Field of the invention This invention relates in general to the field of forming an agent for use as a contrast agent in medical imaging or for radio isotope therapy. The agent has unique chelating properties. The chelating properties enable an easy, very quick and robust labelling with radionuclides or metals metal ions active as contrast agents in a manner that is feasible in practical applications both pre clinically and for clinical purposes. The agent contains nanoparticles as contrast agents in multimodal imaging for diagnostic purposes and for guidance during surgery as well as for use in cancer treatment.

Background General background Nanotechnology

Nanotechnology is based on the use of structures that have one or more dimensions measuring loo nanometers or less. These nanoparticles have a higher surface area to volume ratio than larger particles. Taken together these features turn nanoparticles into interesting structure candidates as novel contrast agents and as drug delivery vehicles. This is due to the fact that they are small enough to reach out in the finest capillaries, yet large enough to display longer circulation times than conventional small molecules and finally the huge surface area/volume provide the possibility to display or carry other molecules on the surface. Imaging and therapy of disease with

radionuclides has been routine clinically for half a century and has developed into molecular imaging in the last decades. In order to combine the benefits of nanoparticle as multimodal contrast agents, drug delivery vehicles or combinations thereof with radionuclides and/or metals, the design of the nanostructures requires a safe, easy and robust method for attaching said metals, ions or radionuclides. Traditionally, it has been accomplished by introducing ligands carrying chelating structures to the nanoparticle surfaces. There are many nanoparticles described in the scientific and patent literature.

Nanotechnology in combination with chemistry generates many various chemical platforms for design of nanostructures. Some examples are nanostructures built up by, dendrimers, liposomes, magnetic nanoparticles, metal nanoparticles, metal oxide nanoparticles, metal hydroxide nanoparticles, metal sulphide nanoparticles, micelles, nano-assemblies, polymeric nanoparticles, and viral nanoparticles.

Medical imaging

Medical imaging is a rapidly evolving and highly multidisciplinary field that involves technologies from a variety of disciplines including physics, chemistry, physiology, and biology as well as engineering and computer science. Information generated through medical imaging is of interest in biology, drug development and in the clinic.

The in vivo preclinical animal market started to take off in the late 1990s when scientists saw that imaging could help them reduce costs and time span of their animal studies while making results more quantitative, reliable and reproducible. Imaging has become an increasingly important tool in drug development/ drug discovery and the understanding of basic pathological physiology, where changes at the organ, tissue, cell or molecular level in animal models must be closely monitored over time. By using imaging the number of animals can be reduced substantially compared with classical studies using histology methods, where animals had to be sacrificed at every time/data point throughout the study. Using imaging it is possible to follow one and the same individual animal over time, over days, weeks and even months. This increases the reliability and accuracy of the imaging since errors due to individual differences or inaccuracies due to non representative choice of tissue slices. The use of medical imaging as tool in clinical trials has increased since it enables rapid diagnosis with visualization and quantitative assessment. Clinical endpoints or outcomes are used to determine whether the therapy is safe and effective. Once a patient reaches the endpoint, he/she is generally excluded from further experimental interaction. Trials that rely solely on clinical endpoints are very costly as they have long durations and tend to need large number of patients. In contrast to clinical endpoints, surrogate endpoints have been shown to cut down the time required to confirm whether a drug has clinical benefits. Imaging is able to reveal subtle change that is indicative of the progression of therapy that may be missed out by more subjective, traditional approaches. Statistical bias is reduced as the findings are evaluated without any direct patient contact. For example, measurement of tumour shrinkage is a commonly used surrogate endpoint in solid tumour response evaluation.

Medical imaging has become an important tool not only in applications such as drug development but also in clinical diagnosis and surgery. Therapy areas including diagnosis of cancer, cardiovascular disease, gastrointestinal condition orthopaedics and trauma are some examples. The imaging technology platform is non-invasive and can reduce health care costs and improve patient management. In the near future therapies such as stem cell therapy and cell transplantation is on the way of exploring the benefits from medical imaging in the process of tracing implanted cells and as a tool to monitor the progress of the therapy related to the transplanted cells.

The imaging instruments that are available to the medical community can thus provide anatomical information using computer tomography (CT), magnetic resonance imaging (MRI) and ultrasound (US) and functional information using positron emission tomography (PET), single photon emission computed tomography (SPECT) and functional magnetic resonance imaging fMRI. In addition, optical imaging can be used for functional information, however as of today mostly utilized in preclinical settings and it is under development for clinical use. The trend in imaging instrumentation is not only to build instruments with higher resolution and performance but also to combine modalities such as MRI and PET in one and the same instrument.

A medical contrast medium (or contrast agent) is a substance used to enhance the contrast of structures or fluids within the body in medical imaging. Traditional contrast agents are often small molecules, for example iodine or bromine as X-ray contrast agents and gadolinium complex in MRI. For PET and SPECT the imaging is based on a radionuclide, which is hence necessary for those imaging modalities. Radionuclides commonly used are 99mTc, niln and 6yGa for both planar and SPECT imaging studies. Several positron emitting radio metals, specifically 64C11, 68Ga and 89Ζ1· have shown significant potential as molecular imaging probes based on PET. Molecular Imaging

The concept Molecular Imaging emerged in the early twenty-first century as a discipline at the intersection of molecular biology and in vivo imaging. It enables the visualisation of the cellular function and the follow-up of the molecular process in living organisms without perturbing them. The multiple and numerous potentialities of this field are applicable to the diagnosis of diseases such as cancer, and neurological and cardiovascular diseases. Molecular imaging differs from traditional imaging in that probes known as biomarkers are used to help image particular targets or pathways. Biomarkers interact chemically with their surroundings and in turn alter the image according to molecular changes occurring within the area of interest.

Sentinel node imaging

Metastatic disease in cancer forms such as breast cancer and malignant melanoma occurs mainly through the lymphatic system. Histological examination of the first lymph node draining the area where the tumor is situated (the sentinel lymph node, SLN) can reveal if the tumor has metastasized. It is a strong prognostic factor governing diagnosis, staging and management of the disease. At present the procedure of SLN-biopsy is performed by 2-24 hours prior to surgery injecting a Tc" ^m -labelled nanocoUoid into or around the primary tumor. In the case of breast cancer the injection may also be placed under the areola. Two to four hours preoperatively a

lymphoscintigram is acquired to visualize the SLN. Between 5 and 15 minutes prior to surgery a blue dye is injected, in the same manner as the nanocoUoid, to help visualize the lymphatic vessels and the SLN. Using the blue dye and a gamma probe as well as the scintigraphic images the surgeon can localize and excise the SLN. As the radioactive colloid, and in some cases the blue dye as well, will have spread to adjacent lymph nodes, it can be difficult to identify the SLN. To manage this uncertainty, more lymph nodes have to be resected for histological examination. While the pathologist examines the SLN, the surgeon proceeds to remove the primary tumor. Before finishing the surgery the histological findings are reviewed and, depending on the presence of metastatic cells, a decision on whether a complete axillary evacuation is necessary is made. A multimodal contrast agent that only targets the SLN in combination with several days of lymphatic retention time would simplify the logistics related to the current method of sentinel lymph node dissection. It could also help visually guide the surgeon intraoperatively and hence avoid the risk of hypersensitivity reactions that can occur when using the blue dye. A nanoparticle consisting of an iron oxide core with a biocompatible coating carrying a radionuclide can be imaged with magnetic resonance imaging (MRI) or PET/SPECT 1-2 days prior to surgery. By coupling different functional groups to the particle, such as fluorescent dyes and antibodies, the particle can be made multifunctional and allows for the surgeon to visualize the nanoparticles by for example hand held optical imaging devices or ultrasound.

The pharmacokinetics of nanoparticles for visualizing SLN depends on the choice of material, surface charge, size, colloidal stability and biological compatibility. Out of these, size has shown to be most significant for the uptake of particles in the lymphatics after subcutaneous injection. Numerous studies have concluded that the ideal size of a particle intended for lymphatic uptake is io to loo nm. A smaller particle will be absorbed directly into the bloodstream while a larger particle will remain at the site of injection. Particles that are taken up in the lymphatic system will travel with the lymph to the regional lymph nodes where they, depending on size and surface characteristics get trapped. Lymph enters the node through the afferent vessel, flows through the subcapsular sinus, followed by the cortical sinus and finally the medullary sinuses before exiting through the efferent lymphatic vessel.

Along the way, particles can get mechanically filtered out in the reticular meshwork of the sinuses and phagocytized by macrophages and dendritic cells. Sufficiently small particles may also be taken up by the endothelial cells through pinocytosis. Particles that are not trapped by the SLN will follow the lymph downstream to the next lymph node, eventually reaching the blood stream via the ductus thorascicus ending up in the reticuloendothelial system.

Radioisotope therapy (RIT)

Systemic radioisotope therapy is a form of targeted therapy. Targeting can be due to the chemical properties of the isotope such as radioiodine which is specifically absorbed by the thyroid gland. Targeting can also be achieved by attaching the radioisotope to another molecule, nanoparticle or antibody to guide it to the target tissue.

In order to reach cancer cells and to optimize the target load of the nanoparticle the nanoparticles will have to carry a specific targeting ligand on their surfaces. One option is to use antibodies specific for the target cells or fragments thereof. The antibody will not carry any radionuclides, rather the nanoparticle coating will be loaded with radionuclides. It is further possible to label each nanostructure with a mixture of multiple radionuclides to increase the therapeutic effects.

A major use of systemic radioisotope therapy is in the treatment of bone metastasis from cancer. The radioisotopes travel selectively to areas of damaged bone, and spare normal undamaged bone. Isotopes commonly used in the treatment of bone metastasis are strontium-89 to yttrium-90.

Other examples of RIT are the infusion of Metaiodbenzylguanidine (MIBG) to treatneuroblastoma of oral iodone-131 to treat thyroid cancer or thyrotoxosis, and of hormone bound lutetium-177 and yttrium-90 to treat neuroendocrine tumours.

Another example is the injection of radioactive glass or resin microspheres into the hepatic artery to radioembolize liver tumours or liver metastases.

Photon activation radiotherapy (PAT) relies on the administration of a drug containing a high-Z element prior to external irradiation with X-rays. Photo-interactions with the administered drug will then produce low-energy photo-electrons and Auger electrons, resulting in a high local absorbed dose. If the drug is targeted to tumour cells, this can potentially yield a very large therapeutic absorbed dose ratio between tumour and normal healthy tissue. Due to the very short range of these low-energy electrons, however, the drug needs to be accumulated into the cell close to the cell nucleus, in the close vicinity of the DNA, in order to be optimally effective which is possible with internalizing nanoparticles. Elements that have been suggested for PAT include indium, gadolinium and gold. Prior art

There exist several methods to produce formulations for use in medical imaging or radio isotope therapy. Most formulations can only be used for specific radio nuclides as described in WO 2011/070203. In other applications the whole nanoparticle need to be prepared in connection with the use as described in WO2007008566. Further, in WO 2011/070203 nanostructures are described which display multiple chelating groups on their surface. Such nanostructures easily react with components in the surrounding media and influence in a negative way the stability of the nanostructure in vivo and it can enhance non-specific interactions. Further in an article by Yu W et al in Nanotechnology 17(2006) pages 4483-4487

"Aqueous dispersion of monodisperse magnetic iron oxide nanocrystals through phase transfer" a method of preparing stable radio nuclides is described.

The invention It has now been unexpectedly found that by modifying the method described by Yu W et al stable multimodal agents ready for one step labeling process with a radio nuclide of choice. The labelling process can be done in a very easy one step process without having to elevate temperatures or perform any chemical reactions. It can be done in vicinity to the source of radionuclide and hence even short half life nuclides (isotopes) are accessible for multimodal imaging using this invention as a therapeutic agent or as an imaging agent in PET/SPECT or in a combinatorial mode using any of the modalities CT/PET/SPECT/MRI/Ultrasound and optical imaging methods. The invention is described in detail in the following:

With the agent produced of nanoparticles designed as contrast agents according to the present invention a biocompatible, stable, functionalized nanostructure that can bind a huge variety of metals and radio nuclides tightly in one single and easy labelling step is provided. The binding of the radionuclides or metals takes place in the interior of the coating avoiding interfering or competition with the surface of the structure or with surface ligands. The outer surface of the coating offers many sites for further conjugation of ligands such as fluorescent dyes, peptides, toxins, antibodies or fragment thereof or other entities. The conjugation chemistry can be chosen from a variety of molecules depending on the polymer chosen to act as the outer component of the surface coating.

Nanoparticles designed as contrast agents differ in many aspects from the traditional contrast agents. The characteristics of the nanoparticles when used as contrast agents open up for other and novel type of imaging studies. Nanoparticle based contrast agents have a size in the range of 5 - 100 nano meter, that is larger than a small molecule, which will influence their physiochemical and pharmacokinetic properties. The surface coatings of the nanostructure can add a huge impact/effect on

physiochemical and pharmacokinetic properties. It is further possible to put recognition molecules or a combination of such molecules, capable of binding to biomarkers, epitopes and cell receptors, or functional groups on the surface of the particle to promote the targeting of the contrast agent to a certain area within the body. The possibility to combine a certain size with coating materials and surface groups provide the designer of the nanoparticle with an interesting toolbox of varieties.

By combining imaging methods more details and information from the same study can be obtained. In many cases multimodal contrast agents are desirable for imaging in a combination of modalities like MRI/PET/SPECT/optical imaging, ultrasound and CT. One example is MRI in combination with PET. MRI has a high spatial resolution, however a low sensitivity. PET/ SPECT on the other hand has a very high sensitivity and poor spatial resolution. The combination of the two can overcome their individual drawbacks and multimodality instruments have recently been commercialized on the preclinical market as well as on the clinical market. If the contrast agent is multimodal, that is it gives rise to contrast in more than one imaging modality, the combined multimodal imaging study can be performed rather convenient using one contrast agent.

One example is the combination of MRI and PET. By using a MRI contrast agent such as iron oxide or gadolinium together with an isotope and a fluorescent dye in one and the same embodiment the imaging study can be performed first in PET which have very low detection limits, followed by MRI to get the best spatial resolution. If one wants to follow up the imaging results using histology, the fluorescent dye can be used to track the contrast agent.

Another example is to use gold nanostructures carrying a radionuclide of choice, for instance ⁶⁴ Cu in order to create a contrast agent for combined CT/PET imaging.

It is very important that the binding of radionuclide or metal compound can take place in an easy operation carried out by the customer in direct connection with the treatment. Radionuclides sometimes have a very short half life and need to be prepared close to the site of intended use or close to the patient. With the agent produced according to the present invention an agent labelled with a radio nuclide can be prepared within a few minutes at room temperature. The multisite chelating coating enables a high concentration of isotopes within a small local volume, which in turn increases the effect and is promising for therapeutic purposes. It is further possible to combine targeting ligands, radionuclides, fluorescent molecules or drugs on the nanoparticle surface.

Furthermore, it is possibilities to design multipurpose or multimodal nanostructures that are contrast agents interesting in molecular imaging combined with surgical procedures such as surgery of tumour. Due the possibility to vary the size of the nanostructure it is possible to use size as a targeting properly in lymph node imaging and surgery. A nanoparticle able to target and image the sentinel lymph node (SLN) of patients with breast cancer or malignant melanoma or other metastazing tumors can greatly improve and facilitate the surgical procedure. Using multimodal nanoparticles designed to have a retention time in the SLN of for at least 24 hours - up to a week, allowing for diagnostic imaging prior to surgery. The size of the nanoparticles are normally 5-100 nm, especially 10 to 100 nm.

Utilizing the possibility of creating multimodal nanostructures enables a combination of drug (radionuclide) delivery and the possibility to follow the delivery process with medical imaging. Combining a core of a metal or metal oxide compound like iron oxide, gold or a gadolinium or manganese, with a well designed coating, that is capable of carrying radionuclides and other molecules as well as displaying targeting units with a minimum of nonspecific interactions, gives on hand a multimodal contrast reagent useful for combined multimodal imaging and therapeutic applications.

To be useful as contrast agents in such applications the nanoparticle has to be: - designed to give contrast in the chosen imaging modality (ies)

- biocompatible with a surface that minimize unspecific interactions

- stable over time during storage as well during handling

- capable of being functionalized in order to directed the nanostructure to specific target sites in the body following systemic administration

- capable of being functionalized in order to carry additional imaging components following systemic administration

To be useful as drug delivery vehicle they have to be: - designed to give contrast in the chosen imaging modality (ies)

- biocompatible with a surface that minimize unspecific interactions

- stable over time during storage as well during handling

- capable of carrying a drug component and directed to specific target sites in the body following systemic administration - capable of being functionalized in order to carry additional imaging components following systemic administration

To be of commercial interest regardless of application the production process, quality control and cost of production has to be reasonable in relation to the price of the final product. The contrast agent or drug delivery vehicle has to be stable enough to be shipped and stored with a reasonable long shelf life. Furthermore, it should be easy for the end-user to add a biomarker of or any other ligand of choice. This requires robust coating, which provides accessible functional groups for further modification.

The general object of the invention is to solve the problem of producing a new agent which can be used for administering a high local concentration of an isotope to a site in the body. The chelating properties enable an easy, very quick and robust labelling with radionuclides in a manner that is feasible in practical applications both preclinically and for clinical purposes. The coating material can be utilized to produce nanoparticles as contrast agents in multimodal imaging for diagnostic purposes and for guidance during surgery. The nanostructures can further be very useful as drug delivery vehicles in radiotherapy and in combinations of therapy and imaging. This innovation describes a coating material with the inherent capability to chelate metal ions and radionuclides in the interior of the coating. This is an important difference in comparison with earlier nanostructures carrying nuclides, where isotopes have been trapped inside the interior of liposomes or are bound in an inner shell. In both these previously described cases the whole nanostructure has to be synthesized from the ground up for each single use. This innovation provides a method in which the nanostructure can be pre-manufactured and then labelled with a metal ion or a radionuclide just prior to use in a quick one step protocol.

Another method tested previously is to attach chelators on the surface of a

nanostructure. This surface modification can induce non specific interactions of the nanostructure when used as a contrast or therapeutic agent. The surface modification can impact the stability of the nanostructure. Furthermore, various nuclides may require different radionuclide specific chelator and in turn the labelling may involve elevated temperatures and unfavourable reaction conditions for the nanostructure. Using this invention a more general chelator has been created and the labelling procedure is done in a few minutes, usually between 1-15 minutes, preferably 1-10 minutes. In addition the chelating unit is in the interior of the coating leaving the surface unchanged and intact.

The coating is useful for coating surfaces. According to the present invention a coated nanostructure is predominantly being used. However in other areas it could also be used for surfaces on analytical chip, implants etc.

Summary of the invention

The surface to be coated needs to carry a hydrophobic surface film. Examples of this are metal oxide cores, iron oxide cores, upconverting crystals etc. Such cores are produced by seed growth in a solvent protected by a fatty acid such as oleic acid or fatty acid coated structures for instance gold nanoparticles conjugated with thiol-functionalized oleic acid. The object of the invention is achieved by forming a coating material having an outer surface of a polymer and an inner chelating surface, which easily chelates metal ions and radionuclides by a) mixing and reacting a poly maleic anhydride having a hydrophobic tail of the formula (I) with

b) an amino terminal polymer (II) to form

c) a multi-amid structure with amides and carboxylic groups side by side d) whereafter said structure is added to a hydrophobic film coated surface whereby the interior of the coating layer forms a poly-chelator network.

Specifically, an agent in the form of nanoparticles in the range of 5-100 nanometer capable of chelating radionuclides or metal entities is prepared from cores in the size of 2 -50 nanometers having a hydrophobic layer from a fatty acid are further coated by

a) mixing and reacting a poly maleic anhydride having a hydrophobic tale

(compound I) with

b) an amino terminal polymer (compound II) in an amount of at least one amino group from the amino terminal polymer( compound II) to at least one malemide anhydride group from (compound I) to form

c) a multi-amid structure with amides and carboxylic groups vicinally

d) whereafter said structure is added to the hydrophobic layer coated surface of the cores, whereby the hydrophobic layer is made to get in contact with the

hydrophobic tail of compound I and whereby the core is coated and a poly-chelator network is formed around the core during the coating process to form the final product which contains nanoparticles with the polymer(compoundll) as the outer component brought in close proximity with a radionucleotide or metal ion for a short period of time(i-i5 minutes), preferably 1-10 minutes, to form the agent for medical imaging or radiotherapy.

It is important that the relation between the number of amino groups in formula (I) to the number of malemide groups in formula(II) are at least 1:1 in order to create as many chelating sites as possible. This is even more important if the formula (II) contains more than one free amino group. Otherwise there will be a risk of cross binding of the polymer. Preferably, there are an excess of amino groups. A suitable excess is 2-100 times depending on the chemical composition of the polyamine compound.

Metal chelates are well known in the art. Some well known are known as DOTA, DIPA, TETA NOTA. The above are only examples, also other macrocyclic metal chelates may be used.

In one embodiment of the invention the compound of the formula (I) is a poly maleic anhydride (see example PMAO (poly maleic anhydride-alt-i-octadecene)

(I)

wherein the open bonds each carries H2, n is between 80 and 135 and x is 16 reacted with an aminoterminal polymer, see example below with a Jeffamine® a

polyetheramine, 0,0-Bis(2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol ), having the formula (II)

H, N— Pol (ID wherein Pol stands for an otherwise inert non toxic hydrophilic polymer. Otherwise inert means that no unwanted reaction takes place. Examples of suitable polymers are amino polyethylene glycol (m-PEG-NH2),polyetheramines, amino polyethylene amines (NH2-PEG-NH2), amino dextrans, 0,0-bis(2-aminopropyl) polypropylene glycol- block-polyethylene glycol-block-polypropylene glycol of various lengths .

This last structure is exemplified by formula (III) below

wherein v+z is 2 - 39 and y is 1-6.

The reaction results in a multi-amide structure with amides and carboxyl group side by side. This reaction product is then added to the hydrophobic film coated surface of the core particles. The hydrophobic film is formed by an unsaturated fatty acid found in nature. One example is oleic acid. The hydrophobic tale of the compound of formula I interacts with the fatty acid and forms an inner hydrophobic layer. This in turn displays the anhydride part connected to the hydrophilic polymer towards the surrounding hydrophilic solvent. A multi site chelator is formed next to the hydrophobic layer. This chelator is strong and the amide and carboxylic groups in three dimensions create a network for metal (or nuclide) binding. The resulting coating on the nano particle has an outer surface of the polymer of choice. It may carry functional groups displayed on the outer surface. The inner space creates a poly-chelator network. It has been proven by binding various metal ions and isotopes. The binding takes place by contacting the nanoparticle coated with the polymer II as the outer surface component with a radionucleotide or metal ion. The inventors have also proven that the coating chelates strongly and the resulting nanostructure is perfect as combined contrast agent for MRI/PET (or SPECT) or optical imaging/PET (or SPECT) or CT/PET (or SPECT) or ultrasound/PET (or SPECT) or CT/PET (or SPECT) if the metal that is chelated is a radionuclide or multiple various radionuclides.

Suitable polymers in the outer layer are amino polyethylene glycol (m-PEG-NH2), polyether amines, amino polyethylene amines (NH2-PEG-NH2), amino dextranes, 0,0-bis (2-aminopropyl) polypropylene glycol-block-polyethylene glycol-block- polypropylene glycol of various lengths .

In another embodiment, the present invention relates to chelating metal ions that give rise to a contrast in MRI such as gadolinium, manganese, cobalt and iron etc. in a coating of upconverting crystals or quantum dots or gold nanoparticles to create a multimodal agent for MRI/optical imaging or CT/optical imaging.

Detailed description of the invention

Examples of core materials:

The size of the core is between ι and 50 nm, preferably between 2 and 50 nm. The core is coated with a micelle like structure of various molecules having one hydrophobic part and one hydrophilic part, the whole particle size is preferably between 2 and 100 nanometer.

Production procedure of cores:

This is common knowledge. The synthesis of cores, or production methods are important from a commercial stand point in order to handle scaling and GMP processes. The core material is often essential for one imaging modality.

There are many possible combinations of metal oxides/hydroxides for use as core material. As long as the resulting cores are hydrophobic on their surfaces (carrying molecules like oleic acid) they exhibit the appropriate surface for coating using the described coating material. Suitable hydrophobic compounds are unsaturated fatty acids found in the nature, especially oleic acid and variants thereof as well as similar compounds.

1. Hydrophobic cores:

a. Iron oxide cores:

Seed growth method by reducing FeO(OH) in i-octadecene and protected by oleic acid. All three ingredients are mixed and then heated to 320 C for 120 minutes under cooling and reflux of solvents. After 120 minutes the mixture is allowed to slowly cool down to room temperature. The cores are washed with diethyl ether and then precipitated with ether. This is a difference compared with earlier methods, where chloroform is used. Since chloroform is listed as an environmental hazard other solvents have to be tested in order to develop a sustainable production process. The procedure is repeated twice and the nanostructures are finally resuspended in diethyl ether.

Iron oxide cores are used to produce MRI contrast agents. b. Up-converting crystals

Upconverting crystals can be made using similar methods as described above as hydrophobic nano cores using oleic acid as protecting agent. An upconverting crystal contains a mixture of, Ytterbium and Yttrium and in combination with small amounts of other ions like for instance Erbium or Thulium in order to set the wavelength optimum of adsorbed and emitted light from these crystals, one example of

composition is: Na ₄ :Yb ³ VTm ³⁺ , which gives rise to emission of 8oo nm when illuminated with 980 nm light source.

Upconverting crystals are used as contrast agents in optical imaging, prefearably Near- IR fluorescence imaging.

c. Gold nanostructures

The production of gold nanostructure is well known and was pioneered by J. Turkevich et al. in 1951 and it is the simplest known method. The method is used to produce monodisperse spherical gold nanoparticles suspended in water of around 10-20 nm in diameter. It involves the reaction of small amounts of hot chlorauric acid with small amounts of sodium citrate solution. The colloidal gold will form because the citrate ions act as both a reducing agent, and a capping agent. The citrate ions can be exchanged with thiol groups known as the thiol ligand exchange method. To produce gold nanoparticles ready for the coating procedure according to the invention the thiol modified oleic acid is made to replace the citric acid on the nanostructure and the oleic acid stabilized gold nanostructures are then ready for the coating procedure. Gold cores are used as contrast agent in CT and optical imaging. d. Quantum dots

Typical colloidal quantum dots are made of binary alloys such as cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide. These quantum dots are produced in a similar process like a and b above where the synthesis of colloidal quantum dots is based on a three-component system composed of precursors of the binary alloy, organic surfactants such as oleic acid, and solvents such as octadecene. When heating a reaction medium to a sufficiently high temperature, the precursors chemically transform into monomers. Once the monomers reach a high enough supersaturation level, the nanocrystal growth starts with a nucleation process. The nanostructures are washed using similar process as described under a for iron oxide nanostructures. Quantum dot cores are used to create contrast (or signal) in fluorescence optical imaging.

Iron oxide core particles are important components in multimodal contrast nanostructures since the magnetic core give rise to contrast in MRI and ultrasound (in combination with an oscillating magnetic field or a magnetic pulse). It is further possible to label their coatings with fluorophors and/or nuclides for imaging in optical imaging and PET/SPECT respectively in combination with MRI and ultrasound - hence the expression multimodal nanoparticles. Gold cores gives rise to a contrast effect in CT and in some optical imaging applications and labelling their coatings with fluorophors and/or nuclides for imaging combinations optical imaging and

PET/SPECT respectively. Upconverting crystals coated according to the invention can act as multimodal agent for optical imaging in combination with PET/SPECT if a radionuclide is chelated in the coating. In another embodiment the cores mentioned above in combination with the coating can assemble chelated metal ions such as gadolinium and manganese or other metals that give rise to a specific contrast in MRI.

Coating procedure:

This production stems from the scientific paper by Yu W et al. mentioned in the prior art part. The coating is built up with PMAO (poly(maleic anhydride-alt-i-octadecene) and PEG (polyethylene glycol). The use of amino PEG is a good choice from an academic point of view, however expensive in commercial production. According to the present invention polyether amines such as the Jeffamine® molecules, 0,0-bis(2- aminopropyl) polypropylene glycol-block-polyethylene glycol-block-polypropylene glycol of various lengths are being used instead of PEG molecules. This lowers the production costs dramatically and results in an even more robust coating. The polymer length can further be adjusted by using various polymers of various lengths to the coating and provide various thickness of the coatings in a very controlled process. The size distribution is still kept very narrow and that is a necessity in order to perform quality control and batch-to-batch consistency.

The coating can be made thicker if that is of importance to the final application of the particle by adding polymers to the functional amino groups on the particle surfaces.

The choice of length of the first Jeffamine/ amino dextran coating step can obviously be varied as well in order to obtain a certain coating thickness and final radius of the nanostructure.

The amino terminal polymer used in the reaction can have amino group on each side of the polymer. With the amino groups not reacted with PMAO the polymer then have free amino groups are generated on the surface of the nanostructure and these amino groups are free to serve as conjugation candidates with other functional groups, e.g. antibodies.

In principle all types of polymers having at least one free amino group, which can react with the anhydride, may be used. Jeffamine is trade mark for a series of products that are polyether diamines. The ED Series are polyether diamines products based on PEG, which imparts complete water solubility to each of the products in the series. It is also possible to use terminal -OH groups on the polymer, which can react readily with the anhydride. PMAO has two functions, the first is carrying octadecene with a fatty tag which can form a micelle like inner coating close to the core and be the anchor for the coating. PMAO may vary in length and composition. The second important part is the anhydride and PMAO contains in between a few to up to loo such units.

It is important that the relation between the number of amino groups (or hydroxide groups) in formula (II) to the number of malemide groups in formula(I) are at least i:i in order to create as many chelating sites as possible. In the case of the molecule described by the formula (II) it is critical that it contains more than one free amino group. Otherwise there will be a risk of cross binding of the polymer. Preferably, there are an excess of amino groups. A suitable excess is 2-100 times depending on the chemical composition of the polyamine compound.

The coating has as characteristics that it is very stable in pH from 2 to 12, in serum and in molar range salt. It can be made with various coating thickness, which opens up for designing nanostructures with various size which can be crucial for the final application. Coating thickness between 3 and 30 nm are favourable, however other size ranges can be of interest as well.

Isotopes of interest to chelate with the agent in the form of nanoparticles (the list is not limiting):

Copper ( ^6l Cu, ⁶ 4Cu, and ⁶⁷ Cu), Indium ( ⁿ Tn), Technetium (99 ^m Tc), Rhenium ( ^l86 Re, ^l88 Re),

Gallium (^Ga, ⁶⁸ Ga), Strontium ( ⁸⁹ Sr), Samarium (^Sm), Ytterbium ( ^l6 9Yb), Thallium ( ²⁰¹ T1),

Astatine ( ²¹¹ At), Lutetium ( ¹⁷⁷ Lu), Actinium ( ²²⁵ Ac), Yttrium (9°Y), Antimony ( ⁿ 9Sb), Tin ( ⁱⁱ ySn, ⁿ 3Sn), Dysprosium ( ¹⁵⁹ Dy), Cobalt ( ⁵⁶ Co), Iron ( ⁵⁹ Fe), Ruthenium (97Ru, ¹⁰ 3Ru),

Palladium ( ¹⁰³ Pd), Cadmium ( ¹¹⁵ Cd), Tellurium ( ^ll8 Te, ¹²³ Te), Barium (^Ba, ^Ba),

Gadolinium (^Gd, ^Gd), Terbium ( ^l6o Tb), Gold 0 ⁸ Au, ^Au), Lanthanum (^La), and Radium ( ²²³ Ra, ²² 4Ra).

Metal ions of interest to chelate with the nanoparticles in the agent according to the invention (but not limited to):

Mn2+, C02+, Cu2+ and Gd2+

It is proven that the chelating effect is strong for multiple ions and isotopes. It is further shown that: a) The chelating effect is not depending on any surface functional groups such as for instance amino groups b) The chelating effect is not depending on the length of the hydrophilic polymer c) It is possible to combine the chelating interior without disturbing chemistry on functional surface groups where we have added fluorophores, antibodies etc. d) The chelating effect is stable at physiological pH and in serum e) The reaction is extremely quick, and normally takes 1-15 minutes, especially 1- 10 minutes. It is formed by simply mixing the metal ion or isotope at low pH with the nanostructure of choice. No incubation time is needed.

The temperature may be raised to 40-50 C, but this is not necessary.

The nanostructure is then separated from nuclides by magnetic separation or HPLC or desalting chromatography or any other suitable separation process that can be applied. The ready to use nanostructures are eluted in physiological salt solution.

Proof of concept has been shown in combined MRI, PET, SPECT, ultrasound and optical imaging detecting lymphnodes in animal models.

In the following several examples are given according to which the invention may be accomplished.

Example 1. Synthesis of nanoparticle cores (iron oxide) and upconverting cores

The iron oxide cores of the nanoparticles were produced according to the process described by Yu W et al. Briefly, 12.7 g Octadecene (Sigma- Aldrich, St Louise, MO, USA), 4.5 g Oleic acid (Sigma-Aldrich) and 356 mg Iron(III) oxide-hydroxide (Sigma- Aldrich) were mixed in a reaction vessel. The mixture was heated to 323 °C for 60 minutes with constant stirring and then allowed to cool to 35 °C, before removing it from the reaction vessel. A sample of the cores was dissolved in hexane and passed through a 0.1 μηι PTFE filter (Whatman, Maidstone, UK). The size of the filtered particles was determined with Dynamic Light Scattering (DLS) in a Malvern Zeta Sizer Nano Series (Malvern Instruments Ltd, Worcestershire, UK) using an acrylic cuvette and measurement parameters for Fe ₃ 0 ₄ . The core size was also confirmed by transmission electron microscopy (TEM). Particles were deposited on a carbon grid and imaged with a FEI Tecnai Spirit BioTWIN transmission electron microscope.

Example 2:

Coating of the particles with various coating thickness

Three variants of nanoparticles were designed using the same core size but varied coating thickness. The three nanoparticles had a final hydrodynamic diameter of 15 nm, 34 nm and 57 nm respectively. They were produced according to the following protocol.

The iron oxide cores were washed by dissolving the reaction mixture in

diethylether (Sigma- Aldrich). The cores were percipitaded by gradual addition of reagent grade absolute ethanol (Scharlau, Barcelona, Spain). The percipitaded cores were collected at the side of the vial with a permanent magnet and the ether-ethanol mixture was removed by decanting. This process was repeated once more. The cores were then resuspended in chloroform (Sigma- Aldrich). O, 0'-Bis(2-aminopropyl) polypropylene grycol-WocA:-poryethylene glycol-WocAi-porypropylene glycol 1900 (Jeffamine ED-2003) (for the 15 nm particle Jeffamine ED-900 was used) and

Poly(maleic anhydride-alt-i-octadecene) (PMAO) were weighed in separate glass flasks and mixed with chloroform. The flasks were heated in a 61 °C water bath until the

Jeffamine and PMAO had dissolved. The Jeffamine was added to a round bottom flask and the PMAO was mixed in in smaller fractions while the flask was swirled to avoid areas with a locally high concentration of PMAO cross binding with the Jeffamine. The flask was placed on a rotary evaporator and spun for 5 minutes after which the iron oxide cores were added and the mixture was spun for an additional 5 minutes. An equal amount of Milli-Q (MQ) water and chloroform was added and the flask was lowered into a 61 °C water bath. The flask was spun at 230 rpm and the pressure was gradually lowered to 100 mbar to evaporate the chloroform.

After removal of the chloroform the volume of water solution containing the particles was measured and an equal volume of 300 mM NaCl was added, to make the SPIONs solution ready for injection. The particles were passed through a filter paper (454, VWR) to remove larger debris. Excess coating material and large complexes were removed by diafiltration in three steps (KrosFlo Research Hi TFF System, Spectrum Laboratories, Inc.). First the particles were passed through a 0.2 μηι PES filter (X32E-300-02N, Spectrum

Laboratories, Inc.) followed by a 500 kD PES filter (P-D1-500E-100-01N, Spectrum Laboratories, Inc.), removing unwanted complexes and larger particles. Finally a 300 kD mPES filter (P-D1-300-E-100-01N, Spectrum Laboratories, Inc.) was applied that retains the particles but excess coating material will pass through. The pump was run at the maximum flow rate (480 mL/ min) with the inlet pressure of the membrane kept at approximately 28 psid for the 0.2 μηι and 500 kD filters. The filtration was run at a constant volume and was allowed to proceed until the permeate was clear. For the 300 kD filter the volume was first reduced to approximately 50 mL and the particles were then washed over night against 5 L 150 mM NaCl at constant volume mode. The next day the volume was reduced to the void volume of the system and the filter was exchanged for a smaller surface area filter with the same rating (C02-E300-05-N, Spectrum Laboratories, Inc.). The tubing of the system was also exchanged for a smaller size tubing. The volume was further reduced to a final volume of 4-5 mL.

To enable detection of the nanoparticles in histology sections, the fluorescent dye DY- 647 (Dyomics) was conjugated to the particles. Amino groups in the coating are used to conjugate the dye through N-Hydroxysuccinimide (NHS) chemistry. For the smallest particle 3 mL of SPIONs (724 μg Fe/mL) were mixed with 3 mL of carbonate buffer (0.2 M NaHC0 ₃ , 0.5 M NaCl) pH 8.3. To a glass vial 50 μΐ, 13 mM NHS-DY-647 in Dimethyl-Sulfoxide (DMSO) was added, followed by the SPION mixture giving an excess of approximately 350 dye molecules per particle. For the particles coated with Jeffamine ED-2003 5 mL SPIONs (1216 μg Fe/mL) was mixed with 5 mL carbonate buffer. The SPION mixture was divided into two vials containing 40 μί, i3mM NHS- DY-647 in DMSO, each. The vials were incubated for 3 hours at a tipping table. For the construction of the 58 nm particle, after 3 hours incubation, 155.2 mg 30 kD mPEG- SMC (Creative PEGWorks) was added to one of the vials giving an excess of 2000 PEG molecules per particle. The reactions were allowed to continue for an additional 3 hours before the SPIONs were purified using diafiltration. The three different sized agents mentioned above having a hydrodynarnic diameter of 15, 34 and 52 nanometer, respectively were used to identify the sentinel node in a animal model using MRI, briefly it could, be concluded that there were clear differences in lymphatic uptake and drainage between the three sizes. In conclusion, the thickness of the coating can be used in the design of a targeted nanostructure.

Example 3 Coating load

Evaluation of the chelating properties of a coating based on PMAO and Jeff amine ED-2003 according to the invention Ironoxide coated nanostructures with a diameter of 34 nm was synthesised according to the protocol described under Example 2 using Jeffamine ED-2003 (O, 0'-bis(2- aminopropyl) polypropylene glycol-WocA:-polyethylene glycol-WocAi-polypropylene glycol 1900) as Formula II and PMAO as Formula I according to the invention. The resulting nanostructures are herein referred to as SPIO34.

Method A

Triplicates of 400 μΐ of SPIO34 (2 mg Fe/ml) were buffer exchanged using a magnetic separation column, M Column (Miltenyi Biotech, Germany) to trap the particle and then elute them in 0.05 M ammonium acetate buffer pH 4.0 (Buffer A). The samples were each divided into two vials. To the first vial 200 μΐ of 100 mM GdCl3 dissolved in Buffer A was added and to the second vial only Buffer A was added. All samples were incubated for 5 minutes.

After incubation the samples were all washed according to the following scheme:

1. The particles were trapped in an M column

2. The particles trapped in the column were then washed using 5χ400μ1 0.05 NaCl 3. The particles trapped in the column were then washed using 5χ400μ1 0.05 NaCl + 0,1 % (v/v) glycerol.

4. The particles trapped in the column were then washed using 5χ400μ1 0.15 M NaCl Finally the particles were eluted in 200 μΐ of 0.15 M NaCl. The eluted sample was dissolved in 2 M HCL and diluted into a total volume of 5 ml. All samples including blanks were analyzed using ICP-OES (Inductively Coupled Plasma-Optical Emission Spectroscopy) for iron and gadolinium content. The analysis was performed on an Optima 8300 (Perkin Elmer, USA) calibrated against freshly prepared standard solutions (SPEX CertiPrep, UK).

The results showed that each particle can bind at least 1000 Gd ³⁺ ions in the coating. Other metals tested were Cu ²⁺ , Al ³⁺ and Ga ²⁺ , which showed similar results.

To test if the labelling was affected by incubation in serum (in vivo like conditions) the samples were incubated in NCF (natal calf serum) overnight at 37°C. After incubation the SPIO34 were washed and analysed according to the protocol described above. The result showed that the particles carried the same amount of metal ions/particle even after this incubation in serum.

To evaluate if the terminal amino groups on the surface are involved in the chelating procedure these groups were quenched on SPIO34 using 0-[(N-succinimidyl)succinyl- amino ethyl] -O'-methyl polyethylene glycol 2000. The amount of free amino groups was measured using a fluorometric assay with Fluram (a reagent that reacts with primary amino groups) and it was concluded that the free amino groups had been eliminated after the quenching reaction. This nanostructure is herein refereed to as SPI034Met.

Metal ions were added and analysed according to Method A above using SPI034Met instead of SPIO34 and it was concluded that the SPI034Met bound the metal ions equally well compared with the SPION34 and hence the primary amino groups did not have a critical role in the chelating effect of the invention. Example 4. Detection of Sentinel Node in combined PET/MRI and Cherenkov imaging (optical imaging)

Iron oxide (Fe ₃ 0 ₄ ) crystals with a diameter of 10 nm ± mm were synthesized and coated with according to the method described in example 1 and example 2 using PMAO in combination with Jeffamine ED-2003. ⁶⁸ Ga was generated from a ⁶⁸ Ga/ ⁶⁸ Ge - generator system (IDB, Holland) and the ⁶⁸ Ga was eluted in 0.6 M HC1. A fraction containing 40-80 Mbq of ⁶⁸ Ga was used for labeling 4x1ο ¹⁴ nanostructures. The nanostructures were labeled with ⁶⁸ Ga in various pH from 3.5 to pH 5.0 with successful results. Labeling in pH above pH 5.0 reduced the labeling ratio per nanostructure most probably due to insoluble metal hydroxides formed by ⁶⁸ Ga ²⁺ . The labeling efficiency and stability of the labeling in human serum were determined using instant thin layer chromatography. An amount of 0.07-0.1 mL (-5-10 MBq, 0.13 mg Fe) of ⁶⁸ Ga -SPIONs was subcutaneously injected in the hind paw of 9 normal rats. The animals were imaged with PET/CT and 9.4T MR systems at 0-3 h and 25 h post injection (p.i.) in vivo. Three rats were imaged ex-vivo with a CCD-based Cherenkov optical system. A biodistribution study was performed by dissecting and measuring the radioactivity in lymph nodes, kidneys, spleen, liver and the injection site.

The labeling yield was 97.3 % after 15 min and the ⁶⁸ Ga -SPIONs were stable in human serum. All three imaging modalities, PET/MR together with Cherenkov luminescence imaging, clearly visualized the SLN. The mean uptake of ⁶⁸ Ga-SPIONs after 3 h p.i. was 123 % IA/g in SLN (popliteal node), 47 % IA/g in the inguinal node, a mean of 0.05 % IA/g in kidneys, 0.1 % IA/g in the spleen and 0.3 % IA/g in the liver.

Example 5 Synthesis of Upconverting nanoparticles ready for tandem imaging in fluorescence imaging and PET/SPECT or fluorescence/MRI

1. 146.3 mg YCI3, 70.0 mg YbCl3 and i.,o mg TmCl3 were mixed with 4 ml oleic acid and 17 ml l-Octadecene at room temperature for 10 minutes. The solution was put under vacuum and the temperature was elevated to 130°C, 3°C/min. The temperature was then held at iso°C for 30 minutes. The mixture was removed from the heat and cooled to room temperature.

2. Into a second reaction vessel 148.6 mg NH4F and 100.6 mg NaOH were mixed with 10 ml methanol. All components were mixed and dissolved using an ultrasonic batch. 3. Solution number 2 was added drop wise to solution 1. The temperature was then heated slowly to evaporate the methanol at ioo°C. When the solution had reached ioo°C an Argon flow was allowed to bubble through the solution. The solution was then heated to 320 C as fast as possible and then maintained at 320°C for 25 minutes. The mixture was then cooled down fast to room temperature using a cool water bath.

4. The solution from 3 was washed according to a procedure, where ether and ethanol were added to precipitate the cores. After a centrifugation step at 5 min, 4000xg the supernatant was discarded and the precipitated nanostructures were resuspended in Hexane. The core size was approximately 35 nano meters and they showed a high emission peak at 800 nano meters when illuminated with a 980 nm laser source.

5. The cores were then washed once more and resuspended in chloroform (Sigma- Aldrich). O, 0'-bis(2-aminopropyl) polypropylene glycol-WocAi-polyethylene glycol- WocA:-polypropylene glycol 1900 (Jeffamine ED-2003) and Poly(maleic anhydride-alt- l-octadecene) (PMAO) were weighed in separate glass flasks and mixed with chloroform. The flasks were heated in a 61 °C water bath until the Jeffamine and PMAO had dissolved. The Jeffamine was added to a round bottom flask and the PMAO was mixed in in smaller fractions while the flask was swirled to avoid areas with a locally high concentration of PMAO cross binding with the Jeffamine. The flask was placed on a rotary evaporator and spun for 5 minutes after which the upconverting cores were added and the mixture was spun for an additional 5 minutes. An equal amount of Milli- Q (MQ) water and chloroform was added and the flask was lowered into a 61 °C water bath. The flask was spun at 230 rpm and the pressure was gradually lowered to 100 mbar to evaporate the chloroform.

6. After removal of the chloroform the volume of water solution containing the particles was measured and an equal volume of 300 mM NaCl was added. The particles were passed through a filter paper (454, VWR) to remove larger debris. Excess coating material and large complexes were removed by diafiltration in three steps (KrosFlo Research Hi TFF System, Spectrum Laboratories, Inc.). First the particles were passed through a 0.2 μηι PES filter (X32E-300-02N, Spectrum Laboratories, Inc.) followed by a 500 kD PES filter (P-D1-500E-100-01N, Spectrum Laboratories, Inc.), removing unwanted complexes and larger particles. Finally a 300 kD mPES filter (P-D1-300-E- 100-oiN, Spectrum Laboratories, Inc.) was applied that retains the particles but excess coating material will pass through. The pump was run at the maximum flow rate (480 mL/min) with the inlet pressure of the membrane kept at approximately 28 psid for the 0.2 μηι and 500 kD filters. The filtration was run at a constant volume and was allowed to proceed until the permeate was clear. For the 300 kD filter the volume was first reduced to approximately 50 mL and the particles were then washed over night against 5 L 150 mM NaCl at constant volume mode. The next day the volume was reduced to the void volume of the system and the filter was exchanged for a smaller surface area filter with the same rating (C02-E300-05-N, Spectrum Laboratories, Inc.). The tubing of the system was also exchanged for smaller sized tubing. The volume was further reduced to a final volume of a few ml in 0.15 M NaCl and was ready for imaging using optical near IR imaging in combination with PET or SPECT.