WO2014090313A1

WO2014090313A1 - Nanoparticle with a molecularly imprinted coating

Info

Publication number: WO2014090313A1
Application number: PCT/EP2012/075424
Authority: WO
Inventors: Thomas Simmet; Berthold BUECHELE; Oleg LUNOV
Original assignee: Universitaet Ulm
Priority date: 2012-12-13
Filing date: 2012-12-13
Publication date: 2014-06-19

Abstract

The invention relates to a structure with a core and a coating imprinted with a specific molecule, wherein the structure is a nanoparticle. The invention further relates to the use of such a structure in an imaging method, in a method for the localised induction of hyperthermia and for the separation and/or isolation of cells, protein and/or nucleic acids. Furthermore, the invention comprises a method for the production of such a structure and a kit containing such a structure.

Description

Nanoparticle with a molecularly imprinted coating

Background of the invention

The invention relates to a structure with a core and a coating imprinted with a specific molecule. Furthermore, the invention relates to the use of such a structure in an imaging method, in a method for the localised induction of hyperthermia and in a method for the separation and/or isolation of cells, protein and/or nucleic acids. In addition to this, the invention comprises a method for the production of the structure and a kit containing the structure.

Prior Art

In sample analysis and compound purification it is often desirable to specifically adsorb a target molecule to a solid phase sorbent. One way to produce such a sorbent is by molecular imprinting. In this method, the target molecule is brought into contact with a monomer which is subsequently polymerised to yield a solid surface with the tightly embedded target molecules. Using a suitable solvent, the target molecules can then be washed out of the surface leaving behind surface cavities with a high affinity to the target molecule on account of a favourable charge distribution and three-dimensional structure of the cavities. When a sample is brought into contact with that surface, the target molecule fills the cavities in a highly selective manner. Thereafter, the target molecule can be washed off the molecularly imprinted polymer in order to be analysed. In the publication "Molecularly-imprinted polymers: Useful sorbents for selective extractions", Trends in Analytical Chemistry, vol. 29, no. 11 , 2010, A. Beltran et al. describe the synthesis and application of molecularly imprinted polymers. According to Beltran, the main

components involved in the production of a molecularly imprinted polymer are the template molecule, the functional monomer and the crosslinking agent. Although there are many different commercially available functional monomers to choose from, the most widely used to date have been methacrylic acid and 4-vinylpyridine. The most widely used crosslinking agent is ethylene glycol dimethacrylate (EGDMA). When molecularly imprinted polymers are used in sample analysis, a dramatic improvement in extraction selectivity can be obtained, as the imprinted polymer sorbent retains the target analyte more strongly than the rest of the compounds also present in the sample. The high selectivity of molecularly imprinted polymers enables compounds of interest to be detected at concentration levels that would not have been obtained with conventional solid phase extraction solvents.

H. H. Weetall et al. report the production of polymer-coated electrodes in "Preparation and characterisation of molecularly imprinted electro-polymerised carbon electrodes", Talanta, vol. 62, 2004, p. 329 to 335. The authors have combined the use of molecularly imprinted polymers and electropolymerisation to produce a sensing electrode that is capable of detecting small molecules. The chosen template molecules were fluorescein in one case and rhodamine in the other. Each electrode was shown to selectively retain fluorescein or rhodamine from a solution containing both compounds.

Furthermore, superparamagnetic nanoparticles, which have been conjugated to streptavidin in order to bind to biotinylated proteins, in particular biotinylated antibodies, are already known. Such superparamagnetic antibodies conjugated nanoparticles can be used in cell, protein and/or nucleic acid purification, detection and analysis. While streptavidin shows a high affinity to biotin, it also binds proteins unspecifically due to its charge and glycosylation. Additionally, sepharose bead coupled protein A or G has been used in co- immunoprecipitation. In this method, an antibody against an epitope on a target protein is used to precipitate the protein-antibody complex by binding of the complex to the sepharose bead coupled protein A or G. This is followed by centrifugation or magnetic separation. As protein A and G predominantly bind to immunoglobulins, this approach can mainly be used to attach nanoparticles to antibodies and not to other proteins. The unspecific binding of proteins to protein A and G requires numerous controls and pre-absorption of cell lysates which may cause an undesired loss of the target proteins. It is well known that superparamagnetic nanoparticles can be formed from ferromagnetic or ferrimagnetic materials. When used in medical imaging as well as compound separation and purification, a high magnetisation (as measured in emu/g) of the nanoparticles is often desirable to increase imaging sensitivity and purification efficiency. While high

magnetisations have been reached in nanoparticles containing cobalt, such nanoparticles are of limited use in biological systems due to the inherent toxicity of cobalt.

In the publication "TEMPO supported on magnetic C/Co nanoparticles: A highly active and recyclable organocatalyst", Chem. Eur. J. 2008, 14, 8262 - 8266, A. Schatz et al. report graphene coated nanobeads with a magnetic cobalt core, which were created on a large scale by reducing flame synthesis. TEMPO was grafted on the nanobeads using a "click"- chemistry protocol. The heterogeneous TEMPO-nanobeads can function as a highly active catalyst for the chemoselective oxidation of primary and secondary alcohols using bleach as terminal oxidant.

The patent application US 2008 / 0213189 A1 discloses nanocrystals comprising metals and metal alloys, which are formed by a process that results in a layer of graphite in direct contact with the metallic core. Preferred metals include iron, gold, cobalt, platinum, ruthenium and mixtures thereof, for example FeCo and AuFe. The nanocrystals may be used in vivo as MRI contrast agents, X-ray contrast agents, near IR heating agents, in drug delivery, protein separation or catalysis. The nanocrystals may be further functionalised with a hydrophilic coating, which improves in vivo stability. The nanocrystals are prepared by chemical vapour deposition and exhibit a high saturation magnetisation, high optical absorbance by the graphitic shell in the near-infrared and remarkable chemical stability. The saturation magnetisation of 7 nm FeCo graphite coated nanocrystals was 215 emu/g, close to bulk FeCo (235 emu/g). The international patent application WO 2012 / 001579 A1 describes a method for forming iron oxide nanoparticles. The disclosed iron oxide nanoparticles are water-soluble and show superior performance in magnetic particle imaging and magnetic particle spectroscopy due to the high saturation magnetisation of 107 emu/g, which is reached in one embodiment. Problem according to the invention

It is the problem according to the invention to provide an improved structure with a core and an imprinted coating. A further problem is to provide a use for the structure. In particular, it is desirable for the structure to have a high affinity towards a specific molecule and low toxicity in biological applications.

Solution according to the invention

The problem according to the invention is solved by a structure with a core and a coating imprinted with a specific molecule, wherein the structure is a nanoparticle. Furthermore, the problem is solved by the use of such a structure in an imaging method, in a method for the localised induction of hyperthermia and in a method for the separation and/or isolation of cells, protein and/or nucleic acids. Another solution is provided by a method comprising the steps of producing the core, coating of the core in the presence of the specific molecule and removal of the specific molecule by using a suitable solvent. Moreover, a kit containing structures according to the invention and a protein labelled with a specific molecule and/or a protein labelling solution that contains the specific molecule solves the problem according to the invention.

The nanoparticle according to the invention consists of a core and a coating. The core can consist of one or a mixture of two or more materials. The core can also consist of a layered structure. The layers can, e.g., be arranged in a substantially concentric pattern. Each layer may consist of one or a mixture of two or more materials. The coating is situated on the outside of the core. Preferably, the coating surrounds the core entirely.

The preferred nanoparticle has a substantially spherical shape. Preferably, the core and the coating each take substantially spherical shapes that are preferably in a substantially concentric arrangement. The invention, however, is not limited to spherical nanoparticles. Rather, the nanoparticles according to the invention can assume any shape, they can, e.g., be rod-like or irregularly shaped. The nanoparticle according to the invention measures less than 500 nm across. Within the scope of this document, the diameter of the nanoparticle is defined as the equivalent spherical diameter, that is, the diameter of a sphere of equivalent volume.

The coating according to the invention is imprinted with a specific molecule. The imprinted coating is produced from a starting material that is brought into contact with the specific molecule. Subsequently, the starting material is induced to harden around the specific molecule. Thereafter, the specific molecule can be removed and may leave behind cavities in the surface of the coating. Preferably, the resulting cavities show a high affinity for the specific molecule. Without prejudice, the inventors attribute the high affinity to a favourable distribution of functional groups and charges in the cavities. Frequently, to achieve high affinity to a target molecule, that very molecule is used to imprint the surface of the coating. However, it is also possible to use another molecule, preferably a molecule that is similar to the target molecule, in the imprinting process. In principle, the specific molecule can be any molecule. Preferably, the specific molecule contains a suitable amount of polar functional groups for a sufficient number of hydrogen bonds to be formed with the imprinted coating to achieve a high affinity between the coating and the specific molecule. The nanoparticle according to the invention can be used in an imaging method. An imaging method is any method suitable for the production of an image from a sample or test subject, such as an animal or human being. Furthermore, the nanoparticle can also be used in a method for the localised induction of hyperthermia. The coating of the nanoparticles may be designed in such a way that when the nanoparticles are injected into the bloodstream, they preferentially distribute to specific target sites, such as tumour sites. Another possibility is to provide an adapter that shows a high affinity to the target sites and the coating. Such an adapter can, e.g., be a protein, in particular, an antibody against characteristic epitopes of the target sites. Preferably, the adapter is conjugated to the specific molecule. The adapter may be attached to the nanoparticle before the introduction into the organism. Alternatively, the adapter and the nanoparticle can be introduced separately to only bind to each other within the organism. By introducing the adapter separately from the nanoparticles, the delivery at the target sites may be improved as, characteristically, the adapter and the nanoparticle by themselves are smaller and pass through vessel walls and tissues more easily than when conjugated to each other. By having a separate adapter and nanoparticle it is also possible to introduce each at distinct points in time. The adapter can, e.g., be injected into the blood stream first, the injection of the nanoparticle is then delayed until a desired enrichment of the adapter at the target sites has taken place. If the nanoparticle contains a suitable material, e.g., a magnetic material, it can be detected in an imaging technique. Furthermore, if the nanoparticle contains a magnetic material, particularly a

superparamagnetic material, the application of an external, alternating magnetic field can induce a rotatory torque in the nanoparticle, which can heat the nanoparticle and its surroundings, causing the target tissues to be damaged. This approach may be especially successful in cancer therapy as many cancer tissues show less heat tolerance than the surrounding healthy tissue.

The nanoparticle according to the invention can also be used in a method for the separation and/or isolation of cells, protein and/or nucleic acids. In this case, the coating is either imprinted to have a high affinity to proteins, nucleic acids or surface structures of cells. Alternatively, the coating shows a high affinity to an adapter, which in turn binds to the cells proteins and or nucleic acids. The adapter can itself be a protein, in particular an antibody. Preferably, the adapter is conjugated to the specific molecule, with which the coating is imprinted. In this way, many different types of adapters can be made to bind to a nanoparticle imprinted with just one specific molecule.

The nanoparticle according to the invention can be produced by a method comprising the steps of producing the core, coating of the core in the presence of the specific molecule and removal of the specific molecule by using a suitable solvent. The core may be produced first as a solid, homogenous structure or as a layered structure consisting of two or more layers. After that, the coating may be brought into contact with the core in the presence of the specific molecule. After the coating is hardened, the specific molecule can be removed with a suitable solvent, preferably leaving behind cavities with a high affinity for the specific molecule. Another aspect of the invention is a kit containing the nanoparticles and a protein labelled with a specific molecule. The nanoparticles in this kit preferably show a high affinity for the specific molecule and therefore also to the protein contained in the kit. Another kit according to the invention contains nanoparticles and a protein labelling solution that contains the specific molecule. This kit may be used to label a protein with the specific molecule to produce a protein to which the nanoparticle shows a high affinity.

The invention makes it possible to provide nanoparticles that show a high affinity to a specific molecule. Advantageously, such nanoparticles can be used in diagnostic imaging techniques such as magnetic resonance imaging and magnetic particle imaging. Furthermore, the nanoparticles according to the invention can be designed to bind specifically to certain antigens and to home to certain target site in the body when injected into the blood stream. The nanoparticles according to the invention can also be applied in vitro to separate nucleic acids, proteins and cells. Proteins can, e.g., be separated from and/or analysed in cell lysates, tissues and bodily fluids. The coating can be imprinted with a specific, small molecule. In order to conjugate a protein, such as an antibody, to the nanoparticle the protein can be labelled with the same specific, small molecule. In this way, almost any protein can easily be made to bind to the surface of the nanoparticle. Necessary reagents may be provided in the kit according to the invention.

Specific embodiments according to the invention

The diameter of the core and/or the entire nanoparticle according to the invention is preferably > 1 nm, more preferably > 2 nm, more preferably > 5 nm, more preferably > 10 nm and most preferably > 15 nm. At the same time, it is preferred that the core and/or the entire nanoparticle according to the invention is < 400 nm, more preferably < 250 nm, more preferably < 150 nm, more preferably < 100 nm, more preferably < 80 nm, more preferably < 60 nm, more preferably < 40 nm and most preferably < 30 nm in diameter.

In one embodiment according to the invention, the imprinted coating contains a polymer. A polymer may be suited particularly well for the production of the coating as many polymers can be hardened from malleable precursors. A polymer that is suitable for the production of the coating is polyvinylpyrrolidone. It is preferred that the coating contains at least one acrylic acid derivative. Experiments have shown that acrylic acid derivatives such as polyacrylate, polymethacrylate, poly(methyl methacrylate), polyhydroxyethylmethacrylate, polyacrylamide and mixed polymers of acrylic acid and vinyl pyridine or ε -caprolactone can be used to produce the molecularly imprinted coating. The preferred coating contains methacrylic acid and ethylene glycol dimethacrylate (EGDMA). The preferred coating is produced from the monomer methacrylic acid and the crosslinker EGDMA. The combination of these two compounds can yield a coating which is inert and can be imprinted to achieve high affinities to target molecules. Moreover, acrylic acid polymers show low toxicity and are widely used in pharmaceutical tablets.

The preferred nanoparticle according to the invention is magnetic. Preferably, the

nanoparticle contains a ferromagnetic or ferrimagnetic material. Examples for such materials are iron, cobalt, nickel and iron oxide. When the nanoparticles are used in cell, protein and/or nucleic acid separation and analysis, it is a great advantage if the nanoparticle is magnetic as the structures bound to the nanoparticle can be separated from a suspension by applying a magnetic field. Furthermore, magnetic nanoparticles can easily be detected in magnetic resonance imaging and magnetic particle imaging. In one embodiment according to the invention, the nanoparticle is superparamagnetic.

Preferably, the core of the nanoparticle contains a superparamagnetic material. More preferably, the entire core is made up from a superparamagnetic material. In the case, in which the core consists of two or more layers, at least one of those layers is made from a superparamagnetic material. Preferably, the nanoparticle is small enough for

superparamagnetism to occur. Advantageously, superparamagnetic nanoparticles have a large, positive magnetic susceptibility, as the entire particle may align with and strengthens the applied magnetic field leading to a local disturbance. When magnetic resonance imaging is performed, the local disturbance can lead to a rapid dephasing of surrounding protons, generating a detectable change in the magnetic resonance signal. Thus, superparamagnetic nanoparticles may easily be detected on magnetic resonance imaging. Furthermore, superparamagnetic nanoparticles can be sufficiently small for the Brownian motion to demagnetise the particles once an applied field is taken away. Thereby, the aggregation of the superparamagnetic nanoparticles in solution due to magnetic attraction can be

prevented.

The preferred nanoparticle according to the invention contains iron. By using iron, it is possible to produce a ferromagnetic and/or superparamagnetic nanoparticle, which is biocompatible. According to the invention it is preferred that the nanoparticle contains iron oxide, more preferably Fe₃0₄. The use of iron oxide allows for the production of

biocompatible nanoparticles with a high magnetisation. Preferably, the core is made up of a mixture of materials containing > 10%, more preferably > 25%, more preferably > 50%, more preferably > 75%, more preferably > 90%, more preferably > 95%, and most preferably >

99% (weight/weight) iron oxide. It is preferred that the core or at least one layer of the core of the nanoparticle consists entirely of iron oxide, of which preferably > 99% (weight/weight) is Fe₃0₄, most preferably, the entire core or at least one entire layer of the core consist of Fe₃0₄. It is preferred that the innermost layer of the core contains iron oxide, more preferably Fe₃0₄. Most preferably, the entire innermost layer is made up of Fe₃0₄.

It is preferred that the nanoparticle according to the invention shows a magnetisation of > 120 emu/g. Preferably, the magnetisation is > 150 emu/g, more preferably > 180 emu/g, more preferably > 190 emu/g, more preferably > 200 emu/g and most preferably > 205 emu/g. A large magnetisation may allow for the easy detection in magnetic resonance imaging and magnetic particle imaging. Furthermore, a large magnetisation can considerably facilitate the use of the nanoparticles in cell, protein and nucleic acid separation and analysis. Additionally, a large magnetisation can lead to an efficient generation of heat in alternating magnet fields. A preferable nanoparticle according to the invention has a relaxivity r1 in T1 of >1 .2, preferably >1.3, more preferably >1.4 and most preferably >1.5 m ^"1s^"1. The preferred nanoparticle according to the invention shows a relaxivity r2 in T2 of > 250, preferably > 500, more preferably > 750, more preferably > 800 and most preferably > 845 mM^'V. The preferred nanoparticle according to the invention has a relaxivity r2^* in T2^* of > 750, preferably > 800, more preferably > 900, more preferably > 1000, more preferably > 1 100, more preferably > 1200, more preferably > 1300, more preferably > 1400 and most preferably > 1500 mM^"1s^"1. High relaxivities allow for high sensitivity and spatial resolution in magnetic resonance imaging and magnetic particle imaging.

In one embodiment of the invention the specific molecule is a fluorescent dye. A fluorescent dye is a compound that absorbs light or other electromagnetic radiation of a first frequency and then emits light of a second frequency, lower than the first frequency. By using a fluorescent dye as the specific molecule, its fluorescence can be used to verify whether the specific molecule has been washed out of the coating after the imprinting step, whether a protein has been labelled with the dye and whether the protein has been attached to the nanoparticle successfully. Furthermore, many fluorescent dyes are sufficiently large, polar molecules to allow for an efficient and highly specific imprinting.

According to the invention, it is preferred that the specific molecule is fluorescein or a derivative thereof. The inventors have found out that a highly specific imprinting can be produced when using fluorescein or a derivative. Without prejudice, fluorescein may be particularly suited for imprinting for its size and characteristic charge distribution to allow strong and reversible van der Waals and ionic interaction. Furthermore, the use of fluorescein as a specific molecule facilitates the conjugation of proteins to the nanoparticles as fluorescein and, in particular, its derivatives and 6-fluorescein-5(6)-carboxamido hexanoic acid-n-hydroxysuccinimide ester (fluorescein-NHS) and fluorescein isothiocyanate (FITC) can, in general, be easily attached to proteins. In addition to this, many antibodies and other proteins are already commercially available in their FITC conjugated form so that they can be easily attached to the fluorescein imprinted nanoparticles according to the invention.

Moreover, fluorescein is already used in human diagnostics and has been shown to have a low toxicity. Acrylic acid derivatives can form molecular imprints with a high affinity for fluorescein as they both contain free carboxylic groups. By variation of the amount of the crosslinker EGDMA, the thickness of the coating and the cavity size can be varied.

It is preferred that the specific molecule contains at least one carboxylic group, at least one hydroxyl group, at least one heterocycle, at least one xanthene and/or at least one ketone. Experiments have shown that molecules containing the aforementioned groups yield molecular imprints of the core with an especially high affinity.

According to the invention, it is preferred that at least one layer of the core comprises graphene. Graphene can serve to protect the core from aggressive substances that could otherwise degrade the core.

Preferably, the outermost layer of the core is a graphene envelope that covers at least 50% of its underlying layer with between 1 and 5 layers of graphene. Preferably, at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably 100% of the surface area of the layer of the core beneath the graphene envelope is covered with between 1 and 5 layers of graphene. Preferably, the entire graphene contained in the nanoparticle is situated in the graphene envelope. Preferably, no part of the graphene envelope contains more than 20, more preferably more than 10 and most preferably more than 5 layers of graphene.

Experiments have shown that a thin graphene envelope of between 1 and 5 layers of graphene creates a high magnetisation in iron oxide nanoparticles.

The preferred nanoparticle according to the invention has a graphene envelope that covers at least 50% of the surface area of the underlying core with three layers of graphene.

Preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably 100% of the surface area of the underlying core is covered with three layers of graphene. The inventors have found that a nanoparticle with three layers of graphene in the graphene envelope shows a high magnetisation while being relatively small. The preferred nanoparticle according to the invention has a graphene envelope that covers at least 50% of the surface of the underlying core. Preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99% and most preferably 100% of the surface area of the underlying core is covered with the graphene envelope. By completely covering the underlying core with the graphene envelope, a very good magnetisation is achieved. In addition to this, a good approximation to a spherical shape can be yielded.

The nanoparticle according to the invention is preferably functionalised with a protein and/or a nucleic acid. By functionalising the nanoparticle with a protein, such as an antibody, other proteins or cells can be labelled with the nanoparticle. Nucleic acids can be labelled with nanoparticles conjugated to complementary nucleic acids. By application of a magnetic field, cells, proteins and nucleic acids can then be separated or analysed. The functionalisation can be carried out by conjugating the protein and/or the nucleic acid with the specific molecule, e.g., fluorescein or FITC.

The preferred nanoparticle according to the invention is functionalised with a drug. The drug can be the specific molecule and can thus be absorbed at the surface of the coating of the nanoparticle. The drug can also be reversibly or irreversibly be conjugated to the specific molecule to be able to attach the drug to the nanoparticle. If desired, the molecular imprinting may be engineered in such a fashion that the affinity of the imprinted coating for the drug is sufficiently low for the drug to be released when desired. In principle, the nanoparticle according to the invention can be functionalised with any drug. Drugs that are targeted against localised disorders such as infections or neoplastic lesions are especially suited for the functionalisation on nanoparticles. The drug conjugated nanoparticles can work as a theranostic, a portmanteau of the words therapeutic and diagnostic. In one embodiment, the nanoparticles according to the invention can be injected into the human body, e.g. via an intravenous route. They then distribute themselves from the injection site to the target tissue. The distribution can be controlled by a suitable imprinting of the coating of the nanoparticles with a specific molecule present at the target site. The nanoparticles can also be conjugated to a protein - directly or via the specific molecule - allowing for the endocytosis of the nanoparticle in a particular, targeted species of cells. Alternatively, adapters - such as antibodies or other proteins - can be employed, which can bind to epitopes in the target sites. The adapters are conjugated to the specific molecule such that they can bind to the coating. The antibody can be, e.g., an antibody directed against a cancer antigen. A two-step approach is possible, in which the proteins conjugated to the specific molecule are injected first, independently of the nanoparticles. After a sufficient time has passed for the proteins to distribute themselves to the target tissues, the nanoparticles are injected. The nanoparticles can then attach themselves to the antibodies in the human body through the specific molecule attached to the antibody. By injecting the antibody independently of the

nanoparticles, the distribution into the target site may be facilitated. Usually, the antibody and the nanoparticle by themselves pass much more easily through the vessel wall than a conjugate of nanoparticle and antibodies. If the core contains a magnetic or

superparamagnetic material, the location of the nanoparticles can be monitored by magnetic resonance imaging or magnetic particle imaging to find out whether they have distributed to the target site with sufficient precision representing the diagnostic part of the theranostic. If the nanoparticles are conjugated with a drug, this can be released at the target site in a second step. The drug release can either occur simply through the passage of time as the drug slowly dissociates itself from the nanoparticle. Alternatively, an external alternating magnetic field can be applied to superparamagnetic nanoparticles. The alternating magnetic field can cause the nanoparticles to be heated and thereby release the drug. Such alternating magnetic fields can be created using specialised machinery or magnetic resonance coils adapted to the purpose. Alternatively, nanoparticles not conjugated with a drug can equally be heated at the target site to cause hyperthermia in order to destroy diseased tissues. Thus, in one embodiment, the nanoparticles according to the invention represent a bona fide theranostic in that they can be used to both find pathologies as well as to cure them. Furthermore, the nanoparticle according to the invention can be used in a method for the analysis of protein-protein and/or protein-nucleic acid interactions. In medical research, it is often desirable to find protein-protein interaction partners, in basic research as well as in drug design. To this end, a first protein, which is a putative interaction partner of a second protein in a solution, can be conjugated with the specific molecule, e.g., fluorescein. The nanoparticle imprinted with the specific molecule can then be added to that solution and subsequently, by using centrifugation or the application of a magnetic field in the case of a magnetic nanoparticle, the nanoparticle can be separated from the solution again. Using an antibody specific for the second protein, it can be detected, whether the nanoparticle has bound to the second protein via the specific molecule and the first protein. Similarly, this method can be used employing an antibody conjugated to the specific molecule, wherein the antibody is specific for the first protein.

In addition to this, it may be of interest at which exact position certain proteins interact with nucleic acids. In particular, the localisation of transcription factor binding sites on DNA is a common object of scientific inquiry. If an antibody to a transcription factor or the transcription factor itself is conjugated to the specific molecule, the binding site of the transcription factor can be found by adding the nanoparticle imprinted with the specific molecule to a pool of genomic DNA, denaturing the protein to make the DNA-protein-connection irreversible, subsequently removing the nanoparticle bound to DNA by the specific molecule and the antibody using a magnetic field (in the case of magnetic nanoparticles) or centrifugation and applying a DNA identification technology such as sequencing to characterise the DNA bound to the transcription factor. The high achievable magnetisation of the nanoparticle according to the invention can increase yield and/or sensitivity in the aforementioned applications. In one embodiment of the invention, the nanoparticles are used in magnetic resonance imaging or magnetic particle imaging. The nanoparticles according to the invention can be produced to have a large magnetisation. Such nanoparticles, when introduced into the magnetic field of a magnetic resonance scanner can cause a large, localised disturbance in the magnetic field, which in turn can cause surrounding protons to dephase rapidly, leading to a loss of T2 signal. As the disturbance induced by a single nanoparticle according to the invention can be greater than in biocompatible nanoparticles already known, a higher sensitivity in magnetic resonance applications may be achieved. That is, in order to produce a detectable change in a magnetic resonance image, fewer nanoparticles according to the invention are needed than would be necessary when using nanoparticles known in the art. Advantageously, the nanoparticles according to the invention can also be used in magnetic particle imaging. Magnetic particle imaging can measure the localisation of magnetic material in a given volume. To achieve high temporal as well as spatial resolution in magnetic particle imaging, it is desirable to utilise a nanoparticle with a very large magnetisation, which can be supplied by the invention.

According to the invention, it is preferred that the core is produced by co-precipitating of iron oxide and graphene. Preferably, the method for the production of the nanoparticle comprises the co-precipitation of iron oxide and graphene using FeCI₂ and FeCI₃ as precursors.

Experiments have shown that the precipitation of FeCI₂ and FeCI₃ and graphene in an aqueous solution by the addition of ammonia solution is a very efficient method to produce the nanoparticle according to the invention. However, the invention is not limited to the production of iron oxide nanoparticles with a graphene envelope by means of co- precipitation. Rather, any feasible method for the production of such nanoparticles is part of the invention. Graphene is preferably produced by the oxidation of graphite yielding graphene oxide followed by the reduction of graphene oxide to graphene. Preferably, the core is coated in a polymerisation reaction. The use of polymers can yield a highly specific molecular imprint.

In a preferred method according to the invention, the specific molecule is fluorescein or a derivative thereof. The inventors have found that by using fluorescein or a derivative thereof in producing the nanoparticle according to the invention, a high affinity to the specific molecule can be achieved.

The invention provides an improved nanoparticle, several uses for the nanoparticle and a method to produce such a nanoparticle. Furthermore, the invention provides a kit containing the nanoparticle. In particular, the invention allows for the production of a nanoparticle with a low toxicity and a high magnetisation.

Brief description of the Figures The invention is explained in detail in the following figures.

The figures show:

Fig. 1 The nanoparticle according to the invention in a schematic representation; Fig. 2a an electron micrograph of iron oxide nanoparticles; an electron micrograph of iron oxide nanoparticles with a graphene envelope; an electron micrograph of iron oxide nanoparticles with a graphene envelope and a molecularly imprinted coating; a diagram depicting size measurements obtained by dynamic light scattering; a schematic representation of the nanoparticle according to the invention with a graphene envelope; a schematic representation of the nanoparticle according to the invention with a graphene envelope and a molecularly imprinted coating and Fig. 3 relaxivities in T1 , T2 and T2^* of the nanoparticle according to the invention compared to commercially available nanoparticles.

Description of specific embodiments of the invention

Fig. 1 depicts a spherical nanoparticle 1 with a core 2 consisting of two layers 3, 10. The outer layer is a graphene envelope 3, which contains several layers of graphene. The underlying, inner layer 10 of the core consists of iron oxide. By applying the graphene envelope 3, a previously unattainable magnetisation of 215 emu/g can be achieved. The nanoparticle also contains a coating 4, which is applied on top of the graphene envelope 3. The coating 4 is created by the polymerisation of methacrylic acid and ethylene glycol dimethacrylate. The coating 4 shields the nanoparticle 1 from its environment and protects it from decay. The coating 4 is polymerised in the presence of fluorescein creating cavities 9 with a high affinity for fluorescein. In other words: the coating 4 is imprinted with fluorescein. The nanoparticle 1 can be functionalised by attaching proteins 5, such as antibodies 6, to the coating. The functionalisation can be carried out by first conjugating fluorescein to the protein 5. After that, the fluorescein conjugated protein 5 is brought into contact with the imprinted coating 4, which causes the protein 5 to attach itself to the coating 4 via fluorescein.

Alternatively or in addition to proteins 5, the nanoparticles 1 can also be conjugated to nucleic acids 7 and drugs 8, particularly antibiotic or antineoplastic drugs, either directly or via fluorescein. According to the invention, the structure 11 is a nanoparticle 1. Fig. 2a shows a scanning electron microscopic image of iron oxide nanoparticles 1 containing no graphene envelope 3 and no coating 4. The uncoated nanoparticles 1 have a mean diameter of 12 nm. Fig. 2b displays the nanoparticles 1 according to the invention with a graphene envelope 3 in a scanning electron micrograph. The graphene envelope 3 has added to the size of the nanoparticles 1 , which now measure 17 nm across on average.

The microscopic image in fig. 2c depicts the nanoparticles 1 according to the invention with a core 3 made up of an inner layer 10 of iron oxide and an outer graphene envelope 3. The core 2 is surrounded by a coating 4. The nanoparticle 1 in fig. 2c has a mean diameter of 28 nm. The methods described herein are, however, not limited to the production of

nanoparticles 1 of these sizes. Rather, nanoparticles 1 of virtually any size may be produced using the methods according to the invention.

Fig 2d contains a diagram of the result of a dynamic light scattering experiment, The nanoparticle diameter is shown on the x-axis while the y-axis displays light intensity as a percentage value. The dashed curve on the left corresponds to the nanoparticles 1 without graphene envelope 3 and without coating 4 as displayed in fig. 2a. The nanoparticles 1 with the graphene envelope 3, but without a molecularly imprinted coating 4, as seen in fig. 2b, correspond to the continuous curve slightly to the right. The dotted curve on the right shows the results from nanoparticles 1 with graphene envelope 3 and coating 4. From this diagram it is clear to see that the graphene envelope 3 has added to the mean diameter of the iron oxide nanoparticles 1 and the coating 4 further adds to the diameter. Fig. 2e is a schematic representation of an iron oxide nanoparticle 1 according to the invention with a graphene envelope 3 with its characteristic hexagonal, honeycomb lattice composition. Fig. 2f shows a nanoparticle 1 with a graphene envelope 3 and a fluorescein imprinted coating 4 onto which fluorescein conjugated antibodies 6 have been attached.

Fig. 3a, 3b, 3c and 3d compare the relaxation rates of the commercially available

superparamagnetic magnetic resonance imaging iron oxide nanoparticles 1 with the nanoparticles 1 according to the invention with a polymer coating and a core containing iron oxide and a graphene envelope. The table in Figure 3d lists the relaxivities of the

commercially available Resovist (labelled here and in the diagrams with the number 1) and Supravist (labelled with the number 2). The last row of the table shows the relaxivities of the nanoparticles 1 according to the invention with a polymer coating and a core containing iron oxide and a graphene envelope (labelled with the number 3). Relaxation rates for T1 , T2 and T2^* are plotted against iron concentration in fig. 3a, 3b and 3c, respectively. The numbers of the fitted lines in Figure 3a, 3b and 3c correspond to the numbers in Figure 3d (1 : Resovist, 2: Supravist, 3: nanoparticle according to the invention). The relaxivities r1 , r2 and r2^* are far higher for the nanoparticles 1 according to the invention than each of the two commercially available nanoparticles 1.

The nanoparticles 1 according to the invention can be used in a variety of applications in vivo and in vitro. In one embodiment, the nanoparticles 1 can be functionalised with proteins 5, in particular with antibodies 6, and can be utilised, for example, in protein isolation, kinase assays, detection of protein-protein interactions, protein-nucleic acid interactions, such as chromatin immunoprecipitation and to separate cells from suspensions, such as blood.

Furthermore, the nanoparticles 1 can be employed in vivo in imaging methods, such as magnetic resonance imaging and magnetic particle imaging. The high magnetisation that can be achieved in the nanoparticles 1 according to the invention can improve sensitivity as well as temporal and spatial resolution in these imaging modalities. In addition to this, the fluorescein imprinted coating 4 allows for any fluorescein conjugated protein 5 to be easily attached to the nanoparticle 1 , thereby providing a great versatility in in vivo as well as in vitro applications.

Protocols for the production of iron oxide nanoparticles with a graphene coating and molecularly imprinted coating with high affinity for fluorescein The following protocols demonstrate one method for the production of the nanoparticles according to the invention. By adhering to the protocols below, nanoparticles covered in three graphene layers, with a high magnetisation of 215 emu/g and with a molecularly imprinted coating specific for fluorescein may be produced. The invention, however, is not limited to the method outlined in these protocols. Rather, other production methods are equally feasible. The production of graphene - as described below - comprises the steps of the synthesis of graphene oxide and the subsequent reduction of graphene oxide to graphene. In a third step, superparamagnetic iron oxide particles with a graphene coating are produced by co-precipitation. Subsequently, the iron oxide nanoparticles are covered with a fluorescein sensitive molecularly imprinted coating. The nanoparticles can then be attached to proteins conjugated to fluorescein. As an example, a protocol for the conjugation of an antibody to fluorescein is detailed below. Synthesis of graphene oxide

60 mg graphite powder (size <20 μπι) and 45 mg sodium nitrate are placed in a 100 ml round-bottom flask, the flask is closed with a drying tube. 4.5 ml of sulphuric acid is added during 10 minutes while stirring and cooling. Further stirring is performed on ice for 2 h. After that, stirring is carried out for 6 days at room temperature. Subsequently, 7 ml 5% sulphuric acid in water (w/w) is added. The mixture is stirred for 2 h at 98 °C. Then, the temperature is lowered to 60 °C. 0.2 ml hydrogen peroxide is added, stirring is performed for 1 h. The solution is centrifuged at 16,060 g for 2 minutes. The sediment is homogenised in 1.7 ml 3% sulphuric acid, 0.5% hydrogen peroxide (w/w). Homogenisation and centrifugation is repeated 15 times. The sediment is then spun in a centrifuge at 16,060 g and washed in 1 ,7 ml 3% sulphuric acid for a total of 3 times. Next, the sediment is suspended in water and homogenised in an ultrasonic bath. After that, the suspension is spun at 16,060 g for 15 minutes and the sediment is dried at 10^"1 torr. The achievable yield is approximately 50 mg graphene oxide.

Synthesis of graphene from graphene oxide by chemical reduction with hydrazine

10 mg graphene oxide from the previous step is suspended in 10 ml ultra-pure water and placed in an ultrasonic bath for 1 minute. 112 μΙ 32% ammonia solution is added within 5 minutes. 18 μΙ 62% hydrazine solution is added within 5 minutes. The suspension is stirred for 1 h at 90 °C under reflux. The entire preparation is then transferred into Falcon tubes and spun in a centrifuge for 3 minutes at 4,600 g. The supernatant is transferred into 20 ml round-bottom flasks und concentrated under vacuum at 80 °C in a rotary evaporator. The residue is dried at 10^"1 torr. The achievable yield is approximately 7 mg graphene.

Production of superparamagnetic iron oxide nanoparticles with a graphene coating 25 mg FeCI₂ x 4 H₂0 (final concentration 5 mM) and 68 mg FeCI₃x 6 H₂0 (final concentration 10 mM) are dissolved in 25 ml ultra-pure water saturated with nitrogen. To the resulting solution, 1 ml graphene solution (5 mg/ 10 ml 1 % (v/v) ammonia solution, pH 8.5) is added under nitrogen saturation. 5.8 ml 32% ammonia solution is added drop by drop within 30 minutes while thoroughly mixing with nitrogen. The mixture is kept at 70 °C for 30 minutes, during which time the nanoparticles precipitate. The nanoparticles are separated using an external magnet. The supernatant is decanted, the nanoparticles are washed twice in 10 ml water and twice in methanol. After that, the nanoparticles are homogenised in 10 ml toluene in an ultrasonic bath (2 mg Fe per ml solvent). For stabilisation and storage, the

nanoparticles are kept in a nitrogen atmosphere at 4 °C.

Coating of superparamagnetic iron oxide-graphene nanoparticles with a fluorescein sensitive molecularly imprinted poly(methacrylate) phase

1 ml iron oxide graphene nanoparticle suspension in toluene (2 mg Fe/ml) is homogenised in an ultrasonic bath for 3 min, separated from the toluene using an external magnet, resuspended in 1 ml ethyl acetate and homogenised again in an ultrasonic bath for 2 min. The following substances are weighed in in a 5 ml glass vessel with screw top: 13 mg methacrylic acid (0.15 mmol), 9 mg ethylene glycol dimethacrylate (EGDMA, 0.05 mmol), 1 mg ex, a' azoisobutyronitrile (0.006 mmol) and 2.5 mg fluorescein sodium salt (0.007 mmol). All components are weighed in and are homogenised using ultrasound for 1 min in order to achieve the dissolution of fluorescein sodium salt. 1 ml iron oxide graphene nanoparticle suspension in ethyl acetate is added and homogenised briefly using ultrasound. The nanoparticle suspension is then kept under exposure to nitrogen gas for 30 min at 55 °C. After that, the nanoparticle suspension is kept at 55 °C for six hours without continuous exposure to nitrogen gas. Subsequently, the nanoparticles are separated from the reaction solution by means of an external magnet and washed three times in ethyl acetate. The nanoparticles are then separated from ethyl acetate using an external magnet and washed three times with 1 ml methanol. The nanoparticles are separated from methanol using an external magnet and washed twice with 1 ml methanol / 0.1 N sodium hydroxide 80/20 (v/v). The nanoparticles are then separated from the suspension using an external magnet and washed twice in 1 ml methanol. After that, the nanoparticles are separated from methanol using an external magnet and washed twice with 1 ml water.

Method for the production of fluorescein labelled antibodies using cetuximab

Cetuximab is transformed with activated fluorescein (6-fluorescein-5(6)-carboxamido hexanoic acid-n-hydroxysuccinimide ester = fluorescein-NHS) and stored at -20 °C, in inert gas and protected from light. 100 μΙ cetuximab (= 500 g antibody) is added to 10 μΙ fluorescein NHS in 0.5 M NaHC0₃ buffer, pH 8.5 (0.7 mg/100 μΙ buffer, pH 8.5) and 40 μΙ NaHC0₃ buffer 0.5 M at room temperature and protected from light. The solution is left at room temperature for 1 hour. A Sephadex G25 column (lllustra NAP-5, GE Healthcare) is equilibrated in 0.9 % NaCI. Non-reacted fluorescein NHS is separated using the lllustra NAP 5 column. To achieve this, 150 μΙ sample solution is added onto the column. After absorption into the column, 350 μΙ NaCI 0.9% is added for the sample to be taken up into the column. After the absorption into the column, elution is carried out with 200 μΙ 0.9 % NaCI as the precursor fraction, followed by elution with 700 μΙ 0.9 % NaCI, releasing the main fraction followed by elution with 100 μΙ 0.9 % NaCI as the control fraction. The precursor fraction yielded 0.015 mg protein / 200 μΙ. The main fraction contained 0.572 mg protein / 700 μΙ and the final control fraction contained 0.005 mg protein / 100 μΙ.

The nanoparticles produced according to the preceding protocols may reach a high affinity of approximately K_d=55 nM for fluorescein labelled antibodies, comparable to the affinity for antibodies of protein A (K_d= 33 nM) and superior to the affinity of protein G (K_d=300 nM). Furthermore, the nanoparticles such produced can show a very high magnetisation of up to 215 emu/g.

Reference numbers

1. Nanoparticle

2. Core

3. Graphene envelope

4. Coating

5. Protein

6. Antibody

7. Nucleic acid

8. Drug

9. Imprinted cavities in the coating

10. Inner layer of the core

11. Structure

Claims

I . Structure (11) with a core (2) and a coating (4) imprinted with a specific molecule, characterised in that the structure (11) is a nanoparticle (1).

2. Structure (11) according to claim 1 , characterised in that the coating contains a polymer.

3. Structure (11) according to any of the preceding claims, characterised in that the coating (4) contains at least one acrylic acid derivative.

4. Structure (11) according to any of the preceding claims, characterised in that the coating (4) is produced from methacrylic acid and ethylene glycol dimethacrylate.

5. Structure (11) according to any of the preceding claims, characterised in that the nanoparticle is magnetic.

6. Structure (11) according to any of the preceding claims, characterised in that the nanoparticle is superparamagnetic.

7. Structure (11) according to any of the preceding clams, characterised in that the nanoparticle contains iron.

8. Structure (1 1) according to any of the preceding claims, characterised in that the nanoparticle contains iron oxide.

9. Structure (11) according to any of the preceding claims, characterised in that the nanoparticle contains Fe₃0₄.

10. Structure (11 ) according to any of the preceding claims, characterised in that the nanoparticle shows a magnetisation > 120 emu/g.

I I . Structure

(11) according to any of the preceding claims, characterised in that the specific molecule is a fluorescent dye.

12. Structure (11) according to any of the preceding claims, characterised in that the specific molecule is fluorescein or a derivative thereof.

13. Structure (11) according to any of the preceding claims, characterised in that the specific molecule contains:

- at least one carboxylic group

and/or

- at least one hydroxyl group

and/or

- at least one heterocycle

and/or

- at least one xanthene

and/or

- at least one ketone.

14. Structure (11) according to any of the preceding claims, characterised in that at least one layer of the core (2) comprises graphene.

15. Structure (11) according to any of the preceding claims, characterised in that the outermost layer of the core (2) is a graphene envelope (3) and covers at least 50% of the surface area of its underlying layer (10) with between 1 and 5 layers of graphene.

16. Structure (1 1) according to claim 15, characterised in that the graphene envelope (3) covers at least 50% of the surface area of its underlying layer (10) with 3 layers of graphene.

17. Structure (1 1) according to claim 15 or 16, characterised in that the graphene envelope (3) covers at least 50% of the surface area of its underlying layer (10).

18. Structure (1 1) according to any of the preceding claims, characterised in that the structure (11) is functionalised with protein (5) and/or nucleic acid (7).

19. Structure (1 1) according to any of the preceding claims, characterised in that the structure (11) is functionalised with a drug (8).

20. Use of a structure (1 1) according to any of the preceding claims in an imaging method.

21. Use of a structure (11) according to claim 20, characterised in that the imaging method is magnetic resonance imaging or magnetic particle imaging.

22. Use of a structure (11 ) according to any one of the claims 1 to 19 in a method for the localised induction of hyperthermia.

23. Use of a structure (11) according to any one of the claims 1 to 19 in a method for the separation and/or isolation of cells, protein (5) and/or nucleic acids (7).

24. Method for the production of a structure (11) with a molecularly imprinted coating (4) according to any one of the claims 1 to 19, characterised in that the structure (1 1 ) is a nanoparticle and comprising the steps of

- producing the core (2);

- coating of the core (2) in the presence of the specific molecule

and

- removal of the specific molecule by using a suitable solvent.

25. Method according to 24, characterised in that at least one part of the core (2) is produced by the co-precipitation of iron oxide and graphene.

26. Method according to claim 24 or 25, characterised in that the core (2) is coated in a polymerisation reaction.

27. Method according to any one of the claims 24 to 26, characterised in that the specific molecule is fluorescein or a derivative thereof.

28. Kit containing structures (11) according to at least one of the claims 1 to 19 and a protein (5) labelled with the specific molecule.

29. Kit containing structures (11) according to at least one of the claims 1 to 19 and a protein labelling solution that contains the specific molecule.