility to benefit from the synergistic effect of the combined therapy. Bioluminescence imaging is an optical imaging technique that is based on the sensitive detection of photons emitted during the bioluminescence reaction from bioreporter cells expressing Luciferase. A novel magnetic nanocomposite composed of a superparamagnetic iron oxide core and a pH- and thermo-responsive polymer shell that can be used as both mediators of heat and drug carriers for the treatment of cancer was previously developed. The dual stimuli-responsive behaviour provides advanced features offering spatial and temporal control over the release of the anti-cancer drug doxorubicin in response to magnetic hyperthermia and tumour acidic pH, therefore limiting unwanted side effects. In vitro experiments performed on a murine prostate carcinoma RM1 cell line genetically modified to express the enzyme Firefly Luciferase confirmed that thermo-chemotherapy treatment applied via the developed smart delivery system exhibits a substantial increase in cytotoxicity as compared to chemotherapy or magnetic hyperthermia as standalone therapies. Cell viabilities were determined by monitoring Luciferase activity using the bioluminescence imaging method (Figure 1). For a treatment applied after internalisation of the nanoparticles inside the cells and subsequent washing, the relative cell viabilities 24 h post-treatment are 65 % for hyperthermia, 82 % for chemotherapy and 44 % for thermo-chemotherapy. For direct treatment which consists in subjecting the cells to an alternating magnetic field directly after mixing them with the nanoparticles, the relative cell viabilities 24 h post-treatment are 93 % for chemotherapy, 89 % for hyperthermia and 57 % for thermo-chemotherapy at 42 °C, 70 % for hyperthermia and 26 % for thermo-chemotherapy at 43 °C. In each case, the combined effects were found to be synergistic in nature.


Towards the synthesis of magnetic-Large Hybrid Vesicles (m-LHVs)

 In this project we developed novel membrane stabilized nanocomposites, with magnetic nanoparticles (MNPs) bound directly into the membrane to maximize hyperthermic response. Induced heat should affect the vesicle permeability, allowing for specific, localized drug release in tumor tissue. The approaches below were developed to produce a library of m-LHVs. A signifiant positive is that these processes can be easily expanded to prepare hybrid vesicles, including lipids.

1. Synthesis of PB-PEO or PDMS-(PEO)2 based polymer vesicles

a. Rehydration method for the formation of Large Unilameller Vesicles (LUVs); as multilamellar vesicles were formed these were extruded. The method developed can be used for a range of materials; including m-LUVs (magnetic LUCs), LHVs (hybrids) and m-LHVs.

b. Emulsion-Evaporation Method for LUVs; the vesicles formed were typically less monodisperse than from rehydration, however as no multilameller vesicles are formed, extrusion was not needed. Again a range of sizes were formed.

c. Electroformation of Giant Unilameller Vesicles (GUVs); importantly this procedure can be easily applied to obtain GHVs.

2. Synthesis of PDMS-PEO2 based polymer vesicles

d. Emulsion-evaporation was successfully applied, however the vesicles were less monodisperse.

In summary vesicles of three different compositions were successfully prepared; (i) PDMS-g-(PEO)2 (3KDa) (ii) PDMS(5kDa)-b-(PEO)2 vesicles; (iii) OA-coated MNPs in PBut-b-PEO vesicles, with typical MNP feed weight ratio of 10, but up to 3wt% (iii). Primary characterisation of the suspensions was completed at LCPO, see Figure. Over the coming months the use of smaller MNPs and improved coating will be evaluated. AC-field hyperthermia and NMR measurements will be undertaken to confirm the particles are localised in the membrane and to evaluate the effect of composition/structure on heating efficiency and MRI contrast potential. We will also assess domain formation at nanoscale by FRET and SANS.


Italian Institute of Technology, Genoa, Italy

Iron oxide nanoparticles are often used as a heating source for hyperthermia applications. After applying alternating magnetic fields, two relaxation mechanisms take place in iron oxide nanoparticles (IONPs): Néel relaxation and Brownian relaxation. The heating ability of IONPs can be expressed by the specific absorption rate (SAR), which is maximized when both relaxation heatings contribute to a final heating. The value of SAR strongly depends on size of IONPs and medium, in which these nanoparticles are distributed. For example, “frozen” (with suppressed Brownian relaxation) IONPs heat less than free nanoparticles.

Polyelectrolyte (PE) capsules are widely used as carrier systems due to their low toxicity, low cost production, and robust synthesis. Cubic IONPs with different size (14, 18 nm) were loaded into the cavity of sub-micrometric polyelectrolyte capsules, so that they can freely move inside the capsule. Additionally, the same IONPs were embedded into a CaCO3 template and in the wall of polyelectrolyte capsule in order to “freeze” them and suppress the Brownian relaxation.

According to the obtained results, free iron oxide nanoparticles (14 and 18 nm) show higher SAR in water. Hysteresis loops of multilayer polyelectrolyte capsules with the iron oxide nanocubes in the cavity are larger than hysteresis loops of calcium carbonate cores or polyelectrolyte capsules with embedded nanocubes. Interestingly, this effect take place for both 14 and 18 nm nanocubes and proofs that nanoparticles have certain freedom to move inside the capsules, and thus Brownian relaxation is not suppressed.

 


Magnetic properties and hyperthermia effect in magnetic nanoparticles

The goal of this STSM was to deepen the knowledge on the techniques used for the synthesis and characterisation of magnetic nanoparticles, the determination of their magnetic properties and their characterisation from the hyperthermia effect perspective trough the determination of the Specific Absorption Ratio (SAR). For this purpose, the first couple of days were dedicated to becoming familiar with the constructions details and operation of a locally built apparatus dedicated to the determination of SAR of liquid samples. The rest of the time was dedicated to the characterisation of locally produced poly-L-lysine (PLL) functionalized magnetic nanoparticles that are used as carriers of specific antibodies to detect and/or target cancer cells. Magnetic nanoparticles with the optimal PLL content were conjugated with antibody specific for the cancer biomarker carbonic anhydrase IX (CA IX). CA IX is localised on the cell surface with the antibody-binding epitope facing the extracellular space and is therefore suitable for antibody-based targeting of tumour cells. The purpose of the essays was to determine if the SAR of the particles was affected by the antibody conjugation. For this purpose, a series of trials were conducted, where the time evolution of the sample temperature was measured for different values of AC magnetic field intensities and different frequencies. From this, the SAR dependence with H was calculated.

Using the opportunity provided by this STSM, we also measured ZFC-FC and magnetization curves of Co doped TiO2 nanoparticles synthesized at the University of the Algarve, using a recently installed 18T cryomagnet equipped with a VSM head. This was a very satisfying experience, since it was the first time I had a hands-on contact with such a technique. This allow me to get a better understanding of the strengths and weakness of VSM when applied to MNP characterisation.


Biodistribution studies of gold-coated magnetite nanoparticles conjugated with Trastuzumab, in tumor- and non-tumor-bearing mice

The main purpose of Short Term Scientific Mission was to carry out detailed biodistribution studies of multimodal nanocarriers that could be used simultaneously for targeted radionuclide therapy and hyperthermia. This kind of nanosystem is based on magnetic nanoparticles (Fe3O4 NPs) radiolabelled with lutetium-177 (t1/2=6.7 d) and conjugated with monoclonal antibodies (Trastuzumab). Trastuzumab is a recombinant humanized monoclonal antibody that targets Her-2 receptors (Human Epidermal Growth Factor Receptor 2) overexpressed on breast and ovarian cancer cells. The most important goal of this research project was to verify if monoclonal antibodies grafted onto the nanoparticles surface would improve tumour-cell targeting in xenografts.

Magnetite nanoparticles were labelled during synthesis by addition of 177Lu as the chloride salt (6 MBq). The yield of labelling was 96.7±1.7% (n=5). Then, radioactive nanoparticles were functionalized using3-phosponopropionic acid and coupled to monoclonal antibodies using carbodiimide chemistry. The overall radiochemical yield of synthesis of radioactive nanoparticles conjugated to monoclonal antibodies was 40%.

Radiolabelled carboxylated and conjugated with monoclonal antibodies nanoparticles were injected mostly intravenously. Only for one experiment the nanoparticles were injected directly into tumour to observe their possible clearance/leakage from cancer tissue. After chosen time-points (24h, 48h and 72h) mice were sacrificed and organs of interest were collected.

Performed experiments confirmed that intravenously injected nanoparticles were quickly eliminated from the blood system and were mainly accumulated in reticuloendothelial cells of the liver and spleen. Monoclonal antibodies grafted on the surface of nanoparticles slightly improved tumour cells targeting in xenografts, but still targeting efficacy was low and needs to be improved. In the case of direct injection of nanoparticles into tumour tissue high retention of nanoparticles coupled with monoclonal antibodies was observed after 3 days.


UCL Healthcare Biomagnetic Labs

Magnetic nanocarriers have attracted increasing attention for multimodal cancer therapy due to the possibility to deliver heat and drugs locally and the possibility to benefit from the synergistic effect of the combined therapy. Bioluminescence imaging is an optical imaging technique that is based on the sensitive detection of photons emitted during the bioluminescence reaction from bioreporter cells expressing Luciferase. A novel magnetic nanocomposite composed of a superparamagnetic iron oxide core and a pH- and thermo-responsive polymer shell that can be used as both mediators of heat and drug carriers for the treatment of cancer was previously developed. The dual stimuli-responsive behaviour provides advanced features offering spatial and temporal control over the release of the anti-cancer drug doxorubicin in response to magnetic hyperthermia and tumour acidic pH, therefore limiting unwanted side effects. In vitro experiments performed on a murine prostate carcinoma RM1 cell line genetically modified to express the enzyme Firefly Luciferase confirmed that thermo-chemotherapy treatment applied via the developed smart delivery system exhibits a substantial increase in cytotoxicity as compared to chemotherapy or magnetic hyperthermia as standalone therapies. Cell viabilities were determined by monitoring Luciferase activity using the bioluminescence imaging method. For a treatment applied after internalisation of the nanoparticles inside the cells and subsequent washing, the relative cell viabilities 24 h post-treatment are 65 % for hyperthermia, 82 % for chemotherapy and 44 % for thermo-chemotherapy. For direct treatment which consists in subjecting the cells to an alternating magnetic field directly after mixing them with the nanoparticles, the relative cell viabilities 24 h post-treatment are 93 % for chemotherapy, 89 % for hyperthermia and 57 % for thermo-chemotherapy at 42 °C, 70 % for hyperthermia and 26 % for thermo-chemotherapy at 43 °C. In each case, the combined effects were found to be synergistic in nature. 


School of Chemistry, University College Dublin, Ireland

In this project we developed novel membrane stabilized nanocomposites, with magnetic nanoparticles (MNPs) bound directly into the membrane to maximize hyperthermic response. Induced heat should affect the vesicle permeability, allowing for specific, localized drug release in tumor tissue. The approaches below were developed to produce a library of m-LHVs. A signifiant positive is that these processes can be easily expanded to prepare hybrid vesicles, including lipids.
1. Synthesis of PB-PEO or PDMS-(PEO)2 based polymer vesicles
a. Rehydration method for the formation of Large Unilameller Vesicles (LUVs); as multilamellar vesicles were formed these were extruded. The method developed can be used for a range of materials; including m-LUVs (magnetic LUCs), LHVs (hybrids) and m-LHVs.
b. Emulsion-Evaporation Method for LUVs; the vesicles formed were typically less monodisperse than from rehydration, however as no multilameller vesicles are formed, extrusion was not needed. Again a range of sizes were formed.
c. Electroformation of Giant Unilameller Vesicles (GUVs); importantly this procedure can be easily applied to obtain GHVs.
2. Synthesis of PDMS-PEO2 based polymer vesicles
d. Emulsion-evaporation was successfully applied, however the vesicles were less monodisperse.
 
In summary vesicles of three different compositions were successfully prepared; (i) PDMS-g-(PEO)2 (3KDa) (ii) PDMS(5kDa)-b-(PEO)2 vesicles; (iii) OA-coated MNPs in PBut-b-PEO vesicles, with typical MNP feed weight ratio of 10, but up to 3wt% (iii). Primary characterisation of the suspensions was completed at LCPO, see Figure. Over the coming months the use of smaller MNPs and improved coating will be evaluated. AC-field hyperthermia and NMR measurements will be undertaken to confirm the particles are localised in the membrane and to evaluate the effect of composition/structure on heating efficiency and MRI contrast potential. We will also assess domain formation at nanoscale by FRET and SANS.

Italian Institute of Technology, Via Morego, 30, 16163 Genoa, Italy

Iron oxide nanoparticles are often used as a heating source for hyperthermia applications. After applying alternating magnetic fields, two relaxation mechanisms take place in iron oxide nanoparticles (IONPs): Néel relaxation and Brownian relaxation. The heating ability of IONPs can be expressed by the specific absorption rate (SAR), which is maximized when both relaxation heatings contribute to a final heating. The value of SAR strongly depends on size of IONPs and medium, in which these nanoparticles are distributed. For example, “frozen” (with suppressed Brownian relaxation) IONPs heat less than free nanoparticles.

Polyelectrolyte (PE) capsules are widely used as carrier systems due to their low toxicity, low cost production, and robust synthesis. Cubic IONPs with different size (14, 18 nm) were loaded into the cavity of sub-micrometric polyelectrolyte capsules, so that they can freely move inside the capsule. Additionally, the same IONPs were embedded into a CaCO3 template and in the wall of polyelectrolyte capsule in order to “freeze” them and suppress the Brownian relaxation.  


Faculty of Technology, University of Novi Sad, Novi Sad, Serbia

Aim of the STSM was to use facilities and available equipment at the Institute Jožef Stefan to investigate ferrite and silica coated ferrite particles synthesised at the Faculty of Technology, Novi Sad, in order to determine their structural and functional properties. Nickel and zinc ferrite nanoparticles were synthesised using co-precipitation method and stabilised with citric acid (CA), poly(diallyl dimethylammonium)chloride (PDDA) and tetramethylammonium hydroxide. Then core/shell structure was made by adjusting zeta potential of as-synthesised particles by choosing appropriate stabilization compound. The other method was to synthesise nickel ferrite and then add zinc and iron nitrate salts in the reaction mixture and continue the synthesis. In this way, zinc ferrite shell would form on the core particles of nickel ferrite. Additionally, ferrite core particles with silica layer on top of them were obtained using Stöber process. All together, 16 different samples were used for magnetic measurements using vibrating sample magnetometry (VSM). Magnetization was measured in the range from -16 kG to 16 kG. Five samples were used for TEM investigations to determine structural characteristics of the synthesised nanoparticles. TEM results showed that samples mainly consisted of non-crystalline nickel ferrite with the presence of FeOOH secondary phase. This was consistent with magnetic measurements which showed that as-synthesised samples were paramagnetic. On the contrary, sample with NiFe2O4 core/ZnFe2O4 shell showed superparagnetic S-curve of magnetization. This indicates that prolonged synthesis time enables crystallization of NiFe2O4. All samples with silica shell were also paramagnetic.


Laboratory of Separation and Reaction Engineering - Laboratory of Catalysis and Materials (LSRE-LCM), Polytechnic Institute of Bragança, Bragança, Portugal

In this short term scientific mission (STSM), carbon-coated yolk-shell magnetic nanoparticles (CYSMNPs) – hybrid materials comprising a superparamagnetic core coated with a carbon shell surrounding a hollow region (i.e., Fe3O4@void@C) – were investigated as multifunctional nanoparticles suitable for biomedical applications, namely for hyperthermia applications and drug delivery systems. This concept was based on the fact that carbon-coated nanoparticles have several advantages in comparison to polymer or silica coatings, since they usually offer higher chemical and thermal stability, larger surface area, biocompatibility and easier functionalization (1, 2). To ensure the colloidal stabilization of these CYSMNPs in aqueous solutions, a two-step procedure was investigated and optimized. Firstly, with the incorporation of carboxylic acid groups using an acid treatment (nitric acid at mild conditions) without compromising the magnetic core, and secondly, by chemical-functionalization of the activated CYSMNPs with 11-aminoundecanoic acid (11-AUA), via amidation. The resulting highly hydrophilic CYSMNPS, obtained during this STSM, revealed good hyperthermia performance, high loading capacity for doxorubicin hydrochloride (DOX) – an anticancer drug that is extensively used in anthracycline for several types of cancer – and strong triggered drug release response at acid pH 5 (similar to tumour environment). Overall, the main tasks proposed to this STSM were all accomplished, proving the great ability to use these new biomaterials in biomedical applications, which fulfils one of the main objectives of the Memorandum of understanding (MoU) of the COST Action RADIOMAG, related to the investigation of new hybrid biomaterials with great potential to be used as multifunctional nanomaterials. 


Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland

 Lately, magnetic nanoparticles have been the focus of intense research, especially as heating sources for tumour treatment by hyperthermia. Previously, we developed new iron oxide nanoparticles (IONPs) with high heating efficiencies and good clinical perspectives. However, the clinical translation of such IONPs is still hindered by a lack of understanding of certain of their features. One key issue that needs to be solved is the amount and distribution of heat at the interface between IONPs and cells. Unfortunately, this cannot be measured with currently used macroscopic temperature probes, which only give data at the bulk level. To reach the nanoscopic resolution, we combined a luminescent molecular thermometer developed at the University of Zaragoza with our IONPs. The purpose of the present work was to measure the temperature generated at the IONPs’ surface during cellular hyperthermia and to correlate the temperature with the cellular response. In order to reach that goal, IONPs were coupled to lanthanide ions (europium and terbium), which display temperature-dependent emission intensities. Lanthanide-coupled IONPs were fully characterized and their luminescent emission was calibrated as a function of temperature. Magnetic field intensities used for the calibration were those applicable to humans (H*f < 4.85*108 Am-1s-1 for a 30cm coil). Ultimately, temperatures were mapped with a high spatial resolution in a cellular assay. Cells with internalized IONPs were subjected to hyperthermia and cell death was studied. Such assays were repeated with different cell lines and different IONPs’ concentrations. In summary, this work proves the feasibility of using a molecular thermometer to characterize the heat generated around IONPs during cellular hyperthermia, addressing a recurrent problem in the field. This precise information sets the ground to further improve the therapeutic efficacy of IONPs while simultaneously minimizing the thermally induced damages to the surrounding healthy tissue.


Institut de Physique et de Chimie des Matériaux de Strasbourg, IPCMS UMR CNRS UDS 7504, 23 rue du Loess, BP 43, 67034 Strasbourg cedex 2, France

The structure of iron oxide nanoparticle (IONPs) scaffold influences their pharmacokinetics and biodistribution behavior and provides some guidance on solid tumors uptake, absorption across membrane barriers and targeting organs of the mononuclear phagocyte system (liver, spleen, lungs and lymphoid tissue). We have successfully synthesized superparamagnetic dendronized IONPs and performed in vitro cytotoxicity and in vivo biodistribution studies, as a function of different coatings (Figure 1). 
In vitro MTT and LDH bioviability tests on HepaRG cell line showed no or low toxicity after 48h of incubation for all dendronized NPs, in a large concentration range and up to 80mg/L. Furthermore, in high concentrations, dendronized IONPs promoted cell growth, due to their ability to diminish intracellular H2O2. 
In vivo MRI studies were performed on mice administered with the triple dose used in clinical trials for humans (15µmoles [Fe3+]/kg body weight). Those studies showed a strong negative contrast in the liver in T2-weighted images 90 min after intravenous injection. The negative contrast persisted over 10h in liver and for 6h in bladder. At 24h post injection of dendronized IONPs, the negative contrast for both liver and bladder appeared underneath 20%, suggesting their susceptibility to renal and hepato-biliary excretion. Furthermore, despite the administered dose, all mice showed no toxic signs over 48h, e.g. mice showed regular breathing, good quality walkways, no surrounding tissue irritation.
Results obtained in the present work provide a strong basis for future cooperation with Dr. Sophie Laurent on in vivo MRI studies, in developing nano-conjugates with higher contrast enhancement effect and with active targeting for apoptosis.
 

 


Department of Physics and Astronomy, UCL, UK

This STSM was between the Department of Physics and Astronomy of UCL, UK and bioengineering department of Bar-Ilan university, Israel. The aim was to conjugate bioactive molecules on magnetic nanoparticles for enhanced accumulation at malignant tissue.

Discriminating between healthy and malignant cells is at the frontiers of cancer research. Actively targeting malignant cells occurs when a targeting moiety is present on the cancer-fighting entity. Such molecules include some sugars, vitamins, antibodies, aptamers etc.

Hyperthermia, the fourth leg of cancer treatment, exploits the ability of magnetic nanoparticles to release heat when placed in an alternating magnetic field. The heat released can be used to either kill cells directly, control the release of drugs and/or sensitise cells for coming radiotherapy. One of the issues with these types of therapies is to get sufficient amount of crystals on the site of interest to achieve a beneficial effect. For this purpose, we attempted antibody and sugar conjugation to single and multi-core crystals, in-house made and commercially available, with short, medium and long coatings. All samples have been tested on an A431 cell line and the concentration of iron was determined using flame spectroscopy. All results were compared to gold nanoparticle antibody conjugates (expertise of hosting institution). Although results varied with some coatings being up-taken more efficiently compared to their antibody/sugar conjugate analogues, there was a lively discussion and gained inside on the importance of the spacer used between the surface of the particles and the bioactive itself. With the gained knowledge on conjugation methods and critical aspects of it we can continue towards the development of actively targeting magnetic nanoparticles and make this therapy a viable alternative.


Italian Institute of Technology, Genova, Italy

This study aims to broaden and deepen the knowledge about the underlying physics of magnetic hyperthermia heating mechanisms in iron oxide nanocubes. The nanocubes with an exchange-biased FeO/Fe3O4 antiferromagnetic-ferrimagnetic (AFM-FiM) core-shell structure were synthesized via a thermal decomposition method. Having employed a stepwise thermal oxidation process, we have tuned the fraction of AFM and FiM phases in these particles in a systematic manner. Within this STSM, we have performed AC hysteresis and specific absorption rate (SAR) measurements on the particle aqueous solutions having different magnetic domain sizes in both water and viscos media, as an attempt to mimic the magnetic response of these nanocubes after being bound to proteins and biomolecules in the body. The AC magnetometry results showed that the magnetization dynamics of the nanocubes alter substantially as went through longer thermal annealing process.  The studies on the viscos mixture of the nanocubes revealed that 18 nm sized nanocubes are almost entirely viscosity independent, yet showing a high heating performance at the field and frequency values below the biological limit i.e. H*f< 5x109 A/m s. The larger nanocubes showed a more significant response to the viscosity change, as reflected in a substantial drop in their heating performance. To complete this study, a series of in-vitro hyperthermia experiments on cancer cell lines is planned to be carried out. These experiments will enable us to understand if the dynamics of the nanocubes in an actual viscos medium such as the cells would be similar to a rather trivial viscos medium like Glycerol. The in-vitro hyperthermia treatment efficacy of the optimal nanocubes being marginally affected by viscosity will be analysed. 


Fondazione Istituto Italiano di Tecnologia, Genova (Italy)

The objective of this short-term scientific mission was to obtain a more complete magnetic characterization of different magnetic nanoparticles developed in my group at the Istituto Italiano di Tecnologia (IIT) of Genova. In particular objective of the study were cubic shaped iron oxide (Fe3O4) or cobalt ferrite (CoxFe3-xO4) nanoparticles, and gold iron oxide dimers (AuFe3O4) with a characteristic dumbbell-like structure. These magnetic materials presented outstanding heating ability under the effect of an alternating magnetic field (AMF) resulting very promising tools for hyperthermia applications. For this reason it is of great interest to understand in deep the relaxation processes which regulate the heating response of these magnetic nanoparticles and in particular how size, chemical composition and also solvent viscosity affect these mechanisms. The consolidate experience of Teran’s group in magnetic characterization and in particular in inductive magnetometry and calorimetric measurements allowed to monitor the hysteresis loops of these magnetic materials under different colloidal and AMF conditions and at the same time compare these data with their heating ability shedding light on the relaxation mechanisms involved. At the same time, this experience was also a great opportunity for me to extend my knowledge in the field of magnetism learning new magnetic characterization techniques.


University College London

During the course of my PhD project at UCL, we developed and optimized the synthesis of iron oxide nanoparticles (IONPs) coated with 3,4-dihydroxyhydrocinnamic acid (DHCA). During my stay at KU Leuven,  we determined the suitability of iron oxide nanoparticles synthesized as potential MRI contrast agents and hyperthermia agents. We were able to demonstrate their efficient uptake by hMSCs within 24 h by TEM and iron-specific Prussian Blue staining, and quantify that up to several hundreds of g of Fe per cell could be taken up by a colorimetric method. This is essential to ensure sufficient contrast by MRI and sufficient heating by MRI. Finally, an important aspect to consider is their toxic effects on cells. Conventional colorimetric MTT and MTS assays were initially used to assess this; but these proved flawed as we observed interference from the IONPs. Within the MoSAIC laboratory in KU Leuven, we were able to test the impact of the IONPs on several factors such as cell viability, mitochondrial activity and actin cytoskeleton network by validated multiparametric high-content imaging analysis. No considerable toxic effects were noticed, although a slight rounding of the cells could be observed. Furthermore, at 10 and 50 µg Fe/ml, an increase in ROS production was observed but could not be correlated to impaired mitochondria and was limited to these two IONP concentrations. Also, we were able to confirm their cellular uptake by in vitro MRI relaxivity measurements of hMSC phantoms at 9.4 T. Finally, their potential as MRI contrast agents was confirmed in vivo. IONP-DHCA were administered in female Swiss model mice and could be visualized for up to two weeks post injecton, thus confirming their potential as T2 weighted MRI contrast agents. 


ICMM – Institute of Material Science of Madrid (CSIC)

Among other functional nanomaterials, magnetic iron oxide nanoparticles (MNPs) are especially promising as contrast enhancement agents for magnetic resonance imaging (MRI), drug carriers for disease treatment and heat mediators for cancer treatment by magnetic hyperthermia. Each of these biomedical applications requires MNPs with specific properties, and therefore it is of great importance to be able to control the shape, size and magnetic interaction of MNPs. 

Nowadays, just a few synthesis strategies and coating methods allow the production of single core nanoparticles, by preventing aggregation and minimizing the inter-particle interactions. However not all the size ranges and varieties of shapes are covered by the synthesis of direct magnetic cores.

We present a three-step aqueous approach to obtain Fe3O4 single-core particles based on the synthesis of antiferromagnetic nanoparticles, its coating and subsequent transformation to magnetite. This method has the advantage of the low inter-particle interactions of as-synthesized nanoparticles. In addition, it covers different size ranges and allows obtaining different morphologies that antiferromagnetic materials exhibit. 

This opens up new possibilities and interesting magnetic behaviours. Regarding hyperthermia for cancer treatment, different geometric shape leads to distinct shape anisotropy, resulting in significant              difference in hysteresis loss and Specific Absorption Rates (SAR) values. Recently, Serantes et al. found that the chain-like arrangement biomimicking magnetotactic bacteria could exhibit better heating performance than the randomly distributed system, implying the crucial role of shape anisotropy.


University College London, UK

Magnetic nanoparticles have been widely investigated as a promising nano-medicine agent since last decade. A number of in vitroand in vivo results suggest that the chemo-thermotherapy via multifunctional magnetic nanoparticle has positive effect in cancer treatment, which means the vector carrying both hyperthermia reagent and chemotherapy drugs has potential in boosting the therapeutic effect. Thus, the in vivo biodistribution of the carrier is vital for evaluating the efficiency and translating our result from bench to bedside. Hence in this STSM project, radiopharmaceutical has been used to evaluate the in vivo biodistribution. Both the superparamagnetic nanoparticles and two chemo-drugs: doxorubicin and gemcitabine loaded thermo-sensitive liposome were labeled with radioisotope Y90 . The in vitro stability and quality studies were conducted by using instant thin layer chromatography before going into animal work. The percentage of injected activity per gram of every organ was calculated by comparing the activities with appropriate standards for injection dose. The biodistribution of magnetic nanoparticle in rats and mice model showed the similar results to that of previous published papers, which mainly captured by the liver. The liposome capsulated carriers exhibit higher accumulation manner in liver, spleen and kidney. This implies that the potential high organ specific toxicity of thermoliposome needs to be taken account for the further application. And the further experiments need to be done to acquire significant statistic results.


Institute of Chemistry, University of Bialystok, Bialystok, Poland

Ferrofluid systems have been found to possess very interesting thermophysical properties such as thermal conductivity, thermal diffusivity, viscosity or connective heat transfer. Detailed analysis of magnetic properties of magnetic nanoparticles is of crucial importance from the application’ point of view. That is why magnetization measurements significantly contribute into studies on magnetic nanoparticles, and can expand material characterization.

During my Short Term Scientific Mission, I have measured different types of previously prepared samples of iron oxide (Fe3O4 – magnetite) nanoparticles. Tested nanostructures were obtained via the same synthetic method, but in every case amount of: metallic precursor, or stabilizng coating layer was different. Because of that, obtained nanoparticles possess various composition and thicknesses of the shell (other iron oxide- maghemite, hematite, or metallic e.g. silver). During my stay, Zero Field Cooled/ Field Cooled (ZFC/FC) at 50 Oe, in temperature range 1.8-300K, and hystereses at 1.8K, 10K, and 300K, were taken. All experiments were undertaken in the liquid helium environment. Such studies were performed to see how magnetic properties changes due to size of: magnetic core, stabilizing coating layer, and addition of silver shell.

 Results obtained druing STSM magnetization correspond very well with other data obtained for analyzed systems (X-ray diffraction, Mössbauer spectroscopy). Therefore, all studies will be presented in further publication.

 

Test and validation of a prototype of AC magnetic field generator for magnetic hyperthermia

The present Shot Term Scientific Mission aimed to test and validate a prototype of Alternating Magnetic Field Generator (amfG) for in vitro magnetic hyperthermia experiments. Such system, with a working range of 10-300kHz and field intensities up to 50 mT, features remarkable advantages compared with current commercial equipments of which should be highlighted the portability, friendly-use interface and an accurate control of cell media thermal conditions. The amfG is based on a ferrite core wired with Litz type wire liquid cooled and automatized via software. Its arrangement permits placing 4 wells Petri dishes inside, applying a quasi-uniform magnetic field on a single well where cells have been previously seeded. The University Hospital of Jena group led by Prof. Ingrid Hilger provided us a suitable framework for testing our prototype, performing during the stay two in-vitro magnetic hyperthermia experiments with MCF-7 cell line as well as subsequent viability and complementary staining test (Prussian Blue). In addition, UHJ research members showed a deep interest on the prototype capabilities for hyperthermia experiments to track the thermal stress on the cell morphology. Just to conclude, from a personal point of view, the present Mission represented for me an inflection point for my PhD work due to the acquired abilities in cell biology during my stay in UHJ will facilitate to achieve the goals of my thesis.


Evaluation of interactions of magnetic nanoparticles with T-lymphocytes

Iron oxide nanoparticles have broadened novel treatment approaches in cancer therapy due to their ability to function at the cellular and molecular level. According to literature there are various types of iron nanoparticles being used in cancer therapy improving current therapeutic approaches, such as chemotherapy 1 and radiotherapy 2. Based on that, many scientific groups investigate the interactions between iron oxide nanoparticles and different cell lines. Therefore, it is crucial to unravel the cellular internalization mechanism, the biocompatibility and the cellular hyperthermia response. The purpose of this short term scientific mission was to evaluate the cellular interactions of iron oxide nanoparticles, such as biocompatibility and cellular internalization mechanism in human Jurkat cell lines. To accomplish this, viability assays and flow cytometry (FACS) analysis were performed. Two different samples of iron oxide nanoparticles were studied during this period. The toxicity of the nanoparticles was studied by the MTT assay and the flow cytometry technique. The obtained results differed, as the first sample was nontoxic for the studied concentration range, whereas the other one was toxic at higher concentrations. Subsequently, the cellular uptake was studied after 1, 2, 4 and 24_h of incubation in Jurkat cells. The appropriate studied concentrations for each sample were chosen based on their cytotoxic profile. It is worth mentioning that a significant cellular uptake was observed and the uptake percentage increased as a function of incubation time for both samples. Finally, the apoptosis assay was carried out after 1-2_h of incubation for the most toxic sample and about 35% of apoptotic cells were observed.

References

1. Biomaterials Volume 31, Issue 18, June 2010, Pages 4995–5006

2. Theranostics 2015, Vol. 5, Issue 9, 1030-1045

 

Liposomal Encapsulated Iron Oxide Nanoflowers for Magnetic Hyperthermia

Iron oxide Nanoflowers (IONFs) are monocrystalline maghemite nanoparticles which have been shown to possess great heating capacity stemming from their superparamagnetic behavior that can be exploited by Magnetic Hyperthermia, for the treatment of cancer [1]. The purpose of this project is to prepare liposomal thermosensitive drug delivery vehicles for Hyperthermia treatment of glioblastoma, which will be conducted on xenografted SCID mice with U87MG cells [2]. The proposed vehicles are thermosensitive liposomes, in which IONFs are encapsulated, that can be loaded with the appropriate drug combination. The idea behind this project is to trigger the IONFs within the liposome with Magnetic Hyperthermia, causing a disruption of the lipid bilayer, thus allowing the drug to be released in the tumor. Synthesis and characterization of the liposomal encapsulated IONFs have been done during the STSM period, whereas the biological evaluation is currently under investigation.

In detail, the IONFs were synthesized according to a revised polyol method [1] and coated with tri-sodium citrate in order to increase their aqueous colloidal stability, which is required for bioapplications. The IONFs were about 28 nm and exhibited a magnetization saturation (Ms) of 77 emu/g of Iron Oxide content.  The liposomes were synthesized with a standard method of hydration of a thin lipid film by mixing the desirable lipids (DSPE-PEG2000, DPPC and DSPE) with cholesterol. The encapsulation of IONFs took place as a part of the hydration step, where a citrate buffer (pH= 5) solution was selected as the optimum medium for the preparation of stable monodispersed liposomes of around 148nm.

References 

1. Journal of Physical Chemistry C, 2012, 116, 15702-15712

2. Journal of Colloid and Interface Science, 433, 2014, 163-175


Assessment of the stability measurement of CNT-MNPs

The visit allowed me to exchange of protocol for assessing the stability measurement of CNT-MNPs, this novel type of materials is likely to get approval for advanced stage cancers. During my visit, I had a chance to discuss with Prof Tombacs and her team how to measure surface charge of CNT coated with magnetic nanopariticles using their home made GIMET1 pontentiometic acid –base titration and observing the experiments her laboratory. We have made measurement of single walled CNT-MNPs during my visit, subsequently we sent multiwalled CNT-MNPs for the measurements.


Relaxivity of the magnetoliposomes

The objective of this STSM was to measure the relaxivity of the magnetoliposomes (ML) developed for thermo-chemotherapy (TC) for metastatic breast cancer. Relaxivity is a measure of the sensitivity of a MRI contrast agent. The measurement of the relaxivity of the MLs is crucial to the project since the ML could provide individualized monitoring of tissue drug concentration distribution during or after treatment. The sensitivity of a T2 MRI contrast agent is generally expressed by the transverse relaxivity r2. The results indicated that r2 relaxivity of SPIOs increases with increasing size of the nanoparticles. Similarly, MLs that encapsulated larger SPIOs also had higher r2 relaxivity. To verify the effectiveness of the ML as negative T2 contrast agents, we obtained T2-weighted images of the control phantom and phantoms with MLs encapsulating SPIOs of different sizes. The MR contrast properties of the MLs were evaluated using 1% agarose phantoms.  The signal intensity of phantoms with MLs were significantly diminished on the T2-weighted MR scans compared to the control phantom. These results show that these MLs are high-performance MRI contrast agents that enable highly sensitive T2-weighted MRI measurements. 


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