Final Report Summary - MULTIFUN (Multifunctional Nanotechnology for selective detection and Treatment of cancer)
Executive Summary:
During the last four years the MULTIFUN consortium has focused its activity on the development and validation of new systems based upon minimal invasive nanotechnology for the early and selective detection and elimination of breast and pancreatic cancer. The project has successfully produced multifuntionalised magnetic iron oxide nanoparticles (MNP) that combine diagnostic and therapeutic features against these two types of cancer.
The therapeutic approach developed within the project includes a synergistic effect between the therapeutic effect produced by magnetic hyperthermia and that due to the intracellular drug delivery selectively targeted to tumour cells. Some of the designed formulations have proven their efficiency, safety and non-toxicity in in vivo models, making them promising candidates to produce new nanomedicines against breast and pancreatic cancer.
As a result of the project research, the consortium has characterised 11 exploitable results with a high potential impact and profitability from a commercial point of view. For the most promising exploitable results, 6 out of 11, we have depicted a Business plan. From these 6 Exploitable Results, 2 have already been protected by a patent.
In addition, we have described 6 project key outcomes that could be very promising and be used as a basis for future research and innovation activities for long-term final exploitation.
The consortium has delivered dissemination activities across all the aspects of the project; thus been actively contributing and participating to more than 330 separate events. Activity beyond the end of the project has been also foreseen for 2016 and 2017 with publication manuscripts, reviews projects and reports, booklet, proceedings, book chapters and also academic degree thesis.
Project Context and Objectives:
1. Introduction and Objectives
The MULTIFUN consortium focuses on the development and validation of new systems based upon minimal invasive nanotechnology for the early and selective detection and elimination of breast and pancreatic cancer with reduced side effects. The project will deploy a strategy based on the multifuntionalisation of magnetic iron oxide nanoparticles (MNP), combining diagnostic and therapeutic features against breast and pancreatic cancer and cancer stem cells.
The MULTIFUN therapeutic approach is multimodal and combines the magnetic heating mediated by MNP when subjected to alternating magnetic fields with intracellular drug delivery. In this manner the therapeutic outcome is reinforced by synergies within the different therapeutic modalities. Since MNP can be detected through magnetic resonance imaging (MRI), they can also be used as contrast agents for cancer cell detection. In this way, MULTIFUN combines therapeutic and diagnostic aspects leading to a potential “theragnostics” tool.
A key point of the project is to assess the safety and toxicity of the developed nanoparticles. Thus, MULTIFUN includes a broad set of in vitro and in vivo toxicity and biodistribution tests in different animal models including mice, rats and pigs.
Finally, in order to improve the translation of the outcomes and their economic potential, the consortium also will study the scale-up of the production methods for the main components.
The MULTIFUN project will meet several scientific and technological objectives indicated below:
Scientific objectives
• Study of cell internalisation routes of Multifuntionalised Magnetic Nanoparticles (MF-MNP).
• Evaluation of cyto-toxicity of MF- MNP in vitro.
• Distribution and toxicity analysis of MF-MNP in vivo.
• Evaluation and validation of breast and pancreatic cancer multimodal therapeutic strategies via in vitro and in vivo models.
• Demonstration of a cancer ‘theragnostic’ approach for early stage cancer targeting stem cells.
• Design of innovative targeting peptides and antibodies for a higher effectiveness in tumour localization and drug dose reduction.
Technological objectives
• Scale-up of production of biocompatible MNP with optimised MRI contrast properties.
• Multifunctionalisation of MNP for the delivery of anti-cancer agents.
• Instrumentation development for MNP detection and quantification in biological samples.
• The development of improved magnetic heating instrumentation for in vitro and in vivo applications.
2. Originality of MULTIFUN Project
The MULTIFUN project has several objectives which differentiate it from other FP7 NMP projects aimed to develop nanotechnology solutions for cancer therapy. These original objectives can be grouped by their scientific and technological contents.
Scientific MULTIFUN originality
The MULTIFUN consortium has developed a comprehensive strategy in order to generate highly selective and efficient multifunctional nanoparticles for detecting and treating cancer. The combination of magnetic heating and other therapeutic modalities for cancer allows new therapeutic approaches. MULTIFUN nanoparticles will target cancer cells including the highly tumorigenic stem cells as the putative root of cancer. These types of cancer cells are associated with drug resistance and tumour relapse and hence their targeting may lead to a significant improvement in therapeutic outcomes. Several members of the consortium have already demonstrated that it is feasible to target cancer stem cells efficiently using multimodal strategies which lead to tumour regression and dramatically enhanced long-term survival in relevant tissue xenograft models. Therefore, the fact that MULTIFUN nanoparticles may also result in the elimination of cancer stem cells represents a major advantage over other nanoparticle-based approaches.
Figure 1: MULTIFUN approach.
MULTIFUN Technological originality
The multidisciplinary framework related to MULTIFUN activities provides a suitable opportunity for the development of scientific instrumentation aimed to the use of magnetic nanoparticles in biomedical applications. One of the proposed technologies is the development and validation of an exvivo spectrometer to detect and quantify the presence of magnetic nanoparticles in tissues and biological fluids (blood and urine). PEPRIC is the partner involved in this task to supply a device that will ease the biodistribution studies. In addition, INSA partner has been involved in the development of a AC magnetic field generator to perform in vitro and in vivo magnetic hyperthermia experiments. The device, supplied to 4 other partners, combines the versatility to locate cells or animals into a coil with all requirements needed to accomplish the magnetic hyperthermia studies with a wider magnetic field conditions. In addition, MULTIFUN has developed multifunctional nanoparticles preserving their biological activity from all conjugated molecules (anticancer agents or ligands). The strategy employed for the conjugation of anticancer agents (chemotherapeutic drugs, siRNAs and peptides) is quite unique allowing the drug release exclusively under intracellular conditions (i.e. reductive environment) . Considering that Magnetic resonance detection allow for the detection and quantification of magnetic nanoparticles , this method is selective and sensitive with reduced heat dissipation in biological tissues. As the magnetic particles are stable this will allow longitudinal monitoring over several days and weeks. The device will be compared and benchmarked to other quantitative methods. This technique involves the use of low magnetic fields and hence the heat generation within the tissue is reduced. This will allow the implementation of ex-vivo measurements to be explored during the project, for which no sample preparation is necessary such as freeze drying or homogenization or mineralization of the sample. This reduces the sampling error and manipulation time for the lab technicians.
Figure 2: New detection equipment for quantitative analysis of MNP’s in blood and tissue samples
The presence of one industrial nanoparticle manufacturer (LRL) and two academics suppliers (IMDEA and ICMM-CSIC) has ensured the supply of materials for MULTIFUN activities with the consortium. Furthermore, the production of biocompatible magnetic nanoparticles with optimized contrast and heat induction features for oncological applications has been scaled up. At the end of the project, among the different scale up processes developed the one performed by the industrial partner provides up to 20 g of MNPs of excellent quality.
Project Results:
The Multifun consortium has employed superparamagnetic nanoparticles with optimal magnetic and magneto-thermal properties. Two chemical routes have been chosen to get nanoparticles with narrow size distribution, high saturation magnetization and high specific absorption rate (SAR) values. The thermal decomposition of an iron precursor in organic media provides suitable magnetite nanoparticles with the best size control (standard deviation lower than 10%). Multifun partners have analyzed which reaction parameters lead to improve the magnetic and magneto-thermal properties of the resulting nanoparticles. Thus, magnetite nanoparticles can be synthesized in a wide particle size range from 7 to 22 nm by varying some chemical reaction parameters related to nucleation and growth processes such the reaction time, and the oleic acid concentration in the reaction mixture. Second, a narrow size distribution is obtained when the reaction mixture is highly homogeneous when the temperature is increased (see Figure 3).
Figure 3: a) Transmission Electronic Microscopy micrographs of 18 nm size magnetite nanoparticles synthesized a) without, b) with stirring the reaction at the beginning of the heat Ramp. Arrows indicate smaller size nanoparticles. Images kindly provided by G. Salas and M.P. Morales.
Thus, a short nucleation and later a uniform particle growth are favoured. Moreover, the resulting nanoparticles are highly crystalline; their morphology is highly uniform and differs from sphere-like for sizes below 15 nm to cube-like for sizes higher than 15 nm. Multifun partners have shown the tight relationship among size distribution, magnetic and magneto-thermal properties: narrower size distribution lead to higher saturation magnetization and SAR values as shown in Figure 4. More info in: G. Salas et al. J. Mater. Chem. 22, 21065 (2012). We have established a relationship between physico-chemical properties of magnetic fluids and their heating capacity. Salas, Gorka et al., International journal of hyperthermia, 29, 768-776 (2013),
We have also been able to scale up the synthesis of larger particles and coating them with polymers keeping a very high saturation magnetization values through green aqueous synthesis, M. Marciello, V. Connord, S. Veintemillas-Verdaguer, M. Andres-Vergés, J. Carrey, M. Respaud, C. J. Serna and M. P. Morales. Journal of materials chemistry B, 1, 5995-6004 (2013).
A detailed series of papers has also been published using nanoparticles synthesized by CGP for the mechanisms of magnetic hyperthermia. These papers detail the mechanisms particles use to generate heat, also details the parameters that are key for controllable magnetic hyperthermia. More info in: G. Fernandez-Vallejo et al. J. Phys. D: Appl. Phys. 46, 312001 (2013) and G. Fernandez-Vallejo et al. Appl. Phys. Lett. 103, 142417 (2013).
Figure 4: a) Transmission Electron Microscopy micrographs of particles produced by the Controlled Growth Process. b) High Resolution TEM (HRTEM) image showing the high crystallinity of the particles produced by the controlled Growth Process. Images kindly provided by Liquids Research Ltd.
The standard characterization techniques were applied to determine important structural and magnetic parameters of the nanoparticles. Both the ferrofluids and powders were used for characterization; a novel protocol for magnetic property measurement of the ferrofluid samples was established.
Phase composition of samples was determined by powder X-ray diffraction (PXRD) and Mӧssbauer spectroscopy (Figure 4 a & c), which served for the precise specification of the maghemite/magnetite content. Real particle size, dTEM and size distribution were determined from the Transmission Electron Microscopy (TEM) images. Size of the crystalline part of the particle, dXRD was refined from the PXRD patterns. Magnetic property measurements were used for determination of the so-called blocking temperature of the particles (TMAX and Tirr, Figure 4d) and estimation of distribution of particle energy barriers. Parameters such as the saturation magnetization, Ms, remanence, Mr and coercivity, μ0Hc were obtained from the magnetization isotherms (M(H) curves, Figure 4 e). Magnetic moments of the particles, μ their distribution, σ and magnetic diameter, dMAG were refined from the un-hysteretic M(H) curves (Figure 4 f).
Comparison of individual particle diameters, dTEM, dXRD and dMAG serves as the perfect indicator of particle crystallinity and internal spin order (Figure 4 g). Any changes in the determined parameters, comparing the bare and coated nanoparticles, then reveal the effect of coating on physical properties of the nanoparticles. More details can be found in Kubickova et al, Appl. Phys. Lett. 104 (2014) 2231051 and Pacakova et al, arXiv.2015.
Figure 5: The example of typical experiments for determination of the properties of magnetic nanoparticles: a) Powder X-ray Diffraction pattern from which the phase composition and the particle diameter (dXRD) are determined. b) Transmission Electron Microscopy, which is used for direct observation of the apparent particle size (dTEM) and its distribution. c) Example of Mӧssbauer spectra. Refinement of spectra can easily distinguished between the presence of the Fe2+ and Fe3+ ions, thus method serves for precise determination of the maghemite/magnetite phase content within the examined sample.
Bottom panel: Magnetic property measurements: d) example of the ZFC-FC curve from which the blocking temperatures, TMAX and Tirr can be determined. e) Example of the M(H) curve and f) the fit of the unhysteretic M(H) curve in Langevin scaling from which the mean and meadian magnetic moments of the particles can be obtained together with the distributions of magnetic moment, σ and median particle diameter (dMAG). g) Illustration of the internal particle structure for the ideal and real particles and relation of individual particle diameters.
Figure 6: SAR values obtained for 18nm size nanoparticles with different size distributions (yellow ±2 nm, red ±4 nm) when exposed to the same HAC conditions (50 mT and 77 kHz). Images kindly provided by G. Salas, M.P. Morales and F.J. Teran.
On the other hand, the magnetic and thermal characterizations of nanoparticles have been analyzed obtaining some preliminary results related to establish the superparamagnetic phase of the synthesized particles (Salas, Gorka et al. Journal of Physical Chemistry C 118, 19985 (2014)). Huge efforts are paid to determine which are the optimal particle sizes and magnetic field conditions leading to optimal contrast agent and hyperthermia mediator features. In this sense, the control and the quantification of heat losses is crucial to reduce error sources providing SAR values with higher accuracy. Multifun partners have reported on the development of an experimental set-up that diminishes heat losses with the surrounding. In this way, the time variation of temperature can be modelled (see Figure 7) what leads to more accurate determination of SAR values. More info: F.J. Teran et al., Appl. Phys. Lett. 101, 062413 (2012). We have also observed that the optimum assembling of magnetic nanoparticles lead to an enhancement of the magnetic hyperthermia, D. Serantes, J. Phys. Chem. C 2014 dx.doi.org/10.1021/jp410717m.
Figure 7: a) Time evolution of SPION temperature when subjected to HAC (78 kHz and 25 mT) (a) at Teq= 30ºC, b) Numerical derivative curves of data shown in previous graph. Solid circles indicate the maximun and minimun dT/dt values. C) Comparison of numerical simulations and experimental data. Shadow and white time zones are exposed to HAC on and off, respectively. Red and blue lines correspond to temperature rise and decay. Images kindly provided by F.J. Teran.
The parameters involved in the heating efficiency related to MNP interacting phenomena were analyzed in order to clarify which are the conditions to control the MNP magnetic heat release and how to control the heat exposure. This is mandatory for clinical purposes. Thus, we have assessed the magnetic heating dependence with field conditions, MNP concentration, aggregation and size for determining the effect of dipolar interactions (Figure 8).
Figure 8: Concentration dependence of SAR for different particle sizes, field frequency and amplitude. Figures kindly provided by D. Cabrera and F.J. Teran
The functionalization of MNPs with different bioactive molecules has afforded novel nanostructures with antitumoral properties. Particularly, we have used tailored linkers to conjugate drugs (Doxorubicin and Gemcitabine) to the nanoparticles. The system developed is stable under physiological conditions and release the cargo mainly inside the cells. (Latorre, A., Couleaud, P., Aires, A., Cortajarena, A. L. & Somoza, Á. Multifunctionalization of magnetic nanoparticles for controlled drug release: A general approach. Eur. J. Med. Chem. 82, 355–362 (2014).)
Figure 9: Intracellular controlled release
In addition we have used targeting molecules such as the Nucant peptide and antibodies to direct the nanostructure to the tumoral cells. Another biomolecules employed in the functionalization of MNPs are nucleic acids, particularly siRNAs.
The discovery of the RNA interference mechanism has triggered the development of nucleic acids derivatives to generate novel medical treatments. Although the use of synthetic small interfering RNA (siRNA) has been demonstrated, still several issues remain to be solved in order to ensure the potential of this novel medicinal avenue. One of the problems that have been addressed during the last years is the vulnerability of oligonucleotides to nucleases, which limits their practical use. Specifically, we explored the effects of the acyclic L-threoninol backbones in place of natural ribose rings on the biological properties of siRNAs. The introduction of these novel derivatives at the 3’-protuding ends of siRNAs confers a strong nuclease resistance, greater potency by a better affinity to PAZ domain and a lower degree of innate immunostimulation. Finally the introduction of these derivatives at the 2on position near the 5’-end of the sense strand (Figure 10) avoids the loading of the sense strand into RISC lowering off-target effects. These results will be valuable in the design of therapeutic siRNAs.
Figure 10: Beneficial effects of the substitution of natural nucleobases by L-threoninol derivatives in siRNAs.
The assessment of toxicity and internalization of iron oxide nanoparticles into breast and pancreatic cancer cells have been performed allowing to test the interaction between cancer cells and derived from co-precipitation in water and thermal decomposition synthesis methods. Thus, nanoparticles with different coatings, and surface charges has been tested in different established cancer cell lines to evaluate cell viability and uptake under nanoparticles concentrations up to 0.2 mg/ml during incubation period. MultiFun partners have reported that nanoparticles may induce different effects on the cell viability and permeability and their lysosomal mass depending on coating, surface and cell line. More info: Adriele Prina-Mello et al. IEEE Transactions on magnetics, 49(1), January 2013. The efficient and safe internalization of some of these magnetic iron oxide nanoparticles designed for biomedical applications have been highlighted in a recent publication: M. Calero et al. Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 733–743.
The MNPs internalization process in different cell types has been characterized using electron microscopy. The internalization pathways follow two routes: One is clathrin-mediated uptake and the other is the macropinocitic route. The particles accumulate in endosome vesicles in the cytoplasm and never reach the nucleus interior.
Figure 11: Internalization of OD15 by MCF-7 cells. Cells incubated at different times with MNPs were studied by electron microsopy. Clathrin-mediated endocytosis was observed for MNP aggregates <200 nm, while macropinocytosis was found for larger aggregates. Cargo vesicles were fused in early endosomes (EE). Multivesicular bodies (MVD) and late endosomes (LE)containing intraluminar vesicles (ILVs) fuse together with Lysosomes (LYS) for final degradation vesicles (Journal of Nanobiotechnology 2015, 13:16. doi:10.1186/s12951-015-0073-9).
Figure 12: Analysis of cytoskeleton. (A) Representative images of cells immunostained for tubulin (green) and DNA counterstained with Hoechst-33258 (blue). Left panel: (a) Interphase control cells. (a′) Metaphase control cell. Middle panel: (b-b′) Interphase cells incubated for 24 h with 0.5 mg ml−1 AD and observed by fluorescence and bright-field microscopy, respectively; (c-c′) Cells treated with DMSA; (d-d′) Cells incubated with APS. Right panel: (e-e′) Mitotic spindle of cells incubated with AD; (f-f′) DMSA or (g-g′) APS. (B) Merged images of F-actin labeled with Phalloidin-TRICT (red), vinculin immunostaining (green) and DNA counterstained with Hoechst-33258 (blue). (a) Control cells. (b-b′) Cells treated with AD and observed by fluorescence and bright-field microscopy, respectively. (c-c′) Cells treated with DMSA. (d-d′) Cells incubated with APS. Scale bars = 10 μm.
The functionalisation of the nanoparticles provided by academic and industrial partners has led to the first generation of functionalized nanostructures. NUCANT has been the molecule to start with. This pseudo-peptide is highly interesting due to its double potential: 1) to interact with protein receptor on cancer cell membrane, 2) to inhibit cancer cell proliferation due to its proapoptotic activity. Preliminary results show that Nucant adsorbed onto nanoparticle surface favour the nanoparticle internalization into cancer cells in some cases. Further investigations to control the internalization mediated by cell receptor are in progress. In this sense, we have investigated the ultrasound generation and high-frequency motion of magnetic nanoparticles in an alternating magnetic field: toward intracellular ultrasound therapy, J. Carrey, Appl. Phys. Lett. 102, 232404 (2013). On the other hand, we were able to target a G-protein coupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death, C. Sanchez, ACS NANO 8, 1350 (2014)
Preliminary in vivo studies related to the hyperthermia effects on tumour evolution have shown promising results. The hyperthermia therapeutic approach has been tested on animal models using nanoparticles that under high frequency field conditions lead to SAR values larger than 200W/g. Thus, tumour volume has been shown to be reduced after few magnetic field sessions. Further investigations are going on to optimise the hyperthermia therapeutic approach.
The role of anesthesia in the magnetic nanoparticle biodistribution among different organs after intravenous injection has been studied in a murine model. Animals were anesthetized by inhalation with isoflurane (0,5 % in oxygen) or by intraperitoneal injection with a mixture of ketamine and xilazine. Then, a single dose of monodisperse dimercaptosuccinic acid coated magnetic nanoparticles (9.2 nm diameter ±13% SD) was administered to the animals. Lung and liver tissues were collected 30 min after the particle administration and the amount of particles in each tissue was determined by AC magnetic susceptibility measurements. Whereas the amount of particles that reaches the liver seems not to be affected by the anesthesia used, the amount of particles that reaches the lungs for inhaled isoflurane is three times less than for the intraperitoneally injected anesthetic. Lucía Gutiérrez et al., IEEE Transactions on magnetics, 49(1), January 2013. We have also studied long term biotransformation and toxicity of dimercaptosuccinic acid-coated magnetic nanoparticles after intravenous injection, Mejias, Raquel; et al. Journal of controlled release 171, 2, 225-233 (2013). A review on prospects in the synthesis, detection and quantification of magnetic nanoparticles following systemic administration has been recently published by L. Gutiérrez, Physical chemistry chemical physics, DOI: 10.1039/c3cp54763a 2014.
Potential Impact:
1. Exploitation
The consortium has characterised 11 exploitable results with a high potential impact and profitability from a commercial point of view.
Table 1: List of Exploitable Results
Exploitable Result / Type / Contact
Production of biocompatible MNPs - Organic Route / product & process / Bonifacio Vega (IMDEA)
Production of biocompatible MNPs - Aqueous Route / product & process / Vijay Patel (LRL)
Cancer Cell (including CSC) biomarkers – Pancreatic Cancer / knowledge / Christopher Heeschen (CNIO)
Cancer Cell (including CSC) biomarkers – Breast Cancer / knowledge / Rob Clarke (UNIMAN)
Novel drugs based on nucleic acids to suppress cancer cell invasion and metastasis / knowledge & product / Ramon Eritja (IQAC-CSIC)
Functionalisation of MNPs with PM01183 / product / Pilar Calvo (PM)
Functionalisation of MNPs with an antagonist of nucleolin as a tool for target cancer cells. / product / Jose Courty (CRRET)
Multifunctional MNPs (MF-MNPs) for cancer treatment and diagnosis / product & process / Bonifacio Vega (IMDEA)
Magnetic heating equipment for in vitro and in vivo evaluation / product / Marc Respaud (INSA)
Device/instrumentation for detection and quantification of MNPs based on EPR method / product & process / Stephanie Teughels (PEPRIC)
High content screening analysis for MNP toxicity / product & process / Yuri Volkov (TCD)
The information of these results is confidential. To know more about them, please, contact the suitable person (contact person).
In addition to these exploitable results, we have described 6 project key outcomes that could be very promising and be used as a basis for future research and innovation activities for long-term final exploitation:
1. siRNAs miRNA molecules for cancer treatment
Partners involved: CSIC
Description: A series of chemical modifications for siRNAs have been developed. The modified siRNA have a large resistance to degradation by nucleases present in sera, resulting in an increase of the efficiency and duration of the gene silencing properties. In addition one of these modifications when introduced at a particular site reduced off-target effects increasing the specificity of the gene silencing activity.
Future uses and applications: The results will be useful for the development of more potent siRNAs and miRNA mimetics for cancer treatment in research projects.
References:
“Effect of North bicyclo[3.1.0]hexane pseudosugars on RNA interferente. A novel class of siRNA modification”. Terrazas, M., Ocampo, S.M. Perales, J.C. Marquez, V., Eritja, R. ChemBioChem, 12, 1056-1065 (2011).
“Functionalization of the 3’-ends of DNA and RNA strands with N-ethyl-N-coupled nucleosides: A promising approach to avoid 3’-exonuclease-catalyzed hydrolysis of therapeutic oligonucleotides”. Terrazas, M., Alagia, A., Faustino, I., Orozco, M., Eritja, R. ChemBioChem, 14, 510-520 (2013).
“RNA/aTNA Chimeras: RNAi effects and nuclease resistance of single and double stranded RNAs”. Alagia, A., Terrazas, M., Eritja, R. Molecules, 19, 17872-17896 (2014).
“Modulation of the RNA interference activity using central mismatched siRNAs and acyclic threoninol nucleic acids (aTNA) units”. Alagia, A., Terrazas, M., Eritja, R. Molecules, 20, 7602-7619 (2015).
2. Understanding the role of Magnetic parameters in the production of biocompatible MNPs
Partners involved: IMDEA, CAS.
Description: IMDEA and CAS have been able to correlate key parameters in iron oxide nanoparticles such size, size distribution, or crystallinity defects that strongly influence their magnetic properties. These parameters can be used for tuning the magnetic properties of nanoparticles.
Future uses and applications: The knowledge gained in the project is useful to understand better how magnetic properties of nanoparticles can be customised for specific applications.
References:
“Structural disorder versus spin canting in monodisperse maghemite nanocrystals” S. Kubickova, D. Niznansky, M. P. Morales Herrero, G. Salas, J. Vejpravova Appl. Phys. Lett. 104 (2014) 223105(1) - 223105(4).
“Modulation of Magnetic Heating via Dipolar Magnetic Interactions in Monodisperse and Crystalline Iron Oxide Nanoparticles” G. Salas, J. Camarero, D. Cabrera, H. Takacs, M. Varela, R. Ludwig, H. Dähring, I. Hilger, R. Miranda, M. P. Morales, F. J. Teran. J.Phys.Chem. C 118 (2014) 19985-19994.
“Controlled synthesis of uniform magnetite nanocrystals with high-quality properties for biomedical applications” G. Salas , C. Casado , F. J. Teran , R. Miranda , C.J. Serna and M.P. Morales. J. Mater. Chem. 22 (2012) 21065-21075.
3. MR acquisition and post-processing software
Partners involved: KCL
Description: KCL has implemented several MR imaging sequences for T1, T2 and T2* mapping and validated those in vials with known magnetic nanoparticle (MNP) concentration and in cells incubated with non-specific and functionalised nanoparticles provided by the Multifun consortium. They subsequently applied those imaging techniques to measure nanoparticle accumulation in a model of breast cancer in-vivo. They also implemented several positive contrast sequences including GRASP and IRON and the post-processing method SGM to visualise nanoparticles with positive contrast. They also developed a positive contrast technique based on the DIXON sequence and validated all these sequences in concentration phantoms demonstrating good accuracy for MNP quantification.
Future uses and applications: They will use the developed methods to quantify MNP’s for tissue characterization in models of atherosclerosis, myocardial infarction, deep venous thrombosis and cancer. KCL intends to use some of the techniques in a H2020 project recently submitted.
References:
“T1 and T2 Mapping of Superparamagnetic Iron Oxide Nanoparticles for the Detection of Breast and Pancreatic Cancer Cells”. D. Krüger, G. Salas, M. Calero, S. Lorrio González, A. Villanueva, M. P. Morales, R. M. Botnar. Poster presented at Annual Meeting of the International Society of Magnetic Resonance in Medicine 2013, Salt Lake City, Utah.
“A Dixon Method for Positive Contrast Imaging of Very Small Superparamagnetic Iron Oxide Nanoparticles in MRI”. D. Krüger, S. Lorrio González, R. M. Botnar. Abstract accepted for the Annual Meeting of the International Society of Magnetic Resonance in Medicine 2015, Toronto, Canada.
“Dixon Fat Suppression for Off-resonant Water Imaging of Superparamagnetic Iron Oxide Nanoparticles”. D. Krüger, S. Lorrio González, R. M. Botnar. Abstract accepted for the Annual Meeting of the International Society of Magnetic Resonance in Medicine 2015, Toronto, Canada.
4. Protocols for safety and handling MNPs
Partners involved: TCD
Description: guidelines and protocols for the preparation and the administration (in vitro and in vivo) of MNPs. These protocols intend to create some homogeneity within the handling of MNP, for addressing safety and efficacy of MNPs in biomedical applications.
Future use and applications: Protocols and know-how developed are enabling to advance any future collaborative research projects and funding opportunities; training and know-how transfer within biomedical and pharmaceutical industry (SMEs and Multinational) are also to be considered in order to provide educational and training on the responsible use of nanotechnology applied to medicine. Trinity College is currently leveraging this library of knowledge and know how within the recently funded EUNCL H2020 infrastructure project in nanomedicine, the European Union Nanomedicine Characterisation Laboratory.
References:
Bergamaschi E., Poland C., Guseva Canu I., Prina-Mello A., The role of biological monitoring in nanosafety, (2015), Nano Today doi: 10.1016/j.nantod.2015.02.001 (accepted), (News and Opinions, Senior Author)
Prina-Mello, A., Crosbie-Staunton, K., Salas, G., Del Puerto Morales, M., Volkov, Y., Multiparametric toxicity evaluation of SPIONs by high content screening technique: Identification of biocompatible multifunctional nanoparticles for nanomedicine, (2013) IEEE Transactions on Magnetics, 49 (1), art. no. 6392408, pp. 377-382.
Movia D., Poland C., Tran L., Volkov Y., Prina-Mello A. Multilayered Nanoparticles for Personalized Medicine: Translation into Clinical Markets, Editors: Bawa R., Audette G.F. and Rubinstein I.: Handbook of Clinical Nanomedicine – From Bench to Bedside, Pan Stanford Series in Nanomedicine (Raj Bawa, Series Editor), Volume 1, Pan Stanford Publishing, Singapore, 2015, ISBN-10: 9814316172. (BOOK CHAPTER)
Prina-Mello A., B.M. Mohamed, N.K. Verma, Y. Volkov, Advanced Methodologies and Techniques for Assessing Nanomaterials Toxicity: from manufacturing to nanomedicine screening, Nanotoxicology, Second Edition, Editors: Nancy A. Monteiro-Riviere & C. Lang Tran, Taylor & Francis, Informa Healthcare Books, February 2014. (BOOK CHAPTER)
Movia D., Gerard V., Maguire C.M. Jain N., Bell A.P. Nicolosi V., O Neill T., Scholz D., Gun’ko Y., Volkov Y. and A. Prina-Mello, A safe-by-design approach to the development of gold nanoboxes as carriers for internalization into cancer cells, Biomaterials (2014), 35:9 (LEAD OPINION PAPER)
Hole P, Sillence K, Hannell C. Maguire CM, Roesslein M, Suarez G, Capracotta S., Magdolenova Z, Horev-Azaria L, Dybowska A, Cooke L, Haase A, Contal S, Manø S, Vennemann A, Sauvain JJ, Crosbie Staunton K, Anguissola S, Luch A, Dusinska M, Korenstein R, Gutleb AC, Wiemann M, Prina-Mello A. *, Riediker M, Wick P, Interlaboratory comparison of size measurements on nanoparticles using Nanoparticle Tracking Analysis (NTA), Patrick Hole, Katherine Sillence, Claire Hannell, (2013) Journal of Nanoparticle Research, 15:2101 (*Joint Senior Authors)
Gobbo OL, Wetterling F, Vaes P, Teughels S, Markos F, Edge D, Shortt CM, Crosbie-Staunton K, Radomski MW, Volkov Y and Prina-Mello A. Biodistribution and Pharmacokinetic studies of SPION using particle Electron Paramagnetic Resonance, MRI and ICP-MS. Nanomedicine (in press, DOI: 10.2217/NNM.15.22).
5. Knowledge regarding pharmacokinetics and bio-distribution of MNPs
Partners involved: UCC
Description: WP4 in MULTIFUN gave us detailed knowledge about the influence of the size, coating and other chemical parameters in the pharmacokinetics and bio-distribution of MNPs in mice (Gobbo et al 2015), rats and pigs. Small diameter iron particles administered i.v. were rapidly (in under 10 minutes in pigs) scavenged from the circulation by the cells in the liver and spleen (likely reticuloendothelial cells), as would be expected. In addition, the small diameter meant that there was significant accumulation in the lungs and the particles were also filtered by the kidneys. Therefore, these results suggest that these particles, allied with the anti-cancer attachments developed by the consortium, may be useful in targeting cancers associated with the liver and bone.
Future use and applications: Exploiting mammalian handling of iron-MNPs developed by MULTIFUN to treat less common cancers (e.g. bile duct cancers, primary or secondary bone cancers, liver cancer).
References:
“Biodistribution and Pharmacokinetic studies of iron oxide nanoparticles using particle Electron Paramagnetic Resonance, MRI and ICP-MS.” Gobbo OL, Wetterling F, Vaes P, Teughels S, Markos F, Edge D, Shortt CM, Crosbie-Staunton K, Radomski MW, Volkov Y, Prina-Mello A (2015). Nanomedicine (IN PRESS).
6. Knowledge regarding the higher impact that the MULTIFUN systems (Multifunctionalised MNP) in combination with magnetic heating has on cancer cell viability
Partners involved: UHJ
Description: UHJ has been able to study the impact of the combinational therapy consisting of nonfunctionalised as well as multifunctionalised MNPs in combination hyperthermia in vitro and in vivo. This combination showed a strong reduction of cell viability of breast and pancreatic cancer cells in vitro. Furthermore, combined with hyperthermia treatment the multifunctionalised MNP exhibited a strong regression of tumor volumes in subcutaneously xenografted breast MDA-MB231 cells and pancreatic BxPC-3 cells. The results clearly emphasize the high therapeutic potential of the multifunctionalised MF66 in combination with hyperthermia and underline the beneficial outcomes provided by this combination therapy if translated to the clinic.
UHJ first established a Standard Operating Procedure (SOP) for the application of magnetic heating in vivo.
Future use and applications: The gained knowledge will be used to:
1. transfer the therapeutic modality to the clinical situation
2. to extend the approach to other diseases and drug formulations
3. to open up new collaborations with pharma industry and research institutions
4. to provide a platform for testing functionalised MNP for pharma industry and other research institutions
References:
Dähring H, Grandke J, Teichgräber U, Hilger I. Improved Hyperthermia Treatment of Tumors Under Consideration of Magnetic Nanoparticle Distribution Using Micro-CT Imaging. Molecular imaging and biology: Mol Bio Imaging. 2015.
Kossatz S, Grandke J, Couleaud P, Latorre A, Aires A, Crosbie-Staunton K, et al. Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery. Breast cancer research: Breast Cancer Res. 2015;17(1):66.
Kossatz S, Ludwig R, Dähring H, Ettelt V, Rimkus G, Marciello M, et al. High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature dosages in the tumor area. Pharm Res. 2014;31(12):3274-88.
Pömpner N, Stapf M, Hilger I. (2015) Heterogeneous response of different tumor cell lines to hyperthermia in presence of MTX-coupled nanoparticles. IJONM (submitted).
2 Dissemination
The MULTIFUN dissemination objectives from the DOW have been fully achieved across all the fields of expertise the consortium has developed. To date, the consortium has already been actively contributing and participating to more than 300 separate events where MULTIFUN project gained visibility and also reputation as a comprehensive project focused in delivering theranostics treatments for breast and pancreatic cancers.
The MULTIFUN partners have also individually or as groups engaged in public events where to provide comprehensive information and awareness on how nanotechnological solution can provide better targeting or personalised treatment to the patients affected by incurable cancers. This objective was achieved by informing the community across the media by press releases, pan-European multimedia events, broadcasting interviews with the key players in the field of theranostics, to presenting MULTIFUN and nanomedicine to young students and the public at Scientific Open days across the geographical spreading that MULTIFUN is involved.
MULTIFUN Stakeholders have been also engaged and informed of MULTIFUN progress and achievements across the many events the project was involved, as invited speakers, chairpersons, event organisers, trade company/ies or exploitation partner/s. Furthermore, the project has also contributed to the shaping up of Funding schemes and opportunities at regional, national and international level by providing scientific input and expertise and working with the relevant organisations involved (e.g. European Commission for Horizon 2020, European Technology Platforms for key enabling and future emerging technologies,
MULTIFUN has truly achieved the dissemination objectives of the project by providing next generation scientific knowledge to the scientific and non-scientific community, and also by training and inspiring the next generation of scientists and technology experts which they all contribute to the improvement of the quality of life and future to the eradication of cancer.
Societally, MULTIFUN has developed a responsible attitude towards the use of nanotechnology in the biomedical industry for the improvement everyone life.
Breakdown summary and details of the dissemination activity of the project can be found in the here below summarised in the Table 1 and histogram plots (Figure 13) where it is shown how and where the project delivered in the past four years. Details are provided for each reporting period and also as overall project activities.
Table 2: MULTIFUN project 48 months dissemination Outcome
Figure 13: Overall details for MULTIFUN dissemination activity outcome (Period June 2011 – June 2015)
It has been planned that after EuroNanoForum 2015 the project dissemination activities will continue further into 2016 and 2017 as part of evaluation process or publication of submitted manuscripts (currently 14 manuscripts either under submission or under revision), reviews projects and reports, booklet (1 under final preparation), proceedings, book chapters and also academic degree thesis (9 pending PhD award titles to be awarded by 2016).
List of Websites:
http://multifun-project.eu/
Prof. Dr. Rodolfo Miranda
contacto.multifun@ imdea.org
MULTIFUN Scientific Coordinator and IMDEA Nanociencia Director.
Ms. Blanca Jordan – blanca.jordan@atos.net
MULTIFUN Administrative Coordinator and Health Sector Manager at Atos Research & Innovation.
During the last four years the MULTIFUN consortium has focused its activity on the development and validation of new systems based upon minimal invasive nanotechnology for the early and selective detection and elimination of breast and pancreatic cancer. The project has successfully produced multifuntionalised magnetic iron oxide nanoparticles (MNP) that combine diagnostic and therapeutic features against these two types of cancer.
The therapeutic approach developed within the project includes a synergistic effect between the therapeutic effect produced by magnetic hyperthermia and that due to the intracellular drug delivery selectively targeted to tumour cells. Some of the designed formulations have proven their efficiency, safety and non-toxicity in in vivo models, making them promising candidates to produce new nanomedicines against breast and pancreatic cancer.
As a result of the project research, the consortium has characterised 11 exploitable results with a high potential impact and profitability from a commercial point of view. For the most promising exploitable results, 6 out of 11, we have depicted a Business plan. From these 6 Exploitable Results, 2 have already been protected by a patent.
In addition, we have described 6 project key outcomes that could be very promising and be used as a basis for future research and innovation activities for long-term final exploitation.
The consortium has delivered dissemination activities across all the aspects of the project; thus been actively contributing and participating to more than 330 separate events. Activity beyond the end of the project has been also foreseen for 2016 and 2017 with publication manuscripts, reviews projects and reports, booklet, proceedings, book chapters and also academic degree thesis.
Project Context and Objectives:
1. Introduction and Objectives
The MULTIFUN consortium focuses on the development and validation of new systems based upon minimal invasive nanotechnology for the early and selective detection and elimination of breast and pancreatic cancer with reduced side effects. The project will deploy a strategy based on the multifuntionalisation of magnetic iron oxide nanoparticles (MNP), combining diagnostic and therapeutic features against breast and pancreatic cancer and cancer stem cells.
The MULTIFUN therapeutic approach is multimodal and combines the magnetic heating mediated by MNP when subjected to alternating magnetic fields with intracellular drug delivery. In this manner the therapeutic outcome is reinforced by synergies within the different therapeutic modalities. Since MNP can be detected through magnetic resonance imaging (MRI), they can also be used as contrast agents for cancer cell detection. In this way, MULTIFUN combines therapeutic and diagnostic aspects leading to a potential “theragnostics” tool.
A key point of the project is to assess the safety and toxicity of the developed nanoparticles. Thus, MULTIFUN includes a broad set of in vitro and in vivo toxicity and biodistribution tests in different animal models including mice, rats and pigs.
Finally, in order to improve the translation of the outcomes and their economic potential, the consortium also will study the scale-up of the production methods for the main components.
The MULTIFUN project will meet several scientific and technological objectives indicated below:
Scientific objectives
• Study of cell internalisation routes of Multifuntionalised Magnetic Nanoparticles (MF-MNP).
• Evaluation of cyto-toxicity of MF- MNP in vitro.
• Distribution and toxicity analysis of MF-MNP in vivo.
• Evaluation and validation of breast and pancreatic cancer multimodal therapeutic strategies via in vitro and in vivo models.
• Demonstration of a cancer ‘theragnostic’ approach for early stage cancer targeting stem cells.
• Design of innovative targeting peptides and antibodies for a higher effectiveness in tumour localization and drug dose reduction.
Technological objectives
• Scale-up of production of biocompatible MNP with optimised MRI contrast properties.
• Multifunctionalisation of MNP for the delivery of anti-cancer agents.
• Instrumentation development for MNP detection and quantification in biological samples.
• The development of improved magnetic heating instrumentation for in vitro and in vivo applications.
2. Originality of MULTIFUN Project
The MULTIFUN project has several objectives which differentiate it from other FP7 NMP projects aimed to develop nanotechnology solutions for cancer therapy. These original objectives can be grouped by their scientific and technological contents.
Scientific MULTIFUN originality
The MULTIFUN consortium has developed a comprehensive strategy in order to generate highly selective and efficient multifunctional nanoparticles for detecting and treating cancer. The combination of magnetic heating and other therapeutic modalities for cancer allows new therapeutic approaches. MULTIFUN nanoparticles will target cancer cells including the highly tumorigenic stem cells as the putative root of cancer. These types of cancer cells are associated with drug resistance and tumour relapse and hence their targeting may lead to a significant improvement in therapeutic outcomes. Several members of the consortium have already demonstrated that it is feasible to target cancer stem cells efficiently using multimodal strategies which lead to tumour regression and dramatically enhanced long-term survival in relevant tissue xenograft models. Therefore, the fact that MULTIFUN nanoparticles may also result in the elimination of cancer stem cells represents a major advantage over other nanoparticle-based approaches.
Figure 1: MULTIFUN approach.
MULTIFUN Technological originality
The multidisciplinary framework related to MULTIFUN activities provides a suitable opportunity for the development of scientific instrumentation aimed to the use of magnetic nanoparticles in biomedical applications. One of the proposed technologies is the development and validation of an exvivo spectrometer to detect and quantify the presence of magnetic nanoparticles in tissues and biological fluids (blood and urine). PEPRIC is the partner involved in this task to supply a device that will ease the biodistribution studies. In addition, INSA partner has been involved in the development of a AC magnetic field generator to perform in vitro and in vivo magnetic hyperthermia experiments. The device, supplied to 4 other partners, combines the versatility to locate cells or animals into a coil with all requirements needed to accomplish the magnetic hyperthermia studies with a wider magnetic field conditions. In addition, MULTIFUN has developed multifunctional nanoparticles preserving their biological activity from all conjugated molecules (anticancer agents or ligands). The strategy employed for the conjugation of anticancer agents (chemotherapeutic drugs, siRNAs and peptides) is quite unique allowing the drug release exclusively under intracellular conditions (i.e. reductive environment) . Considering that Magnetic resonance detection allow for the detection and quantification of magnetic nanoparticles , this method is selective and sensitive with reduced heat dissipation in biological tissues. As the magnetic particles are stable this will allow longitudinal monitoring over several days and weeks. The device will be compared and benchmarked to other quantitative methods. This technique involves the use of low magnetic fields and hence the heat generation within the tissue is reduced. This will allow the implementation of ex-vivo measurements to be explored during the project, for which no sample preparation is necessary such as freeze drying or homogenization or mineralization of the sample. This reduces the sampling error and manipulation time for the lab technicians.
Figure 2: New detection equipment for quantitative analysis of MNP’s in blood and tissue samples
The presence of one industrial nanoparticle manufacturer (LRL) and two academics suppliers (IMDEA and ICMM-CSIC) has ensured the supply of materials for MULTIFUN activities with the consortium. Furthermore, the production of biocompatible magnetic nanoparticles with optimized contrast and heat induction features for oncological applications has been scaled up. At the end of the project, among the different scale up processes developed the one performed by the industrial partner provides up to 20 g of MNPs of excellent quality.
Project Results:
The Multifun consortium has employed superparamagnetic nanoparticles with optimal magnetic and magneto-thermal properties. Two chemical routes have been chosen to get nanoparticles with narrow size distribution, high saturation magnetization and high specific absorption rate (SAR) values. The thermal decomposition of an iron precursor in organic media provides suitable magnetite nanoparticles with the best size control (standard deviation lower than 10%). Multifun partners have analyzed which reaction parameters lead to improve the magnetic and magneto-thermal properties of the resulting nanoparticles. Thus, magnetite nanoparticles can be synthesized in a wide particle size range from 7 to 22 nm by varying some chemical reaction parameters related to nucleation and growth processes such the reaction time, and the oleic acid concentration in the reaction mixture. Second, a narrow size distribution is obtained when the reaction mixture is highly homogeneous when the temperature is increased (see Figure 3).
Figure 3: a) Transmission Electronic Microscopy micrographs of 18 nm size magnetite nanoparticles synthesized a) without, b) with stirring the reaction at the beginning of the heat Ramp. Arrows indicate smaller size nanoparticles. Images kindly provided by G. Salas and M.P. Morales.
Thus, a short nucleation and later a uniform particle growth are favoured. Moreover, the resulting nanoparticles are highly crystalline; their morphology is highly uniform and differs from sphere-like for sizes below 15 nm to cube-like for sizes higher than 15 nm. Multifun partners have shown the tight relationship among size distribution, magnetic and magneto-thermal properties: narrower size distribution lead to higher saturation magnetization and SAR values as shown in Figure 4. More info in: G. Salas et al. J. Mater. Chem. 22, 21065 (2012). We have established a relationship between physico-chemical properties of magnetic fluids and their heating capacity. Salas, Gorka et al., International journal of hyperthermia, 29, 768-776 (2013),
We have also been able to scale up the synthesis of larger particles and coating them with polymers keeping a very high saturation magnetization values through green aqueous synthesis, M. Marciello, V. Connord, S. Veintemillas-Verdaguer, M. Andres-Vergés, J. Carrey, M. Respaud, C. J. Serna and M. P. Morales. Journal of materials chemistry B, 1, 5995-6004 (2013).
A detailed series of papers has also been published using nanoparticles synthesized by CGP for the mechanisms of magnetic hyperthermia. These papers detail the mechanisms particles use to generate heat, also details the parameters that are key for controllable magnetic hyperthermia. More info in: G. Fernandez-Vallejo et al. J. Phys. D: Appl. Phys. 46, 312001 (2013) and G. Fernandez-Vallejo et al. Appl. Phys. Lett. 103, 142417 (2013).
Figure 4: a) Transmission Electron Microscopy micrographs of particles produced by the Controlled Growth Process. b) High Resolution TEM (HRTEM) image showing the high crystallinity of the particles produced by the controlled Growth Process. Images kindly provided by Liquids Research Ltd.
The standard characterization techniques were applied to determine important structural and magnetic parameters of the nanoparticles. Both the ferrofluids and powders were used for characterization; a novel protocol for magnetic property measurement of the ferrofluid samples was established.
Phase composition of samples was determined by powder X-ray diffraction (PXRD) and Mӧssbauer spectroscopy (Figure 4 a & c), which served for the precise specification of the maghemite/magnetite content. Real particle size, dTEM and size distribution were determined from the Transmission Electron Microscopy (TEM) images. Size of the crystalline part of the particle, dXRD was refined from the PXRD patterns. Magnetic property measurements were used for determination of the so-called blocking temperature of the particles (TMAX and Tirr, Figure 4d) and estimation of distribution of particle energy barriers. Parameters such as the saturation magnetization, Ms, remanence, Mr and coercivity, μ0Hc were obtained from the magnetization isotherms (M(H) curves, Figure 4 e). Magnetic moments of the particles, μ their distribution, σ and magnetic diameter, dMAG were refined from the un-hysteretic M(H) curves (Figure 4 f).
Comparison of individual particle diameters, dTEM, dXRD and dMAG serves as the perfect indicator of particle crystallinity and internal spin order (Figure 4 g). Any changes in the determined parameters, comparing the bare and coated nanoparticles, then reveal the effect of coating on physical properties of the nanoparticles. More details can be found in Kubickova et al, Appl. Phys. Lett. 104 (2014) 2231051 and Pacakova et al, arXiv.2015.
Figure 5: The example of typical experiments for determination of the properties of magnetic nanoparticles: a) Powder X-ray Diffraction pattern from which the phase composition and the particle diameter (dXRD) are determined. b) Transmission Electron Microscopy, which is used for direct observation of the apparent particle size (dTEM) and its distribution. c) Example of Mӧssbauer spectra. Refinement of spectra can easily distinguished between the presence of the Fe2+ and Fe3+ ions, thus method serves for precise determination of the maghemite/magnetite phase content within the examined sample.
Bottom panel: Magnetic property measurements: d) example of the ZFC-FC curve from which the blocking temperatures, TMAX and Tirr can be determined. e) Example of the M(H) curve and f) the fit of the unhysteretic M(H) curve in Langevin scaling from which the mean and meadian magnetic moments of the particles can be obtained together with the distributions of magnetic moment, σ and median particle diameter (dMAG). g) Illustration of the internal particle structure for the ideal and real particles and relation of individual particle diameters.
Figure 6: SAR values obtained for 18nm size nanoparticles with different size distributions (yellow ±2 nm, red ±4 nm) when exposed to the same HAC conditions (50 mT and 77 kHz). Images kindly provided by G. Salas, M.P. Morales and F.J. Teran.
On the other hand, the magnetic and thermal characterizations of nanoparticles have been analyzed obtaining some preliminary results related to establish the superparamagnetic phase of the synthesized particles (Salas, Gorka et al. Journal of Physical Chemistry C 118, 19985 (2014)). Huge efforts are paid to determine which are the optimal particle sizes and magnetic field conditions leading to optimal contrast agent and hyperthermia mediator features. In this sense, the control and the quantification of heat losses is crucial to reduce error sources providing SAR values with higher accuracy. Multifun partners have reported on the development of an experimental set-up that diminishes heat losses with the surrounding. In this way, the time variation of temperature can be modelled (see Figure 7) what leads to more accurate determination of SAR values. More info: F.J. Teran et al., Appl. Phys. Lett. 101, 062413 (2012). We have also observed that the optimum assembling of magnetic nanoparticles lead to an enhancement of the magnetic hyperthermia, D. Serantes, J. Phys. Chem. C 2014 dx.doi.org/10.1021/jp410717m.
Figure 7: a) Time evolution of SPION temperature when subjected to HAC (78 kHz and 25 mT) (a) at Teq= 30ºC, b) Numerical derivative curves of data shown in previous graph. Solid circles indicate the maximun and minimun dT/dt values. C) Comparison of numerical simulations and experimental data. Shadow and white time zones are exposed to HAC on and off, respectively. Red and blue lines correspond to temperature rise and decay. Images kindly provided by F.J. Teran.
The parameters involved in the heating efficiency related to MNP interacting phenomena were analyzed in order to clarify which are the conditions to control the MNP magnetic heat release and how to control the heat exposure. This is mandatory for clinical purposes. Thus, we have assessed the magnetic heating dependence with field conditions, MNP concentration, aggregation and size for determining the effect of dipolar interactions (Figure 8).
Figure 8: Concentration dependence of SAR for different particle sizes, field frequency and amplitude. Figures kindly provided by D. Cabrera and F.J. Teran
The functionalization of MNPs with different bioactive molecules has afforded novel nanostructures with antitumoral properties. Particularly, we have used tailored linkers to conjugate drugs (Doxorubicin and Gemcitabine) to the nanoparticles. The system developed is stable under physiological conditions and release the cargo mainly inside the cells. (Latorre, A., Couleaud, P., Aires, A., Cortajarena, A. L. & Somoza, Á. Multifunctionalization of magnetic nanoparticles for controlled drug release: A general approach. Eur. J. Med. Chem. 82, 355–362 (2014).)
Figure 9: Intracellular controlled release
In addition we have used targeting molecules such as the Nucant peptide and antibodies to direct the nanostructure to the tumoral cells. Another biomolecules employed in the functionalization of MNPs are nucleic acids, particularly siRNAs.
The discovery of the RNA interference mechanism has triggered the development of nucleic acids derivatives to generate novel medical treatments. Although the use of synthetic small interfering RNA (siRNA) has been demonstrated, still several issues remain to be solved in order to ensure the potential of this novel medicinal avenue. One of the problems that have been addressed during the last years is the vulnerability of oligonucleotides to nucleases, which limits their practical use. Specifically, we explored the effects of the acyclic L-threoninol backbones in place of natural ribose rings on the biological properties of siRNAs. The introduction of these novel derivatives at the 3’-protuding ends of siRNAs confers a strong nuclease resistance, greater potency by a better affinity to PAZ domain and a lower degree of innate immunostimulation. Finally the introduction of these derivatives at the 2on position near the 5’-end of the sense strand (Figure 10) avoids the loading of the sense strand into RISC lowering off-target effects. These results will be valuable in the design of therapeutic siRNAs.
Figure 10: Beneficial effects of the substitution of natural nucleobases by L-threoninol derivatives in siRNAs.
The assessment of toxicity and internalization of iron oxide nanoparticles into breast and pancreatic cancer cells have been performed allowing to test the interaction between cancer cells and derived from co-precipitation in water and thermal decomposition synthesis methods. Thus, nanoparticles with different coatings, and surface charges has been tested in different established cancer cell lines to evaluate cell viability and uptake under nanoparticles concentrations up to 0.2 mg/ml during incubation period. MultiFun partners have reported that nanoparticles may induce different effects on the cell viability and permeability and their lysosomal mass depending on coating, surface and cell line. More info: Adriele Prina-Mello et al. IEEE Transactions on magnetics, 49(1), January 2013. The efficient and safe internalization of some of these magnetic iron oxide nanoparticles designed for biomedical applications have been highlighted in a recent publication: M. Calero et al. Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014) 733–743.
The MNPs internalization process in different cell types has been characterized using electron microscopy. The internalization pathways follow two routes: One is clathrin-mediated uptake and the other is the macropinocitic route. The particles accumulate in endosome vesicles in the cytoplasm and never reach the nucleus interior.
Figure 11: Internalization of OD15 by MCF-7 cells. Cells incubated at different times with MNPs were studied by electron microsopy. Clathrin-mediated endocytosis was observed for MNP aggregates <200 nm, while macropinocytosis was found for larger aggregates. Cargo vesicles were fused in early endosomes (EE). Multivesicular bodies (MVD) and late endosomes (LE)containing intraluminar vesicles (ILVs) fuse together with Lysosomes (LYS) for final degradation vesicles (Journal of Nanobiotechnology 2015, 13:16. doi:10.1186/s12951-015-0073-9).
Figure 12: Analysis of cytoskeleton. (A) Representative images of cells immunostained for tubulin (green) and DNA counterstained with Hoechst-33258 (blue). Left panel: (a) Interphase control cells. (a′) Metaphase control cell. Middle panel: (b-b′) Interphase cells incubated for 24 h with 0.5 mg ml−1 AD and observed by fluorescence and bright-field microscopy, respectively; (c-c′) Cells treated with DMSA; (d-d′) Cells incubated with APS. Right panel: (e-e′) Mitotic spindle of cells incubated with AD; (f-f′) DMSA or (g-g′) APS. (B) Merged images of F-actin labeled with Phalloidin-TRICT (red), vinculin immunostaining (green) and DNA counterstained with Hoechst-33258 (blue). (a) Control cells. (b-b′) Cells treated with AD and observed by fluorescence and bright-field microscopy, respectively. (c-c′) Cells treated with DMSA. (d-d′) Cells incubated with APS. Scale bars = 10 μm.
The functionalisation of the nanoparticles provided by academic and industrial partners has led to the first generation of functionalized nanostructures. NUCANT has been the molecule to start with. This pseudo-peptide is highly interesting due to its double potential: 1) to interact with protein receptor on cancer cell membrane, 2) to inhibit cancer cell proliferation due to its proapoptotic activity. Preliminary results show that Nucant adsorbed onto nanoparticle surface favour the nanoparticle internalization into cancer cells in some cases. Further investigations to control the internalization mediated by cell receptor are in progress. In this sense, we have investigated the ultrasound generation and high-frequency motion of magnetic nanoparticles in an alternating magnetic field: toward intracellular ultrasound therapy, J. Carrey, Appl. Phys. Lett. 102, 232404 (2013). On the other hand, we were able to target a G-protein coupled receptor overexpressed in endocrine tumors by magnetic nanoparticles to induce cell death, C. Sanchez, ACS NANO 8, 1350 (2014)
Preliminary in vivo studies related to the hyperthermia effects on tumour evolution have shown promising results. The hyperthermia therapeutic approach has been tested on animal models using nanoparticles that under high frequency field conditions lead to SAR values larger than 200W/g. Thus, tumour volume has been shown to be reduced after few magnetic field sessions. Further investigations are going on to optimise the hyperthermia therapeutic approach.
The role of anesthesia in the magnetic nanoparticle biodistribution among different organs after intravenous injection has been studied in a murine model. Animals were anesthetized by inhalation with isoflurane (0,5 % in oxygen) or by intraperitoneal injection with a mixture of ketamine and xilazine. Then, a single dose of monodisperse dimercaptosuccinic acid coated magnetic nanoparticles (9.2 nm diameter ±13% SD) was administered to the animals. Lung and liver tissues were collected 30 min after the particle administration and the amount of particles in each tissue was determined by AC magnetic susceptibility measurements. Whereas the amount of particles that reaches the liver seems not to be affected by the anesthesia used, the amount of particles that reaches the lungs for inhaled isoflurane is three times less than for the intraperitoneally injected anesthetic. Lucía Gutiérrez et al., IEEE Transactions on magnetics, 49(1), January 2013. We have also studied long term biotransformation and toxicity of dimercaptosuccinic acid-coated magnetic nanoparticles after intravenous injection, Mejias, Raquel; et al. Journal of controlled release 171, 2, 225-233 (2013). A review on prospects in the synthesis, detection and quantification of magnetic nanoparticles following systemic administration has been recently published by L. Gutiérrez, Physical chemistry chemical physics, DOI: 10.1039/c3cp54763a 2014.
Potential Impact:
1. Exploitation
The consortium has characterised 11 exploitable results with a high potential impact and profitability from a commercial point of view.
Table 1: List of Exploitable Results
Exploitable Result / Type / Contact
Production of biocompatible MNPs - Organic Route / product & process / Bonifacio Vega (IMDEA)
Production of biocompatible MNPs - Aqueous Route / product & process / Vijay Patel (LRL)
Cancer Cell (including CSC) biomarkers – Pancreatic Cancer / knowledge / Christopher Heeschen (CNIO)
Cancer Cell (including CSC) biomarkers – Breast Cancer / knowledge / Rob Clarke (UNIMAN)
Novel drugs based on nucleic acids to suppress cancer cell invasion and metastasis / knowledge & product / Ramon Eritja (IQAC-CSIC)
Functionalisation of MNPs with PM01183 / product / Pilar Calvo (PM)
Functionalisation of MNPs with an antagonist of nucleolin as a tool for target cancer cells. / product / Jose Courty (CRRET)
Multifunctional MNPs (MF-MNPs) for cancer treatment and diagnosis / product & process / Bonifacio Vega (IMDEA)
Magnetic heating equipment for in vitro and in vivo evaluation / product / Marc Respaud (INSA)
Device/instrumentation for detection and quantification of MNPs based on EPR method / product & process / Stephanie Teughels (PEPRIC)
High content screening analysis for MNP toxicity / product & process / Yuri Volkov (TCD)
The information of these results is confidential. To know more about them, please, contact the suitable person (contact person).
In addition to these exploitable results, we have described 6 project key outcomes that could be very promising and be used as a basis for future research and innovation activities for long-term final exploitation:
1. siRNAs miRNA molecules for cancer treatment
Partners involved: CSIC
Description: A series of chemical modifications for siRNAs have been developed. The modified siRNA have a large resistance to degradation by nucleases present in sera, resulting in an increase of the efficiency and duration of the gene silencing properties. In addition one of these modifications when introduced at a particular site reduced off-target effects increasing the specificity of the gene silencing activity.
Future uses and applications: The results will be useful for the development of more potent siRNAs and miRNA mimetics for cancer treatment in research projects.
References:
“Effect of North bicyclo[3.1.0]hexane pseudosugars on RNA interferente. A novel class of siRNA modification”. Terrazas, M., Ocampo, S.M. Perales, J.C. Marquez, V., Eritja, R. ChemBioChem, 12, 1056-1065 (2011).
“Functionalization of the 3’-ends of DNA and RNA strands with N-ethyl-N-coupled nucleosides: A promising approach to avoid 3’-exonuclease-catalyzed hydrolysis of therapeutic oligonucleotides”. Terrazas, M., Alagia, A., Faustino, I., Orozco, M., Eritja, R. ChemBioChem, 14, 510-520 (2013).
“RNA/aTNA Chimeras: RNAi effects and nuclease resistance of single and double stranded RNAs”. Alagia, A., Terrazas, M., Eritja, R. Molecules, 19, 17872-17896 (2014).
“Modulation of the RNA interference activity using central mismatched siRNAs and acyclic threoninol nucleic acids (aTNA) units”. Alagia, A., Terrazas, M., Eritja, R. Molecules, 20, 7602-7619 (2015).
2. Understanding the role of Magnetic parameters in the production of biocompatible MNPs
Partners involved: IMDEA, CAS.
Description: IMDEA and CAS have been able to correlate key parameters in iron oxide nanoparticles such size, size distribution, or crystallinity defects that strongly influence their magnetic properties. These parameters can be used for tuning the magnetic properties of nanoparticles.
Future uses and applications: The knowledge gained in the project is useful to understand better how magnetic properties of nanoparticles can be customised for specific applications.
References:
“Structural disorder versus spin canting in monodisperse maghemite nanocrystals” S. Kubickova, D. Niznansky, M. P. Morales Herrero, G. Salas, J. Vejpravova Appl. Phys. Lett. 104 (2014) 223105(1) - 223105(4).
“Modulation of Magnetic Heating via Dipolar Magnetic Interactions in Monodisperse and Crystalline Iron Oxide Nanoparticles” G. Salas, J. Camarero, D. Cabrera, H. Takacs, M. Varela, R. Ludwig, H. Dähring, I. Hilger, R. Miranda, M. P. Morales, F. J. Teran. J.Phys.Chem. C 118 (2014) 19985-19994.
“Controlled synthesis of uniform magnetite nanocrystals with high-quality properties for biomedical applications” G. Salas , C. Casado , F. J. Teran , R. Miranda , C.J. Serna and M.P. Morales. J. Mater. Chem. 22 (2012) 21065-21075.
3. MR acquisition and post-processing software
Partners involved: KCL
Description: KCL has implemented several MR imaging sequences for T1, T2 and T2* mapping and validated those in vials with known magnetic nanoparticle (MNP) concentration and in cells incubated with non-specific and functionalised nanoparticles provided by the Multifun consortium. They subsequently applied those imaging techniques to measure nanoparticle accumulation in a model of breast cancer in-vivo. They also implemented several positive contrast sequences including GRASP and IRON and the post-processing method SGM to visualise nanoparticles with positive contrast. They also developed a positive contrast technique based on the DIXON sequence and validated all these sequences in concentration phantoms demonstrating good accuracy for MNP quantification.
Future uses and applications: They will use the developed methods to quantify MNP’s for tissue characterization in models of atherosclerosis, myocardial infarction, deep venous thrombosis and cancer. KCL intends to use some of the techniques in a H2020 project recently submitted.
References:
“T1 and T2 Mapping of Superparamagnetic Iron Oxide Nanoparticles for the Detection of Breast and Pancreatic Cancer Cells”. D. Krüger, G. Salas, M. Calero, S. Lorrio González, A. Villanueva, M. P. Morales, R. M. Botnar. Poster presented at Annual Meeting of the International Society of Magnetic Resonance in Medicine 2013, Salt Lake City, Utah.
“A Dixon Method for Positive Contrast Imaging of Very Small Superparamagnetic Iron Oxide Nanoparticles in MRI”. D. Krüger, S. Lorrio González, R. M. Botnar. Abstract accepted for the Annual Meeting of the International Society of Magnetic Resonance in Medicine 2015, Toronto, Canada.
“Dixon Fat Suppression for Off-resonant Water Imaging of Superparamagnetic Iron Oxide Nanoparticles”. D. Krüger, S. Lorrio González, R. M. Botnar. Abstract accepted for the Annual Meeting of the International Society of Magnetic Resonance in Medicine 2015, Toronto, Canada.
4. Protocols for safety and handling MNPs
Partners involved: TCD
Description: guidelines and protocols for the preparation and the administration (in vitro and in vivo) of MNPs. These protocols intend to create some homogeneity within the handling of MNP, for addressing safety and efficacy of MNPs in biomedical applications.
Future use and applications: Protocols and know-how developed are enabling to advance any future collaborative research projects and funding opportunities; training and know-how transfer within biomedical and pharmaceutical industry (SMEs and Multinational) are also to be considered in order to provide educational and training on the responsible use of nanotechnology applied to medicine. Trinity College is currently leveraging this library of knowledge and know how within the recently funded EUNCL H2020 infrastructure project in nanomedicine, the European Union Nanomedicine Characterisation Laboratory.
References:
Bergamaschi E., Poland C., Guseva Canu I., Prina-Mello A., The role of biological monitoring in nanosafety, (2015), Nano Today doi: 10.1016/j.nantod.2015.02.001 (accepted), (News and Opinions, Senior Author)
Prina-Mello, A., Crosbie-Staunton, K., Salas, G., Del Puerto Morales, M., Volkov, Y., Multiparametric toxicity evaluation of SPIONs by high content screening technique: Identification of biocompatible multifunctional nanoparticles for nanomedicine, (2013) IEEE Transactions on Magnetics, 49 (1), art. no. 6392408, pp. 377-382.
Movia D., Poland C., Tran L., Volkov Y., Prina-Mello A. Multilayered Nanoparticles for Personalized Medicine: Translation into Clinical Markets, Editors: Bawa R., Audette G.F. and Rubinstein I.: Handbook of Clinical Nanomedicine – From Bench to Bedside, Pan Stanford Series in Nanomedicine (Raj Bawa, Series Editor), Volume 1, Pan Stanford Publishing, Singapore, 2015, ISBN-10: 9814316172. (BOOK CHAPTER)
Prina-Mello A., B.M. Mohamed, N.K. Verma, Y. Volkov, Advanced Methodologies and Techniques for Assessing Nanomaterials Toxicity: from manufacturing to nanomedicine screening, Nanotoxicology, Second Edition, Editors: Nancy A. Monteiro-Riviere & C. Lang Tran, Taylor & Francis, Informa Healthcare Books, February 2014. (BOOK CHAPTER)
Movia D., Gerard V., Maguire C.M. Jain N., Bell A.P. Nicolosi V., O Neill T., Scholz D., Gun’ko Y., Volkov Y. and A. Prina-Mello, A safe-by-design approach to the development of gold nanoboxes as carriers for internalization into cancer cells, Biomaterials (2014), 35:9 (LEAD OPINION PAPER)
Hole P, Sillence K, Hannell C. Maguire CM, Roesslein M, Suarez G, Capracotta S., Magdolenova Z, Horev-Azaria L, Dybowska A, Cooke L, Haase A, Contal S, Manø S, Vennemann A, Sauvain JJ, Crosbie Staunton K, Anguissola S, Luch A, Dusinska M, Korenstein R, Gutleb AC, Wiemann M, Prina-Mello A. *, Riediker M, Wick P, Interlaboratory comparison of size measurements on nanoparticles using Nanoparticle Tracking Analysis (NTA), Patrick Hole, Katherine Sillence, Claire Hannell, (2013) Journal of Nanoparticle Research, 15:2101 (*Joint Senior Authors)
Gobbo OL, Wetterling F, Vaes P, Teughels S, Markos F, Edge D, Shortt CM, Crosbie-Staunton K, Radomski MW, Volkov Y and Prina-Mello A. Biodistribution and Pharmacokinetic studies of SPION using particle Electron Paramagnetic Resonance, MRI and ICP-MS. Nanomedicine (in press, DOI: 10.2217/NNM.15.22).
5. Knowledge regarding pharmacokinetics and bio-distribution of MNPs
Partners involved: UCC
Description: WP4 in MULTIFUN gave us detailed knowledge about the influence of the size, coating and other chemical parameters in the pharmacokinetics and bio-distribution of MNPs in mice (Gobbo et al 2015), rats and pigs. Small diameter iron particles administered i.v. were rapidly (in under 10 minutes in pigs) scavenged from the circulation by the cells in the liver and spleen (likely reticuloendothelial cells), as would be expected. In addition, the small diameter meant that there was significant accumulation in the lungs and the particles were also filtered by the kidneys. Therefore, these results suggest that these particles, allied with the anti-cancer attachments developed by the consortium, may be useful in targeting cancers associated with the liver and bone.
Future use and applications: Exploiting mammalian handling of iron-MNPs developed by MULTIFUN to treat less common cancers (e.g. bile duct cancers, primary or secondary bone cancers, liver cancer).
References:
“Biodistribution and Pharmacokinetic studies of iron oxide nanoparticles using particle Electron Paramagnetic Resonance, MRI and ICP-MS.” Gobbo OL, Wetterling F, Vaes P, Teughels S, Markos F, Edge D, Shortt CM, Crosbie-Staunton K, Radomski MW, Volkov Y, Prina-Mello A (2015). Nanomedicine (IN PRESS).
6. Knowledge regarding the higher impact that the MULTIFUN systems (Multifunctionalised MNP) in combination with magnetic heating has on cancer cell viability
Partners involved: UHJ
Description: UHJ has been able to study the impact of the combinational therapy consisting of nonfunctionalised as well as multifunctionalised MNPs in combination hyperthermia in vitro and in vivo. This combination showed a strong reduction of cell viability of breast and pancreatic cancer cells in vitro. Furthermore, combined with hyperthermia treatment the multifunctionalised MNP exhibited a strong regression of tumor volumes in subcutaneously xenografted breast MDA-MB231 cells and pancreatic BxPC-3 cells. The results clearly emphasize the high therapeutic potential of the multifunctionalised MF66 in combination with hyperthermia and underline the beneficial outcomes provided by this combination therapy if translated to the clinic.
UHJ first established a Standard Operating Procedure (SOP) for the application of magnetic heating in vivo.
Future use and applications: The gained knowledge will be used to:
1. transfer the therapeutic modality to the clinical situation
2. to extend the approach to other diseases and drug formulations
3. to open up new collaborations with pharma industry and research institutions
4. to provide a platform for testing functionalised MNP for pharma industry and other research institutions
References:
Dähring H, Grandke J, Teichgräber U, Hilger I. Improved Hyperthermia Treatment of Tumors Under Consideration of Magnetic Nanoparticle Distribution Using Micro-CT Imaging. Molecular imaging and biology: Mol Bio Imaging. 2015.
Kossatz S, Grandke J, Couleaud P, Latorre A, Aires A, Crosbie-Staunton K, et al. Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery. Breast cancer research: Breast Cancer Res. 2015;17(1):66.
Kossatz S, Ludwig R, Dähring H, Ettelt V, Rimkus G, Marciello M, et al. High therapeutic efficiency of magnetic hyperthermia in xenograft models achieved with moderate temperature dosages in the tumor area. Pharm Res. 2014;31(12):3274-88.
Pömpner N, Stapf M, Hilger I. (2015) Heterogeneous response of different tumor cell lines to hyperthermia in presence of MTX-coupled nanoparticles. IJONM (submitted).
2 Dissemination
The MULTIFUN dissemination objectives from the DOW have been fully achieved across all the fields of expertise the consortium has developed. To date, the consortium has already been actively contributing and participating to more than 300 separate events where MULTIFUN project gained visibility and also reputation as a comprehensive project focused in delivering theranostics treatments for breast and pancreatic cancers.
The MULTIFUN partners have also individually or as groups engaged in public events where to provide comprehensive information and awareness on how nanotechnological solution can provide better targeting or personalised treatment to the patients affected by incurable cancers. This objective was achieved by informing the community across the media by press releases, pan-European multimedia events, broadcasting interviews with the key players in the field of theranostics, to presenting MULTIFUN and nanomedicine to young students and the public at Scientific Open days across the geographical spreading that MULTIFUN is involved.
MULTIFUN Stakeholders have been also engaged and informed of MULTIFUN progress and achievements across the many events the project was involved, as invited speakers, chairpersons, event organisers, trade company/ies or exploitation partner/s. Furthermore, the project has also contributed to the shaping up of Funding schemes and opportunities at regional, national and international level by providing scientific input and expertise and working with the relevant organisations involved (e.g. European Commission for Horizon 2020, European Technology Platforms for key enabling and future emerging technologies,
MULTIFUN has truly achieved the dissemination objectives of the project by providing next generation scientific knowledge to the scientific and non-scientific community, and also by training and inspiring the next generation of scientists and technology experts which they all contribute to the improvement of the quality of life and future to the eradication of cancer.
Societally, MULTIFUN has developed a responsible attitude towards the use of nanotechnology in the biomedical industry for the improvement everyone life.
Breakdown summary and details of the dissemination activity of the project can be found in the here below summarised in the Table 1 and histogram plots (Figure 13) where it is shown how and where the project delivered in the past four years. Details are provided for each reporting period and also as overall project activities.
Table 2: MULTIFUN project 48 months dissemination Outcome
Figure 13: Overall details for MULTIFUN dissemination activity outcome (Period June 2011 – June 2015)
It has been planned that after EuroNanoForum 2015 the project dissemination activities will continue further into 2016 and 2017 as part of evaluation process or publication of submitted manuscripts (currently 14 manuscripts either under submission or under revision), reviews projects and reports, booklet (1 under final preparation), proceedings, book chapters and also academic degree thesis (9 pending PhD award titles to be awarded by 2016).
List of Websites:
http://multifun-project.eu/
Prof. Dr. Rodolfo Miranda
contacto.multifun@ imdea.org
MULTIFUN Scientific Coordinator and IMDEA Nanociencia Director.
Ms. Blanca Jordan – blanca.jordan@atos.net
MULTIFUN Administrative Coordinator and Health Sector Manager at Atos Research & Innovation.
