Technical field of the invention

The present invention relates to nanoparticles as well as methods for preparing said nanoparticles as well as use of the nanoparticles as contrast agents or other uses. The invention specifically relates to contrast agents for visualizing or imaging biological 5 material.

Technical Background

Magnetic resonance Imaging, MRI, is a medical imaging modality where the soft tissues of the body are visualized by utilization of the magnetization of atomic nuclei. Normally the abundant hydrogen nuclei of the water molecules of the body are

10 imaged. The strength of the MRI signal depends on the nature of the nucleus, its abundance and its local magnetic environment. These factors affect the longitudinal (Tl) and transverse (T2) relaxation times, which in turn affect the signal strength. Thus, the source of contrast in MRI is a combination of the local concentration of nuclei and their magnetic environment. Various morphological features can be

15 enhanced by emphasizing the Tl or the T2 contrast. The local magnetic environment can be modified by the presence of contrast agents and, depending on their magnetic properties, the signal can be increased (positive contrast) or decreased (negative contrast). Positive contrast agents are often preferred because interpretation of the images becomes simpler.

20 The market is currently dominated by water soluble gadolinium chelates. Because of their small physical size (< 1 nm) they rapidly distribute into the extracellular space (the blood plus the interstitial space between the cells of the tissues) which somewhat limits the contrast effect. A problem with the in-vivo use of paramagnetic metal ions, such as gadolinium, is their toxicity and the chelates in the currently marketed

25 contrast agents rather sucessfully adresses this. Lately it has been discovered that the chelates releases small amounts of gadolinium which becomes apparent in patients with non-existent of very poor kidney function, where a serious side effect called Nephrogenic Systemic Fibrosis, NSF, has been discovered (Grobner et al. Nephrology, Dialysis and Transplantation 2006, 21, 1104; Sieber et al. Invest.

Radiol. 2008, 43, 65).

Nanoparticles based on crystalline gadolinium oxide as contrast agents are known. Bridot et al. discloses a method for production of crystalline particles of gadolinium oxide, in which larger particles than 2.2 nm have to be produced by a multistep procedure (Bridot et al. J. Am. Chem. Soc. 2007, 129, 5076).

Additionally, nanoparticles of other materials containing gadolinium ions are known, examples thereof are described in Gadolinium phosphate (H. Hifumi. S. Yamoka, A. Tanimoto, D. Citterio, K. Suzuki, J. Am. Chem. Soc. 2006, 128, 15090), gadolinium fluoride (F. Evanics, P. R. Diamente, F. C. J. M van Veggel, G. J. Stanisz, R. S.

Prosser Chem. Mater. 2006, 129, 5076) and gadolinium terephtalate (M.D. Rowe, C- C Chang, D. H. Thamm, S. L. Kraft, J. F. Harmon, Jr., A. P. Vogt, B. S. Sumerlin, S. G. Boyes).

Additionally nanoparticles of gadolinium hydroxide formed inside the hollow protein ferritin is known (Sanches, P., Dalton Trans., 2009, 800). The nanoparticles will be in rapid equilibrium with the surrounding medium and hence be unsuitable for in-vivo use in humans since it will release toxic gadolinium ions in the blood.

Summary of the invention

The object of the present application is to provide a novel nanoparticle, methods for it preparation and use of the nanoparticle.

In one aspect the object of the present invention is achieved by a nanoparticle comprising a core of amorphous rare earth element hydroxide, and an organic coating comprising silicon atoms and phosphorus atoms.

In one aspect the object of the present invention is achieved by a composition comprising a nanoparticle comprising a core of amorphous rare earth element hydroxide, and an organic coating comprising silicon atoms and phosphorus atoms. In one aspect the object of the present invention is achieved by using the nanoparticle or a composition comprising the nanoparticle as a contrast agent or a marker, for example as MRI contrast agent or X-ray contrast agent. In one aspect the present invention relates to a method for obtaining nanoparticles, said method for obtaining a coated nanoparticle comprises a) providing a rare earth element salt and a hydroxide source in solution in the presence of a capping agent b) forming an intermediate nanoparticle rare earth element hydroxide; c) adding a base, one or more organo-oxysilane residue(s) according to any one of formulas I-VI, , and one or more a phosphorus containing compound (s) in the presence of a non-aqueous solvent; and d) obtaining coated rare earth element hydroxide nanoparticle, wherein the coating comprises silicon atoms and phosphorus atoms. Short description of drawings

Fig 1 is a schematic illustration of nanoparticles 1 according to the present invention.

Fig 2 is a schematic illustration of nanoparticles 2 according to the present invention.

Fig 3 is a schematic illustration of how the organo-oxysilane may be bound to the core. Definitions of terms

The term "nanoparticle" is used to describe a particle with a total diameter from 1- 100 nm of essentially spherical shape, i.e. excluding flakes, rods and ribbons. The term "lanthanide" is considered synonymous to the term "rare earth" and includes the elements La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

The term "transition metal" includes the elements Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, Hf, Ta, W, Re, Os, Ir, Pt, Au and Hg.

"Bio-inert" refers to a material that is bio-compatible, i.e. harmless to a living organism and at the same time stable to degradation in-vivo.

"Monolayer" refers to a one molecule thick layer. Examples of monolayer in the present invention are fatty acid cappings and organo-oxysilanes. When the

nanoparticle is conjugated the conjugate in not considered to be a part of the monolayer.

"Oriented" in the context of coatings refers to a layer of coating molecules where all the heads and tails (as arbitrarily defined from case to case but as intended in the present invention we consistently refer to the silane, where present, as the head) of the coating molecules are oriented in the same way in relation to the particle core surface.

"Polydispersity" is a term relating to the distribution of molecular weight in a polymeric material. Polymers are normally produced by methods that produce a variety of chain lengths with different molecular weight. They will have an average molecular weight which can be calculated in different ways. The polydispersity describes how wide the distribution is around the average molecular weight. There are mathematical ways to define a polydispersity index to quantify this property as can be found in a standard polymer chemistry textbook (J. R. Fried, Polymer Science and Technology, Prentice Hall, 1995) but for the purposes of understanding the present invention, it suffices with a qualitative picture. High polydispersity is a situation where the polymer mixture has a wide distribution of molecular weight and low polydispersity describes the opposite. The term monodisperse is normally considered synonymous to low polydispersity but it may also describe the situation where the material has been produced by a method that gives absolute control of the chain length. Typically such a material would be produced by the costly process of iterative chemical reactions and purifications. This product may also be referred to as a material of "defined molecular weight". For the purpose of producing coatings for nanoparticles, in particular for nanoparticles of the present invention, for

pharmaceutical use, it is preferable to use material of defined molecular weight to produce coatings since the regulatory process will be simplified if the material is as uniform as possible.

"TOPO" is an acronym for tri-n-octylphosphine oxide "DLS" is an acronym for dynamic light scattering, a particle sizing method, and may also be referred to as Photon Correlation Spectroscopy or Quasi-Elastic Light Scattering.

"Hydrophilic organic residue" refers to an organic residue that promote solubility in aqueous solvents and in the current invention it is implicit that they are bio-inert, which excludes polypeptides and complex carbohydrates. Examples of suitable hydrophilic organic residues are any group containing carbon with a molecular composition (aO+bN)/(cC+dS+eSi+fP) > 0.3 where a, b, c, d, e and f are the mol percentage of oxygen (O), nitrogen (N), carbon (C), sulfur (S), silicon (Si) and phosphorus (P), respectively. "Capping agent" refers to any surfactant as defined in any standard text on detergents (see e.g. Rosen, M. J.; "Surfactants and interfacial phenomena", 2 ^nd ed. Wiley 1989. Within the present invention we mostly refer to oleic acid, oleyl alcohol and other unsaturated long chain fatty acids or any molecule of the type X-R where X is a hydrophilic organic residue and R is a straight or branched hydrocarbon chain optionally containing one or more sulfur atom, but no nitrogen or oxygen atoms. The capping agent becomes a removable capping, for example fatty acid capping "Activated silane" refers to a silane of the following type R _n Si(X)4_ _n , where X is an alkoxy group, aryloxy group, a halogen, a dialkylamino group, a nitrogen containing heterocycle or an acyloxy group.

"Oxysilane" refers to any organic compounds with one or more oxygen atoms attached to the silicon atom. Non-limiting examples thereof are:

"Organosilane" refers to organic compounds containing one or more carbon silicon bonds.

"Organo -oxysilane" refers to organic compounds containing one or more carbon atoms and one or more oxygen atoms attached to the silicon atom. Non-limiting examples thereof are:

"Hydrocarbon" or "hydrocarbon chain" is an organic residue consisting of hydrogen and carbon. As used in the present invention a hydrocarbon may, when indicated, comprise heteroatoms selected from O, S and N. This means that one or more of the carbon atoms have been replaced by a heteroatom selected from O, S or N. The hydrocarbon may be fully saturated or it may comprise one or more unsaturations. The hydrocarbon may contain any number of carbon atoms between 1 and 50. "Alkyl" refers to a straight or branched hydrocarbon chain fully saturated (no double or triple bonds) hydrocarbon group. The alkyl group may have 1 to 8 carbon atoms. The alkyl group of the compounds may be designated as "Ci_s alkyl" or similar designations. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.

Whenever it appears herein, a numerical range such as "1 to 8" or "1-8" refer to each integer in the given range; e.g. , "1 to 8 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. , up to and including 8 carbon atoms.

As used herein, "alkoxy" refers to the formula -OR wherein R is a Ci_8 alkyl, e.g. methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, amyloxy, iso-amyloxy and the like. An alkoxy may be optionally substituted.

As used herein, "aryloxy" refers to RO- in which R is an aryl wherein, "aryl" refers to a carbocyclic (all carbon) ring or two or more fused rings (rings that share two adjacent carbon atoms) that have a fully delocalized pi-electron system. The aryl ring may be a 4-20 membered ring. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. An aryl group may be optionally substituted, e.g., phenoxy, naphthalenyloxy, azulenyloxy, anthracenyloxy, naphthalenylthio, phenylthio and the like. An aryloxy may be optionally substituted

As used herein, "acyl" refers to a carbonyl group, i.e. -C(=0)-. As used herein, "acyloxy" refers to an oxygen atom connected via a carbonyl group, i.e. -C(=0)-0-.

As used herein, "heterocycle" refers to a stable 3- to 18 membered ring which consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. The heterocycle may be monocyclic, bicyclic or tricyclic.

"Strong base" refers in the current context to bases that are stronger than hydroxide and not compatible with aqueous environments. "Hydrodynamic diameter" refers to the diameter of the hypothetical hard sphere that diffuses at the same speed as the particle. Hydration and shape is included in the behavior of the sphere. The term is also known as "Stokes diameter" or "Stokes- Einstein diameter.

"Conjugate" refers to a molecular entity that is a fluorescence marker, dye, spin-label, radioactive marker, ligand to a biological receptor, chelate, enzyme inhibitor, enzyme substrate, antibody or anti-body related structure. See e.g. "Bioconjugate

Techniques", Greg T. Hermanson second edition, Elsevier 2008, ISBN 978-0-12- 370501-3 for background on the subject.

"Handle for conjugation" or "attachment point" refers to a bifunctional molecule that can bind to, or be incorporated in, the silane coating but leaving one reactive group that can be linked to a conjugate, as defined above. A typical, but not exclusive, example would be (EtO) ₃ SiCH ₂ CH ₂ CH ₂ NH ₂ .

Detailed description of the invention

The present invention relates to paramagnetic nanoparticles which may be used as contrast agents for visualizing or imaging biological material or for other purposes known to the skilled person, such as supporting electrolyte in capillary

electrophoresis, in-vitro diagnostics, drug delivery, sensor applications etc. In some aspects the nanoparticles comprise a particle core and coating. Individual particle cores consist of amorphous rare earth element hydroxide, wherein said rare earth element is a lanthanide or transition metal ion. In certain embodiments the core may contain additional material. The particle core is paramagnetic and the metal ion is in embodiments of the invention a lanthanide (+ΙΠ) or transition metal ion or a mixture thereof. In some embodiments at least 50 % of the rare earth elements are Y, Eu, Gd, Tb or Dy. In some embodiments, the rare earth elements comprise Gd. In some embodiments, at least 50 % of the rare earth elements are Gd. In some embodiments more than 95 % of the rare earth elements are Gd, such as more than 99 %.In some embodiments of the invention the lanthanide is gadolinium (+ΙΠ). In some

embodiments the core is amorphous. In some embodiments the core is amorphous and composed of gadolinium hydroxide. Gadolinium hydroxide means that minor amounts of other elements may be present as impurities.

In some embodiments the core of the particle has a diameter between 1- 95 nm; such as 1-50 nm; such as 1-20 nm; such as 1-10 nm, 2-6 nm or 3-6 nm. In some

embodiments 2-6 nm or 3-6 nm are preferred. The diameter of the core is measured by TEM (transmission electron microscopy) analysis.

The coating is organic and comprises both silicon atoms and phosphorus atoms. In some embodiments the organic coating comprises a capping agent. In some embodiments this capping agent is oleic or linoleic acid. In some embodiments the organic coating is polymeric. In some embodiments the organic coating comprises hydrophilic residues. In some embodiments the organic coating comprises one or more organosilanes. In some embodiments the organic coating is crosslinked. In some embodiments the organosilanes are crosslinked. In some embodiments the

organosilanes are crosslinked via Si-O-Si bonds. In some embodiments the organic part of the crosslinked organosilane coating is hydrophilic and bio-inert.

The present invention relates to nanoparticles as disclosed herein for the visualization of biological material. The invention also relates to compositions of the nanoparticles. Some aspects of the invention relates to methods for preparing the nanoparticles comprising a core and a coating; and methods for preparing the core particles to be coated. In some aspects of the invention the above mentioned nanoparticles are used to manufacture a composition for in vivo visualization or imaging of biological material or for other purposes known to the skilled person. The nanoparticles and compositions of said nanoparticles according to the present invention are especially suited for magnetic resonance imaging (MRI) and other imaging techniques such as X-ray, computer tomography (CT) etc. In particular the invention has the advantage of being a positive contrast agent, i.e. giving a bright image. They also have advantages for dual energy CT where the favorable characteristics of the X-ray absorption spectrum of gadolinium give enhanced contrast.

A contrast agent based on the nanoparticles of the present invention may be useful in the clinical use of MRI. It may be used to enhance the visibility of blood vessels or blood-rich tissues in general. This may be useful for diagnosing problems in the vascular system like stenoses or malformations. It may also be particularly useful for the detection of cancerous tumors since it is known that the endothelial walls of the capillaries in tumors are less organized than in healthy tissue. This allows

nanoparticles, which are too large to pass through normal vessel walls, to selectively escape into tumor tissue to give an enhanced depiction of the tumor. This effect may be further enhanced by the attachment of biomarkers on the nanoparticles, which would further increase the local concentration of nanoparticles. The nanoparticles may also be linked to a biomarker that labels a disease marker on the inside of blood vessels. It would be of interest with e.g. markers for angiogenesis or inflammation to light up areas with tumor growth and vulnerable atherosclerotic plaque, respectively.

In some embodiments of the present invention the nanoparticles is a metal ion containing core that is encapsulated in a cross-linked, bio-inert silane mesh which brings about an enhanced stability while allowing sufficient contact with the surrounding water molecules to give a good magnetic relaxivity. In a one aspect the present invention a nanoparticle according to Fig 1 having "A" an inorganic core of paramagnetic metal hydroxide. In some embodiments the core is amorphous. In one embodiment the metal hydroxide is gadolinium hydroxide, such as Gd(OH) ₃ . In some embodiments the core is amorphous, paramagnetic Gd(OH) ₃ . The particles have been determined to be amorphous by TEM (Transmission electron microscopy). According to the aspect according to Fig 1 "B" is a layer or coating of organic polymeric material most commonly composed by organo-oxysilanes that may be covalently attached to the oxide core via Gd-O-Si bonds; or via ionic binding and/or forming Gd-OH ₂ -0-Si bonds (ionic binding via a hydration layer); or "B" is kept in place by non-covalent interactions or by the meshwork that is formed by the cross- linking of the organo-oxysilanes; or a combination of those binding modes.

Furthermore, said organo-oxysilane layer is advantageously crosslinked to form a mesh of Si-O-Si bonds surrounding the particle core, preventing the escape of metal ions from the core. The organic part of the silanes is hydrophilic organic residues attached to the silanes via Si-C bonds. The composition of the layer (or coating) is typically a mixture of different organo-oxysilanes but the present invention also relates to the layer being composed of the same organo-oxysilane.

In some embodiments the thickness of the coating is between 1 and 20 nm, or between 1 and 10 nm, or between 1 and 5 nm or preferably between 1 and 3 nm. The diameter of the coating is determined by the difference between the hydrodynamic diameter, determined by DLS (dynamic light scattering), and the core size as measured by TEM (transmission electron microscopy) analysis. Additionally elemental composition may be used to assist the determination. It should be emphasized that this is the contribution to the radius so the contribution to the particle diameter is twice as large. In certain embodiments the thickness of the coating is between 1 and 3 nm. The coating does not necessarily have the same thickness within a particle.

In some embodiments, the organo-oxysilane residue has the structure O ₃ S1-R ¹ where the oxygens may bind to the surface of the core or to other silanes or to a hydrogen or Ci_8 alkyl group. R ¹ may be a straight or branched hydrophilic organic residue.

Typical examples are listed below, see Formulas I-VI. where

A, B, and C are independently from each other selected from the group consisting of hydrogen, Ci_8 alkyl, a bond to another silane or organo-oxysilane derivative forming an oxygen-silane bond, or is absent in which case the oxygen is directly attached to the particle core;

n, o, p, q, r are independently from each other selected from 1-6;

m, s, t are independently from each other selected from 1-20;

X ¹ , X ² , X ^3a ' X ^3b , X ^4a ' X ^4b , X ^5a ' X ^5b X ^6a , X ^6b , X ^6c are independently from each other selected from H or Ci_s alkyl;

Y ² , Y ³ , Y ⁵ are independently from each other absent or NH; and

Y ⁶ is O, S, -NHCO- or -NHCONH-.

The organo-oxysilanes according to formulas I-VI may be directly bound to the particle core, for example by one or more of A, B, and C being absent and the corresponding oxygen is attached to the particle core, for example by forming a bond to the core. Additionally the organo-oxysilanes may be bound indirectly to the core, for example by forming one or more bonds to another organo-oxysilane which is attached to the particle core.

A, B, and C may be bound to the core as disclosed above, a schematic example can be seen in Fig 3. Fig 3 schematically illustrates how silanes may be crosslinked at the surface of a gadolinium hydroxide particle.

Whenever a variable, such as Y ² , Y ³ and Y ⁵ , are stated to be absent it means that the there is a bond. For example when Y ² , Y ³ and Y ⁵ are absent the methylene group in any of the compounds according to formulas II-V is bound to the carbonyl group. Whenever "independently" is used for variables it means that the variable may be selected independently other variables, for example "A, B, C are independently..." means that A, B and C may be selected independently of each other. In one embodiment the organo-oxysilane is selected from branched polyethers according to Formula VI where n is selected from 1-6; each m is separately selected from 1-20 and X ^6a , X ^6b , X ^6c are independently from each other selected from H or Ci_ 8 alkyl. In one embodiment X ^6a , X ^6b , X ^6c are methyl. Preferred is the group of molecules where n is 3 and each m is separately from each other selected from 3, 4 or

When particles are coated with the organo-oxysilane and the obtained particles have good properties, such as good water solubility and good stability. The coating molecule is monodisperse and relatively low-cost to produce, as opposed to most conventionally available coating molecules. The coating with organo-oxysilanes results in a material which is easy to develop, for example into pharmaceutical applications since characterisation of starting materials and final product becomes easier than with conventional materials. In some embodiments, the coating further comprises a phosphosilane of the general formula

(X ^7a O)(X ^7b O)PO-(CH ₂ )„Si(OX ^7c )(OX ^7d )(OX ^7e ) (VII), where X ^7a , X ^7b , X ^7c , X ^7d , X ^7e are independently from each other selected from H, Ci_8 alkyl or benzyl and n is selected from an integer from 1 to 6. The

phosphosilane improves the aqueous stability.

In one embodiment of the present invention the coating contains a coating comprising organo-oxysilanes and a phosphosilane In some embodiments, a second silane of the formulas VIII or IX

(X ^8a O) ₂ PO-(CH ₂ ) _n Si(OX ^8b ) ₃ VIII,

(X ^9a O) ₃ Si(CH ₂ ) _n Si(OX ^9b ) ₃ IX, wherein n is selected from 1 to 6; and X ^8a is selected from hydrogen or Ci_s alkyl;

X ^8b , X ^9a , X ^9b are independently from each other selected from Ci_s alkyl, acyl, aryl, or X ^{1 1} ; wherein X ^{1 1} is (Si(OX ¹² ))„X ¹³ , wherein X ¹² is selected from Ci_ ₈ alkyl, acyl, or aryl; and X ¹³ is hydrogen or Ci_s alkyl,

is added to the coating. The second silane improves the aqueous stability

dramatically and may be added any time, but is in some embodiments of the present invention added in a second step, i.e. after the formation of the coating discussed above. This silane binds in between the larger organo-oxysilanes and improves the stabilizing effect of the coating without adding to the diameter of the particles. Within the present invention this is termed "hardening". It has proven that the conditions for a successful hardening must be very precise in order to obtain enhanced stability. An unsuccessful hardening results in aggregation of particles making them unsuitable for the purposes such as MRI. According to the present invention an aqueous polar solvent is used as the solvent for this procedure. A useful mixture is n-propanol containing 3-30 vol% or, preferably containing 10-25 vol%, and even more preferably 18-22 vol% water. Solvents like ethanol, 2-propanol, butanol, DMF (N,N- dimethylformamide), NMP (N-methylpyrrolidone) and other amide solvents, glymes and water miscible ethers, glycols and DMSO (dimethyl sulfoxide) can also be used. According to the present invention the heating scheme for the hardening has also proven critical in order to obtain a product having a desired balance between yield and stability. In one embodiment the hardening is performed at a scheme with 1-100 h at a temperature between 40 and 100 °C followed by 1-48 h at 100-140 °C. In one embodiment a temperature between 60 and 100 °C is kept for 24-60 h followed by 10-30 h at 1 10-130 °C. In one embodiment the first temperature is kept between 90 and 100 °C for 24-60 h. Suitable silanes are dimethylphosphatoethyl triethoxysilane, diethylphosphatoethyl triethoxy silane, and bis(triethoxysilyl)methane but the more easily hydro lyzed methoxy silanes only cause aggregation. Neither does a mixture of triethoxy ethyl silane and ethylphosphonic acid diethyl ester work so it is important that there are two functionalities in the same molecule for the hardening to be efficient.

In one embodiment of the present invention the coating contains a coating comprising organo-oxysilanes and a phosphosilane and is hardened with a second silane as described above.

In a preferred embodiment of the present invention, the nanoparticle has an elemental composition of 10-30 weight% of gadolinium, 2-10 weight% of silicon, 1-8 weight% of phosphorus, and 20-50 weight% of carbon of the total weight of the nanoparticle. Residual ash after burning at red heat is preferably 30-60 weight%. In another embodiment of the nanoparticle according to the present invention, layer B (Fig 1 and Fig 2) is made entirely of an organic, crosslinked polymer preventing the escape of metal ions by the mesh formed by the polymer. Many possible polymers can be envisioned for this purpose some, but not limiting, are based on amides or vinylic groups or aromatic groups or any combination of the above. A typical composition would be polystyrene cross-linked by divinyl benzene which is then further derivatized with polar chains to enhance bio-inertness and aqueous stability. Polymer networks based on polyacrylamide, polyalcohols or polyethers may also be contemplated.

In some embodiments of the invention, the nanoparticle comprises attachment points for introduction of at least one conjugate. In some embodiments, more than one conjugate is present and in some embodiments combinations of conjugates are present. The nanoparticle comprising attachment points for introduction of at least one conjugate may be formed by the treatment of the nanoparticle according to the present invention in a second step, in which a nanoparticle as visualized in Fig 2 is formed. Most attachment points are introduced by an attachment group, which is a third silane according to the following formula

(RO) ₃ Si-X ¹⁰ -Y ¹⁰ (X) where R is Ci_s alkyl, X ¹⁰ is a spacer group containing a backbone of between 1 and 50 C, N, O or S atoms, for example a straight or branched hydrocarbon chain, optionally comprising heteroatoms selected from N, O or S. Y ¹⁰ is the attachment handle. In one embodiment Y ¹⁰ is C(=0)OZ, wherein Z is H or any organic or inorganic residue. Examples of organic or inorganic residues are Ci_s alkyl, Ci_s alkenyl, Ci_s alkynyl, Ci_s alkynyl, Ci_s alkylidene; Ci_s alkoxy, thiols (Z is SH) and a maleimido group. In one embodiment each R is independently from each other selected from Ci_s alkyl, acyl, aryl, or di-Ci_s alkylamino; X ¹⁰ is selected from (CH ₂ ) _n - (OCH ₂ CH ₂ ) _m -, where n is 1-6 and m is 0-50; Y ¹⁰ is selected from -C(=0)Z, S0 ₂ -Z, - SQ, -N ₃ , -NC0 ₂ t-butyl, -NC0 ₂ benzyl, NH ₂ or NHQ, where Z is independently selected from Ci_s alkoxy, acyloxy, aryloxy, or di-Ci_s alkylamino or N- oxysuccinimide, and Q is Ci_8 alkyl. In one embodiment the attachment points are introduced in the form of a primary amino group. In one example the attachment point is introduced by the treatment with 3-aminopropyltriethoxysilane. Also other functional groups can be utilized. A large number of typical strategies for this type of derivatization are well known in the art and described in "Bioconjugate Techniques", Greg T. Hermanson second edition, Elsevier 2008, ISBN 978-0-12-370501-3. The attachment point may be introduced together with the organo-oxysilane; with the phosphosilane, with the second silane or after the hardening.

In some embodiment, the nanoparticle comprises a particle core and coating wherein the coating is obtained by addition of organo-oxysilanes and by performing cross- linking and/or hardening by treatment with a phosphosilane; a disilane; a polysilane; and/or a molecule containing any combination of phosphonates and silanes. Where appropriate, hardening may be carried out thereafter. In one embodiment a conjugate is included in the nanoparticle. In one embodiment the conjugate is at least one biomarker

In the case where the particles of the present invention are to be used for other purposes than MRI, such as those taking advantage of the luminescent properties of certain compositions, the composition may advantageously be lower in gadolinium and correspondingly higher, typically between 5 and 30 mol % of the total metal content, in the luminescent elements europium or terbium. As produced, these particles are not luminescent due to the presence of energy dissipating OH groups but the materials can be heated in a second step to produce luminescent particles. In one aspect, the present invention refers to a method for preparation of a nanoparticle according to the present invention. In one particular aspect, the present invention refers to a method for preparation of the nanoparticle according to fig 1 as disclosed above.

One embodiment relates to a method for obtaining a coated nanoparticle according comprising: providing a rare earth element salt and a hydroxide source in solution in the presence of a capping agent; forming an intermediate nanoparticle rare earth element hydroxide; adding a base, one or more organo-oxysilane residue(s) , and one or more a phosphorus containing compound (s), in the presence of a non-aqueous solvent; and obtaining coated rare earth element hydroxide nanoparticle. The method may be performed by combining the step.

In one embodiment, the method for preparation of a nanoparticle comprises the following main steps of production of particle cores, coating the particle cores with organo-oxysilanes and performing cross-linking and/or hardening by treatment with a phosphosilane; a disilane; a polysilane; and/or a molecule containing any

combination of phosphonates and silanes. If applicable, hardening may be carried out thereafter. In some embodiments a conjugate is added to the particle. In one embodiment the conjugate is at least one biomarker. In a first step, detergent capped precursor particles are produced. Thus, the particle cores are capped by a stabilizing but removable layer of capping agent. An example of a capping agent is oleate. The first step is based on the formation of a metal hydroxide by reaction of a metal salt with a source of hydroxide ions. A specific example is the formation of gadolinium hydroxide by the reaction of gadolinium chloride with tetramethyl ammonium hydroxide in an ethanol solution in the presence of capping agent(s) as described in example 1. By slightly changing the relative and absolute concentrations, the particle size (including the oleate capping, as measured by DLS in cyclohexane) can be tuned from 5 to 20 nm. More base generally give larger particles. This method is very convenient because it directly yields isolatable particles with the correct core size in a tunable way. The gadolinium chloride is a convenient source of gadolinium ions but any of the common salts like gadolinium bromide, gadolinium nitrate, gadolinium acetate or gadolinium acetoacetate etc may be used. Gadolinium chloride may also be generated in situ by the action of hydrochloric acid on gadolinium oxide or gadolinium metal. The source of hydroxide ions is tetramethyl ammonium hydroxide but other sources of hydroxide ions soluble in the medium can be contemplated for this purpose. The solvent may be ethanol, another alcohol, THF (tetrahydrofuran) or any polar organic solvent.

The most convenient capping agent is oleic acid but most low melting fatty acids or other anionic detergents, either alone or in a mixture would be obvious variations to one skilled in the art. According to our experience, the particles turned out to be less polydisperse in the presence of oleyl alcohol, oleyl amine or 1,2-tetradecanediol. This is surprising since analysis of the finished particles show no trace of oleyl alcohol. It was found to be advantageous for the re-suspendability of the particles to add a small amount of trioctyl phosphine oxide to the particle solution before freeze drying. Typically 1-10% (w/w) was sufficient and most commonly 4 % was used. The analysis of the material from this step shows about one half equivalent of chloride relative to gadolinium. This chloride seems to be loosely bound since it is removed in the subsequent reaction steps. The capped nanoparticles may be oleate capped nanoparticles. In one embodiment the capping is oleic acid and/or oleyl alcohol and thus oleate capped nanoparticles are produced and optionally isolated. It is contemplated that any capping agent may be used any capped nanoparticles are produced. The produced capped nanoparticle cores, for example oleate capped nanoparticles, are considered intermediate nanoparticles. The capped nanoparticle cores may be isolated and purified. Addition of an equal volume of acetone selectively precipitates impurities in the form of larger particle aggregates.

In the second step, when the capping agent is exchanged for the permanent organo- oxysilane coating, it is critical to use a strong base, such as KOt-Bu (Potassium tert- butoxide) or LiHMDS (Lithium bis(trimethylsilyl)amide) or sodium hydride or an alkyl lithium or an organomagnesium compound or a metal amide such as LDA (lithium diisopropylamide), in a non-aqueous polar solvent, such as THF, and a trimethoxy silane instead of the more common (and less sensitive) triethoxy silanes to displace the capping agent and form an oriented organo-oxysilane layer (example 2). It is critical for the solubility and stability of the material that the silanes are correctly oriented and bound to the surface rather than first bound to each other and then loosely associated with the surface in a non-oriented fashion. If that happens, like when a too weak base is used, only marginal stability at pH 5.8 is achieved after the hardening step. Conventionally the use such strongly basic conditions to coat metal hydroxide particles have not been used. As gadolinium hydroxide is quite strongly basic vigorous conditions sufficient to deprotonate the hydroxide to enable the surface to react directly with the organo-oxy silanes, thus not requiring hydrolysis and oligomerization prior to surface adsorption that is the conventional procedure for methods of silanization of gadolinium based particles.

To verify that the organo-oxysilanes are indeed bound to the particle surfaces and oriented with the silane facing the core, NMR studies of the chemically very similar but non-magnetic yttrium hydroxide particles (Example 3b) (ionic radius of Gd(III)= 108. 9 pm, Y(III)=104 pm, W. D. Nesse, Introduction to mineralogy, Oxford, 2000) were carried out. Yttrium and gadolinium have very similar crystal structures for most of their analogous compounds so it is reasonable to use these particles as a tool for investigating the coating of the gadolinium hydroxide particles, where NMR spectroscopy is hampered by extensive paramagnetic line broadening. An attempt to record a 'H-NMR spectrum of coated gadolinium hydroxide particles only resulted in a very broad peak with no discernible features. The lack of paramagnetism in yttrium ions allows high resolution NMR spectra to be

recorded from analogous yttrium hydroxide particles. From the 1H-NMR spectrum it can be seen by comparison with the spectrum of coating precursor 1, that there is a broadening of the signals closest to the silicon atom. Progressively, the lines get narrower as we go out towards the end of the chain, with the methyl group marginally broader than for the corresponding signal of 1. This indicates that the coating atoms closest to the silicon atom experience less rotational freedom then the terminal methyl groups (Chapter 5 in Harris, R. K., Nuclear Magnetic Resonance Spectroscopy, Longman, 1986 ). This is in accordance with the expected behavior of surface bound silanes where the silicon is anchored to the surface of a particle. Seen from this perspective, the nanoparticle behaves as a large body. There are also other methods that would be possible to use in order to determine that the organo-oxysilanes is bound to the particle surface.

Furthermore, from the presence of a signal at 3.58 ppm (in acetone-d ₆ ) originating from the protons of the MeOSi (methoxysilane) groups, it can be seen that after the first step of coating, that, while the silicon atoms are indeed bound to the surface, they are not bound to each other, so the meshwork of Si-O-Si bonds is not yet developed. This is in accordance with the stability behavior we see at this stage. As measured by titration of free gadolinium (or yttrium for the particles used for NMR experiments) by an EDTA colorimetric method (Example 7) there is no stability of this material over that of the DEG-coated gadolinium oxide nanoparticles of Bridot et al. (neither is their material stable). All the gadolinium is detected as free, immediately (within a few seconds) at pH 5.8 by this method. However, the presence of the coating is evident from the altered solubility behavior. The oleate capped precursor particles are freely soluble in non-polar solvents like cyclohexane and toluene. After the polar coating has been bound to the particles, the material is insoluble in non-polar solvents but soluble in water, DMF and/or THF. Advantageously, the coated particles can be purified by dissolution in THF and precipitated by diethyl ether or another non- solvent, where unreacted coating precursors are soluble but the coated particles are not.

In the third step, cross-linking/hardening of the organosilane by treatment with a phosphonate-silane, a disilane, or a mixture of the two is achieved. To affect the crosslinking of the organosilane layer it was surprisingly found that treatment of the particles from step 2 with e. g. organo-oxysilane 2, in aqueous propanol at 95 °C for 48 h and then 120 °C for 24 h in a sealed ampoule, affected this in an efficient manner, without causing the aggregation of the particles. Aggregation is a problem under most other conditions. It seems that very few compounds are efficient in this step. It works well with dimethylphosphatoethyl triethoxysilane,

diethylphosphatoethyl triethoxy silane, and bis(trietoxysilyl)methane but the more easily hydro lyzed methoxy silanes only cause aggregation. Neither does a mixture of triethoxy ethyl silane and ethylphosphonic acid diethyl ester work so it is important that there are two functionalities in the same molecule for the hardening to be efficient.

In the NMR spectrum after the particles have been through the cross- linking/hardening step of the coating process it can be seen that the methyl groups are gone and we can detect the presence of the added-phosphono silane. The stability of this material is very different from the unhardened material. The amount of free gadolinium after 24 h at pH 5.8 is between 2 and 50% after this treatment and is essentially 100% before. The reference 100% is the amount of gadolinium that is released after 24 h at pH 1.3 The detailed procedure for the measurements are given in example 7. The hardened coated nanoparticles may be further treated in order to introduce one or more functional groups allowing attachment of at least one conjugate. Handles for conjugation (attachment points) may be introduced. In one embodiment, the functional groups are introduced in the form of a primary amino group by the treatment with 3-aminopropyltriethoxysilane. Conjugates may be selected from biomarkers, fluorescence markers, dyes, spin-labels, radioactive markers, metal chelators, antenna chromophores, ligands to a biological receptor, enzyme inhibitors, enzyme substrates, antibodies and/or anti-body related structures or any combination thereof. In some embodiments, at least one biomarker, fluorescence marker, dye, spin-label, radioactive marker, metal chelator, antenna chromophore, ligand to a biological receptor, enzyme inhibitor, enzyme substrate, antibody and/or anti-body related structure or any combination thereof is attached to at least some of the functional groups that serves as handles for conjugation.

The in- vivo use of the nanoparticles of this invention requires them to be formulated according to best practice well known to those skilled in the art. Water is a preferred solvent but one or more co-solvents or additives may be added in 0.1-10%) to improve stability in solution. Acceptable co solvents would be alcohols like ethanol or glycerol, biocompatible polymers like ethyleneglycol or polyvinyl alcohol, dimethyl sulfoxide, or N-methyl pyrrolidinone. It can also be advantageous to add one or more osmoregulators like mannitol, sorbitol, lactose, glucose or other sugars or sugar alcohols. Many of the above may also fulfill the function of a cryoprotectant, enhancing the efficiency of reconstitution after freeze drying. It may also be advantageous to add electrolytes to lower the physiological effects of the injected solution. Preferred electrolytes would be a combination of NaCl, CaCl ₂ and MgCl ₂ . Regulation of the pH of the injectable solution is preferable and any buffer suitable for injection can be contemplated but preferred is Tris-HCl. Metal ion scavengers can also be contemplated as an additive. Some typical examples would be EDTA

(ethylendiaminetetraacetic acid), DTPA (diethylene triamine pentaacetic acid) and DOTA (1, 4, 7, 10-tetraaza-cyclododecane-N,N',N",N" '-tetraacetic acid). The use of solid-phase ion scavenging resins added to the storage bottle can also be

contemplated.

Examples

Example la. Production of oleate capped nanoparticles of gadolinium hydroxide.

1. We prepared a 50 mM stock solution of gadolinium chloride (9.29 g GdCl ₃ 6 H ₂ 0) in 95% ethanol (500 ml) under nitrogen by first dissolving the material in 200 ml ethanol and heating to 40 °C for 30 minutes. Filter the solution through a glass microfibre filter and dilute to 500 ml in a volumetric flask under nitrogen.

2. We prepared a 90 mM stock solution of tetramethyl ammonium hydroxide pentahydrate (8.15 g) under nitrogen by first dissolving the material in 200 ml refluxing 95% ethanol and, after allowing to attain room temperature, dilute to 500 ml in a volumetric flask by adding ethanol. All operations under nitrogen.

3. Reflux 42 ml of the gadolinium chloride solution above in a three-neck, round bottom flask, under nitrogen. Add 341 μΐ of oleic acid and 1175 μΐ of oleyl alcohol. Heat 48 ml of the tetramethyl ammonium hydroxide solution above to 50 °C and add it rapidly to the gadolinium chloride solution by means of a syringe. Reflux for five more minutes. During this time the mixture first turns milky then gradually it clears and a glass-like material sticks to the sides of the flask. Remove the stirring bar, stopper the flask and leave it in an ice-bath for 20 minutes. Pour off the supernatant and wash the glass-like precipitate with 3 x 2 ml ethanol. Remove the residual ethanol at reduced pressure but don't heat. Dissolve in 6 ml cyclohexane and add trioctyl phosphine oxide (27 mg). Centrifuge to remove a small amount of insoluble residues. Freeze the supernatant in a dry-ice bath and freeze dry. Be observant of the safety precaution s required when freeze drying a flammable substance. The oleate capped nanoparticles form a white powder in a yield of 0.66 g.

Analysis: Diameter was measured by DLS, volume average 7 nm, Gd 36 % w/w (Method according to A. Barge, G. Cravotto, E. Gianolio and F. Fedeli, Contrast Med. Mol. Imaging 1 : 184-188 (2006)), CI w/w 4.5 % according to Mohr, Water (as volatiles below 200 °C by TGA) 10 % w/w, Ash 44% w/w, Oleate (as determined by HPLC according to Example 8) 40.2 % w/w. TOPO (HPLC) 3 % w/w. Oleyl alcohol < 0.01% w/w (HPLC). These data are compatible with a core of Gd(OH)3 with a density of 5.3 g/ml and a diameter of 4 nm (crystalline Gd(OH)3 has a density of 5.41 g/ml), a hydrated surface layer with some chloride ions and a monolayer of oleate with a thickness of 1.4 nm.

Example lb

Substituting the 50 mM gadolinium chloride solution for a mixture of 30 mM GdCl ₃ and 20 mM EuCl ₃ gave analogous nanoparticles with a core of 60% gadolinium hydroxide and 40%> europium hydroxide.

Example lc Substituting the 50 mM gadolinium chloride solution for a mixture of 30 mM GdCl ₃ and 20 mM TbCl ₃ gave analogous nanoparticles with a core of 60% gadolinium hydroxide and 40% terbium hydroxide.

Example Id Substituting the 50 mM gadolinium chloride solution for a mixture of 30 mM GdCl ₃ and 20 mM DyCl ₃ gave analogous nanoparticles with a core of 60% gadolinium hydroxide and 40%> dysprosium hydroxide.

Example le

Substituting the 50 mM gadolinium chloride solution for a mixture of 40 mM GdCl ₃ and 10 mM DyCl ₃ gave analogous nanoparticles with a core of 80% gadolinium hydroxide and 20%> dysprosium hydroxide.

Example If

Substituting the 50 mM gadolinium chloride solution for a mixture of 50 mM YC1 ₃ gave analogous nanoparticles with a core of yttrium hydroxide. Example lg: Preparation of oleate capped nanoparticles in the presence of 1,2- tetradecanediol.

A 20 ml (0,05M GdCl ₃ solution) is heated to 100 °C. During the heating period, 0.1290 g of 1 ,2-Tetradecanediol is added. When the solution reached 100 °C, 177.5 μΐ oleic acid is added and mixed. In a separate flask, 20 ml of TMAH

(tetramethylammonium hydroxide) (0.0825 M solution) is heated to 50 °C. After 5 minutes of waiting period, the TMAH solution is mixed with the hot GdCl ₃ solution containing tetradecanediol and oleic acid. After 2 minutes of mixing, clear flake-like precipitate started to form. Size according to DLS in cyclohexane: 10 nm.

Example lh: Preparation of oleate capped nanoparticles in the presence of oleyl amine. A 20 ml (0,05M GdCl ₃ solution) is heated to 100 °C. When the solution reached 100 °C, 177.5 μΐ oleic acid is added and mixed. In a separate flask, 13.2 ml of TMAH (0.0825 M solution) mixed with 226 μΐ oleyl amine is heated to 50 °C. After 5 minutes of waiting period, the TMAH solution containing oleyl amine is mixed with the hot GdCl ₃ solution containing oleic acid. White precipitate is formed after mixing. Size according to DLS in cyclohexane: 10 nm.

Example 2a. Coating of oleate capped particles with PEG silane.

Oleate capped particles (250 mg) from example 1 was dissolved in dry THF (tetrahydrofuran) (20 ml). Methoxy(polyethyleneoxy)propyltrimethoxysilane (610 μΐ, 1.0 mmol, average Mw 550) was added and the reaction mixture was shaken for a few minutes. Then KOt-Bu (120 mg, 1.0 mmol) was added and the mixture was stirred for 48 h at room temperature under N ₂ . The resulting mixture was filtered and the filtrate was concentrated at reduced pressure until approximately 2 ml remained. Diethyl ether (20 ml) was added to the concentrated solution whereupon a precipitate formed. After 20 h at 4 °C, the supernatant was removed and the remaining precipitate was washed with two portions of diethyl ether. The precipitate was dried at reduce pressure to give a waxy residue (370 mg) of PEG-coated particles.

Analysis: Diameter as measured by DLS, volume average 8 nm, Gd 20 % w/w.

Example 2b Coating of oleate capped yttrium hydroxide particles. The procedure according to example 2a was carried out on yttrium hydroxide particles prepared according to example If

Example 3a: Hardening of PEG-silane coated particles with

Diethylphosphatoethyl triethoxysilane (DPTS).

For 10 mg of PEG-silane coated particles, 2 ml of n-propanol (80% aq. n-PrOH) was used to dissolve the particles. Five minutes of sonication was applied to ensure complete dissolution. Diethylphosphatoethyl triethoxysilane (DPTS), 0.025 mmol was added. The mixture was then subjected to heating and shaking at 95 °C for 48h and thereafter 120 °C for 24h. The room-temp cooled mixture was syringe- filtered through a 0.2 μιη filter before diafiltration purification or DLS measurements.

Example 3b: Hardening of PEG silane coated particles of Yttrium hydroxide with DPTS.

The procedure of example 3a was carried out on material according to example 2b.

Example 4: Hardening of PEG-silane coated particles with

Dimethylphosphatoethyl triethoxysilane (DTEP).

Dimethyl-2-(triethoxysilyl)ethyl phosphonate (180 mg, 0.6 mmol) was added to PEG coated particles (200 mg) (example 2a) dissolved in 40 ml aqueous 80% 1-propanol in a pressure vessel. The reaction mixture was stirred for 48h at 95 °C and then 24h at 120 °C. The temperature was lowered to room temperature and the clear solution was filtered (0.2 μιη syringe filter). The hardened material is purified using diafiltration as described in Example 6. Volume particle size maximum = 9 nm; Composition (ICP, mole ratio): Si/Gd = 2.1, P/Gd = 0.87, Si/P = 2.5; Stability for 24h at pH5,8 = 50%; ti at 81.3 MHz, 26.2°C = 4.19 mM ^"1 Gd ms ^"

Example 5: Hardening of PEG-silane coated particles with

bis(triethoxysilyl)methane.

Bis(triethoxysilyl)methane (306 mg, 0.9 mmol) was added to PEG coated particles (300 mg, example 2a) dissolved in 60 ml aqueous 80%> 1-propanol in a pressure vessel. The reaction mixture was stirred for 48 h at 95 °C and then 24 h at 120 °C. The temperature was lowered to room temperature and the clear solution was filtered (0.2 μιη syringe filter). The hardened material is purified using diafiltration as described in Example 6. Volume particle size maximum = 16 nm; Composition (ICP, mole ratio): Si/Gd = 4.8, P/Gd < 0.04; Stability for 24h at pH5.8 = 92%; ti at 81.3 MHz, 25.4°C = 3.7 mM ^"1 Gd ms ^"1

Example 6: Purification by Diafiltration Hardened material (4 ml) from Examples 3-5 was mixed and sonicated for 5 minutes with 41 ml TRIS/HCl buffer (5 mM at pH7,4; 0,2 μιη filtered) and subjected to diafiltatration. First, a 500k NMWC (Nominal Molecular Weight Cut-off) pore size column (GE Healthcare's Midgee ultrafiltration cartridge Model #: UFP-500-C- MM01A) was used and the permeate was collected. About 2 ml retentate was left after the diafiltration process and 43 ml TRIS/HCl buffer was added for another cycle of diafiltration using the same cartridge. The permeate was again collected for this cycle. The collected permeates (-86 ml) were subjected to a 10k NMWC pore size diafilter column (GE Healthcare's Midgee ultrafiltration cartridge Model #: UFP- 10-C-MMOl A) to remove the free ions. The collected retentate was about 2 ml. A ~7 ml "dead- volume" from the cartridge was washed out with TRIS/HCl buffer, adding up to a total volume of 8-9 ml diafiltered solution. The diafilter columns were operated with a typical inlet pressure of 1 bar overpressure. The diafiltered sample was later subjected to 0.2 μιη syringe filtration before stability or size measurement, to remove unwanted material like dust particles.

Example 7. Stability Measurement

A measured amount of hardened (typically 300 μΐ) or diafiltered material (typically 2 ml) is subjected for stability testing at different incubation conditions. Incubation conditions were used: (a) at pH5.8 for Oh; (b) at pH5.8 after lh; (c) at pH5.8 after 24h; (d) at pHl .3 after 24h. Typically, 300 μΐ of hardened material or 2 ml of diafiltered material was incubated in a 5 -ml acetate buffer solution (at pH5,8). For the pH1.3 condition, aqueous HC1 (1,0 M) is added to adjust the pH of the buffer. After subjecting the sample to the desired incubation period, the pH of the solutions were adjusted to pH5.8 (if needed) before determining the free gadolinium

concentration by titration. The titration method is described in A. Barge, G. Cravotto, E. Gianolio and F. Fedeli, Contrast Med. Mol. Imaging 1 : 184-188 (2006).

Example 8. Quantitative analysis of Oleate in oleate capped particles from example 1. About 10 mg oleate capped particles were weighed in and dissolved in a mixture of 1.0 ml THF, 1.0 ml formic acid and 1.0 ml water. A 1.0 ml aliquot of this solution was further diluted with 2.0 ml acetonitrile and 1.0 ml water and injected on a HPLC system. (The pump was a Varian 9010 and the detector an Alltech ELSD 2000 with the temperature set to 50 °C and the gas flow set to 1.5 1/min. The column was YMC Hydrosphere 150 x 4.6 mm. The mobile phase consisted of 90 % acetonitrile and 10 % Water containing 1 % formic acid. Since this type of detector only can be considered to be linear within a short range, two standards of oleic acid were prepared with a concentration close (one slightly higher and one slightly lower) to the expected concentration of oleic acid in the sample. Three injection of were made for each of the standards and the sample and the areas under the peaks were integrated. The concentration in the sample solution was determined through linear interpolation. The percentage of oleic acid in the particles was calculated from the amount of particles dissolved and the dilution of the sample.

Example 9. Pharmacological formulation of nanoparticles according to example 6.

A solution from the diafiltration process in example 6 was analyzed for gadolinium and the volume was reduced by spin filtration on a lk cut-off filter so the

concentration of 17 mM of gadolinium was achieved. To this solution mannitol was added to reach a concentration of 250 mM. This yields an isotonic solution suitable for animal experiments.

Example 10 Aminopropylation of Gd(OH)3 particles. Aminopropyltrimethoxysilane (0.14 μΐ,, 0.76 μιηοΐ) was added to 2 ml solution of hardened particles (example 4). The solution was shaken at 95 °C for 16 h. Tris/HCl buffer (0.5 ml, pH 7.4) was added to the solution, which was then concentrated in a stream of nitrogen until 1 ml remained. The particles were purified on a Sephadex G- 25 short column (PD-10, GE) eluting with H ₂ 0. In total, 3 ml of an aqueous particle solution was collected.

Example 11 Conjugation of aminopropylated particles to a fluorescence marker.

Example 11a Fluorescence marker 7-(Diethylamino)-coumarin-3-carboxylic acid Aminopropyl coated particles (1 ml) from example 10 were mixed with 7-

(Diethylamino)-coumarin-3-carboxylic acid N-succinimidyl ester (10 μΐ, 2.8 μιηοΐ/ml in DMF) and shaken at room temperature for 18 h. The particles were purified using a Sephadex G-25 short column (PD-10, GE) eluting with H ₂ 0. In total, 3 ml of an aqueous particle solution was collected. DLS analysis indicated an average size of 10- 12 nm of the final particles. The solution of the purified particles was fluorescent under a UV-lamp (365 nm).

Example lib Fluorescence marker N-dansylcysteine

Zinc powder (20 mg) was added to N,N-didansylcystine (2 mg) dissolved in 0.5% trifluoracetic acid in acetonitrile (1 ml). The reaction mixture was shaken at room temperature for 1 h and then filtered. The yellow filtrate was used without any further purification.

6-Maleimidohexanoic acid N-hydroxysuccinimide ester (10 μΐ, 3 μιηοΐ/ιηΐ in DMF) was added to gel- filtrated (H ₂ 0, GE, PD-10) aminopropylated particles (1 ml) and then shaken at room temperature for 18 h. The mixture was gel- filtrated (GE, PD-10, H ₂ 0) and the Gd-positive fractions (2 and 3) were combined. The pH of the combined fractions was corrected with PBS buffer (0.5 ml, pH 7.4) and N- dansylcysteine solution (40 μΐ) was added. The mixture was shaken at room temperature for 18 h and then gel- filtrated (H ₂ 0, GE, PD-10). Gd and UV positive fractions (2-4) were combined. Volume particle size distribution = 10-15 nm. E xam ple 12 : Synth e sis o f ( l-trimethoxysilyl)propyl-3-Tri-m-PEG ₄ (3,3- dimethoxy-9,9-di-2,5,8,ll,14-pentaoxapentadecyl-2,7,ll,14,17 ,20,23-heptaoxa-3- silatetracosane)

Trimethoxysilane toluene

Karstedt's catalyst room temperature

Example 12a: 3-(3-bromo-2,2-bis(bromomethyl)propoxy)prop-l-ene.

Sodium hydride (1.67 g, 42 mmol) was added carefully to 3-bromo-2,2- bis(bromomethyl)propanol (9.75 g, 30 mmol) and allyl bromide (12.9 ml, 150 mmol) in dry and degassed DMF (40 ml) under nitrogen at 0 °C. The temperature was then increased to room temperature (22 °C) and the reaction mixture was stirred for another 3h. The reaction mixture was then carefully added to an aqueous saturated NH ₄ CI (50 ml). The H ₂ 0-phase was then extracted with diethyl ether (2 x 50 ml) and the combined organic phases were washed with H ₂ 0 (5 x 50 ml) and then brine (50 ml). The organic phase was dried with Na ₂ S0 ₄ followed by filtration. The volatile materials were removed at reduced pressure to give a pale yellow oil (9.7 g). Column chromatography on silica (heptane :EtO Ac 9: 1) gave 6.6 g (62%) of the product as a clear oil. 1H-NMR (CDC1 ₃ ); 5.93 (m, 1H), 5.28 (m, 2H), 4.05 (d, 2H), 3.58 (s, 6H), 3.52 (s, 2H).

Example 12b: 16-(allyloxymethyl)-16-2,5,8,ll,14-pentaoxapentadecyl- 2,5,8,11, 14,18,21,24,27,30-decaoxahentriacontane.

Tetraethyleneglycol monomethyl ether (1.91 ml, 9 mmol) dissolved in dry and degassed DMF (3.5 ml, dried 24h, 4A MS) was added carefully to sodium hydride (365 mg, 9 mmol) in dry and degassed DMF (15 ml, dried 24h, 4A MS) under nitrogen at 0 °C using a syringe. The temperature was then raised to room temperature and the reaction mixture was stirred for another 30 min. 3-(3-bromo-2,2- bis(bromomethyl)propoxy)prop-l-ene (730 mg, 2.0 mmol) was then added and the temperature was raised to 100 °C. After 14h the reaction was completed (HPLC- ELSD-C18, 95:5 to 5:95 H ₂ 0/ACN in 25 min, R _t product = 19.5 min) the temperature was decreased to room temperature and the reaction mixture was carefully added to H ₂ 0 (150 ml) and the H ₂ 0-phase was washed with diethyl ether (2x 50 ml). Sodium chloride was then added to the H ₂ 0-phase until saturation. The H ₂ 0-phase was extracted with EtOAc (4x50 ml) and the combined organic phases were washed with brine (2x30 ml). Sodium sulfate and charcoal was added to the organic phase. The clear organic phase was filtered and the volatile material was removed at reduced pressure (8 mmHg, 40 °C then 0.1 mmHg (oil pump) and 40 °C to remove residual DMF). Column chromatography (EtOAc:MeOH 9: 1) gave 1.05 g (70%) of the product. 1H-NMR (CDC1 ₃ ); 5.90 (m, 1H), 5.20 (m, 2H), 3.94 (dt, 2H), 3.70-3.55 (m, 48H), 3.45 (s, 6H), 3.43 (s, 2H), 3.40 (s, 9H).

Example 12c: 3,3-dimethoxy-9,9-di-2,5,8,ll,14-pentaoxapentadecyl-

2,7, 11, 14, 17,20,23-heptaoxa-3-silatetracosane. Karstedt's catalyst (Platinum(0)-l,3-divinyl-l,l,3,3-tetramethylsiloxane) (20 μΐ, 2% in xylene) was added to 16-(allyloxymethyl)-16-2, 5,8,11, 14-pentaoxapentadecyl- 2,5,8,11, 14,18,21,24, -27,30-decaoxahentriacontane (0.75g, 1.0 mmol, example 12b) and trimethoxysilane (255 μΐ, 2.0 mmol) in dry toluene (6 ml) under nitrogen at room temperature. The reaction mixture was shaken at room temperature for 24 h. Charcoal was then added and the reaction mixture was filtered after 2 minutes. The volatile material was removed at reduced pressure, which gave 830 mg (96%) of the title product and 16-2,5,8,11, 14-pentaoxapentadecyl-16-((prop-l-enyloxy)methyl)- 2,5,8,11,14,18,21,24,27,-30-decaoxahentriacontane (HPLC-ELSD-C18, 95:5 to 5:95 H ₂ 0/ACN in 25 min) in a 70 : 30 ratio .

Example 13: TriPEG coated nanoparticles. Oleate capped particles (175 mg) from example la was dissolved in dry THF (15 ml). The material from example 12c; 3,3- dimethoxy-9,9-di-2,5,8, 11 , 14-pentaoxapentadecyl-2,7, 11,14,17,20,23-heptaoxa-3- silatetracosane (830mg, 0.7 mmol) was added and the reaction mixture was shaken for a few minutes. Potassium tert-butoxide (84 mg, 0.7 mmol) was added and the mixture was stirred for 48 h at room temperature under nitrogen. The resulting mixture was filtered and the filtrate was concentrated at reduced pressure until approximately 2 ml remained. Diethyl ether/heptanes 1 : 1 mixture (20 ml) was added to the concentrated and the precipitate was isolated by centrifugation and then washed with two portions of heptane. The precipitant was dried at reduce pressure to give a waxy residue (270 mg) of coated particles. Volume particle size distribution = 11 nm. Gd 19%.

Example 14: Hardening of TriPEG coated nanoparticles. Dimethyl-2- (triethoxysilyl)ethyl phosphate (54 mg, 0.2 mmol) was added to TriPEG coated particles (60 mg) (example 13) dissolved in 7.5 ml aqueous 80%> 1-propanol in a pressure vessel. The reaction mixture was stirred for 48 h at 95 °C and then 24 h at 120 °C. The temperature was lowered to room temperature and the clear solution was filtered (0.2 μηι syringe filter). The hardened material is purified using diafiltration as described in Example 6. Volume particle size maximum = 6.5 nm; Stability for 24h at pH5.8 = 47%; ti at 81.3 MHz, 26.2°C = 5.07 mM ^"1 Gd ms ^"1 .

Example 15a Allyl-3-(2,5,8,ll,14,17,20,23-octaoxapentacosan-25-yl)urea. Allyl isocyanate (400 μΐ, 4.6 mmol) was added to 2,5,8,11,14, 17,20,23-octaoxapentacosan- 25-amine (550 mg, 1.4 mmol) in dry THF (4 ml) under nitrogen. The reaction mixture was shaken at 50 °C for 18h. The volatile materials were removed at reduced pressure to give a dark yellow oil (670 mg, >95% yield), which was used without any further purifications. 1H-NMR (CDC1 ₃ ); 5.85 (m, 1H), 5.12 (m, 2H), 3.80 (d, 2H), 3.75-3.60 (m, 28H), 3.55 (m, 4H) 3.38 (s, 3H).

Example 15b Allyl-3-(2,5,8,ll,14,17,20,23,26,29,32,35-dodecaoxahepta- triacontan-37-yl)urea. Allyl isocyanate (158 μΐ, 1.8 mmol) was added to 2,5,8,11, 14,17,20,23,26,29, 32,35-dodecaoxaheptatriacontan-37-amine (230 mg, 0.4 mmol) in dry THF (4 ml) under nitrogen. The reaction mixture was shaken at 50 °C for 18h. The volatile materials were removed at reduced pressure to give a yellow oil (270 mg, >95%), which crystallized upon standing. 1H-NMR (CDC1 ₃ ); 5.85 (m, 1H), 5.12 (m, 2H), 3.80 (d, 2H), 3.75-3.60 (m, 44H), 3.55 (m, 4H) 3.38 (s, 3H).

Example 15c l-(2,5,8,ll,14,17,20,23-octaoxapentacosan-25-yl)-3-(3-(trime thoxy- silyl)propyl)urea. K ar s t e dt ' s c at a l y s t ( P l at i num ( 0 )-l,3-divinyl-l, 1,3,3- tetramethylsiloxane ) ( 2 0 μ 1 , 2 % i n xylene) was added to Allyl-3- (2,5,8,1 l,14,17,20,23-octaoxapentacosan-25-yl)urea (117 mg, 0.25 mmol, example 15b) and trimethoxysilane (192 μΐ, 1.5 mmol) in dry toluene (1 ml) under nitrogen at room temperature. The reaction mixture was shaken at 70 °C for 3h. Charcoal was then added and the reaction mixture was filtered after 2 minutes. The volatile material was removed at reduced pressure, which gave 130 mg (88%) of the title product.

Example 16: Hardening of PEG-silane coated particles with 75%

Dimethylphosphatoethyl triethoxysilane (DTEP) and 25%

bis(triethoxysilyl)methane Dimethyl-2-(triethoxysilyl)ethyl phosphonate ( 20.3 mg, 0.0675 mmol) and

Bis(triethoxysilyl)methane ( 7.7 mg, 0.0225 mmol) was added to PEG coated particles (30 mg) dissolved in 6 ml aqueous 80% 1-Propanol in a pressure vessel. The reaction mixture was stirred for 46h at 95°C and then 24h at 120°C. The temperature was lowered to room temperature and the clear solution was filtered (0.2 μιη syringe filter). A fraction of the filtrate (5 ml) was diluted with TRIS/HCl buffer (95 ml, 5 mM pH7.4) and then filtered using 500k NMWC pore-size column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-500-C-MM01A). The collected permeates (~96 ml) were then filtered using a 300k NMWC pore size diafilter column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-300-C-MM01 A) to remove the free ions and concentrate the particles. The retentate was collected and then filtered using a syringe filter (0.2 μιη). Volume particle size maximum = 10 nm; Composition (ICP, mole ratio): Si/Gd = 1.96, P/Gd = 0.5, P/Si = 0.26; Stability for 24h at pH5.8 = 68%; ti at 80 MHz = 3.67 mM ^"1 Gd ms ^"1 . Example 17: Hardening of PEG-silane coated particles with 50%

Dimethylphosphatoethyl triethoxysilane (DTEP) and 50%

bis(triethoxysilyl)methane

Dimethyl-2-(triethoxysilyl)ethyl phosphonate (13.5 mg, 0.045 mmol) and

Bis(triethoxysilyl)methane ( 15.3 mg, 0.045 mmol) was added to PEG coated particles (30 mg) dissolved in 6 ml aqueous 80%> 1-Propanol in a pressure vessel. The reaction mixture was stirred for 46h at 95°C and then 24h at 120°C. The temperature was lowered to room temperature and the clear solution was filtered (0.2 μιη syringe filter). A fraction of the filtrate (5 ml) was diluted with TRIS/HCl buffer (95 ml, 5 mM pH7.4) and then filtered using 500k NMWC pore-size column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-500-C-MM01A). The collected permeates (~96 ml) were then filtered using a 300k NMWC pore size diafilter column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-300-C-MM01A) to remove the free ions and concentrate the particles. The retentate was collected and then filtered using a syringe filter (0.2 μιη). Volume particle size maximum = 10 nm; Composition (ICP, mole ratio): Si/Gd = 2.77, P/Gd = 0.37, P/Si = 0.13; Stability for 24h at pH5.8 = 74%; ti at 81.5 MHz, 25.9°C = 3.02 mM ^"1 Gd ms ^"1 .

Example 18: Hardening of PEG-silane coated particles with 75%

Dimethylphosphatoethyl triethoxysilane (DTEP) and 25%

bis(triethoxysilyl)methane

Dimethyl-2-(triethoxysilyl)ethyl phosphonate (6.75 mg, 0.0225 mmol) and

Bis(triethoxysilyl)methane (23 mg, 0.0675 mmol) was added to PEG coated particles (30 mg) dissolved in 6 ml aqueous 80% 1-Propanol in a pressure vessel. The reaction mixture was stirred for 46h at 95°C and then 24h at 120°C. The temperature was lowered to room temperature and the clear solution was filtered (0.2 μιη syringe filter). A fraction of the filtrate (5 ml) was diluted with TRIS/HCl buffer (95 ml, 5 mM pH7.4) and then filtered using 500k NMWC pore-size column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-500-C-MM01A). The collected permeates (~96 ml) were then filtered using a 300k NMWC pore size diafilter column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-300-C-MM01 A) to remove the free ions and concentrate the particles. The retentate was collected and then filtered using a syringe filter (0.2 μιη). Volume particle size maximum = 11.7 nm; Composition (ICP, mole ratio): Si/Gd = 4.32, P/Gd = 0.28, P/Si = 0.06; Stability for 24h at pH5.8 = 82%; ti at 81.5 MHz, 25.9°C = 2.4 mM ^"1 Gd ms ^"1 . Example 19: Hardening of PEG-silane coated particles with 10%

Dimethylphosphatoethyl triethoxysilane (DTEP) and 90%

bis(triethoxysilyl)methane

Dimethyl-2-(triethoxysilyl)ethyl phosphonate (2.7 mg, 0.009 mmol) and

Bis(triethoxysilyl)methane ( 27.6 mg, 0.081 mmol) was added to PEG coated particles (30 mg) dissolved in 6 ml aqueous 80%> 1-Propanol in a pressure vessel. The reaction mixture was stirred for 46h at 95°C and then 24h at 120°C. The temperature was lowered to room temperature and the clear solution was filtered (0.2 μιη syringe filter). A fraction of the filtrate (5 ml) was diluted with TRIS/HCl buffer (95 ml, 5 mM pH7.4) and then filtered using 500k NMWC pore-size column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-500-C-MM01A). The collected permeates (~96 ml) were then filtered using a 300k NMWC pore size diafilter column (GE Heathcare's Midgee ultrafiltration cartridge Model: UFP-300-C-MM01A) to remove the free ions and concentrate the particles. The retentate was collected and then filtered using a syringe filter (0.2 μιη). Volume particle size maximum = 13.5 nm; Composition (ICP, mole ratio): Si/Gd = 4.87, P/Gd = 0.12, P/Si = 0.02; Stability for 24h at pH5.8 = 76%; ti at 81.5 MHz, 25.9°C = 1.79 mM ^"1 Gd ms ^"1 .