WO2012156094A1 - Nanoparticules fonctionnalisées avec du polyglycérolsulfate dendritique - Google Patents

Nanoparticules fonctionnalisées avec du polyglycérolsulfate dendritique Download PDF

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WO2012156094A1
WO2012156094A1 PCT/EP2012/002136 EP2012002136W WO2012156094A1 WO 2012156094 A1 WO2012156094 A1 WO 2012156094A1 EP 2012002136 W EP2012002136 W EP 2012002136W WO 2012156094 A1 WO2012156094 A1 WO 2012156094A1
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nanoparticles
dpgs
nanoparticles according
mol
linker
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Rainer Haag
Jonathan VONNEMANN
Jens Dernedde
Marie Weinhart
Dominic GRÖGER
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Charite Universitaetsmedizin Berlin
Freie Universitaet Berlin
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Charite Universitaetsmedizin Berlin
Freie Universitaet Berlin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/04—X-ray contrast preparations
    • A61K49/0409—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is not a halogenated organic compound
    • A61K49/0414—Particles, beads, capsules or spheres
    • A61K49/0423—Nanoparticles, nanobeads, nanospheres, nanocapsules, i.e. having a size or diameter smaller than 1 micrometer
    • A61K49/0428—Surface-modified nanoparticles, e.g. immuno-nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • 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.
  • the acute inflammatory response is thus the body's first alarm signal designed to contain and eventually eliminate proinflammatory stimuli.
  • 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 cytokines.
  • 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.
  • 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)
  • leukocytes The initial contact of leukocytes with the endothelial cells takes place by interaction of L-selectin carbohydrate ligands of the endothelial cells.
  • 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.
  • 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
  • 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.
  • nanoparticles having an inorganic core and a shell of linker and dendritic Polygycerolsulfaten (dPGS) according to the features of the claims.
  • dPGS dendritic Polygycerolsulfaten
  • the nanoparticles comprise
  • 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). :
  • 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.
  • 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.
  • the degree of branching of the polyglycerol can be adjusted as desired.
  • the degree of branching is 1-100%.
  • the degree of branching is from 30-80%, and more preferably from 55-65%.
  • 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
  • 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
  • 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, Schok 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%.
  • 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.
  • 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.
  • the core of the nanoparticles according to the invention has a diameter of 5-45 nm, preferably 10-30 nm.
  • the linkers may be covalently or coordinately attached to the nucleus via the terminal thio groups or phosphonate groups.
  • 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
  • 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.
  • 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.
  • thio groups such as thiol groups or sulfur-containing heterocycles.
  • 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.
  • 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.
  • 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.
  • the linker generally forms an inner layer around the inorganic core around which there is an outer layer of dPGS.
  • the linker has a length of 0.5-10 nm, preferably 1-5 nm.
  • 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.
  • the nanoparticles according to the invention have a very high affinity for selectins such as L-selectin.
  • 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.
  • conjugates of the nanoparticles according to the invention with active substances are likewise the subject matter of the present invention.
  • substances can be transported very selectively into inflamed tissue.
  • nanoparticles according to the invention are 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.
  • MSOT Multispectral Optoacoustic Tomography
  • 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.
  • 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.
  • 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
  • IFN cytokinin y
  • macrophages which in turn produce hydrolytic enzymes and reactive oxygen and nitrogen species, resulting in the destruction of surrounding tissue.
  • TNFa is released, causing the
  • 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.
  • 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.
  • diagnostics e.g. as a contrast agent for use in imaging techniques such as MRI and MSOT.
  • 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.
  • 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).
  • MSOT Multispectral Opto
  • 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.
  • dyes in particular fluorophores such as e.g. Aminomethylcoumarin, fluorescein, cyanine, rhodamine and their derivatives can be used.
  • 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.
  • conjugates with signaling molecules are:
  • 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 ).
  • 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.
  • chemokines are, for example, proinflammatory cytokines, in particular TNF ⁇ , IL-1, IL-6, as well as IL-8 and MIP-1 ⁇ .
  • proinflammatory cytokines in particular TNF ⁇ , IL-1, IL-6, as well as IL-8 and MIP-1 ⁇ .
  • 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.
  • the nanoparticles according to the invention can preferably also be used in in vitro applications.
  • 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.
  • 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.
  • AuNP gold nanoparticles
  • DIPEA diisopropylethylamine
  • mPEG-SH 1000 methoxy-terminated polyethylene glycol thiol with a
  • CTAB cetyltrimethylammonium bromide
  • MSOT Multispectral Optoacoustic Tomography
  • 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 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 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 Reaction scheme for coupling a linker such as a lipoic acid linker to dPGS; Functionalization of dPGS with a lipoic acid linker.
  • FIG. 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 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 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
  • the linker 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, Mosk 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.
  • the dPGS is coupled to the core particles using the linker.
  • the linker 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).
  • 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.
  • 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.
  • 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.
  • the drugs or signaling molecules can be covalently linked to the TA-dPGS.
  • 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.
  • 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.
  • 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
  • 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;
  • LPS mouse lipopolysaccharide
  • 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
  • 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.
  • an allergic contact dermatitis model e.g. be demonstrated by an allergic contact dermatitis model.
  • TMA trimellitic anhydride
  • 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:
  • UV / Vis absorption spectra were recorded on a UV / Vis spectrophotometer from Scinco TM Co., LTD. Analysis of the data was performed on the associated LabProPlus TM software.
  • 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
  • a mixture of dimethylformamide (15 mL) and distilled water 13 mL
  • lipoic acid-NHS 15.15 mg, 0.05 mmol
  • DIPEA 0.017 mL, 0.1 mmol
  • 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%).
  • 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
  • ultrapure water 340 mL
  • a trisodium citrate solution (187 mg, 0.524 mmol) in ultrapure water (20 mL) was added to the solution.
  • 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.) ,
  • 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.
  • Example 5 Synthesis of AuNR with a length of 40 nm and a
  • 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.
  • 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.
  • 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.
  • Example 8 Measurement of the binding affinity of AuNP-TA-dPGS 10,000 g / mol and AuNP-TA-dPGS 5,300 g / mol:
  • the IC 50 value indicates that concentration of a substance at which a half-maximal inhibition is observed. It was measured as follows.
  • 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.
  • the IC 50 Values are calculated.
  • the IC 50 values indicate the point of the curve at 50 percent relative binding of the control.
  • 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.
  • IC S0 value of 136 pM resulted. This value is very low compared to the nanomolar IC 50 value of dPGS (Dernedde
  • 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.

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Abstract

L'invention concerne des nanoparticules caractérisées par a) un noyau composé de matériau inorganique et b) une enveloppe composée de coupleurs et de polyglycérolsulfate dendritique, le polyglycérolsulfate étant constitué d'unités glycérine de formule (RO-CH2)2CH-OR qui se répètent, avec R = H, ou d'autres unités glycérine, un ou plusieurs groupes OH des unités glycérine étant remplacés par des groupes sulfate de formule -OSO3X, X étant H, un atome de métal alcalin tel que Li, Na ou K, ou un ion ammonium, et le coupleur reliant le polyglycérolsulfate au noyau. Les nanoparticules servent à des applications médicales, par ex. au traitement et au diagnostic de maladies inflammatoires.
PCT/EP2012/002136 2011-05-18 2012-05-18 Nanoparticules fonctionnalisées avec du polyglycérolsulfate dendritique Ceased WO2012156094A1 (fr)

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CN107457412A (zh) * 2017-08-03 2017-12-12 吉林大学 一种高稳定的金纳米花制备方法
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Cited By (13)

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Publication number Priority date Publication date Assignee Title
CN103226103A (zh) * 2013-04-04 2013-07-31 哈尔滨师范大学 一种汞离子比色检测探针及其应用方法
US11826438B2 (en) 2014-05-09 2023-11-28 Yale University Hyperbranched polyglycerol-coated particles and methods of making and using thereof
EP3140269A4 (fr) * 2014-05-09 2018-01-03 Yale University Particules enrobées dans un polyglycérol hyper-ramifié, leurs procédés de production et d'utilisation
EP3139909A4 (fr) * 2014-05-09 2018-01-03 Yale University Formulation topique de particules enrobées de polyglycérol hyperramifié
US10272019B2 (en) 2014-05-09 2019-04-30 Yale University Topical formulation of hyperbranched polyglycerol-coated particles thereof
US10758459B2 (en) 2014-05-09 2020-09-01 Yale University Topical formulation of hyperbranched polyglycerol-coated particles thereof
EP3721875A1 (fr) * 2014-05-09 2020-10-14 Yale University, Inc. Particules revêtues de polyglycérol hyperramifié et leurs procédés de fabrication et d'utilisation
US11896686B2 (en) 2014-05-09 2024-02-13 Yale University Hyperbranched polyglycerol-coated particles and methods of making and using thereof
US11918695B2 (en) 2014-05-09 2024-03-05 Yale University Topical formulation of hyperbranched polymer-coated particles
EP4331618A3 (fr) * 2014-05-09 2024-06-12 Yale University Particules enrobées de polyglycérol hyperramifié et leurs procédés de fabrication et d'utilisation
US10525141B2 (en) 2015-04-15 2020-01-07 Freie Universität Berlin Polyglycerol derivative and a method for manufacturing the same
CN105223347A (zh) * 2015-09-22 2016-01-06 福州大学 一种半定量可视化酶联免疫分析方法
CN107457412A (zh) * 2017-08-03 2017-12-12 吉林大学 一种高稳定的金纳米花制备方法

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