WO2012018290A1

WO2012018290A1 - Discrete coated nanoparticles

Info

Publication number: WO2012018290A1
Application number: PCT/SE2011/000092
Authority: WO
Inventors: Maria Kempe; Henrik Kempe
Original assignee: Maria Kempe; Henrik Kempe
Priority date: 2010-05-26
Filing date: 2011-05-26
Publication date: 2012-02-09
Also published as: SE1230153A1

Abstract

This invention pertains in general to the field of nanoparticles. More particularly the invention relates to a method for silanization of a magnetic nanoparticle so that the nanoparticle stays discrete, i.e. is not agglomerated during the silanization process. The invention also pertains to such discrete nanoparticle and a composition comprising discrete nanoparticles and various uses of the particle or composition.

Description

DISCRETE COATED NANOP ARTICLES

Technical Field

This invention pertains in general to the field of nanoparticles. More particularly the invention relates to a method for silanization of a magnetic nanoparticle.

Background

Nanoparticles are particles with a diameter in the size range 1 nm to 1000 nm. Magnetic nanoparticles are commonly used within medicine, biomedicine, and biotechnology for purification, separation, drug delivery, magnetofection, MRI examinations, and hyperthermia therapy. Magnetic nanoparticles are also useful for the preparation of magnetic ink used in the MICR (magnetic ink character recognition) technology, and for preparation of electromagnetic shielders, materials for high-density digital storage, and magnetic tapes.

The term magnetic nanoparticles refers here to nanoparticles that are superparamagnetic, paramagnetic, diamagnetic, ferrimagnetic, or ferromagnetic . The magnetic nanoparticles are either permanently magnetic or become magnetic when placed in a magnetic field. Magnetic nanoparticles are normally composed of a core of one or more of the elements iron, nickel, and cobalt in the form as a metal, an alloy, an oxide, or mixtures thereof. Magnetic nanoparticles made of magnetic iron oxide have gained popularity due to their low toxicity. Examples of magnetic iron oxides are magnetite (Fe30₄) and maghemite (Fe₂0₃).

It is desirable that the prepared Fe30₄ and Fe₂03 nanoparticles are stabilized to maintain a high magnetic saturation and to prevent aggregation, oxidation by air, and degradation. Stabilization of nanoparticles may be achieved by coating with silica or silica derivatives. Coating with silica or silica derivatives by the Stober method [Stober, W. et al. J. Colloid Interface Sci. 1968, 26, 62-69] means that a silane is hydrolyzed, oligomerized/polymerized, and condensed/coupled to OH-groups at the surface of the nanoparticles [Yamaura, M. et al. J. Magn. Magn. Mater. 2004, 279, 210-217; Ma, M. et al. Colloids and Surfaces A: Physicochem. Eng Aspects 2003, 212, 219-226; Bruce, I.J.; Sen, T. Langmuir 2005, 21, 7029-7035; Lu, Y. et al. Nanoletters 2002, 2, 183-186; Ma, D. et al. Chem. Mater. 2006, 75, 1920-1927]. A layer based on silica or silica derivatives is thereby formed on the surface. Silanization with alkoxysilanes such as tetraethoxysilane or tetramethoxysilane has proven especially effective in providing a protective layer/coating on the surface of the nanoparticles. However, due to the inherent tendency of magnetic nanoparticles to aggregate, there is a large risk that the layer formed during the silanization reaction will cover aggregates, i.e. clusters of two or more nanoparticles, rather than discrete nanoparticles. Aggregation is here defined as clustering of two or more nanoparticles. The silanization glues the nanoparticles together permanently, resulting in a larger apparent particle size of the preparation.

For medical applications in vivo involving transportation of the nanoparticles in the vascular system, aggregates of nanoparticles may obstruct the blood vessels. For applications ex vivo and in vitro, aggregates of nanoparticles may obstruct microfluidic channels, tubings, nozzles, and other types of small-sized devices.

Thus, there is a need for a new method for the preparation of silanized magnetic nanoparticles, in which nanoparticles are not silanized as aggregates.

Summary of the Invention

The present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art singly or in any combination and solves at least the above mentioned problems by providing a method for the preparation of discrete silanized magnetic nanoparticles according to the appended patent claims.

The general solution according to the invention is to subject nanoparticles to one or more specific compound/s before silanization. This gives a colloidal solution of nanoparticles, which, when the nanoparticles are subsequently silanized, results in discrete silanized nanoparticles.

According to a first aspect of the invention, a method is provided, for coating a magnetic nanoparticle with hydroxyl groups on its surface, by forming a layer thereon. The method comprising the steps of subjecting the nanoparticle to a first solution comprising a compound according to formula (I):

I

wherein "n" is an integer in the interval 0 (zero) to 7000. The method also comprises a step of subjecting the nanoparticle to a second solution comprising a silanization agent, and a step of allowing formation of a silanized layer on the magnetic nanoparticle.

According to a second aspect of the invention, a composition is provided, which is obtainable by the method according to the first aspect.

According to a third aspect of the invention, a composition is provided, comprising mainly discrete, silane coated nanoparticles.

Further embodiments of the invention are defined in the dependent claims, as well as in the description.

The present invention has the advantage over the prior art in that it results in discrete silanized nanoparticles, i.e. nanoparticles prepared by formation of the silanized layer around non-aggregated, singular particles. Thus, a composition comprising said nanoparticles will comprise mainly discrete nanoparticles with a silanized layer on each nanoparticle.

Brief Description of the Drawings

These and other aspects, features and advantages of which the invention is capable of will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which

Fig. 1 is cross-sectional schematic illustration of a nanoparticle according to an embodiment of the invention;

Fig. 2 is a graph showing FT-IR spectra of nanoparticles;

Fig. 3 is a transmission electron microscopy (TEM) picture of nanoparticles; Fig. 4 is an overview of immobilization of tPA according to an embodiment; Fig. 5A is a schematic instrumental setup of magnetic targeting of coated nanoparticles in vitro, and Figs 5B to F are graphs showing the influence of the flow rate on nanoparticle capture efficiency (CE);

Fig. 6 is photographic representation of a segment of a capillary tube with inserted coiled wire;

Fig. 7 shows magnetic hysteresis loops of (A) naked magnetite nanoparticles from Example 1, (B) silanized nanoparticles from Example 5, (C) silanized

nanoparticles from Example 6, and (D) tPA-nanoparticle conjugates from Example 29; and

Fig. 8 is a graph showing the remaining enzyme activity of tPA-nanoparticle conjugates from Example 28 (grey bars) and Example 29 (black bars) after (A) ultrasonic treatment for 1 h; or incubation at 4 °C for (B) 24 h, (C) 48 h, (D) 10 days, (E) 21 days, and (F) 40 days. Description of embodiments

Several embodiments of the present invention will be described in more detail below with reference to the accompanying drawings in order for those skilled in the art to be able to carry out the invention. The invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Furthermore, the terminology used in the detailed description of the particular embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention.

In Fig. 1, a schematic cross-section of a nanoparticle according to an embodiment is disclosed. Layer (A) of the nanoparticle is an inner core. Layer (B) is a silanized layer or coating, of silica or a silica derivative, applied around the singular nanoparticle producing a discrete, silanized nanoparticle. Thus, the nanoparticle is silanized as a non-aggregated, singular particle. Layer (C) is an optional additional coating, conjugated to Layer (B).

Since the nanoparticle is silanized as a non-aggregated, singular particle, according to an embodiment, it is provided a composition comprising mainly discrete, silanized nanoparticles, such as above 50%, 60%, 70%, 80% or 90% discrete nanoparticles with a silanized layer on each nanoparticle.

According to an embodiment, said nanoparticle is produced by a method for forming a layer on, or coating, a nanoparticle. The nanoparticle may be any kind of nanoparticle, as long as it has hydroxyl groups on its surface. The method further comprises a step of subjecting the nanoparticle to a first solution comprising a compound according to formula (I):

I

In formula (I), "n" is an integer in the interval 0 (zero) to 7000, preferably in the interval 0 (zero) to 2300 and more preferably in the interval 2 to 800.

In an embodiment, the nanoparticle is a magnetic nanoparticle.

Examples of compounds of formula (I) include, but are not limited to ethylene glycol, diethylene glycol (DEG), methylene glycol (TREG), tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol, and other oligoethylene glycols/polyethylene glycols (also termed polyethylene oxides) of molecular weights up to 300000, such as PEG 400, PEG 2000, PEG 3400, PEG 8000, PEG 20000, PEG 35000, PEG 100000, PEG 200000, and PEG 300000, or a combination thereof.

The subjection of the nanoparticles to the above solution is carried out by allowing the nanoparticles to be placed in contact with the solution under stirring, agitation, shaking, tumbling, and/or sonication, typically during a time period between 1 min and 24 h, preferably for 5 min to 3 h and most preferably for 30 min to 1.5 h, to produce a colloidal solution.

In an embodiment, the solvent of the first solution is selected from the group consisting of: water, methanol, ethanol, n-propanol, iso-propanol, N,N- dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone and acetonitrile, or a combination thereof. However, the first solution may also consist of compound according to formula (I), if compound I in itself is in liquid form, such as TREG.

The first solution may also consist of several kinds of compounds according to formula (I), all of which are in liquid form.

In an embodiment, compound according to formula (I) is a liquid and acts as a solvent of the first solution.

In an embodiment, the first solution comprises several kinds of compounds according to formula (I), and a solvent, such as selected from the group consisting of: water, methanol, ethanol, n-propanol, iso-propanol, Ν,Ν-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone and acetonitrile, or a combination thereof.

In an embodiment, the first solution further comprises at least one base and/or at least one second solvent.

The base may be selected from the group consisting of: ammonia, sodium hydroxide, potassium hydroxide, triethylamine, trimethylamine, dimethylamine, diethylamine, ethylamine, propylamine, Ν,Ν-diisopropylethylamine, n-methyl morpholine, N-methylpyrrolidone, oleylamine, ethanolamine, pyridine, 4- dimethylaminopyridine, methylamine, and piperidine, or a combination thereof.

The second solvent may be selected from the group consisting of: water, methanol, ethanol, n-propanol, iso-propanol, Ν,Ν-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone and acetonitrile, or a combination thereof.

The method further comprises a step of treating the nanoparticle with a second solution comprising a silanization agent, allowing formation of a silanized layer, or coating, on the (magnetic) nanoparticle.

The silanization agent may be a silane.

In an embodiment, the silane is alkoxysilane, such as selected from the group consisting of tetramethoxysilane, tetraethoxysilane, tetra-n-propoxysilane, tetra-iso- propoxysilane, tetra-n-butoxysilane, tetra-t-butoxysilane, trimethoxysilane,

triethoxysilane, tri-n-propoxysilane, tri-iso-propoxysilane, tri-n-butoxysilane, tri-t- butoxysilane, trimethoxychlorosilane, triethoxychlorosilane, tri-n-propoxychlorosilane, tri-iso-propoxychlorosilane, tri-n-bytoxychlorosilane, tri-t-butoxychlorosilane, benzyltrimethoxysilane, benzyltriethoxysilane, dimethyldimethoxysilane,

dimethyldiethoxysilane, and mixtures thereof.

In an embodiment, the silane is a halosilane, such as selected from the group consisting of tetrachlorosilane, trichlorosilane, tetrafluorosilane, trifluorosilane, and mixtures thereof.

In an embodiment, the silane is an aminosilane, such as selected from the group consisting of 3-aminopropyltrimethoxysilane, 3- aminopropylmethyldimethoxysilane, 3-aminopropyldimethylmethoxysilane, N-(2- aminoethyl)-3 -aminopropylmethyldimethoxysilane, N-(2-aminoethyl-3 - aminopropyl)trimethoxysilane, 4-aminobutyldimethylmethoxysilane, 4- aminobutyltrimethoxysilane, aminoethylaminomethylphenethyltrimethoxysilane, N-(2- aminoethyl)-3 -aminoisobutylmethyldimethoxysilane, N-(6- aminohexyl)aminopropyltrimethoxysilane, 3 -(m- aminophenoxy)propyltrimethoxysilane, aminophenyltrimethoxysilane, 3- aminopropyltriethoxysilane, 3-aminopropylmethyldiethoxysilane, 3- aminopropyldimethylethoxysilane, N-(2-aminoethyl)-3 - aminopropylmethyldiethoxysilane, N-(2-aminoethyl-3-aminopropyl)triethoxysilane, 4- aminobutyldimethylethoxysilane, 4-aminobutyltriethoxysilane,

aminoethylaminomethylphenethyl triethoxysilane, N-(2-aminoethyl)-3- aminoisobutylmethyldiethoxysilane, N-(6-aminohexyl)aminopropyltriethoxysilane, 3- (m-aminophenoxy)propyltriethoxysilane, aminophenyltriethoxysilane, and mixtures thereof.

In an embodiment, the silane is an olefin-containing silane, such as selected from the group consisting of 3-(trimethoxysilyl)propylmethacrylate, 3- (triethoxysilyl)propylmethacrylate, methacryloxymethyltrimethoxysilane,

methacryloxymethyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, vinyltrichlorosilane, and mixtures thereof.

In an embodiment, the silane is a fluorescent silane.

In an embodiment, the silane is a radiopaque silane.

The silanization step may be repeated with the same or different silanization agent. An advantage with this is that several silanization layers may be obtained.

Silanization is typically carried out at temperatures of 0°C to 200°C by placement of the bottles or flasks in cold room, at room temperature, in a water-bath, in an oil bath, in a heating block, in a heating mantle, in a microwave oven, in a microwave-accelerated reaction system, or in an oven.

In one embodiment, the silanization is carried out by placement in a microwave oven or a so called microwave-accelerated reaction system. This is advantageous, since the silanization proceeds quickly and efficiently.

The mixtures are optionally stirred, agitated, shaken, tumbled, and/or sonicated. Stirring can be carried out with an overhead stirrer, a magnetic stirrer, or a homogenizer at 50 rpm to 30000 rpm, preferably at 200 rpm to 3000 rpm. The silanization is allowed to proceed between 10 min and 72 h.

After silanization, the magnetic nanoparticles are separated from the solution either by aid of a permanent neodymium magnet, by centrifugation, by sedimentation, or by dialysis. Separation of the nanoparticles from the solutions is either carried out directly, or after addition of ethyl acetate or other organic solvent to help precipitate the nanoparticles. The solutions are optionally cooled before separation. The nanoparticles are washed with water and/or MeOH and/or other organic solvents. The coated nanoparticles are dried in vacuum at room temperature or in a vacuum oven, or used directly for further applications. The coating procedures result in typical mass increases of 5-100%.

In an embodiment, the method further comprises a step of immobilizing a functional entity on the silanized layer.

The functional entity may be at least one enzyme, protein, antibody, peptide, affinity ligand, oligonucleotide, carbohydrate, lipid, surfactant, aptamer, or a pharmaceutically active (drug) molecule to provide derivatized magnetic nanoparticles, and combinations thereof.

This is advantageous, since the nanoparticle may then be suitable for therapy, diagnostics, separation, purification, or MICR (magnetic ink character recognition).

The functional entity may also be a molecularly imprinted polymer layer, to provide molecularly imprinted magnetic nanoparticles. The functional entity may further be a polymer containing functional groups to serve as starting points for either step-wise solid-phase synthesis or further

derivatization by conjugation to an enzyme, protein, antibody, peptide, affinity ligand, oligonucleotide, carbohydrate, lipid, surfactant, aptamer, or drug molecule.

The functional entity may also be a natural or synthetic polymer capable of entrapping or encapsulating drug molecules for later applications in drug delivery, said polymer being coated or grafted on the nanoparticle.

This is advantageous, since the nanoparticle may then suitable for drug delivery.

In an embodiment, a composition obtainable by the method according to some embodiments is disclosed. Said composition comprises mainly discrete nanoparticles with a silanized layer, of silica or silica derivative, on each nanoparticle, such as above 50%, 60%, 70%, 80% or 90% discrete nanoparticles with a silanized layer on each nanoparticle.

In an embodiment, wherein the nanoparticles are magnetic nanoparticles, the composition comprising mainly discrete nanoparticles with a silanized layer on each nanoparticle may be used as a magnetic ink.

Thus, in an embodiment, a magnetic ink is provided, comprising the composition according to embodiments of the invention.

In an embodiment, wherein the nanoparticles are radiopaque nanoparticles or fluorescent nanoparticles, the composition comprising mainly discrete nanoparticles with a silanized layer on each nanoparticle may be used as a contrast agent or marker.

Experimental embodiments

The following experimental embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The embodiments do not limit the invention, but the invention is only limited by the appended patent claims.

The synthesis methods of magnetic iron oxide nanoparticles can be divided into those carried out in aqueous media and those carried out in organic media.

Synthesis of magnetic iron oxide nanoparticles by alkaline hydrolysis of iron salts in aqueous media has been described by Massart [Massart, R. IEEE Trans. Magn. 1981, / 7, 1247-1248]. Massart' s method for the synthesis of magnetite starts with a mixture of iron(II)- and iron(III) salts in a molar ratio corresponding to the oxidation numbers of Fe in magnetite (Fe₃0₄). A number of other publications use variations of this method starting from mixtures of iron(II)- and iron(III) salts to prepare magnetite (Fe^C^) or maghemite (y-Fe₂0₃) [Molday, R.S. US4452773; Liang et al. J. Radioanal. Nuclear Chem. 2006, 269, 3-7; Horak et al. Bioconjugate Chem. 2007, 18, 635-644; Qaddoura, M; Hafeli, U. Polym. Preprints 2007, 48, A25-A26; Sahoo et al. J. Phys. Chem. B 2005, 109, 3879-3885; Ma, M. et al. Colloids and Surfaces A: Physicochem. Eng Aspects 2003, 272, 219-226; Yamaura, M. et al. J. Magn. Magn. Mater. 2004, 279, 210-217; Zheng, W. et al. J. Magn. Magn. Mater. 2005, 288, 403^110; Gu, S. et al. J. Colloid Interface Sci. 2005, 289, 419-426]. When the desired product is Fe₃0₄, the synthesis is sometimes carried out under an inert atmosphere to prevent further oxidation to Fe₂0₃. Synthesis of magnetic nanoparticles in organic media is carried out by thermal decomposition of organometallic compounds, e.g., iron acetyl acetonates or iron acetyl carbonates, in high boiling organic solvents in the presence of surface active agents, e.g., fatty acids, oleic acid or hexadecyl amine [Burke, N.A.D et al. Chem. Mater. 2002, 14, 4752-4761 ; Simenoides, K. et al. J. Magn. Magn. Mat. 2007, 316, e l-e4]. The resulting nanoparticles of the synthesis in organic media are normally covered by hydrophobic molecules that make them soluble only in organic media.

Examples 1 to 4 below regard synthesis of naked magnetite (Fe₃0₄) nanoparticles in water. However, as will be appreciated by a person skilled in the art, other synthesis methods are also possible within the scope of the invention. Example 1

Water was purged with a stream of nitrogen gas for 1 h and then used for the preparation of two solutions: the first solution was prepared by dissolving 0.834 g (3 mmol) of FeS0₄-7H₂0 in 125 mL of water and the second one contained 0.842 g (15 mmol) of KOH and 5.056 g (50 mmol) of KNO₃ in 125 mL of water. The two solutions were sonicated in an ultrasonic bath for 5 min and then mixed in a 250-mL screw- capped bottle at which a green precipitate was formed. The bottle was placed in a preheated (90 °C) water-bath for 2 h. At the end of the reaction time, a black dense precipitate had formed. The bottle was cooled in cold (8 °C) water for 15 min. The precipitate was separated from the solution by aid of a permanent neodymium magnet (N35; 50 x 50 x 30 mm; 0.48 T at the surface) and washed with water (250 mL x 3) and methanol (MeOH) (250 mL x 3). The procedure gave 231 mg of nanoparticles (100% yield). Analysis of the iron content indicated 70.5% and 71.5% Fe by ICP-AES (inductively coupled plasma atomic emission spectrometry) and a colorimetric iron assay, respectively. The nanoparticles caused 0.07% hemolysis of diluted blood after a 24-h incubation, and 0.21% and 0.30% hemolysis of isolated erythrothrocytes after incubations for 1 h and 24 h, respectively.

Fig. 2 A shows a FT-IR spectra of a nanoparticle obtained according to this example.

Example 2

Water was purged with a stream of nitrogen gas for 1 h and then used for the preparation of two solutions: the first solution was prepared by dissolving 0.2 g (0.72 mmol) of FeS(V7H₂0 in 30 mL of water and the second one contained 0.202 g (3.6 mmol) of OH and 1.214 g (12 mmol) of KNO3 in 30 mL of water. The two solutions were sonicated in an ultrasonic bath for 5 min and then mixed at which a green precipitate was formed. The mixture was added to a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 120 °C and then for 15 min at constant temperature (120 °C). The content of the vessel was then cooled to approx. 65 °C. A black dense precipitate was retrieved from the solution by aid of a permanent neodymium magnet. The nanoparticles were washed with 25 mL of water. An amount of 56 mg nanoparticles ( 100% yield) was obtained.

Example 3

Water was purged with a stream of nitrogen gas for 1 h and then used for the preparation of two solutions: the first solution was prepared by dissolving 0.2 g (0.72 mmol) of FeSCv7H₂0 in 15 mL of water and the second one contained 0.202 g (3.6 mmol) of KOH and 1.214 g (12 mmol) of KNO₃ in 15 mL of water. The two solutions were sonicated in an ultrasonic bath for 5 min. Volumes of 15 mL of methylene glycol were added to each solution. The solutions were sonicated briefly. The solutions were mixed at which a green precipitate was formed. The mixture was added to a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 120 °C and then for 15 min at constant temperature (120 °C). The content of the vessel was cooled to approx. 65 °C. A black dense precipitate was retrieved from the solution by aid of a permanent neodymium magnet. The nanoparticles were washed with 25 mL of water. An amount of 56 mg nanoparticles (100% yield) was obtained.

Example 4

One liter of deionized water was heated to 95 °C in a screw-cap bottle. An amount of 600 mg FeCl₂«4 H₂0 was added and the bottle was placed in a heated (95 °C) waterbath. The solution was stirred at 8000 rpm with a dispersing tool (homogenizer) during the synthesis. A volume of 5 mL of 7 M ammonia solution was added at the start of the synthesis. The reaction was allowed to proceed for 1 h. The nanoparticles were separated from the solution with a permanent neodymium magnet and washed by suspension in water 3 times. An amount of 232 mg of nanoparticles (100%) was obtained.

Examples 5 to 27 below regard synthesis of silanized magnetite nanoparticles according to different embodiments of the invention. The following description focuses on an embodiment of the present invention applicable to a magnetic nanoparticle and in particular to a magnetite (Fe₃0₄) nanoparticle. However, it will be appreciated that the invention is not limited to this application but may be applied to many other nanoparticles, as long as they have hydroxyl groups on their surface. Examples of such nanoparticles are maghemite (Fe.C ) nanoparticles, metal iron oxide (MFe₂0₄ wherein M is Co or Mn) nanoparticles, iron (Fe) nanoparticles, iron platinum (FePt) alloy nanoparticles, or silica based nanoparticles.

In addition to the below examples, the first solution may be any solution according to Table 1.

Table 1. Different compositions of the first solution according to the invention.

Example 5 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were subjected to, i.e. added to, a solution containing 2.5 g of PEG 8000 in a mixture of 120 mL of MeOH and 30 mL of ammonia (25%) solution. The mixture was sonicated for 15 min in an ultrasonic bath. The bottles were then placed in room temperature and stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by drop-wise addition during 5 min of 250 μΐ of TEOS (tetraethoxysilane, also called tetraethyl orthosilicate or orthosilicic acid tetraethyl ester), dissolved in 3 mL of MeOH. Silanization proceeded under continuous stirring for 3 h at room temperature. After silanization, the nanoparticles were separated directly from the solution by aid of a permanent neodymium magnet. The solutions were decanted and the nanoparticles were washed with MeOH (100 mL x 2), water (100 mL x 4), and finally with MeOH again (100 mL x 4). Before addition of each next fresh wash solution, the nanoparticles were retrieved with a magnet while the solutions were decanted. The coated nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a mass increase of 26%. Elemental composition: 57.0% Fe (by ICP-AES), 59.6% Fe (by colorimetric iron assay), 3.2% Si (by ICP-AES), 0.7% C (by elemental analysis), 0.5% H (by elemental analysis), 0.3% N (by elemental analysis). The nanoparticles caused 0.06% hemolysis of diluted blood after a 24-h incubation, and 5.92% and 21.15% hemolysis of isolated erythrothrocytes after incubations for 1 h and 24 h, respectively.

Fig. 2 B shows a FT-IR spectra of a coated nanoparticle according to this example and Fig. 3 B shows a transmission electron microscopy (TEM) of a coated nanoparticle according to this example. Example 6 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to 5 g of PEG 400 in a mixture of 240 mL of triethylene glycol and 60 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. The bottle was then placed in a preheated (90 °C) water- bath and stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by drop-wise addition during 5 min of 250 μΐ of TEOS dissolved in 3 mL of MeOH. Silanization proceeded under continuous stirring for 2 h at 90 °C. After silanization, the solution was first cooled and then diluted with ethyl acetate (200 mL) to precipitate the nanoparticles. The latter step was carried out in order to speed up the subsequent magnetic separation. The nanoparticles were washed with MeOH ( 100 mL x 2), water (100 mL x 4), and finally with MeOH again (100 mL x 4). Before addition of each next fresh wash solution, the nanoparticles were retrieved with a magnet while the solutions were decanted. The coated nanoparticles were dried in vacuo at room temperature overnight. The coating procedures resulted in a mass increase of 15%. Elemental composition of nanoparticles: 60.8% Fe (by ICP-AES), 64.4% Fe (by colorimetric iron assay), 2.5% Si (by ICP-AES), 0.7% C (by elemental analysis), 0.4% H (by elemental analysis), 0.3% N (by elemental analysis). The nanoparticles caused no hemolysis of diluted blood after a 24-h incubation, and 3.94% and 22.3% hemolysis of isolated erythrothrocytes after incubations for 1 h and 24 h, respectively.

Fig. 2 C shows a FT-IR spectra of a coated nanoparticle according to this example and Fig. 3 C shows a transmission electron microscopy (TEM) of a coated nanoparticle according to this example.

Example 7 - Silanization with tetraethoxysilane and 3- (trimethoxysilyl)propyl methacrylate

Freshly synthesized magnetite nanoparticles (56 mg) were added to a solution containing 48 niL of methylene glycol, 1 g of PEG 400, and 12 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. A volume of 150 μΐ of TEOS was added. The solution was added to a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 90 °C and then for 15 min at constant temperature (90 °C). The content of the vessel was cooled to approx. 60 °C. A volume of 150 μΐ of 3- (trimethoxysilyl)propyl methacrylate was added and the solution was mixed. The solution was then again subjected to 1200 W microwave treatment with a gradient over 1 min up to 60 °C and then for 15 min at constant temperature (60 °C). After cooling, ethyl acetate (50 mL) was added. The nanoparticles were separated by the use of a permanent neodymium magnet while the solution was decanted. The nanoparticles were washed with MeOH (50 mL x 3). Before addition of each next fresh MeOH wash solution, the nanoparticles were retrieved with a magnet while the solutions were decanted. The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a typical mass increase of 20-60 mg (36- 107%).

Example 8 - Silanization with N-trimethoxysilylpropyl-Ν,Ν,Ν- trimethylammonium chloride

Freshly synthesized magnetite nanoparticles (approx. 463 mg, prepared as described in Example 1) were added to a solution consisting of 300 mL of triethylene glycol and 2 mL of ammonia (25%) solution. The mixture was sonicated for 10 min in an ultrasonic bath. The bottle was then placed in a heated (90 °C) water bath and the solution stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 15 mL of N-trimethoxysilylpropyl-N,N,N- trimethylammonium chloride (50% in methanol). Silanization proceeded under continuous stirring for 2 h at 90 °C. After silanization, the solution was cooled and ethyl acetate (1.2 L) was added to precipitate the nanoparticles. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL x 2). The coated nanoparticles were dried in vacuo at room temperature overnight.

Example 9 - Silanization with

[hydroxy(polyethyleneoxo)propyl]triethoxysilane (8-12 EO)

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to a solution consisting of 150 mL of triethylene glycol and 1 mL of ammonia (25%) solution. The mixture was sonicated for 30 min in an ultrasonic bath. The bottle was then placed in a heated (95 °C) water bath and stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 1 mL of a 50% solution of [hydroxy(polyethyleneoxo)propyl]triethoxysilane (8- 12 EO) in ethanol. Silanization proceeded under continuous stirring for 2 h at 95 °C. After silanization, the solution was cooled and ethyl acetate (350 mL) was added to precipitate the nanoparticles. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with ethyl acetate (100 mL x 2) and MeOH (100 mL x 2). The coated nanoparticles were dried in vacuo at room temperature overnight.

Example 10 - Silanization with tetramethoxysilane and

[hydroxy(polyethyleneoxo)propyl]triethoxysilane (8-12 EO)

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to a solution consisting of 150 mL of methylene glycol and 1 mL of ammonia (25%) solution. The mixture was shaken to disperse the nanoparticles. The bottle was placed in a 95 °C water and the solution was stirred at 900 rpm. Silanization was started by addition of a volume of 250 μΐ of tetramethoxysilane. The stirring was continued at 900 rpm. After 30 min, a volume of 1 mL of a 50% solution of [hydroxy(polyethyleneoxo)propyl]triethoxysilane (8-12 EO) in ethanol was added. Silanization was allowed to proceed with stirring for another 1.5 h at 95 °C. The solution was cooled and ethyl acetate (approx. 350 mL) was added. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with ethyl acetate (100 mL x 2) and MeOH (100 mL x 2). The coated nanoparticles were dried in vacuo at room temperature overnight. The coating resulted in a weight increase of 15 mg (7%). Example 11 - Silanization with fluorescein isothiocyanate-derivatized silane and tetraethoxysilane

Fluorescein isothiocyanate (FITC)-derivatized silane was synthesized by reacting an amount of 50 mg of 5-fluorescein isothiocyanate isomer I with 6 mL of 3- aminopropyltriethoxysilane in 5 mL of ethanol for 24 h. Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in example 1) were added to 360 mL of water, 15 g of PEG 2000, and 90 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. The bottle was then placed in a preheated (90 °C) water-bath and stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 1.0 mL of the FITC-derivatized silane solution prepared above. After 15 min, an amount of 2.25 mL of TEOS was added. Silanization proceeded under continuous stirring at 90 °C for another 45 min and then at room temperature for 13 h. After silanization, the fluorescent nanoparticles were separated using a permanent neodymium magnet and washed with MeOH ( 100 mL x 2), water (100 mL x 4), and finally with MeOH again (100 mL x 4). Before addition of each next fresh wash solution, the nanoparticles were retrieved with the magnet while the solution was decanted. The fluorescent nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a mass increase of 72%.

An advantage with these nanoparticles is that they may be used as markers and contrast agents, since they are fluorescent.

Thus, in an embodiment, a contrast agent is provided, comprising the composition according to embodiments of the invention.

Example 12 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 232 mg, prepared as described in Example 1) were added to a solution consisting of 120 mL of water, 5 g of PEG 2000, and 30 mL of ammonia (25%) solution. The mixture was sonicated for 15 min in an ultrasonic bath and then stirred at 900 rpm with an overhead stirrer.

Silanization of the nanoparticles was started by addition of 0.25 mL of TEOS.

Silanization proceeded under continuous stirring for 40 h at room temperature. After silanization, the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with water (100 mL x 2) and MeOH (100 mL x 2). The coated nanoparticles were dried in vacuo at room temperature overnight.

Example 13 - Silanization with tetraethoxysilane

An amount of 50 mg of commercial iron (II, III) oxide nanopowder > 50 nm

(Sigma- Aldrich catalogue number 637106) were added to a solution consisting of 48 mL of methylene glycol, 12 mL of ammonia (25%) solution, and 1 g of PEG 400. The mixture was sonicated for 1 h in an ultrasonic bath. A volume of 200 μΐ of TEOS was added. The solution was poured into a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 90 °C and then for 15 min at constant temperature (90 °C). After cooling, ethylacetate was added and the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (100 mL 3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a typical mass increase of 25 mg (50%).

Example 14 - Silanization with tetraethoxysilane and 3- aminopropyltriethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to a solution consisting of 600 mL of MeOH, 2.5 g of PEG 8000, and 150 mL of ammonia (25%) solution. The mixture was sonicated for 15 min in an ultrasonic bath and then stirred at 900 rpm in room temperature.

Silanization of the nanoparticles was started by addition of 0.25 mL of TEOS. After 3.5 h, an amount of 0.25 mL of 3-aminopropyltriethoxysilane was added. The silanization was continued for another 1 h. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the

nanoparticles were washed with MeOH (100 mL x 2), water (100 mL x 2), and MeOH (100 mL x 2). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a mass increase of 55 mg (24%).

Example 15 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 232 mg, prepared as described in Example 1) were added to a solution consisting of 600 mL of MeOH, 10 g of PEG 20000, and 1 0 mL of ammonia (25%) solution. The mixture was sonicated for 30 min in an ultrasonic bath and then stirred at 1000 rpm in room temperature.

Silanization of the nanoparticles was started by addition of 0.25 mL of TEOS. After 3 h, the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL x 2), water (300 mL x 3), and MeOH (100 mL x 2). The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a mass increase of 47 mg (20%).

Example 16 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to a solution consisting of 600 mL of MeOH, 10 g of PEG 35 000, and 150 mL of ammonia (25%) solution. The mixture was sonicated for 30 min in an ultrasonic bath and then stirred at 1000 rpm in room temperature.

Silanization of the nanoparticles was started by addition of 0.25 mL of TEOS. After 18 h, the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL), water (500 mL), and MeOH (200 mL). The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a mass increase of 46 mg (20%).

Example 17 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to a solution consisting of 600 mL of MeOH, 5 g of PEG 2000, and 150 mL of ammonia (25%) solution. The mixture was sonicated for 30 min in an ultrasonic bath and then stirred at 1000 rpm in room temperature.

Silanization of the nanoparticles was started by addition of 0.25 mL of TEOS. After 1 h, the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (100 mL), water (300 mL x 4), and MeOH (200 mL). The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating resulted in a mass increase of 35 mg (15%).

Example 18 - Silanization with

[hydroxy(polyethyleneoxo)propyl]triethoxysilane (8-12 EO) and N- trimethoxysilyIpropyl-N,N,N-trimethylammonium chloride Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to a solution consisting of 150 mL of triethylene glycol. A volume of 0.25 mL of ethanolamine was added. The mixture was sonicated for 10 min in an ultrasonic bath. The bottle was then placed in a heated (95 °C) water bath and the solution stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 1 mL of

[hydroxy(polyethyleneoxo)propyl]triethoxysilane (8-12 EO) (50% in ethanol) and 4 mL of N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (50% in methanol). Silanization proceeded under continuous stirring for 2 h at 95 °C. The solution was then cooled and ethyl acetate (300 mL) was added to precipitate the nanoparticles. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with ethylacetate (200 mL x 2) and MeOH (200 mL x 2). The coated nanoparticles were dried in vacuo at room temperature overnight.

Example 19 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 231 mg, prepared as described in Example 1) were added to a solution consisting of 120 mL of MeOH, 10 g of PEG 3400, and 30 mL of ammonia (25%) solution. The mixture was sonicated for 20 min in an ultrasonic bath. The solution was stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 1 mL of TEOS. Silanization proceeded under continuous stirring for 3 h at room temperature. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL x 5), water (200 mL x 5), and MeOH (200 mL x 5). The procedure resulted in a weight increase of 253 mg (109%).

Example 20 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 232 mg, prepared as described in Example 1) were added to a solution consisting of 120 mL of MeOH, 2.5 g of PEG 3400, and 30 mL of ammonia (25%) solution. The mixture was sonicated for 20 min in an ultrasonic bath. The solution was stirred at 1000 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 0.25 mL of TEOS.

Silanization proceeded under continuous stirring for 3 h at room temperature. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL x 5), water (200 mL x 5), and MeOH (200 mL x 5). The procedure resulted in a weight increase of 53 mg (23%).

Example 21 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (approx. 232 mg, prepared as described in Example 4) were added to a solution consisting of 120 mL of MeOH, 5 g of PEG 400, and 30 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. The solution was stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 1 mL of TEOS. Silanization proceeded under continuous stirring for 1 h at 95 °C. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL x 5), water (200 mL x 5), and MeOH (200 mL x 5). The procedure resulted in a weight increase of 30 mg (13%). Example 22 - Silanization with tetraethoxysilane and 3-

(trimethoxysilyl)propyl methacrylate

Freshly synthesized magnetite nanoparticles (approx. 278 mg) prepared as described in Example 3 were added to a solution containing 290 mL of triethylene glycol, 6 g of PEG 400, and 70 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. A volume of 600 μΐ of TEOS was added. The solution was added to six HP-500 Plus microwave vessels (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 90 °C and then for 15 min at constant temperature (90 °C). The content of the vessels were cooled to approx. 55 °C and pooled. A volume of 600 μΐ of 3-(trimethoxysilyl)propyl methacrylate was added, the solution was mixed, and divided to six microwave vessels. The solutions were again subjected to 1200 W microwave treatment with a gradient over 1 min up to 50 °C and then for 30 min at constant temperature (50 °C). After cooling, ethyl acetate (400 mL) was added. The nanoparticles were separated by the use of a permanent neodymium magnet while the solution was decanted. The nanoparticles were washed with MeOH ( 100 mL x 3). Before addition of each next fresh MeOH wash solution, the nanoparticles were retrieved with a magnet while the solutions were decanted. The silanized nanoparticles were dried in vacuo at room temperature overnight. The coating procedure resulted in a typical mass increase of 171 mg (62%). Example 23 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example 3, were added to a solution consisting of 57 mL of triethylene glycol and 3 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. A volume of 150 μΐ of TEOS was added. The solution was poured into a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 90 °C and then for 15 min at constant temperature (90 °C). After cooling, ethylacetate was added and the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (50 mL x 3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a mass increase of 31 mg (56%).

Example 24 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example

3, were added to a solution consisting of 54 mL of triethylene glycol and 6 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. A volume of 150 μΐ of TEOS was added. The solution was poured into a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp.,

Matthews, NC) with a gradient over 1 min up to 90 °C and then for 15 min at constant temperature (90 °C). After cooling, ethylacetate was added and the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (50 mL x 3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a mass increase of 33 mg (59%).

Example 25 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example 3, were added to a solution consisting of 57 mL of triethylene glycol and 3 mL of methylamine (40%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. A volume of 150 μΐ of TEOS was added. The solution was poured into a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 90 °C and then for 1 min at constant temperature (90 °C). After cooling, ethylacetate was added and the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (50 mL x 3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a mass increase of 34 mg (60%).

Example 26 - Silanization with tetraethoxysilane

Freshly synthesized magnetite nanoparticles (56 mg), prepared as in Example 3, were added to a solution consisting of 54 mL of triethylene glycol and 6 mL of methylamine (40%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. A volume of 150 μΐ of TEOS was added. The solution was poured into a HP-500 Plus microwave vessel (CEM Corp., Matthews, NC) and subjected to 1200 W microwave treatment using a MARS 5 microwave-accelerated reaction system (CEM Corp., Matthews, NC) with a gradient over 1 min up to 90 °C and then for 15 min at constant temperature (90 °C). After cooling, ethylacetate was added and the nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (50 mL x 3). The silanized nanoparticles were dried in vacuo at room temperature overnight. The procedure resulted in a mass increase of 45 mg (81%).

Example 27 - Silanization with tetraethoxylsilane and iodinated silane Iodinated silane was synthesized by reacting 1.266 g (1 1 mmol) N- hydroxysuccinimide, dissolved in 50 mL of CH₂C1₂, with 4.998 g (10 mmol) 2,3,5- triiodobenzoic acid, dissolved in 50 mL of CH2CI2, and 2.108 g (1 1 mmol) EDC (water soluble carbodiimide), dissolved in 50 mL of CH2CI2. The reaction was proceeded for 2 days. The solution was extracted with water three times, with saturated sodium chloride solution three times, and finally with water one time. The solution was dried over

MgS0₄. The solution was then evaporated and the solid product was dried in vacuo over night. An amount of 4.363 g (73% yield) of succinimidyl 2,3,5-triiodobenzoate was obtained. An amount of 0.884 g (4 mmol) of 3-aminopropyltrethoxysilane was added to 2.388 g (4 mmol) of succinimidyl 2,3,5-triiodobenzoate dissolved in 8 mL of DMF. The reaction was carried out for 2 days to obtain the iodinated silane.

Freshly synthesized magnetite nanoparticles (approx. 232 mg, prepared as described in Example 4) were added to a solution consisting of 120 mL of MeOH, 5 g of PEG 400, and 30 mL of ammonia (25%) solution. The mixture was sonicated for 1 h in an ultrasonic bath. The solution was placed in a heated (95 °C) water bath and stirred at 900 rpm with an overhead stirrer. Silanization of the nanoparticles was started by addition of 1 mL of TEOS. The silanization proceeded under continuous stirring for 30 min at 95 °C. The iodinated silane reaction mixture, prepared as described above, was then added. The silanization was carried out for another 3 h. The nanoparticles were separated from the solution by aid of a permanent neodymium magnet. The solution was decanted and the nanoparticles were washed with MeOH (200 mL 5), water (200 mL x 5), and MeOH (200 mL x 5).

An advantage with these nanoparticles is that they may be used as X-ray contrast agents or markers, since they are radiopaque.

Thus, in an embodiment, a contrast agent is provided, comprising the composition according to embodiments of the invention. Examples 28 to 30 below regard conjugation of enzyme or peptide to the silanized nanoparticles according to embodiments of the invention.

Example 28 - Immobilization of recombinant human tissue plasminogen activator (tPA) by activation of silica coated nanoparticles with NHSS-EDC

Fmoc-Gly-OH (0.595 g, 2 mmol), dissolved in DMF (1 mL), was added to 200 mg of silanized magnetite nanoparticles, synthesized as described in Example 5.

Coupling was initiated by the addition of DIPCDI (0.252 g, 2 mmol) in DMF (1 mL) and DMAP (25 mg, 0.2 mmol) in DMF (1 mL). The reaction was carried out on a rotator for 3 days at room temperature. The nanoparticles were separated from the solution with a permanent magnet and washed with DMF (5 mL x 10). Magnetic separation was carried out between the washings. The Fmoc groups were removed by treatment with 5 mL of piperidine— DMF (1 :4) for 5 min. After removal of the first cleavage solution, 5 mL of fresh piperidine-DMF (1 :4) was added and the mixture was incubated for another 15 min. The nanoparticles were washed with DMF (5 mL x 10) and CH2CI2 (5 mL x 10). The Kaiser qualitative ninhydrin test detected free amino groups at this point. Succinylation of the free amino groups was carried out by addition of succinic anhydride (0.4 g, 4 mmol) in 6 mL of CtLC^-pyridine (1 : 1 ). The mixture was incubated in room temperature on a rotator for 30 min. After magnetic separation and removal of the solution, the nanoparticles were washed with CH2CI2 (5 mL x 5), DMF (5 mL x 5), and MeOH (5 mL x 5). The Kaiser test was negative, indicating that succinylation was complete. The nanoparticles were dried in vacuo at room temperature overnight. Elemental analysis: 3% C, 0.6% H, 0.6% N. An amount of 125 mg of the dried succinylated nanoparticles was suspended in 1 mL of water. The nanoparticles were activated by addition of NHSS (87 mg, 0.4 mmol) in water (1 mL) and EDC (77 mg, 0.4 mmol) in water ( 1 mL). The esterification proceeded in room temperature on a rotator for 2 h. The nanoparticles were withdrawn with a magnet and the reagent solution was removed. The nanoparticles were washed with water (5 mL x 10) and finally suspended in 2.08 mL of water. Enzyme immobilization was carried out by addition of a solution of recombinant human tissue plasminogen activator, tPA

(marketed by Boeringer Ingelheim under the trademark Actilyse) (12.5 mg dissolved in 6.25 mL of water) to the nanoparticle solution. Coupling proceeded on an orbital shaker (200 rpm) for 4 h at 4 °C. The tPA-nanoparticle conjugates were withdrawn with a magnet, the solution was removed, and washing was carried out with water (5 mL x 5), phosphate-buffered saline (PBS) pH 7.4 (5 mL x 3), and water again (5 mL x 3). The protein concentration of the wash solutions was determined by the method of Bradford using bovine serum albumin as the reference. The mass of tPA immobilized was calculated by subtracting the mass of tPA found in the wash solutions from the mass of tPA fed to the nanoparticles at the start of the immobilization. The immobilization yield was calculated as 100% * (mass of tPA immobilized)/(mass of tPA fed). The immobilization yield was 63%. The tPA loading was calculated as (mass of tPA immobilized)/(mass of nanoparticles). The tPA loading was 63 g tPA/mg

nanoparticles. The enzyme activity of the free and immobilized enzyme was determined by monitoring the formation of p-nitroaniline (pNA) spectrophotometrically at 405 nm during hydrolysis of H-D-Ile-Pro-Arg-pNA used as a substrate. The assay was carried out by mixing 0.25 mL of a solution containing either free tPA or tPA-nanoparticle conjugates, 0.25 mL of 100 mM Tris- HC1 pH 8.4 containing 100 mJVl NaCl, and 0.25 mL of a 1 mM-solution of the substrate in water. The specific enzyme activity was 0.86 U/mg tPA. The enzyme activity yield was calculated as 100%*(total activity of immobilized tPA)/(total activity of fed tPA). The enzyme activity yield was 45%. A reaction scheme is provided in Figure 4A.

Example 29 - Immobilization of tPA by activation of coated nanoparticles with tresyl chloride

An amount of 280 mg of silanized nanoparticles, synthesized as described in Example 6, was first washed with dry acetone (5 mL x 2) and then suspended in 6.7 mL of dry acetone and 0.78 mL of dry pyridine. Activation with tresyl chloride (0.3 mL) was initiated by drop-wise addition to the nanoparticles solution under shaking. The reaction was carried out on an orbital shaker (1 000 rpm) for 2 h at 4°C. The nanoparticles were then retained with a permanent neodymium magnet and the solution was removed. The nanoparticles were washed with acetone (5 mL x 3), acetone-water

(2: 1) (5 mL x 2), acetone-water (1: 1) (5 mL x 2), acetone-water (1 :2) (5 mL x 2), acetone—water (1 :4) (5 mL x 2), and water ( 10 mL x 3). The nanoparticles were then suspended under sonication in 10 mL of water and added dropwise to dialyzed tPA (38 mg) in 80 mL of 0.2 M sodium phosphate buffer pH 8. Coupling of tPA to tresyl chloride activated nanoparticles was carried out at 4 °C for 33 h on an orbital shaker (200 rpm). The nanoparticles were then retrieved with a permanent magnet and washed with 0.2 M Tris-HCl pH 8 (50 mL). Capping of remaining tresyl groups was performed with 0.2 M Tris-HCl pH 8 during 23 h at 4 °C on an orbital shaker (200 rpm). The tPA- nanoparticle conjugates were separated with a magnet and washed with water ( 10 mL x 2), 50 mM sodium phosphate buffer pH 7 (10 mL), 25 mM sodium phosphate buffer pH 7 (20 mL), and 12.5 mM sodium phosphate buffer pH 7 (20 mL x 2). Determination of protein concentration, enzyme activity, and calculations of immobilization parameters were carried out as described in example 28. The enzyme loading was 71 μg tPA/mg nanoparticles. The immobilization yield was 52%. The specific enzyme activity was 0.82 U/mg tPA. The enzyme activity yield was 41%. The tPA-nanoparticle conjugates caused no hemolysis of diluted blood after a 24-h incubation, and 0.15% and 1.03% hemolysis of isolated erythrothrocytes after incubations for 1 h and 24 h, respectively. A reaction scheme is provided in Figure 4B.

Fig. 2 D shows a FT-IR spectra of a coated nanoparticle according to this example and Fig. 3 D shows a transmission electron microscopy (TEM) of a coated nanoparticle according to this example. A reaction scheme is provided in Fig. 4B.

Example 30 - Coupling of an NGR-containing peptide to the coated nanoparticles via a PEG spacer

Fmoc-Gly-OH (0.298 g, 1 mmol), dissolved in DMF (0.5 mL), was added to 100 mg of silanized magnetite nanoparticles, synthesized as described in Example 5. Coupling was initiated by the addition of DIPCDI (0.126 g, 1 mmol) in DMF (0.5 mL) and DMAP ( 13 mg, 0.1 mmol) in DMF (0.5 mL). The reaction was carried out on a rotator for 3 days at room temperature. The nanoparticles were separated from the solution with a permanent magnet and washed with DMF (3 mL x 10). Magnetic separation was carried out between the washings. The Fmoc groups were removed by treatment with 3 mL of piperidine-DMF (1 :4) for 5 min. After removal of the first cleavage solution, 3 mL of fresh piperidine-DMF (1 :4) was added and the mixture was incubated for another 15 min. The nanoparticles were washed with DMF (3 mL x 10) and CH₂C1₂ (3 mL x 10). The Kaiser qualitative ninhydrin test detected free amino groups at this point. Fmoc-NH-(PEG)₂-COOH (48 mg, 0.086 mmol) dissolved in 0.4 mL of DMF, DIPCDI ( 1 1 mg, 0.086 mmol) in 0.2 mL of DMF, and HOBt (12 mg, 0.086 mmol) in 0.2 mL of DMF was added to the nanoparticles. The coupling was carried out on a rotator for 24 h. After the coupling, the Kaiser qualitative ninhydrin test was negative. The Fmoc groups were removed by treatment with 3 mL of piperidine- DMF (1 :4) for 5 min. After removal of the first cleavage solution, 3 mL of fresh piperidine-DMF ( 1 :4) was added and the mixture was incubated for another 15 min. The nanoparticles were washed with DMF (3 mL x 10) and CH₂C1₂ (3 mL x 10). The Kaiser qualitative ninhydrin test detected free amino groups at this point. Ac-Gly- Asn(Trt)-Gly-Arg(Pbf)-Gly-Ahx-Gly-OH (10.8 mg, 9.28 μπιοΐ), DIPCDI (5 mg, 40 μπιοΐ), and HO At (5.4 mg, 40 μπιοΐ) were dissolved in 0.2 mL of DMF and added to the nanoparticles. The reaction was carried out for 3 days. After the coupling, the Kaiser qualitative ninhydrin test was negative. The nanoparticles were dried in vacuo overnight. The protecting groups were removed by treatment with 0.5 mL of TFA- CH₂Cl₂-water (90:5:5) for 2 h. The nanoparticles were washed with 1 mL each of CH₂C1₂, DMF, CH₂C1₂, MeOH, water, and MeOH. The nanoparticles were dried in vacuo overnight.

Characterization of coated magnetic nanoparticles

A number of methods were utilized to study the coated nanoparticles produced according to embodiments of the invention.

Transmission electron microscopy

Size and morphology of the nanoparticles (NPs) were studied by transmission electron microscopy (TEM) using a JEOL JEM- 1230 (Tokyo, Japan) equipped with a Gatan multiscan camera model 791 (Pleasanton, CA, USA). Samples were deposited on Pioloform (polyvinylbutyral) films and images were recorded at accelerating voltages of 80 kV. Fig. 3 show transmission electron microscopy (TEM) of (A) naked magnetite nanoparticles from Example 1, (B) surface coated magnetite nanoparticles from

Example 5, (C) surface coated magnetite nanoparticles from Example 6, and (D) tPA- nanoparticle conjugates from Example 29.

Dynamic light scattering

The hydrodynamic particle size distribution was determined by dynamic light scattering (DLS) using a Nanotrac Ultra Particle Size Analyzer from Microtrac (Montgomeryville, PA, USA). The typical hydrodynamic size in triethylene glycol of naked magnetite particles prepared as in Example 1 was 140 nm. The hydrodynamic size could not be measured in water as the naked nanoparticles aggregated in water. The hydrodynamic size in water of the silanized nanoparticles prepared in Example 5 was 300-365 nm. The hydrodynamic size in water of the silanized nanoparticles prepared in Example 6 was 250-300 nm.

Magnetic characterization

Magnetic properties of NPs embedded in epoxy resin were measured at room temperature with a Princeton Measurement Corp. M2900-2 alternating gradient magnetometer (Princeton, NJ, USA). Magnetic hysteresis loops are shown in Fig. 7.

FT-IR spectroscopy

Fourier-transform infrared (FT-IR) spectra of NPs (in KBr pellets) were measured on a Bruker IFS66 FT-IR spectrometer (Billerica, MA, USA). Spectra are found in Fig. 2.

Elemental analysis

The Fe and Si contents of the nanoparticles (NPs) were analyzed on an Optima 3000 DV ICP-AES (Perkin Elmer, Waltham, MA, USA). Elemental analysis (C, H, and N) was carried out by Mikrokemi AB (Uppsala, Sweden).

Colorimetric iron assay The iron content of the nanoparticles was determined by a modification the method described in Sasikumar PG, Kempe M. Magnetic CLEAR supports for solid- phase synthesis of peptides and small organic molecules. Int J Peptide Res Ther 2007;13(l-2): 129-141. Nanoparticle samples (1-4 mg) were treated with 0.3 mL of HCl (37%) for 30 min. The dissolved samples were transferred quantitatively to 25-mL volumetric flasks and diluted with water. Volumes of 0.5 mL of the diluted solutions were mixed with a hydroxylamine hydrochloride solution (0.25 mL; 0.1 mg/mL), a sodium acetate solution (2.5 mL; 0.1 mg/mL), and a 1,10-phenanthroline monohydrate solution (2.5 mL; 1 mg/mL). The solutions were diluted 2-10 times with water and the absorbance was measured at 508 nm. Standard ferrous ion solutions for calibration were prepared under identical conditions from FeS0₄*7H₂0.

Hemolysis assay on diluted human blood

A volume of 5 mL of human fresh blood anticoagulated with sodium citrate was diluted with 5 mL of PBS. The blood sample was obtained from a healthy human volunteer. Volumes of 0.75 mL of nanoparticles (0.1 mg/mL) in PBS were incubated in Eppendorf microtubes for 30 min at 37 °C. Negative and positive controls were prepared by substituting PBS and water, respectively, for nanoparticle solutions. Each sample was run in triplicates. Diluted blood (0.25 mL) was added and samples were incubated on a rotator for 24 h at 37 °C. The tubes were then centrifuged (10 min, 5 000 rpm). The absorbance of the supernatants was measured in a spectrophotometer at 545 nm. The hemolysis degree was calculated as 100%*(A_sampi_e - A_neg_atlve controi (^Apositive control - Anegative control). The naked magnetite nanoparticles from Example 1 caused 0.07% hemolysis. The silanized nanoparticles from Example 5 caused 0.06% hemolysis. The silanized nanoparticles from Example 6 and the tPA-conjugated nanoparticles from Example 29 caused no hemolysis.

Hemolysis assay on washed isolated human erythrocytes

A volume of 2 mL of PBS was added to 5 mL of human fresh blood anticoagulated with sodium citrate. The mixture was inverted for 2 min on a rocking mixer and was then centrifuged at 5 000 rpm for 5 min. The supernatant was removed and the pellet of erythrocytes was washed twice more with 4 mL of PBS by suspension, centrifugation, and decantation. Finally, an erythrocyte suspension was prepared by adding 5 mL of PBS. Volumes of 1 mL of nanoparticles (0.1 mg/mL) in PBS were incubated at 37 °C in Eppendorf microtubes. Negative and positive controls were prepared by substituting PBS and water, respectively, for nanoparticle solution. Each sample was run in triplicates. After 30 min, 0.1 mL of the erythrocyte suspension was added to each tube. The tubes were incubated on a rotator at 37 °C during either 1 h or 24 h. The tubes were then centrifuged at 10 000 rpm for 10 min. The absorbance of the supernatants was measured in a spectrophotometer at 545 nm. The hemolysis degree was calculated as described above. The naked magnetite nanoparticles from Example 1 caused 0.21% and 0.30% hemolysis after 1 h and 24 h, respectively. The silanized nanoparticles from Example 5 caused 5.92% and 21.15% hemolysis after 1 h and 24 h, respectively. The silanized nanoparticles from Example 6 caused 3.94% and 22.30% hemolysis after 1 h and 24 h, respectively. The tPA-nanoparticle conjugates from Example 29 caused 0.15% and 1.03% hemolysis after 1 h and 24 h, respectively.

Stability study on tPA-nanoparticle conjugates

Solutions of tPA-nanoparticle conjugates from Examples 28 (5.7 mg/mL) and Example 29 (2.4 mg/mL) in water were subjected to enzyme activity assay after sonication in an ultrasonic bath during 1 h and after incubation at 4 °C for up to 40 days. The obtained enzyme activities are shown in Fig. 8.

Examples 31 to 33 regard magnetic particle targeting. Example 31 - In vitro magnetic particle targeting to a coiled wire in a single-pass flow-through set-up experiment

A Kantahl D ferromagnetic wire (length 100 mm, 0 0.13 mm) was looped 15 times to fabricate a coil (length 40 mm, 0 2 mm). The coil was inserted into a Wiretrol II (Drummond Scientific Company, Broomall, PY, USA) glass capillary tube (length 90 mm, 0 2.2 mm). The capillary tube was positioned between two permanent neodymium magnets (N35; 50 x 30 x 30 mm; 0.48 T at the surface), at a distance of 3 cm from each magnet. At this distance, the magnetic field applied to the coil was 0.1 T, as measured with a Gaussmeter model GM-2 (Alphalab, Saltlake City, UT, USA). Both ends of the capillary tube were connected to silicon tubing (inner 0 2 mm, outer 0 4 mm) as shown in the set-up in Fig. 5A. Evaluation of the nanoparticle capture efficiency of the coil during single-pass flow-through experiments was carried out by connecting the experimental set-up to a KDS 100 syringe infusion pump (KD Scientific, Holliston, MA, USA). A volume of 4.7 mL of nanoparticles prepared as described in Example 5 (25 g/πlL water) was pumped into the evacuated system at 0.5-6 mL/min. The dead volume was 0.7 mL. The effluent (4 mL) was collected and the absorbance was measured at 350 nm in a spectrophotometer. A standard curve showed a linear relationship between the absorbance and the nanoparticle concentration. The percentage of nanoparticles retained in the capillary, hereafter referred to as the capture efficiency (CE), was calculated as CE = 100*(A₀ - A)/Ao where A₀ is the initial absorbance of the nanoparticle solution and A is the absorbance of the effluent. The experiment was repeated without the coil present in the capillary tube in order to determine the blank retention. The CE is shown in Figure 5B.

Example 32 - In vitro magnetic particle targeting to a coiled wire in recirculation flow-through set-up experiments

A Kantahl D ferromagnetic wire (length 100 mm, 0 0.13 mm) was looped 15 times to fabricate a coil (length 40 mm, 0 2 mm). The coil was inserted into a Wiretrol II (Drummond Scientific Company, Broomall, PY, USA) glass capillary tube (length 90 mm, 0 2.2 mm). The capillary tube was positioned between two permanent neodymium magnets (N35; 50 x 30 x 30 mm; 0.48 T at the surface), at a distance of 3 cm from each magnet. At this distance, the magnetic field applied to the coil was 0.1 T, as measured with a Gaussmeter model GM-2 (Alphalab, Saltlake City, UT, USA). Both ends of the capillary tube were connected to silicon tubing (inner 0 2 mm, outer 0 4 mm) as shown in the set-up in Fig. 5A. Evaluations of the nanoparticle capture efficiency at recirculation were carried out by connecting the experimental set-up to a Gilson minipuls 2 peristaltic pump. The tubings were arranged in a closed-loop system and nanoparticles from Example 5 (4-10 mL, 25 μg nanoparticles/mL water) was recirculated at flow rates of 1-40 mL/min during 10-60 min (except for the evaluation at 1 mL/min, which was carried out for 90 min). At the end of each experiment, the loop was disconnected and a sample of the nanoparticle solution was collected for measurement of the absorbance and calculation of the CE as in Example 30. The CE's are shown in Figure 5C-F. The deposition of nanoparticles on the wire is shown in Figure 6B, as compared to Figure 6A, which is a naked wire.

Example 33 - In vivo magnetic particle targeting and lysis of in-stent thrombosis by tPA-nanoparticle conjugates

A anthal D wire (length 80 mm, x 0.13 mm) was woven into a NIR Primo™ coronary stent (length 16 mm, x 3 mm; Boston Scientific Scimed, Maple Grove, MN, USA) in a coiled configuration. The stent was mounted manually on a Maverick™ coronary balloon (length 30 mm, x 3 mm; Boston Scientific Scimed), A domestic female pig (40 kg) were pre-sedated, anaesthesized, intubated orally with cuffed endotracheal tubes, ventilated with nitrous oxide and oxygen (7:3), and monitored by electrocardiography (ECG). Radiological procedures were performed in an

experimental catheterization laboratory (Shimadzu Corp., Kyoto, Japan). Angiograms were obtained by injection of iohexol. Heparin (5000 IU) was given intravenously before catheterization. The left femoral artery, the left carotid artery, and the right external jugular vein were surgically exposed and 6F introducer sheaths were inserted into the vessels. A sternotomy was performed on the pig. Through the introducer in the left carotid artery, a guide catheter was advanced into the left main coronary artery. The catheter was used to place a Doppler flow velocity transducer (Jometrics Flowire, Jomed NV), connected to a FloMap monitor (Cardiometrics, Mountain View, CA, USA) and a guide wire into the left anterior descending artery (LAD). The NIR

PrimoTM coronary stent with a Kanthal D wire was placed in the mid portion of the LAD, distal to the first diagonal branch, by inflation of the balloon to 10 atm for 10 s. An intravascular ultrasound probe was advanced over the guide wire to image the stented artery segment at various time points. The baseline flow, measured by the flow wire, was 20 cm s after stent insertion. A permanent neodymium magnet (N48;

50x15x15 mm; 0.48 T at the surface) was applied to the anterior portion of the heart, in contact with the stented segment of the LAD. After spontaneous formation of a thrombus in the stent, the baseline flow decreased to 5 cra/s. A solution of tPA- nanoparticle conjugates from Example 29 (40 mL, 0.14 mg/mL) was injected through the guide catheter into the left main coronary artery. The thrombus was lysed by the action of the tPA-nanoparticle conjugate. The baseline flow increased to 15 cm/s. The pig was sacrificed at the end of the experiment.

According to an embodiment, the composition according to embodiments of the invention is provided, for use as a medicament.

Specifically, according to an embodiment, the composition according to embodiments of the invention is provided, for the treatment of thrombosis.

Thus, according to an embodiment, a method of treatment of thrombosis in a subject is disclosed, comprising a first step of injecting the composition comprising coated magnetic nanoparticles according to some embodiments, into the blood stream, i.e. the cardiovascular system, of the subject. Next, a magnetic field is applied to the place of the thrombosis, where after the nanoparticles are attracted to the thrombosis with the magnetic field, thus resolving the thrombus.

In an embodiment, a method of treatment of in-stent thrombosis in a subject with an implanted magnetizable stent is disclosed, comprising a first step of injecting the composition comprising coated magnetic nanoparticles according to some embodiments, into the blood stream, i.e. the cardiovascular system, of the subject. Next, a magnetic field is applied to the place of the stent, where after the nanoparticles are attracted to the stent with the magnetic field, thus resolving the thrombus.

As described above, the magnetic nanoparticle may be conjugated to tPA, i.e. recombinant human tissue plasminogen activator, further enhancing the anti thrombosis effect.

Although the present invention has been described above with reference to specific embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims and, other embodiments than the specific above are equally possible within the scope of these appended claims . In the claims, the term "comprises/comprising" does not exclude the presence of other elements or steps. Furthermore, although individually listed, a plurality of means, elements or method steps may be implemented by e.g. a single unit or processor. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. The terms "a", "an", "first", "second" etc do not preclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way.

Claims

1. A method for forming a layer on a nanoparticle with hydroxyl groups on its surface, comprising the steps of

subjecting the nanoparticle to a first solution comprising a compound according to formula (I):

I

wherein "n" is an integer in the interval 0 (zero) to 7000;

subjecting the nanoparticle to a second solution comprising a silanization agent; and

allowing formation of a silanized layer on the nanoparticle.

2. The method according to claim 1 , wherein the nanoparticle is a magnetic nanoparticle.

3. Method according to claim 1 or 2, wherein the compound according to the formula (I) is selected from the group consisting of: ethylene glycol, diethylene glycol (DEG), methylene glycol (TREG), tetraethylene glycol, pentaethylene glycol, hexaethylene glycol, heptaethylene glycol, octaethylene glycol, and other oligoethylene glycols/polyethylene glycols/polyethylene oxides of molecular weights up to 300000 (such as PEG 400, PEG 2000, PEG 3400, PEG 8000, PEG 20000, PEG 35000, PEG 100000, PEG 200000, and PEG 300000), or a combination thereof.

4. Method according to any of the preceding claims, wherein the silanization agent is a silane.

5. Method according to claim 4, wherein the silane is an alkoxysilane.

6. Method according to claim 5, wherein the alkoxysilane is selected from the group consisting of tetramethoxysilane, tetraethoxysilane, tetra-n-propoxysilane, tetra-iso-propoxysilane, tetra-n-butoxysilane, tetra-t-butoxysilane, trimethoxysilane, triethoxysilane, tri-n-propoxysilane, tri-iso-propoxysilane, tri-n-butoxysilane, tri-t- butoxysilane, trimethoxychlorosilane, triethoxychlorosilane, tri-n-propoxychlorosilane, tri-iso-propoxychlorosilane, tri-n-bytoxychlorosilane, tri-t-butoxychlorosilane, benzyltrimethoxysilane, benzyltriethoxysilane, dimethyldimethoxysilane,

dimethyldiethoxysilane, and mixtures thereof.

7. Method according to claim 4, wherein the silane is a halosilane.

8. Method according to claim 8, wherein the halosilane is selected from the group consisting of tetrachlorosilane, trichlorosilane, tetrafluorosilane, trifluorosilane, and mixtures thereof.

9. Method according to claim 4, wherein the silane is an aminosilane

10. Method according to claim 9, wherein the aminosilane is selected from the group consisting of 3-aminopropyltrimethoxysilane, 3- aminopropylmethyldimethoxysilane, 3-aminopropyldimethylmethoxysilane, N-(2- aminoethyl)-3-aminopropylmethyldimethoxysilane, N-(2-aminoethyl-3- aminopropyl)trimethoxysilane, 4-aminobutyldimethylmethoxysilane, 4- aminobutyltrimethoxysilane, aminoethylaminomethylphenethyltrimethoxysilane, N-(2- aminoethyl)-3 -aminoisobutylmethyldimethoxysilane, N-(6- aminohexyl)aminopropyltrimethoxysilane, 3-(m- aminophenoxy)propyltrimethoxysilane, aminophenyl trimethoxysilane, 3 - aminopropyltriethoxysilane, 3-aminopropylmethyldiethoxysilane, 3- aminopropyldimethylethoxysilane, N-(2-aminoethyl)-3- aminopropylmethyldiethoxysilane, N-(2-aminoethyl-3-aminopropyl)triethoxysilane, 4- aminobutyldimethylethoxysilane, 4-aminobutyltriethoxysilane,

aminoethylaminomethylphenethyl triethoxysilane, N-(2-aminoethyl)-3- aminoisobutylmethyldiethoxysilane, N-(6-aminohexyl)aminopropyltriethoxysilane, 3 - (m-aminophenoxy)propyltriethoxysilane, aminophenyltriethoxysilane, and mixtures thereof.

1 1. Method according to claim 4, wherein the silane is an olefin-containing silane.

12. Method according to claim 1 1, wherein the olefin-containing silane selected from the group consisting of 3-(trimethoxysilyl)propylmethacrylate, 3- (triethoxysilyl)propylmethacrylate, methacryloxymethyltrimethoxysilane,

methacryloxymethyltriethoxysilane, vinyltrimethoxysilane, vinyltriethoxysilane, allyltrimethoxysilane, allyltriethoxysilane, vinyltrichlorosilane, and mixtures thereof

13. Method according to claim 4, wherein silane is a fluorescent silane.

14. Method according to claim 4, wherein the silanization agent is a radiopaque silane.

15. Method according to any of the preceding claims, wherein the first solution further comprises a base and/or a second solvent.

16. Method according to claim 15, wherein the base is selected from the group consisting of: ammonia, sodium hydroxide, potassium hydroxide, triethylamine, trimethylamine, dimethylamine, diethylamine, ethylamine, propylamine, N,N- diisopropylethylamine, n-methyl morpholine, N-methylpyrrolidone, oleylamine, ethanolamine, pyridine, 4-dimethylaminopyridine, methylamine, and piperidine, or a combination thereof.

17. Method according to claim 15 or 16, wherein the second solvent is selected from the group consisting of: water, methanol, ethanol, n-propanol, iso- propanol, Ν,Ν-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, and acetonitrile, or a combination thereof.

18. Method according to any of the preceding claims, further comprising a step of immobilizing a functional entity on the silanized layer, wherein said functional entity is selected from the group consisting of: enzyme, protein, antibody, peptide, affinity ligand, oligonucleotide, carbohydrate, lipid, surfactant, or a pharmaceutically active molecule.

19. A composition obtainable by the method according to claims 1 to 18.

20. A composition comprising mainly discrete nanoparticles with a silanized layer on each nanoparticle.

21. The composition according to claims 19 or 20, wherein the nanoparticles are magnetic.

22. The composition according to claims 19 to 21 , wherein the nanoparticles are radiopaque.

23. The composition according to claims 19 to 21, wherein the nanoparticles are fluorescent.

24. The composition according to claims 19 to 21 , wherein the nanoparticles are MRI-active.

25. The composition according to any of claims 19 to 24, for use as a medicament.

26. The composition according to claim 21 , for the treatment of thrombosis.

27. A contrast agent, comprising the composition according to any of claims

22 to 24.

28. A magnetic ink, comprising the composition according to claim 21.

29. A method of treatment of thrombosis in a subject, comprising the steps of:

injecting the composition according to claim 21 into the bloodstream of the subject;

applying a magnetic field to the place of the thrombosis; and

attracting the nanoparticles to the thrombosis with the magnetic field, thus resolving the thrombus.

30. A method of treatment of in-stent thrombosis in a subject with an implanted magnetizable stent, comprising the steps of:

applying a magnetic field to the place of the stent; and

attracting the nanoparticles to the stent with the magnetic field, thus resolving the thrombus.