WO2012156094A1 - Nanoparticles functionalized with dendritic polyglycerol sulfate - Google Patents

Abstract

The invention relates to nanoparticles, characterized by a) a core consisting of an inorganic material and b) a shell consisting of a linker and dendritic polyglycerol sulfate, wherein the polyglycerol sulfate consists of repeated glycerol units of formula (RO-CH₂)₂CH-OR, where R = H or other glycerol units, and one or more OH groups of the glycerol units are replaced by sulfate groups of formula -OSO₃X, where X is equal to H, by an alkali metal atom, such as Li, Na or K, or by an ammonium ion, and wherein the linker links the polyglycerol sulfate and the core to each other. The nanoparticles are suitable for medical uses, for example, for the diagnosis and treatment of inflammatory diseases.

Description

T DENDRITICAL POLYGLYCEROLSULFATE FUNCTIONALIZED NANOPARTICLES

The present invention relates to nanoparticles with dendritic polyglycerol sulfates, processes for their preparation and their uses, in particular in the medical field. The dendritic polyglycerol sulfates and the inorganic nanoparticles are linked together via linkers.

Inflammation is a characteristic response of human or animal tissue to a deleterious stimulus or invading pathogens, in which leukocytes play a key role due to their antimicrobial, secretory and phagocytic activities. In all forms of the inflammatory response, recruitment of leukocytes to the vascular endothelium and subsequent migration into the surrounding inflamed tissue is observed.

CONFIRMATION COPY The acute inflammatory response is thus the body's first alarm signal designed to contain and eventually eliminate proinflammatory stimuli. In addition to this desired, protective property of the inflammatory reaction to

However, elimination of antigens in our body may cause malfunction of the immune response, but severe secondary tissue damage if the proinflammatory irritation persists. This is observed in chronic inflammatory diseases such as rheumatism, Crohn's disease, ulcerative colitis, chronic tissue rejection in transplants, asthma, osteoarthritis, berylliosis or

Psoriasis.

The first step of leukocyte emigration is the activation of vascular endothelial cells by signals (cytokines) that originate from the site of inflammation in the tissue. Activated endothelial cells secrete more

Signal molecules (chemokines) that activate leukocytes. Furthermore, activation of the endothelial cells results in the expression of cell adhesion molecules on the vascular surface. Complementary cell adhesion molecules on leukocytes and endothelial cells are presented, which bind to each other and thus initiate the adhesion cascade at the end of which the migration of leukocytes is.

This complex process of recruitment of leukocytes to the endothelium at the site of inflammation is initiated by weak interactions of selectin ligand complexes, which leads to the reduction of the flow velocity of the cells and the subsequent attachment of the leukocytes to the blood vessel wall.

Selectins are transmembrane, homologous glycoproteins classified into L, E and P selectin according to their cellular abundance. E-Selekin is presented on endothelial cells, P-selectin is expressed both by endothelial cells and on platelets (platelets) and L-selectin from the white blood cells

(Leukocytes) formed.

The initial contact of leukocytes with the endothelial cells takes place by interaction of L-selectin carbohydrate ligands of the endothelial cells. An important role as a binding partner is presented on protein and lipids

Tetrasaccharide sialyl LewisX (sLeX), which is used as the standard ligand for structure-activity relationships for the characterization of binding properties as well as for the search for selectin inhibitors. Since selectin-ligand interactions initiate leukocyte emigration, their targeting / blockade provides a means for localization and therapeutic intervention of inflammation. Various selectin inhibitors are known in the art, but so far no therapeutic is on the market.

For example, DE 102006036326 A1 discloses dendritic polyglycerol sulfates and sulfonates which have a high affinity for L- and P-selectin. The IC ₅₀ values of these compounds are between 10 and 40 nM.

EP 2123269 A1 discloses functionalized nanoparticles having a size of 15-50 nm. The particles consist of a gold core and a shell of long-chain linear alkyl groups with sulfated amino alcohols as the functional group. The particles are suitable inhibitors of selectin-ligand interactions and show

IC50 values in the picomolar range.

The object of the invention is therefore to provide improved substances which are particularly suitable for the localization and treatment of inflammation, are non-toxic and can be prepared simply and inexpensively.

The object is achieved by nanoparticles having an inorganic core and a shell of linker and dendritic Polygycerolsulfaten (dPGS) according to the features of the claims.

According to the invention, the nanoparticles comprise

 a) a core of an inorganic material and

 b) a shell of linker and dendritic polyglycerol sulfate,

wherein the polyglycerol sulphate consists of repeating glycerol units of the formula (RO-CH ₂ ) ₂ CH-OR, where R = H, or other glycerol units, one or more OH groups of the glycerol units by sulfate groups of the formula -OSO ₃ X, wherein X is H, an alkali metal atom such as Li, Na or K, or an ammonium ion such as triethylammonium or diisopropylethylammonium, are replaced,

 and wherein the linker links polyglycerol sulfate and core together.

The PGS shell around the core is dendritic (tree-like) branched. Since glycerol has 3 OH groups, it can be used in the polymerization to polyglycerol - unlike, for example, in propanediol - create more or less cross-linked, three-dimensional structures (dendritic polymers). The extent of dPGS branching can be specified by the degree of branching (DB). The degree of branching is in The scope of this invention is defined by Frey (Wilms D., Wurm F., Nieberie J., Bohm P., Kemmer-Jonas U., Frey H., Macromolecules, 2009, 42, 3230-3236) by the following equation (1). :

2D

 DB =

 2D + L

D and L in equation (1) define the number of linear (L) or dendritic (D) glycerol units per nanoparticle. L corresponds to the number of glycerol units (glycerol molecules) per nanoparticle, which are each connected to 2 other glycerol units. D represents the number of

Glycerol units per nanoparticle, which are each connected to 3 other glycerol units. The theoretical maximum according to this formula amounts to 100%, in this case of maximally branched polyglycerols one speaks of so-called polyglycerol dendrimers.

To illustrate the degree of branching, the section of a dPGS molecule is shown by way of example in FIG. T represents terminal (T) glycerol units, that is, glycerol units linked to only one glycerol molecule. The terminal glycerol units form the end of the respective polyglycerol chain and are thus located on the surface of the nanoparticles.

Depending on the polymerization conditions, the degree of branching of the polyglycerol can be adjusted as desired. According to the invention, the degree of branching is 1-100%. Preferably, the degree of branching is from 30-80%, and more preferably from 55-65%.

According to the invention, the dPGS is preferably not perfect, i. it has a degree of branching of less than 100% (so-called dendritic polymers). Dendritic polymers have several advantages over dendrimers (maximum branched). In dendrimers, the steric hindrance increases with increasing

Branching to, so that the growth of the shell theoretically limits are set and the particles can not exceed a certain diameter. This is not the case with dendritic polymers. In addition, dendritic polymers have the advantage of having free functional groups not only on their surface but also internally. These can be used, for example, for others

Coupling molecules, such as medically active substances or other marker molecules. Furthermore, dendritic polymers have greater flexibility than dendrimers. According to the invention, the layer thickness of the sheath of dPGS and linker is about 1-20 nm, preferably 2-10 nm.

The average molecular weight of the dPGS is about 100 to 1,000,000 g / mol, preferably 2,500 to 200,000 g / mol, and more preferably 5,000 to 15,000 g / mol.

The dPGS have sulfate groups -OSOaX; wherein X is H, an alkali metal atom such as Li, Na or K, or an ammonium ion such as triethylammonium or diisopropylethylammonium. The sulfate groups can be introduced into the starting dendritic polyglycerols using suitable sulfation reagents (Türk H, Haag R, Alban S (2004) "Dendritic polyglycerol sulfate as new heparin analogue and potent inhibitor of the complement system", Bioconjugate Chem 15: 162 The dendritic polyglycerols can be prepared, for example, by a one-stage anionic polymerization (eg a so-called anionic multibranching ring-opening polymerization, Haag R, Mecking S, Türk H. DE 1021 1664A1, 2002) ₃ is used with bases, such as pyridine or triethylamine. About the ratio of S0 ₃ to the OH groups of the dendritic polyglycerol, the resulting degree of sulfation can be adjusted.

The degree of sulfation is 1-100%, preferably 50-99%, and more preferably 80-99%, most preferably 84-99%. The term "degree of sulfation" in the context of this invention expresses the percentage of -OH groups of the glycerol units of the dPG (dendritic polyglycerol) which is sulfated in the dPGS If, for example, half of the -OH groups of the glycerol units are sulfated, the degree of sulfation is 50%.

The core consists of a nanoscale inorganic material. In principle, all metals and inorganic metal compounds which are inert and biocompatible, and water-insoluble are suitable. Because of their high biocompatibility, gold and iron oxide are particularly preferred and are therefore particularly suitable for use in the therapeutic or diagnostic field. Very particular preference is given to gold nanoparticles. Typically, the core of the nanoparticles according to the invention has a diameter of 5-45 nm, preferably 10-30 nm.

Suitable as linkers are d-coco-hydrocarbon radicals, for example branched or linear C-C ₂₀ -alkyl chains, optionally interrupted by -NH-, -O-, -S-, - (C =O) -NH-, - (C =O) -O-, -N- (C = O) -N -, - N- (C = S) -N-, -N- (O = O) -O-, -N- (C = S) -0 -, or -triazol-, which have alpha-terminal 1, 2 or 3 thio groups or phosphonate groups and omega-terminal one or more, preferably one reactive group, selected from the group - (C = O) -OH, C- (C = O) -R, with R = N-hydroxysuccinimide, Cl, -N = C = S, -N ₃ , or -alkin.

The linkers may be covalently or coordinately attached to the nucleus via the terminal thio groups or phosphonate groups. In the case of gold nanoparticles, alpha-terminal thio groups are preferred. The binding of the thio groups to the gold nanoparticles preferably takes place via a "soft base, soft acid" bond, the binding energy is generally 126-146 kJ / mol, which is comparable to weak covalent bonds (150-500 kJ / mol In the case of iron oxide nanoparticles, alpha-terminal phosphonate groups are preferred, whereby the binding of the phosphonate group to the iron oxide nanoparticles preferably takes place via a coordinative bond.

Because of the high affinity of sulfur for many metals, thio groups are a preferred function by which the dPGS can be linked to the nucleus.

The linker molecules are preferably coupled to the surface of the inorganic nuclei via thio groups, such as thiol groups or sulfur-containing heterocycles. Examples of sulfur-containing heterocycles are sulfur-containing five-membered rings such as dithiolanes.

Preferred linkers are C ₁ -C ₂ o-hydrocarbon chains, such as Ci-C ₂₀ alkyl chains, for example, propyl, butyl, pentyl, hexyl, octyl or decyl. These can be connected to the nucleus via one or more, in particular 1, 2 or 3, sulfur atoms and to the dPGS via a carboxyl group.

Particularly preferred linkers are disulfides, especially cyclic disulfides such as dithiolanes. These have an adsorption energy that is comparable to that of covalent bonds. This is exemplified in Scheme 1 for lipoic acid as a linker.

A suitable linker is, for example, lipoic acid. It has surprisingly been found that lipoic acid couples dPGS with an M _w in the range from 10,000 g / mol to 15,000 g / mol of gold nanoparticles with high stability. The linker molecule is linked to the dPGS via a covalent bond. The

Binding of the linker to the dPGS occurs via a functional group of the linker which is linked to a free NH ₂ or OH group of the dPGS, for example via an ester bond or a peptide bond. The linking reaction is typically a condensation reaction in which the linker is linked to the dPGS via a functional group, eg, a free carboxyl group.

For lipoic acid, binding to dPGS occurs, for example, via the carboxyl group. The linker generally forms an inner layer around the inorganic core around which there is an outer layer of dPGS.

Typically, the linker has a length of 0.5-10 nm, preferably 1-5 nm.

In a preferred embodiment, the nanoparticle according to the invention comprises a core of gold, a dPGS shell with an average molecular weight M _w of 500-100,000 g / mol and a degree of branching of 30-80% and a thio linker of a d-Ce-alkyl Group having one or more alpha-terminal disulfide groups and an omega-terminal group -C- (C = O) -OH, -C- (C = O) -Cl or -

C- (C = 0) -N-hydroxysuccinimide.

In a particularly preferred embodiment, the nanoparticle according to the invention comprises a core of gold, a dPGS shell with an average molecular weight of 5,000-15,000 g / mol and a degree of branching of 55-65% and a lipoic acid linker coupled to the dPGS via an amide Binding.

The nanoparticles according to the invention may have the same or different dPGS and linker. Preference is given to using a linker type and a dPGS type.

Surprisingly, it has been found that the nanoparticles according to the invention have a very high affinity for selectins such as L-selectin. The high affinity of the nanoparticles according to the invention for selectines makes them excellently suitable substances for use in the medical or diagnostic field, especially in inflammatory diseases.

The nanoparticles according to the invention can be combined with suitable medicinal active compounds to give conjugates. These conjugates of the nanoparticles according to the invention with active substances are likewise the subject matter of the present invention. Thus, for example, substances can be transported very selectively into inflamed tissue.

An advantage of the nanoparticles according to the invention is that no further targeting molecules such as antibodies, antibody fragments, proteins, peptides or oligonucleotides have to be used for the transport of the particles to the site of action, since the dPGS itself has a targeted effect. This reduces the synthetic effort and the associated costs and increases the robustness of the system. Furthermore, the nanoparticles according to the invention do not necessarily require signaling molecules in disgnostic applications, since the inorganic core materials such as gold or iron oxide themselves can act as signal transmitters, for example in MSOT (Multispectral Optoacoustic Tomography) measurements or in MRT.

As a pharmaceutical or pharmaceutical composition with acceptable carrier, the nanoparticles may be e.g. be applied in the form of tablets, capsules, powders, suspensions, and infusions. The pharmaceutical or pharmaceutical compositions may further contain carriers and / or excipients.

Suitable carriers and excipients include binders, suspending agents, lubricants, colors, flavors and preservatives.

For a therapeutic treatment with the nanoparticles according to the invention, e.g. all inflammatory processes in question, both acute and chronic.

The subject of the invention is therefore also the use as prophylactics or therapeutics for inflammatory diseases. The substances according to the invention are preferably used in diseases in which the extravasation of leukocytes into the tissue plays a role and leads to tissue damage.

The nanoparticles according to the invention are particularly suitable for the treatment of chronic inflammatory diseases, in particular rheumatoid arthritis, psoriaris, Crohn's disease, ulcerative colitis, allograft rejection, asthma, beryliosis or autoimmune diseases or tissue rejection.

The nanoparticles according to the invention can also be used for the treatment of inflammatory diseases in which the selectin-mediated leukocyte adhesion is dysregulated. Chronic inflammatory processes cause tissue destruction

Emergence of fibrosis. The two cytokines IFN and TNFa play an important role here. IFN _y is secreted by a certain population of leukocytes and results in the activation of macrophages, which in turn produce hydrolytic enzymes and reactive oxygen and nitrogen species, resulting in the destruction of surrounding tissue. In addition, TNFa is released, causing the

Expression of cell adhesion molecules on adjacent endothelia leads to increased recruitment of leukocytes.

The nanoparticles according to the invention bind selectins such as L- and P-selectin with particularly high affinity and thus block the interaction with their ligands. Of the Leukocyte / endothelial contact is reduced, thus suppressing the increased migration of leukocytes into the inflammatory foci.

The nanoparticles can therefore also be used as selective selectin inhibitors. Surprisingly, IC ₅₀ values of up to 180 fM were observed with the nanoparticles according to the invention, which are lower by a factor of 100 than the IC ₅₀ values for substances known from the prior art (eg EP 2123269 A1). The nanoparticles according to the invention are thus particularly advantageously suitable for inhibiting selectin-mediated leukocyte adhesion.

The nanoparticles of the invention may also be used as diagnostics, e.g. as a contrast agent for use in imaging techniques such as MRI and MSOT. Preferably, the use as diagnostics in inflammatory diseases, e.g. be used as selectin indicators for the diagnosis, localization and visualization of the selectins, especially in vitro in inflamed tissue, in organs, in tissue sections but especially in vivo. Due to the inorganic core of the nanoparticles according to the invention, these are suitable directly as contrast agents, e.g. Gold Nanoparticles in Multispectral Optoacoustic Tomography (MSOT) and Iron Oxide Nanoparticles in MRI (Magnetic Resonance Tomography).

If required or desired, the nanoparticles of the invention may also be loaded with other signaling molecules or bound to signaling molecules.

Preferred signaling molecules are, for example, radioactive isotopes such as iodine-124, iodine-125 or fluorine-18. In addition, dyes, in particular fluorophores such as e.g. Aminomethylcoumarin, fluorescein, cyanine, rhodamine and their derivatives can be used. However, the signaling molecules can also be a fluorescence donor or reporter and a fluorescence acceptor or quencher, which can be used in particular as a pair of respectively one fluorescence donor / reporter and one fluorescence acceptor A quencher.

Examples of such conjugates with signaling molecules are:

 1) gold nanoparticle dPGS covalently conjugated to cyanine dye,

 2) gold nanoparticle dPGS covalently conjugated with rhodamine dye,

3) Gold nanoparticle dPGS covalently conjugated to chelators for radionuclides such as 1, 4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (DOTA), diethylenetriamine-pentaacetic acid (DTPA) or mercaptoacetyltriglycine (MAG ₃ ).

The nanoparticles of the invention are also capable of specifically binding chemokines. These are, for example, proinflammatory cytokines, in particular TNFα, IL-1, IL-6, as well as IL-8 and MIP-1β. In the case of an inhibitory binding of the chemokines, such as INF or TNFa, by the dPGS nanoparticles according to the invention, an interaction with receptors of the chemokines is prevented so that tissue damage and leukocyte extravasation are prevented.

Because of their specific interactions with proteins such as selectins, chemokines and coagulation factors, in particular with L- and P-selectin, the nanoparticles according to the invention can preferably also be used in in vitro applications. For this purpose, the nanoparticles according to the invention-analogously to the commercially available heparin sepharose-are immobilized on a matrix. Preferred matrices or surfaces for immobilization are inorganic and polymeric natural synthetic materials, for example glass, silica, dextran, agarose, sepharose or synthetic hydrophilic polymers. The immobilized nanoparticles of the present invention may be used to fractionate biological samples such as body fluids, plasma, blood, serum, cell suspensions and supernatants from cell cultures, or to purify specific proteins, e.g. L-selectin, P-selectin, chemokines, coagulation factors are used. Finally, the nanoparticles of the invention may also be used as scavengers, e.g. in the

ELISA can be used.

The nanoparticles of the invention thus have many advantages: they are very good biocompatible, easy to prepare, and have a very high affinity for L- and P-selectin.

MANUFACTURING

The preparation of the nanoparticles according to the invention is explained in more detail in the following Schemes and exemplary embodiments.

The following abbreviations are used in the illustrated reaction schemes:

 AuNP = gold nanoparticles

AuNR = gold nanorods

DCC = dicyclohexylcarbodiimide

MsCl = methanesulfonyl chloride

NHS = / V-hydroxysuccinimide

DIPEA = diisopropylethylamine

Pyr = pyridine

TA = lipoic acid

mPEG-SH 1000 = methoxy-terminated polyethylene glycol thiol with a

Molecular weight of 1000 g / mol

CTAB = cetyltrimethylammonium bromide

MSOT = Multispectral Optoacoustic Tomography

OD = optical density

The figures show:

Fig. 1: Structure of dendritic polyglycerol. Terminal (T), linear (L) and dendritic (D) repeat units are grayed out.

Fig. 2 (Scheme 1): lipoic acid as a linker.

Figure 3 (Scheme 2): Reaction scheme for preparing an activated linker molecule exemplified by lipoic acid (NHS activation of lipoic acid) after the synthesis of Liu et al. (Liu, W. Journal of the American Chemical Society, 2008, 1274-1284 ).

Figure 4 (Scheme 3): Reaction scheme for the preparation of an amine-functionalized dendritic polyglycerol (Roller, S., Zhou, H., Haag, R. Molecular Diversity, 2005, 9, 305-316). The dPG starting material was synthesized by a one-step \ "anionic multibranching ring-opening polymerization \" (Haag R, Mecking S, Turk H. DE 10211664A1, 2002); Controlled amine functionalization of dPG.

FIG. 5 (Scheme 4): Reaction scheme for the sulfation of the OH groups of the dendritic polyglycerol (Türk H, Haag R, Alban S (2004) Dendritic polyglycerol sulfates as new heparin analogues and potent inhibitors of the complement system. Bioconjugate Chem 15: 162-167.); Sulfation of partially aminated dPG.

Figure 6 (Scheme 5): Reaction scheme for coupling a linker such as a lipoic acid linker to dPGS; Functionalization of dPGS with a lipoic acid linker.

Figure 7 (Scheme 6): Ligand exchange of citrate-stabilized gold nanoparticles with dPGS in aqueous solution. The number of citrate or dPGS per gold nanoparticle is shown here only schematically (ligand exchange on citrate-stabilized gold nanoparticles with TA-dPGS in aqueous solution).

Figure 8 (Scheme 7): shows the mPEG-SH 1000 functionalization of CTAB-functionalized gold nanorods. The gold nanorods are shown here only schematically; Ligand exchange on gold nanorods from CTAB bilayer to mPEG-SH 1000 layer.

Figure 9 (Scheme 8): shows the TA-dPGS 10,000 g / mol functionalization of mPEG-SH 1000 functionalized gold nanorods. The gold nanorods are shown here only schematically; Ligand exchange on gold nanorods of mPEG-SH 1000 layer to TA-dPGS 10,000 g / mol layer.

Figure 10 (Figure 1): SPR sensorgram of dPGS-TA functionalized gold nanoparticles.

Figure 11 (Figure 2): SPR sensorgram of AuNR-TA-dPGS 10,000 g / mol.

The nanoparticles according to the invention can be prepared by first linking the dPGS with a linker and then reacting the reaction product with the core material of inorganic nanoparticles.

The linker, e.g. Lipoic acid can be activated, e.g. by reacting with N-hydroxysuccinimide in dichloromethane and tetrahydrofuran to give the lipoic acid succinimide (see Figure 3, Scheme 2).

The dPG used as starting material can be obtained, for example, by a one-stage anionic multi-branching ring-opening polymerization "(Haag R, Mecking S, Türk H., DE 10211664A1, 2002) .This can be functionalized before the reaction with the linker, for example with amine groups (FIG 4, Scheme 3). The dPG can be converted to dPGS with sulfating agents such as SCyPyridine (Figure 5, Scheme 4).

The linkage of the amine-functionalized dendritic dPGS with the linker occurs e.g. via a succinimide coupling in dichloromethane with / V, / V-diisopropylethylamine as the base (see Figure 6 (Scheme 5)).

The inorganic nanoparticles which are useful as a core for the preparation of the dPGS nanoparticles of the invention may be e.g. by a method described by X. Lou and C. Wang, C (Biomacromolecules, 2007, 8, 1385-1390), e.g. by reduction of HAuC with sodium citrate in aqueous solution at temperatures around 100 ° C.

The rod-shaped inorganic nanoparticles useful as nuclei for the preparation of the dPGS nanoparticles of the invention may be e.g. are synthesized by a protocol developed by Jana Nikhil (N., Small, 2005, 8, 875-882). The CTAB-functionalized gold nanorods may then be coated e.g. as in the protocol of Xiaoge Hu and Xiaohu Gao (X. Hu, X. Gao, Phys. Chem. Chem. Phys., 2011, 13, 10028-10035) are functionalized with mPEG-SH 1000.

Finally, the dPGS is coupled to the core particles using the linker. For this, e.g. those of Mei, B. C; Susumu, K .; Medintz, I. L .; Delehanty, J. B .; Mountziaris, T.J .; Mattoussi H. (Journal of Materials Chemistry, 2008, 18, 4949-4958), in which the citrate shell of the inorganic nanoparticles in the aqueous system is exchanged for the linker with a linker such as lipoic acid functionalized dPGS overnight at room temperature. Purification then takes place via dialysis in water (FIG. 7, Scheme 6).

For the gold nanorods, the coupling of the TA-dPGS is 10,000 g / mol of mPEG-SH 1000 g / mol, e.g. at 60 ° C for 12 hours while stirring in water. Subsequently, the purification is carried out e.g. about washing by centrifugation.

Synthesis of conjugates of TA-dPGS 10,000 g / mol or 5,000 g / mol of functionalized gold nanoparticles or gold nanorods with drugs or other signaling molecules or chelators for signaling radioactive metals can be accomplished in two ways.

In the first approach, the drugs or signaling molecules can be linked to a linker with one or more alpha-terminal disulfide groups such as lipoic acid, for example via an ester bond, peptide bond or triazole moiety. Active substances can also be used, for example, via acid-labile bonds, such as, for example, acetal bonds. Ester bonds or hydrazone bonds are linked. Subsequently, the linker-providing signaling molecules or drugs can be mixed in the incubation of gold nanoparticles or gold nanorods with the TA-dPGS 10,000 g / mol or TA-dPGS 5,000 g / mol statistically with. Thus, a statistical distribution of drugs, signaling molecules and TA-dPGS on the surface is achieved.

Alternatively, the drugs or signaling molecules can be covalently linked to the TA-dPGS. Here, a dPG is functionalized with several azide groups as described in Scheme 3 (FIG. 4). Subsequently, only a portion of these azide groups is reduced to amine groups. The linker unit, e.g. Lipoic acid is coupled to the amine group as described in Scheme 5 (Figure 6). Subsequently, the remaining azide groups for e.g. an azide-alkyne Huisgen cycloaddition (Huisgen, R., Proc. Chem. Soc., 1961, 357-396) to link the signaling molecule or drug to the TA-dPGS. Alternatively, multiple azide groups can also be reduced and the linker moiety and the drug or signaling molecule randomly linked to the dPGS as in Scheme 5 (Figure 6) via an amide coupling. The TA-dPGS linked to the drug or signaling molecule can then be used to functionalize the gold nanoparticles or gold nanorods. use

Due to their inorganic core, the TA-dPGS functionalized nanoparticles or nanorods according to the invention are outstandingly suitable for use as contrast agents for the diagnosis of inflammatory diseases such as, for example, by MSOT (Multispectral Optoacoustic Tomography). More information about MSOT

Measurements are e.g. included in the review by Vasiiis Ntziachristos. (Ntziachristos, V., Razansky, D. chemical reviews 2010, 110, 2783-2794). To study the efficacy of the TA-dPGS of the invention, nanoparticles or nanorods functionalized as contrast agents for e.g. MSOT measurements may e.g. an LPS-induced arthritis model (Chen, W; Mahmood, U; Weissleder, R;

Tung, C, Arthritis Res Ther. 2005, 310-317). Here, a mouse lipopolysaccharide (LPS) is injected intra-articularly. Subsequently, the mouse eg the AuNR-TA-dPGS 10,000 g / mol suspension (eg 100 μί, 100 OD) is injected intravascularly. Subsequent MSOT examination shows increased contrast in the arthritis-induced joints due to the strong attachment of the TA-dPGS functionalized nanoparticles or nanorods to the L-selectin of the inflamed Endothelium. Labeling of the TA-dPGS functionalized nanoparticles or nanorods according to the invention with signaling molecules such as cyanine dyes, rhodamine dyes or chelators for radionuclides allows the use of further diagnostic methods such as fluorescence tomography, PET (positron emission tomography) or SPECT (single photon

Emission computed tomography) via analog measurements.

The use of the TA-dPGS functionalized nanoparticles or nanorods of the present invention as an anti-inflammatory agent may be e.g. be demonstrated by an allergic contact dermatitis model. Here, as in

(Dernedde J., Rausch A., Weinhart M., Enders S. Tauber R., Licha K., Schirner M. Ruegel U. Von Bonin A. Haag R., Proc Natl Aca., See, USA 2010, 107 , 19679-19684) described a mouse induced by the injection into the ear of the allergen trimellitic anhydride (TMA) acute allergic contact dermatitis. Then, depending on the dose, the ear thickness is measured at equal time intervals after the addition of the anti-inflammatory agent. Based on the reduced ear thickness as compared to the control without addition of anti-inflammatory agent, the efficiency of the anti-inflammatory effect can be determined.

EMBODIMENTS

The invention will be explained in more detail with reference to the following embodiments. Spherical nanoparticles with a gold core (AuNP) and a shell of dendritic

PGS with molecular weights of 5,300 g / mol, or 10,000 g / mol and a degree of sulfation of 85% were prepared using lipoic acid (TA) as a linker as follows: Methods:

 1 H NMR and 13 C NMR were recorded on a Bruker ™ ECX spectrometer at 400 MHz. The chemical shift is reported as δ (ppm) and was related to the solvent signal. Multiplicities are given as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). The associated coupling constants J are given in Hertz (Hz). Analysis of the data was done with MestReNovaTM version 6.0.2.

UV / Vis absorption spectra were recorded on a UV / Vis spectrophotometer from Scinco ™ Co., LTD. Analysis of the data was performed on the associated LabProPlus ™ software. DLS and zeta potential measurements were taken on a Zetasizer Nano ZS analyzer with an integrated 4 mW He-Ne laser, λ = 633 nm (Malvern Instruments ™ Ltd, UK). DLS measurements were taken either in ultrapure water or 10 mM phosphate buffer at pH = 7.4. All zeta potential measurements were taken in a 10 mM phosphate buffer at pH = 7.4.

TEM transmission electron microscopy was recorded on a FEI ™ CM-12 with an accelerating voltage of 100 kV. The data were analyzed with Jlmage vi .43.

 Competitive surface plasmon resonance measurements for the determination of selectin binding were performed on a Biacore ™ X instrument.

Example 1: Preparation of TA-dPGS 10,000 g / mol:

 The functionalization of dPGS with a molecular weight of 10,000 g / mol with lipoic acid NHS was as follows. dPGS 10,000 g / mol (500 mg, 0.05 mmol) was dissolved in a mixture of dimethylformamide (15 mL) and distilled water (13 mL). Subsequently, lipoic acid-NHS (15.15 mg, 0.05 mmol) and DIPEA (0.017 mL, 0.1 mmol) were added and the reaction mixture was stirred for 3 days at room temperature. The solvent was removed in vacuo and the crude product was dialyzed first against methanol, then with sodium chloride solution and then with distilled water. After freeze-drying, the product was obtained as a colorless powder (491 mg, 95%).

¹ H-NMR (400 MHz, D 2 O): δ (ppm) 4.64-3.00 (m, 331 H, PG backbone, SS-CH 2, S-S-CH 2, SS-CH-), 2.38 (m, 1 H, S-CH 2 -CH 2 -), 2.19 (m, 2 H, CH 2 CO-), 1.9 (m, 1 H, S-CH 2 -CH 2 -), 1.52 - 1.52 (m, 10 H, CH 2 -CH 2 - CH2-CH2CO-, -CH2- initiator), 0.78 (m, 3 H, CH3 initiator). ¹³ C NMR (400 MHz, D20): δ (ppm) 80-65 (PG backbone).

Example 2 Preparation of Citrate-Stabilized Gold Nanoparticles with a Diameter of 17 nm:

The preparation of citrate-stabilized gold nanoparticles with a z-average diameter of 17 nm was prepared by a slightly modified protocol from Lou et al. Wang, C; He, L. biomacromolecules., 2007, 8, 1385-1390.) The glassware and the stir bar were washed with aqua regia, purified with Millipore and then dried at 120 ° C. HAuCl 3H 2 O (34.42 mg, 0.087 mmol) in ultrapure water (340 mL) was heated at reflux for 20 minutes at 110 ° C. Subsequently, a trisodium citrate solution (187 mg, 0.524 mmol) in ultrapure water (20 mL) was added to the solution. After 15 minutes, a color change from yellow to deep red occurred. The solution was cooled to room temperature and filtered through a cellulose filter (0.22 pm). The resulting gold colloid suspension was stored at 4 ° C in the dark. Particle analysis via dynamic light scattering in ultrapure water gave a z-average diameter of 17.17 nm with a polydispersity index of 0.06. The Feret diameter determined by the automatic counting of electron microscopy images at an accelerating voltage of 100 kV using the program Jlmage gave a value of 18 nm ± 2.8 nm and confirmed the diameter of the particles. UV-Vis measurement of the particles revealed a plasmon absorption maximum at 520 nanometers. The concentration of the gold nanoparticles was then calculated on the assumption of a 100% gold acid conversion and the geometric factors of the nanoparticles. The resulting concentration of 1.39 nanomolar was in very good agreement with the theory described in (Haiss, W .; Thanh, N., Aveyard, J, Fernig, D., Anal. Chem., 2007, 79, 4215-4221.) ,

Example 3: Preparation of AuNP-TA-dPGS at 5,300 g / mol

For the ligand exchange, a suspension of 17 nm citrated-stabilized gold nanoparticles (20 mL, 1.39E-9 mol / l, 2.79E-1 1 mol with TA-dPGS 5.3 kDa (4.3 mg) (synthesized according to Scheme 5 (FIG. 6)) were incubated overnight at room temperature with stirring and then purified by dialysis against H ₂ O. The amount of TA-dPGS used corresponded to 2 equivalents of TA-dPGS per gold surface atom on the particle, since 15 nm particles according to Mei et al Susumu, K., Farrell, D., Mountziaris, TJ, Mattoussi, H. langmuir., 2009, 25, 10604-1061 1) have approximately 10,000 gold atoms on the surface For this reason, 28,000 equivalents of TA-dPGS per gold nanoparticle were used, giving complete particle occupancy, and after incubation overnight and purification by dialysis against water could be carried out using Dynamic Light scattering in ultrapure water a z-av diameter of 22.68 nm at a polydispersity index of 0.18. A redshift of the plasmon absorption maximum from 520 nanometers to 523 nanometers confirmed the functionalization.

Example 4: Preparation of AuNP-TA-dPGS at 10,000 g / mol

The preparation is analogous to the synthesis in Example 3, except that 7.81 mg of TA-dPGS 10,000 g / mol were used here in order to achieve 28,000 equivalents dPGS 10,000 g / mol per gold nanoparticle. After incubation overnight and purification by dialysis against water, a z-average diameter of 22.90 nm with a polydispersity index of 0.18 could be measured by dynamic light scattering in ultrapure water. Example 5: Synthesis of AuNR with a length of 40 nm and a

Diameter of 10 nm:

 Rod-shaped nanoparticles with a gold core (AuNR) and a shell of dendritic PGS with molecular weights of 5,300 g / mol, and 10,000 g / mol and a degree of sulfation of 85%, respectively, were prepared using lipoic acid (TA) as

Linker prepared as follows:

The synthesis was based on the instructions of Jana Nikhil (N., small, 2005, 8, 875-882). All glassware was first thoroughly cleaned with aqua regia and ultrapure water. HAuCI4-3H ₂ 0 (98 mg, 0:25 mmol) was dissolved in ultrapure water (260 mL). Subsequently, CTAB (18.2 g, 50 mmol), silver nitrate (8.5 mg, 0.05 mmol) and ascorbic acid (88 mg, 0.5 mmol) were added to the reaction mixture. A change of color from slightly yellowish to reddish to colorless occurred. Next, NaBH ₄ (9.5 pg, 2.5E-7 mol) was added to the reaction mixture with vigorous stirring. The reaction solution turned deep purple to purple after 15 minutes and the excess of CTAB was removed by centrifuging twice. The purified CTAB-functionalized gold nanorods were stored at 4 ° C in the dark. Zeta potential measurements by dynamic light scattering in a 10 mM phosphate buffer at pH 7.4 gave a zeta potential of +30 mV and thus confirmed the positively charged CTAB envelope. The proportions of the particles and distribution were determined by electron micrographs at an acceleration voltage of 100 kV and gave a particle length of 40 nm and a diameter of 10 nm. UV-Vis spectra were used to investigate the optical properties of the particles and their dispersion and gave a transversal plasmon absorption maximum of 515 nm and a longitudinal plasmon absorption maximum of 788 nm.

Example 6: mPEG-SH 1000 Functionalization of AuNR-CTAB

 The functionalization of AuNR-CTAB with mPEG-SH 1000 was based on the protocol of Xiaoge Hu and Xiaohu Gao (X. Hu, X. Gao, Phys. Chem. Chem.

Phys., 201 1, 13, 10028-10035). mPEG-SH 1000 (0.134 g, 133 mmol, 1.24E5 eq.) was added to a CTAB-functionalized gold nanorod suspension (4 mL, 2.7E-7 mol / l, 1.08E-9 mol) and for 24 hours at room temperature touched. The 1.24E5 equivalents used are based on an observation by Sanda C. Boca as described in (S. Boca, Nanotechnology, 2010, 21, 235601). Subsequently, the particles were washed by centrifuging twice with ultrapure water. The successful mPEG-SH functionalization was confirmed by UV-Vis spectroscopy using the red shift of 4 nm and the neutral zeta potential in 10 mM phosphate buffer at pH 7.4 using dynamic light scattering. Example 7: TA-dPGS 10,000 g / mol functionalization of AuNR-PEG

 TA-dPGS 10,000 g / mol (20 mg, 2E-6 mol) was added to an AuNR-mPEG-SH 1000 suspension (0.2 ml, 7.23E-8 mol / l, 1.44E-6 mol) and added for 12 hours Stirred 60 ° C. Subsequently, the particles were washed five times by centrifugation with ultrapure water. The successful functionalization was confirmed by the negative zeta potential by means of dynamic light scattering in a 10 mM phosphate buffer at pH = 7.4.

Example 8: Measurement of the binding affinity of AuNP-TA-dPGS 10,000 g / mol and AuNP-TA-dPGS 5,300 g / mol:

To quantify the binding affinity of the produced AuNP-TA-DPGS and AuNR-TA-DPGS to L- selectin, which were IC ₅ o values by surface plasmon resonance spectroscopy (SPR), determined in a competitive binding assay. An exact implementation of the method is described in (Enders S., Bernhard G., Zakrzewicz A., Tauber R., Biochim Biophys Acta, 2007, 10, 1441-1449).

The IC 50 value indicates that concentration of a substance at which a half-maximal inhibition is observed. It was measured as follows.

First, the binding of L-selectin modified gold nanoparticles to a chip surface immobilized synthetic selectin ligand (sLeX, 20 mol% + sulfo-tyrosine 5 mol% coupled on linear polyacrylamide) was measured as a positive control and normalized to 100% binding , The degree of binding was measured in Resonance Units (RU). Subsequently, the selectin functionalized

Gold nanoparticles were pre-incubated with AuNP-TA-dPGS as a binding enhancer in increasing concentration and the relative decrease in binding was measured compared to the control. Finally, the IC ₅₀ values were determined by calculating the concentration of AuNP-TA-DPGS, this leads to 50% of control binding signal.

From the views of the SPR sensorgram for the AuNP-TA-dPGS 10,000 g / mol and AuNP-TA-dPGS 5.300 g / mol (FIG. 10, FIG. 1 and FIG. 2, FIG. 2), the IC ₅₀ Values are calculated. The IC ₅₀ values indicate the point of the curve at 50 percent relative binding of the control.

Here, the 5,300 g / mol and the 10,000 g / mol AuNP-TA-dPGS particles gave IC ₅₀ values of 210 fM, or of 180 fM, which are surprisingly low. For the AuNR-TA-dPGS 10,000 g / mol an IC _S0 value of 136 pM resulted. This value is very low compared to the nanomolar IC ₅₀ value of dPGS (Dernedde

J., Rausch A., Weinhart M., Enders S. Tauber R., Licha K., Schirner M. Reins U. Von Bonin A. Haag R., Proc. Nat. Aca. Be. USA 2010, 107, 19679-19684). The particles can therefore be used to target high affinity selectins and thus provide the basis of the TA-dPGS functionalized nanoparticles or nanorods of the invention as a contrast agent and as an anti-inflammatory agent.

Claims

"Claims" 1. Nanoparticles, characterized by  a) a core of an inorganic material and  b) a shell of linker and dendritic polyglycerol sulfate, wherein the polyglycerol sulphate consists of repeating glycerol units of the formula (RO-CH ₂ ) ₂ CH-OR, where R = H, or other glycerol units, one or more OH groups of the glycerol units by sulfate groups of the formula -OSO ₃ X in which X is replaced by H, an alkali metal atom such as Li, Na or K, or an ammonium ion,  and wherein the linker links polyglycerol sulfate and core together. 2. Nanoparticles according to claim 1, characterized in that the degree of sulfation of the dendritic Polyglycerolsulfats 1-100%, preferably 50- 99 %%, and particularly preferably 80-99%. 3. nanoparticles according to claim 1 or 2, characterized in that the dendritic polyglycerol has an average molecular weight of 100 to 1,000,000 g / mol, preferably 2,500 to 20,000 g / mol and more preferably from 5,000 to 15,000 g / mol. 4. nanoparticles according to one or more of claims 1-3, characterized in that the inorganic core consists of gold or iron oxide, preferably of gold. 5. Nanoparticles according to one or more of claims 1-4, characterized in that the linker represents a C ₁ -C ₂₀ hydrocarbon radical which is branched or linear and is optionally interrupted by -NH-, -O-, -S- , - (C = O) - NH-, - (C = O) -O-, -N- (C = O) -N -, - N- (C = S) -N-, -N- (C = 0) -0-, -N- (C = S) -O- or -triazole-, and the alpha-terminal 1, 2 or 3 thio groups or phosphonate groups and omega-terminal one or more reactive groups selected from the group - (C = O) -OH, C- (C = O) -R, with R = N-hydroxysuccinimide or Cl, -N = C = S, -N ₃ , or -alkyne, preferably lipoic acid , 6. nanoparticles according to one or more of claims 1-5, characterized in that the degree of branching of the dendritic Polyglycerolsulfats 1-100%, preferably 30-80% and particularly preferably 55-65%. Nanoparticles according to one or more of claims 1-6, characterized in that the nanoparticles have a diameter of 10-50 nm. 8. Use of nanoparticles according to one or more of claims 1-7 as a selectin inhibitor. 9. Use of nanoparticles according to one or more of claims 1-7 as a carrier for active substance substances. 10. Conjugate containing nanoparticles according to one or more of claims 1-7 and one or more active substances or proteins. 11. Use of nanoparticles according to one or more of claims 1-7 or conjugates according to claim 10 as a contrast agent. 12. Use of nanoparticles according to one or more of claims 1-7 or of conjugates according to claim 10 for the diagnosis or for the treatment of inflammatory processes.