Final Report Summary - NANOMAGDYE (Magnetic nanoparticles combined with submicronic bubbles and dye for oncologing imaging)
The treatment of cancer is a European wide health and economical problem since over millions of citizens are concerned. Cancer is the second leading cause of death after heart disease. Each year on a worldwide basis, almost seven million people die of the consequences of cancer (Intern. Agency of Cancer database). In France and in Germany respectively approximately 300 000 and 400 000 people are newly diagnosed with cancer. The success of therapy in terms of increasing life expectancy depends on the earliness of the diagnosis and of the grading of the tumour. The detection of the Sentinel node (SN), which is any node receiving lymph drainage from the tumour site and containing most likely malignancy if the tumour has metastasised, has recently improved surgery strategies. The detection of the first draining lymph node is an important prognosis step since a positive metastasis histopathological diagnosis requires the complete lymph node chain exeresis. Nowadays, most teams working on sentinel node biopsy in the treatment of breast cancer inject either radioactive colloid (RuS labelled with Tc99m) or vital blue dye around the primary tumour to label the lymph node system for its peroperative detection. The detection is done by a visual colour inspection or a gamma probe. Usually, preoperative scintigraphy provides imaging but unfortunately at low resolution. If radioisotope injection is widely used today for the detection of sentinel nodes in the most advanced countries, new strategies are being investigated that use non-nuclear detection and imaging methodologies, including the use of light photons and magnetic nanoparticles that receive increasing attention.
The objectives of the NANOMAGDYE project were exactly directed to these new directions. It aimed at developing a novel clinical methodology based on tailored and versatile magnetic and optical nanosystems able to target the sentinel nodes when injected in cancer tumour, and on multimodal detection easy to utilise even with minimum technical environment.
The first objective of the NANOMAGDYE project consisted in developing tailored and versatile biocompatible nanosystems combining magnetic, optical and / or mechanical properties. This combination had to be achieved through hybrid systems made either of an iron magnetic oxide core on which an organic layer had to be grafted (hereinafter called nano-objects), or an organic shell on which nanoparticles had to be attached (hereinafter called magnetic bubbles).
Nano-objects combining magnetic properties and interactive functions at the surface have been previously obtained through the creation of few atomic layers of organic polymer or inorganic metallic (e.g. gold) or oxide surfaces (e.g. silica or alumina) on the nanoparticle, suitable for further functionalisation by attaching various molecules. Such a platform was shown to be suitable for biomedical applications: Superparamagnetic iron oxide (SPIO) and Ultrasmall superparamagnetic iron oxide (USPIO) consisting of nonstoichiometric microcrystalline magnetite cores, coated with dextrans (in ferumoxide) or siloxanes (in ferumoxsil) are already currently used as MRI contrast agents. Stabilisation of iron oxide in suspensions was also previously achieved by using dendrons. But the iron oxide basic units had always sizes below 10 nm.
Regarding the microbubbles, the use of microbubbles in medicine had increased spectacularly, as contrast agents for echosonography (diagnosis by ultrasound imaging), for delivery of drugs and genetic material and intravascular delivery of oxygen. We had shown previously that bubbles stability could be increased much more effectively by using a fluorinated analogue of DMPC (F-GPC) as the shell component. Their half-life, when Perfluorohexane (PFH) is present along with N2 in the internal gas phase reaches ~70 min, which is exceptional. In that context, the synthesis of new nanosystems was the first key issue to address.
Batch of magnetite nanoparticles of sizes between 2 and 70 nm had to be prepared. Magnetite nanoparticles of 12 ± 2nm, and 39 ± 5nm were previously obtained by coprecipitation of iron chlorides in tetramethyl ammonium. Other sizes in the range of 20-25 nm, 28-32 nm, 48-55 nm and 60-70 nm with a dispersion of less than 15 % of the size had to be obtained by changing the organic basis. The oxidation of magnetite nanoparticles could lead to nanoparticles of maghemite with the same size and dispersion. The sizes and the dispersion in size had to be measured by combining X-ray diffraction, granulometry and specific surface measurements and electron microscope observations.
An organic dendrimer molecule had to be grafted on the oxide in order to get a biocompatible entity, or the oxide grafted on an organic bubble leading to various architectures of nanosystems; grafting will be performed through a phosphonate. The grafting rate was to be evaluated by indirect Ultraviolet (UV) spectroscopy, chemical analysis and thermogravimetric analysis.
Bubbles had to be prepared using appropriate combinations of fluorinated and hydrogenated phospholipid analogs. The fluorinated phospholipids, which carry phosphate or a phosphonate polar head, could segregate from the hydrogenated ones within the bubble interfacial membrane, due to the strong lipophobicity of fluorinated chains. The resulting phosphate (or phosphonate)-coated isolated domains could then be used to adsorb the iron nanoparticles. The number of nanoparticles could be tuned by controlling the rate of phosphate or phosphonate polar heads. The stability of the nanoparticle-decorated microbubbles in the aqueous dispersion was to be assessed by static light scattering, optical microscopy and transmission electron microscopy. The segregated morphology of the microbubbles was to be assessed by an atomic force microscopy experiment using a liquid cell, which allows investigation of a sample without drying it.
The second objective of this project was to design and produce magneto-optical nanosystems useful for new cancer diagnostic imaging and therapy strategies. The first nano-objects could be designed to serve as magneto-optical marker of the sentinel node, i.e. to combine magnetic and optical characteristics in a single nanoparticle, in order to be able to detect nodes with two different techniques concurrently. They would be made by combining magnetic nanoparticles and organic dendrimer molecules. The dendrimers molecules could be designed in order to control the interactions between the nanosystems themselves, and / or with the environment, substrate or tissues. They could be made of four parts: the dendron, the hydrophile chains to get biocompatibility, the dye and a molecule ensuring stability of the suspension for at least five days. The optomagnetic contrast agent which conjugates magnetic and optical markers could be used in the method of peroperative detection of the sentinel node. Combining optical and magnetic labelling into a single biocompatible nanosystem would provide higher spatial resolution than presently obtained and avoid using ionising radiation, thus improving medical effectiveness and patient safety and comfort by avoiding multiple injections. In addition, the superparamagnetic behaviour of oxide nanoparticles would allow pre-operative MRI before surgery.
The magnetic bubbles were to be designed to provide a magnetic and mechanical marker. A conventional ultrasound probe could be used at bedside by the surgeon himself after bubble suspension injection, to define on the skin mammary area, the best appropriated access to the nodes chain. The bubbles were to be optimised by controlling their size and the amount of magnetic nanoparticles. The best compromise between magnetic signal (enough magnetic nanoparticles), ultrasound response and stability of the bubbles in solution would have to be reached. Particular attention would be devoted to the localization of nanoparticles around the bubbles that will be achieved by using a variable field - magnetic force microscope on islands or monolayers of microbubbles.
Although all the molecules involved are known to be biocompatible, biological tests in vitro on cells and in vivo on rats had to be performed. Such new material tailored at the nano-scale will provide a multimodal marker for preoperative high spatial resolution localisation by MRI, preoperative non invasive localisation of the nodes at the patient bed during its surgical preparation for the area to lance, and multimodal magneto-optical marker for peroperative sentinel node detection.
The third objective was to test these nanosystems in a medical application. A new dedicated magneto-optical probe had to be fabricated. In fact, no surgical probe existed at that time to detect nodes loaded with magnetic particles. The device was a Fluxset type magnetic field sensor combined with electromagnetic field generation coil(s). The sensibility had to be adapted to the quantity of injected nanoparticles. The geometry had to be designed in agreement with the surgeons' demands. They should be of the same size as the actual nuclear probes used for conventional and endoscopic surgery which diameters are respectively 11 or 16 mm, and 10 mm. Then, the probe had to be tested in vivo on rats.
The objectives of the NANOMAGDYE project were exactly directed to these new directions. It aimed at developing a novel clinical methodology based on tailored and versatile magnetic and optical nanosystems able to target the sentinel nodes when injected in cancer tumour, and on multimodal detection easy to utilise even with minimum technical environment.
The first objective of the NANOMAGDYE project consisted in developing tailored and versatile biocompatible nanosystems combining magnetic, optical and / or mechanical properties. This combination had to be achieved through hybrid systems made either of an iron magnetic oxide core on which an organic layer had to be grafted (hereinafter called nano-objects), or an organic shell on which nanoparticles had to be attached (hereinafter called magnetic bubbles).
Nano-objects combining magnetic properties and interactive functions at the surface have been previously obtained through the creation of few atomic layers of organic polymer or inorganic metallic (e.g. gold) or oxide surfaces (e.g. silica or alumina) on the nanoparticle, suitable for further functionalisation by attaching various molecules. Such a platform was shown to be suitable for biomedical applications: Superparamagnetic iron oxide (SPIO) and Ultrasmall superparamagnetic iron oxide (USPIO) consisting of nonstoichiometric microcrystalline magnetite cores, coated with dextrans (in ferumoxide) or siloxanes (in ferumoxsil) are already currently used as MRI contrast agents. Stabilisation of iron oxide in suspensions was also previously achieved by using dendrons. But the iron oxide basic units had always sizes below 10 nm.
Regarding the microbubbles, the use of microbubbles in medicine had increased spectacularly, as contrast agents for echosonography (diagnosis by ultrasound imaging), for delivery of drugs and genetic material and intravascular delivery of oxygen. We had shown previously that bubbles stability could be increased much more effectively by using a fluorinated analogue of DMPC (F-GPC) as the shell component. Their half-life, when Perfluorohexane (PFH) is present along with N2 in the internal gas phase reaches ~70 min, which is exceptional. In that context, the synthesis of new nanosystems was the first key issue to address.
Batch of magnetite nanoparticles of sizes between 2 and 70 nm had to be prepared. Magnetite nanoparticles of 12 ± 2nm, and 39 ± 5nm were previously obtained by coprecipitation of iron chlorides in tetramethyl ammonium. Other sizes in the range of 20-25 nm, 28-32 nm, 48-55 nm and 60-70 nm with a dispersion of less than 15 % of the size had to be obtained by changing the organic basis. The oxidation of magnetite nanoparticles could lead to nanoparticles of maghemite with the same size and dispersion. The sizes and the dispersion in size had to be measured by combining X-ray diffraction, granulometry and specific surface measurements and electron microscope observations.
An organic dendrimer molecule had to be grafted on the oxide in order to get a biocompatible entity, or the oxide grafted on an organic bubble leading to various architectures of nanosystems; grafting will be performed through a phosphonate. The grafting rate was to be evaluated by indirect Ultraviolet (UV) spectroscopy, chemical analysis and thermogravimetric analysis.
Bubbles had to be prepared using appropriate combinations of fluorinated and hydrogenated phospholipid analogs. The fluorinated phospholipids, which carry phosphate or a phosphonate polar head, could segregate from the hydrogenated ones within the bubble interfacial membrane, due to the strong lipophobicity of fluorinated chains. The resulting phosphate (or phosphonate)-coated isolated domains could then be used to adsorb the iron nanoparticles. The number of nanoparticles could be tuned by controlling the rate of phosphate or phosphonate polar heads. The stability of the nanoparticle-decorated microbubbles in the aqueous dispersion was to be assessed by static light scattering, optical microscopy and transmission electron microscopy. The segregated morphology of the microbubbles was to be assessed by an atomic force microscopy experiment using a liquid cell, which allows investigation of a sample without drying it.
The second objective of this project was to design and produce magneto-optical nanosystems useful for new cancer diagnostic imaging and therapy strategies. The first nano-objects could be designed to serve as magneto-optical marker of the sentinel node, i.e. to combine magnetic and optical characteristics in a single nanoparticle, in order to be able to detect nodes with two different techniques concurrently. They would be made by combining magnetic nanoparticles and organic dendrimer molecules. The dendrimers molecules could be designed in order to control the interactions between the nanosystems themselves, and / or with the environment, substrate or tissues. They could be made of four parts: the dendron, the hydrophile chains to get biocompatibility, the dye and a molecule ensuring stability of the suspension for at least five days. The optomagnetic contrast agent which conjugates magnetic and optical markers could be used in the method of peroperative detection of the sentinel node. Combining optical and magnetic labelling into a single biocompatible nanosystem would provide higher spatial resolution than presently obtained and avoid using ionising radiation, thus improving medical effectiveness and patient safety and comfort by avoiding multiple injections. In addition, the superparamagnetic behaviour of oxide nanoparticles would allow pre-operative MRI before surgery.
The magnetic bubbles were to be designed to provide a magnetic and mechanical marker. A conventional ultrasound probe could be used at bedside by the surgeon himself after bubble suspension injection, to define on the skin mammary area, the best appropriated access to the nodes chain. The bubbles were to be optimised by controlling their size and the amount of magnetic nanoparticles. The best compromise between magnetic signal (enough magnetic nanoparticles), ultrasound response and stability of the bubbles in solution would have to be reached. Particular attention would be devoted to the localization of nanoparticles around the bubbles that will be achieved by using a variable field - magnetic force microscope on islands or monolayers of microbubbles.
Although all the molecules involved are known to be biocompatible, biological tests in vitro on cells and in vivo on rats had to be performed. Such new material tailored at the nano-scale will provide a multimodal marker for preoperative high spatial resolution localisation by MRI, preoperative non invasive localisation of the nodes at the patient bed during its surgical preparation for the area to lance, and multimodal magneto-optical marker for peroperative sentinel node detection.
The third objective was to test these nanosystems in a medical application. A new dedicated magneto-optical probe had to be fabricated. In fact, no surgical probe existed at that time to detect nodes loaded with magnetic particles. The device was a Fluxset type magnetic field sensor combined with electromagnetic field generation coil(s). The sensibility had to be adapted to the quantity of injected nanoparticles. The geometry had to be designed in agreement with the surgeons' demands. They should be of the same size as the actual nuclear probes used for conventional and endoscopic surgery which diameters are respectively 11 or 16 mm, and 10 mm. Then, the probe had to be tested in vivo on rats.