Toxin-binder for hemodialysis

Abstract

The invention relates to new toxin-binders for hemodialysis. The invention relates to an apoferritin nanoparticle for use as a toxin-binder in hemodialysis. The apoferritin nanoparticle comprises apoferritin subunits that, when combined to the apoferritin nanoparticle, form an inner cavity, and the apoferritin nanoparticle does not contain any nanoparticles in its inner cavity. The invention also relates to a composition, a sorption cartridge and a dialysis system.

Description

[0001] TOXIN BINDING AGENT FOR HEMODIALYSIS

[0002] The invention relates to novel toxin binders for hemodialysis.

[0003] Approximately 9% of the world's population suffers from chronic kidney disease (CKD) in various severe forms [1], As the disease progresses, those affected have a significantly higher susceptibility to cardiovascular diseases [2], In 2017, 1.2 million people died from the direct consequences of CKD and another 1.4 million people died from cardiovascular diseases attributable to CKD [1], The high cardiovascular risk could be attributed to various uremic toxins such as indoxyl sulfate (IS) and para-cresyl sulfate (PCS)

[0003] -

[0010] . These toxins can only be separated from the plasma to a limited extent, even with prolonged or more frequent treatments within the framework of conventional hemodialysis

[0011] , Due to their partially hydrophobic nature, the toxins bind to plasma proteins (e.g.Serum albumin), which prevents efficient diffusion through the pores of the dialysis membranes [12, 13]. Therefore, they are also referred to as protein-bound uremic toxins (PBUT). On average, 95% of IS and pCS are protein-bound

[0014] , which explains their low separation efficiency in hemodialysis procedures.

[0004] One way to improve the separation of PBUTs is the addition of oral or intravenous supplements. Intravenous supplements are intended to displace the toxins from the binding sites of plasma proteins, thus increasing their soluble and dialysis-separable fraction. This can be achieved, for example, with ibuprofen ((α5)-2-(4-isobutylphenyl)propionic acid)

[0015] , mesna (sodium 2-sulfanylethanesulfonate)

[0016] , or acetylcysteine ​​

[0017] . However, the effective separation of pCS and IS has only been investigated with the addition of ibuprofen. Oral supplements such as the carbon-based AST-120 (Kremezin®) adsorb the toxins produced by microbial processes in the intestine before they are absorbed into the bloodstream

[0018] or inhibit the production of corresponding toxins, such as the synbiotic NatuREN G

[0019] . In a systematic comparison of all studies on the reduction of protein-bound uremic toxins up to January 2nd.In 2020

[0020] , the above-mentioned methods showed a reduction rate for the toxins IS and pCS of up to 44%

[0018] . However, significantly higher reduction rates of between 71 and 78% were achieved with a combination of fractionated plasma separation, adsorption, and dialysis (FPAD) [20, 21]. This shows that direct adsorption of protein-bound toxins is one of the most effective methods for toxin reduction in patient plasma. The disadvantage of this system, however, is the complex plasma separation and filtration required to separate albumin from the remaining blood components, which must not come into contact with the adsorption materials

[0022] , making routine use very costly.

[0005] There is still a lack of effective adsorption materials for PBUTs that possess sufficient biocompatibility and hemocompatibility to allow them to be brought into direct contact with the blood and thus more easily integrated into conventional hemodialysis treatments. Existing PBUT adsorption agents lack a combination of a) high affinity for the toxins and b) suitable hemocompatibility and biocompatibility. The state of the art only includes studies on common adsorption materials such as activated carbon

[0023] , metal-organic frameworks

[0024] , or zeolites

[0025] , although these studies primarily address the affinity of these materials to uremic toxins. Only a few studies also consider the biocompatibility of the materials

[0023] .

[0006] DE 10 2016 108 017 A1 describes loaded protein containers for constructing nanostructured materials. The protein containers are formed from ferritin complexes, within which nanoparticles can be enclosed. Ferritin complexes containing such nanoparticles are said to be suitable for use, among other things, in dialysis for the removal of hydrophobic toxins. However, no corresponding experiments have been described. WO 2004 / 001019 A1 describes nanoparticles comprising protein cages, for example, bacterial ferritin-like protein cages, which are loaded with at least one guest material, for example, metals.The object of the present invention is to provide an adsorbent for the separation of protein-bound uremic toxins, PBUTs, during dialysis, which has the highest possible affinity to PBUTs, but at the same time has sufficient bio- and hemocompatibility to be brought into direct contact with the blood.

[0007] To achieve this object, the present invention provides, in a first aspect, an apoferritin nanoparticle for use as a toxin binding agent in hemodialysis, wherein the apoferritin nanoparticle comprises apoferritin subunits which, when assembled to form the apoferritin nanoparticle, form an internal cavity, and wherein the apoferritin nanoparticle does not contain any nanoparticles in its internal cavity.

[0008] It has surprisingly been found that apoferritin nanoparticles are well suited for use as adsorbents for toxins in the blood, for example, for the separation of protein-bound uremic toxins (PBUTs). The apoferritin nanoparticles according to the invention have an internal cavity (hereinafter also referred to as a cavity) in which toxins can be bound, and furthermore exhibit high biocompatibility and hemocompatibility. This makes them very well suited for removing toxins, for example PBUTs or other toxins such as heavy metals, from the blood during dialysis, for example hemodialysis. They can therefore be advantageously used, for example, for the treatment of chronic kidney disease (CKD) and / or for alleviating the consequences of chronic kidney disease (CKD). The protein shell of the apoferritin nanoparticles according to the invention has channels orPores through which small molecules such as PBUTs can penetrate into the interior of the particle, while larger macromolecules such as proteins or cells are retained. The affinity of the ferritin nanoparticles according to the invention for the toxins to be bound can be specifically adjusted and optimized, for example by altering the amino acid sequence of at least one of the subunits in the region of the inner cavity so that, for example, a suitable number of hydrophobic amino acid residues or a suitable mixture of hydrophobic and hydrophilic amino acid residues is present, or by functionalizing one or more amino acid residues with chemical groups. Furthermore, the mass transport in the ferritin nanoparticles can be improved by enlarging or changing the surface charge in the region of the pores, for example by deliberately exchanging sterically demanding or charged amino acids with smaller hydrophobic amino acids.The inner surface and pore area of ​​the nanoparticles (protein cages) can thus be altered through sequence modification or chemical functionalization to increase affinity for toxins and / or facilitate the transport of toxins into the nanoparticles without compromising assembly or biocompatibility. Furthermore, the protein cages can be assembled into a well-defined crystalline material with uniformly distributed solvent channels. This ensures high material purity, facilitates handling and use as sorption materials, and is also helpful for material characterization.

[0009] "Ferritin" (abbreviated "Ftn") is a globular protein complex (spheroidal protein) composed of 24 protein subunits forming a hollow nanocage, which naturally binds iron. The terms "apoferritin" or "apoferritin nanoparticles" refer to the non-iron-bound protein complex. The protein complex has an inner cavity of approximately 6-8 nm in diameter, while the outer diameter is approximately 12-13 nm (see, for example,

[0028] ,

[0029] ). Apoferritin can be composed of identical subunits, for example, exclusively of the so-called heavy chain (H chain), or of varying proportions of a light chain (L chain) and a heavy chain (H chain). The H chain (abbreviated hFTN-H, HFt or FTH) of human ferritin is encoded by the FTHl gene and is a naturally occurring protein of approximately 21 kDa and 183 amino acids (see UniProtKB No. P02794, 2007-01-23 v2; NCBI NP 002023.2).The L-light chain (abbreviated hFTN-L, LFt, or FTL) of human ferritin is encoded by the FTL gene and is a naturally occurring protein of approximately 19.5 kDa and 175 amino acids (see UniProtKB No. P02792, 2007-01-23 v2; NCBI NP_000137.2). Both the light chain and the heavy chain exhibit a tertiary structure with a bundle of four alpha helices (A, B, C, and D), with the B and C helixes connected by an 18-amino acid non-helical loop (BC-loop). In addition, a fifth alpha helix (E) is present at the C-terminus, which extends into the cavity. The N-terminus and the A and C helixes are located on the outside of the ferritin, while the B and D helixes are directed toward the interior of the ferritin. Ferritin has channels or pores in its protein shell that connect the inner cavity with the external environment (

[0028] ).Triple channels (triple axis channels) are formed at locations where three apoferritin subunits collide due to the spatial arrangement of the apoferritin subunits, and fourfold channels (quadruple axis channels) are formed at locations where four apoferritin subunits collide (see, for example,

[0028] ). An apoferritin nanoparticle composed of 24 subunits typically comprises eight triple channels and six quadruple channels (see, for example,

[0028] ).

[0010] The terms "human H-chain ferritin" or "human heavy chain ferritin," abbreviated HuHF, refer to a human ferritin whose subunits consist exclusively of the human heavy chain, and not a mixture of a heavier (H-chain) and lighter (L-chain) chain. The term also includes variants with H chains whose amino acid sequences are modified compared to the wild-type H-chain sequence (see UniProtKB No. P02794, 2007-01-23 v2; NCBI NP_002023.2 for the amino acid sequence of the human ferritin heavy chain).

[0011] The term "toxin binder" in relation to apoferritin nanoparticles means that the apoferritin nanoparticles bind a toxin in their hollow space, i.e., the inner cavity, so that it can be removed from, for example, a body fluid, e.g., blood, while bound to the apoferritin nanoparticle. The bond between the toxin and the apoferritin nanoparticle is preferably non-covalent and can occur, for example, through hydrophobic interactions, electrostatic interactions, or hydrogen bonds. Possible toxins include, for example, protein-bound uremic toxins such as indoxyl sulfate (IS) and para-cresyl sulfate (pCS), organic heavy metal compounds, heavy metal ions, and other chemical substances and compounds that have properties that allow binding preferably via non-covalent bonding. However, covalent bonding is not excluded.The terms “protein-bound uremic toxins” or “protein-bound uremic toxins”, PBUTs, refer to generally poorly water-soluble (hydrophobic) chemical compounds that are present in serum bound to protein. Examples of PBUTs are indoxyl sulfate (IS), para-cresyl sulfate (p-cresyl sulfate, also p-cresyl sulfate, pCS), phenyl acetate (PheAc, PhAc), p-hydroxyhippuric acid, kynurenine, kynurenic acid, indole-3-acetic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) and p-cresyl glucuronide (see e.g.

[0037] ; “Uremic Solutes Database”, “List of uremic solutes”, https: / / database. uremic-toxins. org / soluteList.php?sortkey=class&direction=asc, accessed on 25.07.2022, which lists 33 PBUTs).

[0012] "Hemodialysis" refers to the extracorporeal treatment of blood in which metabolic waste products (e.g., urea, uric acid, creatinine, excess phosphate, etc.) and / or excess fluid are removed from the blood. Hemodialysis is regularly used when a person's kidneys are no longer sufficiently functional, for example, in the case of chronic kidney disease (CKD). The term "for use in hemodialysis" includes use in hemapheresis, particularly whole blood apheresis. "Hemapheresis" refers to a procedure for the extracorporeal removal of certain blood components or pathogenic substances, microorganisms, or the like from blood (see, for example, "Standard of Therapeutic Apheresis 2019, March 29, 2019, German Society of Nephrology).

[0013] The term "chronic kidney disease (CKD)" refers to a chronic kidney disease characterized by a gradual and prolonged (>3 months) impairment of kidney function, leading to permanent kidney failure or complete kidney failure (ICD-10 code N18). The term specifically refers to stage 5 chronic kidney disease (ICD-10 code N18.5), in which a glomerular filtration rate (GFR) of <15 mL / min requires renal replacement therapy for blood purification. The term "chronic kidney failure" is used here as a synonym for "chronic kidney disease" where appropriate.

[0014] The expression that the apoferritin nanoparticle “does not contain any nanoparticles in its internal cavity” means that the internal cavity of the apoferritin nanoparticles is empty, i.e. it does not contain any separate nanoparticles designed, for example, to bind toxins.

[0015] The term "in the region of the inner cavity" with reference to an apoferritin subunit refers here to the part of the apoferritin subunit, specifically the amino acid residues of this apoferritin subunit, that faces the inner cavity of the apoferritin nanoparticle. These are primarily the B helix (amino acids 50-77 in the human apoferritin heavy chain), the D helix (amino acids 128-159 in the human apoferritin heavy chain), the E helix (amino acids 165-174 in the human apoferritin heavy chain), and the C-terminal region (175-183) following the E helix toward the C-terminus.

[0016] The term "in the region of a triple channel" with reference to an apoferritin subunit here means the part of the apoferritin subunit, in particular the amino acid residues of this apoferritin subunit, which in an assembled apoferritin nanoparticle are located at a triple channel of the apoferritin nanoparticle, or amino acid residues that have a direct influence on the transport of a toxin into the interior of the apoferritin nanoparticle, i.e., whose exchange leads to a facilitation of toxin transport into the interior of the apoferritin nanoparticle. These can, for example, be the amino acids at the end of the C-helix and the beginning of the D-helix (in the heavy chain of human apoferritin, for example, amino acids 118 to 141, preferably 120-140). The term also includes amino acids on the outside of the apoferritin subunit."Facilitated toxin transport" (or "enhanced toxin transport") here means that, when incubated with a given toxin concentration under suitable conditions within a given period of time, a larger number of toxin molecules enter the cavity of an apoferritin nanoparticle modified in the triple channel region compared to an apoferritin nanoparticle not modified in the triple channel region. Instead of the term "triple channel," the term "pore" may also be used synonymously here.

[0017] The term "in the region of a quadruple channel" with reference to an apoferritin subunit here means the part of the apoferritin subunit, in particular the amino acid residues of this apoferritin subunit, which are located on a quadruple channel of the apoferritin nanoparticle. For example, the amino acids M 158, L 165, Y 168, L 169, and H 173 are located in the region of the quadruple channel (see, for example,

[0041] ). The term includes amino acid residues of the respective subunit that are located in an assembled apoferritin nanoparticle on a quadruple channel of the apoferritin nanoparticle, for example, lining the quadruple channel in an assembled apoferritin nanoparticle or located at the openings of the channel, or amino acid residues that have a direct influence on the shape, for example, the diameter, or the physicochemical properties of the channel.

[0018] The terms "redesigned apoferritin nanoparticle" or "redesigned apoferritin subunit" are used herein to refer to an apoferritin nanoparticle or apoferritin subunit with an amino acid sequence altered from the wild-type sequence. This term includes an apoferritin nanoparticle or apoferritin subunit with an altered amino acid sequence in which some or all of the exchanged amino acid residues are additionally functionalized with chemical groups.

[0019] The term "functionalized" means that an additional chemical group or linking residue is covalently bonded to a suitable amino acid residue, such as cysteine. An example is a cysteine ​​residue to which an N-phenylacetamide residue is bonded via its thiol group (SH group).

[0020] The term "organic compound residue" refers to carbon-containing chemical side groups or molecules that are covalently bonded, for example to a cysteine ​​residue. It can be an aliphatic or aromatic compound residue, whereby the terms here are not restricted to pure hydrocarbon compounds, but also include heteroaliphatic or heteroaromatic compound residues, i.e. compound residues with heteroatoms such as nitrogen, oxygen, phosphorus or sulfur. An "aromatic compound residue" is understood to mean any compound residue that has an aromatic or heteroaromatic moiety. An "aliphatic compound residue" is understood to mean any aliphatic or heteroaliphatic compound residue, i.e. any organic compound residue that does not contain an aromatic group.

[0021] The term "aliphatic compound residue" encompasses cyclic or acyclic, linear (straight-chain) or branched, saturated or unsaturated carbon compound residues that are not aromatic residues. The term "heteroaliphatic residue" refers to aliphatic residues in whose carbon skeleton one or more carbon atoms are replaced by heteroatoms, for example, oxygen, sulfur, nitrogen, or phosphorus.

[0022] The term "aromatic compound residue" (also called "aryl") refers to chemical groups with aromaticity, including multi-membered single-ring aromatic groups and multicyclic systems with at least one aromatic ring. Examples of aromatic compound residues (aryl groups) include benzyl and phenyl. The term "heteroaromatic compound residue" (also "heteroaryl") refers to aromatic compound residues in whose carbon skeleton one or more C atoms are replaced by heteroatoms, for example oxygen, sulfur, nitrogen, or phosphorus.

[0023] The term "dialyzer" refers to a replaceable blood purification unit of a dialysis system. A dialyzer can, for example, be a hollow-fiber membrane module in which biocompatible semipermeable polymer membranes in the form of hollow fibers are arranged in a housing. This allows, for example, blood to flow through the hollow fibers and a dialysate to flow through the interior of the housing along the outer sides of the hollow fibers (for example, countercurrent to the blood flow). Preferably, low-molecular-weight compounds such as salts, protein, or nucleic acid fragments can diffuse from the blood across the membrane into the dialysate and thus be transported away.

[0024] The specification of a range such as "1-10" is to be understood as also disclosing every intermediate value. For a specification that can only refer to whole numbers, such as a number of atoms, this also means that only whole numbers are disclosed. Any narrower range from a broader range is also disclosed by specifying the broader range, whereby the narrower range also includes ranges that do not encompass any of the limit values ​​of the broader range (e.g., a range of 2-5 from a range of 1-10).

[0025] Amino acids are listed here using their three-letter or one-letter codes, where appropriate. A list of amino acids with their corresponding codes is given below:

[0026] Amino acid three-letter code one-letter code

[0027] Alanine Ala A

[0028] Arginine Arg R

[0029] Asparagine Asn N

[0030] Asparagus Asp D

[0031] Cysteine ​​Cys C

[0032] Glutamine Gin Q

[0033] Glutamine fat Glu E

[0034] Glycin Gly G

[0035] Histidine His H

[0036] Isoleucin Level I

[0037] Leucine Leu L

[0038] Lysine Lys K

[0039] Methionine Met M

[0040] Phenylalanine Phe F Proline Pro P

[0041] Sulfur Ser S

[0042] Threonine Thr T

[0043] Tryptophan Trp W

[0044] Tyrosine Tyr Y

[0045] Valin Val V

[0046] Mutations in protein sequences in the form of amino acid substitutions are given in the format X1PX2, where Xi denotes the amino acid originally located at a position P and replaced (in a single-letter code), X2 denotes the amino acid that replaces the original amino acid at the same position after the substitution (in a single-letter code), and P denotes the position within the amino acid sequence at which the substitution was made.

[0047] The apoferritin nanoparticle for use as a toxin binder in hemodialysis can have an amino acid sequence in the inner cavity that is different from the wild-type sequence, for example, it can have cysteine ​​residues at positions where no cysteine ​​residue is present in the wild-type sequence. The cysteine ​​residues can be further functionalized. Alternatively or additionally, the apoferritin nanoparticle for use as a toxin binder in hemodialysis can have an amino acid sequence in the region of a triple or quadruple channel that is different from the wild-type sequence, for example, an amino acid exchange that leads to easier transport of a toxin into the cavity of the apoferritin nanoparticle.

[0048] In a preferred embodiment, at least one of the apoferritin subunits of the apoferritin nanoparticle has an amino acid sequence in the region of the inner cavity that is different from the wild-type sequence. In the case where the apoferritin nanoparticle according to the invention is composed of a light (L) and a heavy (H) chain with the general structure H n L m, where n and m are integers and n + m = 24, this means that at least one of the chains, preferably one of the heavy chains, has an amino acid sequence that is altered compared to the respective wild-type sequence. If the at least one of the apoferritin subunits with the altered amino acid sequence is a subunit from the heavy chain, this is altered compared to the wild-type sequence of the heavy chain; if the at least one of the apoferritin subunits with the altered amino acid sequence is a subunit from the light chain, this is altered compared to the wild-type sequence of the light chain. In the case of a human apoferritin nanoparticle, i.e. an apoferritin nanoparticle composed of the respective human protein subunits, the corresponding reference is the wild-type sequence of the respective human subunit (see UniProtKB No. P02794, 2007-01-23 v2 or NCBI NP_002023.2 for the heavy chain, UniProtKB No. P02792, 2007-01-23 v2 or NCBI NP_000137.2 for the light chain). The wild-type sequence of the human heavy chain is also reproduced in the present application as sequence with SEQ ID NO: 1, and the wild-type sequence of the human light chain is also reproduced as sequence with SEQ ID NO: 2, whereby, unlike the above-mentioned database sequences, the N-terminal methionine has been omitted since it is not part of the sequence of the finished protein.

[0049] SEQ ID NO: 1: Human ferritin heavy chain. TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQ SHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLL ELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKH TLGDSDNES

[0050] SEQ ID NO: 2: Human ferritin light chain.

[0051] SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEK REGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLH ALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLK HD

[0052] In a preferred embodiment of the apoferritin nanoparticle according to the invention for use as a toxin binding agent in hemodialysis, the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence is modified in the region of the cavity in its amino acid sequence compared to the wild-type sequence such that the apoferritin nanoparticle has a higher binding affinity to a toxin compared to an apoferritin nanoparticle composed of apoferritin subunits with an amino acid sequence unchanged compared to the wild-type sequence.In this embodiment of the apoferritin nanoparticle according to the invention, the change in the amino acid sequence of at least one of the apoferritin subunits in the region of the inner cavity is preferably designed such that the binding affinity of an apoferritin nanoparticle with the at least one subunit modified in its amino acid sequence for a toxin to be removed from blood is higher than that of an apoferritin nanoparticle with subunits whose amino acid sequence is unchanged from the wild-type sequence. The change can, for example, provide for an increase in the number of hydrophobic amino acid residues. Examples of amino acids with a hydrophobic amino acid residue are alanine, valine, methionine, leucine, isoleucine, proline, tryptophan, and phenylalanine. However, it can also be advantageous to provide a higher proportion of polar-neutral, acidic, or basic amino acids in the at least one apoferritin subunit for binding certain toxins.Examples of amino acids with polar / neutral amino acid residues are tyrosine, threonine, glutamine, glycine, serine, cysteine, and asparagine. Examples of acidic amino acids are glutamic acid and aspartic acid. Examples of basic amino acids are lysine or arginine. Furthermore, it may also be advantageous to provide a specific mixture and / or distribution of amino acids with hydrophobic and polar / neutral amino acid residues for binding certain toxins. The binding affinity of modified apoferritin nanoparticles to toxins, for example, PBUTs, compared to unmodified apoferritin nanoparticles can be easily determined by one skilled in the art.

[0053] In a preferred embodiment of the apoferritin nanoparticle according to the invention for use as a toxin binding agent in hemodialysis, the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence has, in the region of the inner cavity, at least one additional cysteine ​​residue compared to the wild-type sequence or a cysteine ​​residue at a different position in the sequence, wherein the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence is functionalized or functionalizable by covalently binding an organic compound residue, e.g., an aliphatic or aromatic compound residue, to the cysteine ​​residue. Functionalization preferably serves to increase the binding affinity of the apoferritin nanoparticle according to the invention to toxins to be removed from the blood, for example, protein-bound uremic toxins.

[0054] Particularly preferably, in the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence, any cysteine ​​residues naturally present in the region of the inner cavity are replaced by other amino acid residues, for example by alanine residues. This allows for targeted positioning of the cysteine ​​residues to be introduced and thus of the sites at which the apoferritin subunit according to the invention can be functionalized. Therefore, in the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence, any cysteine ​​residues present in the region of the inner cavity are each preferably replaced by a different amino acid residue, e.g., an alanine residue, and at least one cysteine ​​residue is introduced in the region of the inner cavity at a position where no cysteine ​​residue is present in the native protein.

[0055] Particularly preferably, the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence in the apoferritin nanoparticle according to the invention for use as a toxin binding agent in hemodialysis has, compared to the wild-type sequence, two, three or four cysteine ​​residues in the region of the inner cavity, to each of which an organic compound residue is or can be covalently bound. In this embodiment, it is preferred that three or four cysteine ​​residues are present in the region of the inner cavity, each of which is identically or differently functionalized or functionalizable. In the event that all 24 subunits of an apoferritin nanoparticle according to the invention are functionalized or functionalizable in this way, this results in a number of 72-96 functionalization sites, i.e. sites within the apoferritin nanoparticle that are functionalizable or functionalizable.As described above, any cysteine ​​residues of the native protein in the region of the inner cavity are preferably replaced by, for example, alanine residues, and at least one of the introduced cysteine ​​residues is introduced at a position where no cysteine ​​residue is present in the native protein. The functionalization sites present in an apoferritin nanoparticle according to the invention can be identically or differently functionalized, i.e., functionalized with the same or different chemical groups.

[0056] The organic linking moiety can be hydrophobic, hydrophilic, or amphiphilic. Hydrophobic linking moieties are preferred for binding, for example, hydrophobic uremic toxins. However, even for binding hydrophobic uremic toxins, it can be advantageous to provide an amphiphilic linking moiety for functionalization, or to functionalize different cysteine ​​residues in the region of the internal cavity with different linking moieties, e.g., with a mixture of hydrophilic and hydrophobic linking moieties. The adjustment of the hydrophobicity or hydrophilicity of a linking moiety is known to the person skilled in the art and can, if necessary, be determined through routine experiments. For example, the hydrophobicity of a linking moiety can be influenced by the size or length of a hydrocarbon moiety, and the hydrophilicity by providing polar or charged moieties.

[0057] The organic linking residue can, for example, be an acetamidyl compound of the general structure -CH2-C(O)-NH-R. The R residue can be an aliphatic, heteroaliphatic, aromatic, or heteroaromatic residue. In the case of aliphatic and heteroaliphatic residues, a maximum chain length, i.e., a maximum number of atoms composing the linking residue, of 35 atoms is preferred, particularly preferably a maximum chain length of 34, 33, 32, 31, or 30 atoms. In the case of aromatic and heteroaromatic residues, electron-poor aromatic systems are preferred, which allow effective 7t-7t interactions with electron-rich aromatic systems of the toxins. Examples of electron-poor aromatic residues are, for example, 3,4,5-trinitrophenyl, 3,4,5-trihalogenyl (e.g. 3,4,5-trifluorophenyl) or 3,4,5-tris(trihalogenylmethyl)phenyl residues (e.g. 3,4,5-tris(trifluoromethyl)phenyl residues).Examples of electron-poor heteroaromatic groups are pyridine, quinoline, or isoquinoline, whose electron-poor character can be further enhanced with electron-withdrawing substituents such as nitro groups, tertiary amino groups, positively charged groups, or halogen atoms. For aromatic and heteroaromatic radicals, a molecular size of a maximum of 45, preferably a maximum of 44, 43, 42, 41, or 40 atoms is preferred, i.e., radicals composed of a maximum of 45, preferably a maximum of 44, 43, 42, 41, or 40 atoms, including heteroatoms. Further preferred for aromatic and heteroaromatic radicals is a molecular size of a maximum of 39 atoms, particularly preferably a maximum of 38, 37, 36, 35, 34, 33, 32, 31, or 30 atoms. Since the majority of PBUTs carry a negative formal charge under physiological conditions, amphiphilic linker residues with a combination of hydrophobic groups with positively charged groups are preferred.Examples of groups that are positively charged under physiological conditions are primary, secondary, tertiary, and quaternary amines. In the case of heteroaliphatic compounds, it is preferred if these groups are present in the chain. Depending on the geometry of the toxin to be bound, these groups could also be incorporated alternately in the chain, preferably not exceeding a maximum chain length of 35 atoms, particularly preferably 34, 33, 32, 31, or 30 atoms. In the case of aromatic and heteroaromatic residues, the charged groups are preferably arranged with appropriate symmetry to the aromatic or heteroaromatic system.Here, too, it is preferred if a maximum molecular size of the connecting residue is not exceeded, preferably a molecular size of 45, preferably 44, 43, 42, 41, or 40 atoms, more preferably a molecular size of 39 atoms, particularly preferably 38, 37, 36, 35, 34, 33, 32, 31, or 30 atoms. For toxins such as cresyl sulfate or phenylacetic acid, for example, l-amino-2-phenylethyl or l,l-diamino-2-phenylethyl residues are suitable.Preferred linking residues for binding protein-bound uremic toxins, which can be or are covalently bound to the at least one additional cysteine ​​residue or one arranged at a different position in the sequence, are selected from the group consisting of N-phenylacetamidyl, N-decylacetamidyl, N-(l-amino-2-phenylethyl)acetamidyl, N-(l-amino-2-phenylpropyl)acetamidyl, N-(l-amino-2-phenylbutyl)acetamidyl, N-(l,1-diamino-2-phenylethyl)acetamidyl, N-(l,l-diamino-2-phenylpropyl)acetamidyl, N-(l,1-diamino-2-phenylbutyl)acetamidyl, N-(3,4,5-tris(trifluoromethyl)phenyl)acetamidyl, N-(3,4,5-trinitrophenyl)acetamidyl, N-(l-amino-2-(3,4,5-trinitrophenyl)ethyl)acetamidyl and N-(2-(heptylamino)ethyl)acetamidyl. These compounds are preferably covalently bound to the cysteine ​​residue via the acetamide group. Halogenated acetamide derivatives can be used for this purpose, which react with the thiol group of cysteine.Furthermore, it is of course also possible to use other suitable coupling groups, for example haloalkyl or maleimide derivatives, for binding to the thiol group.

[0058] The organic compound residue can, for example, also be a chelating agent that binds heavy metals such as lead, antimony, arsenic, cadmium, nickel, mercury, thallium, or uranium. Examples of chelating agents (complexing agents) are ethylenediaminetetraacetic acid (EDTA), dimercaptosuccinic acid (DMSA), or dimercaptopropanesulfonic acid (DMPS). The term "Schwerin metal" includes heavy metal ions.

[0059] In a preferred embodiment of the apoferritin nanoparticle according to the invention for use as a toxin binding agent in hemodialysis, the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence - alternatively or in addition to a change in the amino acid sequence in the region of the inner cavity - is modified in its amino acid sequence in the region of a triple channel compared to the wild-type sequence in such a way that the transport of a toxin into the cavity of the apoferritin nanoparticle through the triple channel is facilitated compared to an apoferritin nanoparticle with an unchanged amino acid sequence.In this embodiment, the amino acid sequence of at least one apoferritin subunit can be altered compared to the wild-type sequence only in the region of a triple channel, for example, to merely facilitate transport into the apoferritin nanoparticle without also having changes to the amino acid sequence in the region of the inner cavity, or the amino acid sequence of at least one apoferritin subunit can be altered compared to the wild-type sequence also in the region of a triple channel, in addition to a change to the amino acid sequence in the region of the inner cavity. It is preferred that at least two, preferably three, apoferritin subunits forming a triple channel have at least one change to the amino acid sequence in the region of the triple channel.For example, one, two, three or more, for example up to 12, amino acid changes can be present in one, two, or three of the apoferritin subunits forming a triple channel. In the case of changes to two or three of the apoferritin subunits, these changes can be identical or different for each subunit, but are preferably identical. Particularly preferred is an apoferritin nanoparticle for use as a toxin binding agent in hemodialysis that is composed of identical subunits, each of which has identical changes to the amino acid sequence compared to the wild-type sequence in the region of a triple channel, for example at least at one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve amino acid positions in the region of the triple channel.As already mentioned, these changes can occur alone or in combination with a change in amino acids in the inner cavity.

[0060] The toxin to be bound during hemodialysis with the aid of the apoferritin nanoparticle according to the invention is preferably a protein-bound uremic toxin, which is preferably selected from the group consisting of indoxyl sulfate, para-cresyl sulfate, phenyl acetate and p-hydroxyhippuric acid.

[0061] In the apoferritin nanoparticle according to the invention for use as a toxin binder in hemodialysis, preferably at least two, more preferably at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10, more preferably at least 12, at least 14, at least 16, at least 18, at least 20 or at least 22, particularly preferably all 24 subunits have an amino acid sequence that is altered compared to the wild-type sequence in the region of the inner cavity and / or in the region of a triple channel and / or in the region of a quadruple channel. The subunits of which the apoferritin nanoparticle according to the invention is composed are preferably identical in terms of their amino acid sequence.Further preferably, an apoferritin nanoparticle according to the invention as described above is modified according to one embodiment by introducing cysteine ​​residues which are functionalized with suitable chemical linking residues, wherein preferably at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10, further preferably at least 12, at least 14, at least 16, at least 18, at least 20 or at least 22, particularly preferably all 24 subunits are modified and functionalized in the same way.In an embodiment in which the apoferritin nanoparticle has a modified amino acid sequence in the region of a triple channel, whether alone or in combination with a modified amino acid sequence in the region of the inner cavity, preferably at least two, more preferably at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10, more preferably at least 12, at least 14, at least 16, at least 18, at least 20 or at least 22, particularly preferably all 24 subunits have an amino acid sequence that is modified compared to the wild-type sequence in the region of a triple channel.

[0062] In a preferred embodiment of an apoferritin nanoparticle according to the invention for use as a toxin binder in hemodialysis, preferably at least two, more preferably at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10, more preferably at least 12, at least 14, at least 16, at least 18, at least 20 or at least 22, particularly preferably all 24 subunits have an amino acid sequence which is modified compared to the wild-type sequence in the region of the inner cavity and / or in the region of a triple channel and / or in the region of a quadruple channel and which is selected from one of the amino acid sequences according to SEQ ID NO: 13-24. It is preferred that the subunits with an amino acid sequence modified compared to the wild-type sequence each have the same sequence.For example, if an apoferritin nanoparticle according to the invention for use as a toxin binding agent in hemodialysis has 24 subunits with an amino acid sequence that is altered compared to the wild-type sequence in the region of the inner cavity and / or in the region of a triple channel and / or in the region of a quadruple channel, the 24 subunits preferably have the same amino acid sequence, for example the amino acid sequence according to SEQ ID NO: 13, SEQ ID NO: 17 or SEQ ID NO: 24. Preferably, the subunits have an amino acid sequence that is selected from one of the amino acid sequences according to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, or SEQ ID NO: 24.

[0063] It is particularly preferred that the apoferritin nanoparticle according to the invention is composed exclusively of subunits consisting of the heavy chain, particularly preferably the human heavy chain. The chain can be modified with regard to the amino acid sequence, including the protein regions located on the outside (e.g., helices A and C), compared to the wild-type sequence, for example to produce a specific external charge, for example, a negative or positive charge. Furthermore, it is particularly preferred that the apoferritin nanoparticle according to the invention does not have any cysteine ​​residues on the outside of the nanoparticles, but optionally exclusively in the inner cavity, so that functionalization with an organic compound residue occurs exclusively at the cysteine ​​residues in the inner cavity.Functionalization can be achieved by disassembling the apoferritin nanoparticle into its subunits under suitable conditions, and then functionalizing the individual subunits. After the original conditions are restored, the subunits spontaneously reconstruct the apoferritin nanoparticle. Suitable conditions are known to the person skilled in the art and are described, for example, in

[0031] ,

[0038] ,

[0039] and further below.

[0064] In a second aspect, the invention also relates to a composition for use as a toxin binder in hemodialysis, comprising a plurality of apoferritin nanoparticles according to the invention for use as a toxin binder in hemodialysis according to the first aspect of the invention described above. Such a composition can advantageously be used as an adsorption material for binding toxins in the blood, for example, for binding protein-bound uremic toxins (PBUTs) or for binding heavy metals.

[0065] In the composition according to the invention for use as a toxin binder in hemodialysis, the plurality of apoferritin nanoparticles according to the invention for use as a toxin binder in hemodialysis can be present in amorphous form or in a defined arrangement, for example in a crystalline form. A composition of apoferritin nanoparticles present in amorphous form is understood here to mean a composition of apoferritin nanoparticles in which the apoferritin nanoparticles are interconnected only by forming a disordered or indeterminate, in particular non-crystalline, structure. A defined, e.g., crystalline, arrangement of apoferritin nanoparticles according to the invention can be achieved in a manner known to the person skilled in the art (see, for example,

[0031] ).In the case of an arrangement in crystalline form, for example, the apoferritin nanoparticles contained in the arrangement are preferably cross-linked with one another for use as toxin binders in hemodialysis. Such an adsorption material comprises apoferritin nanoparticles according to the invention as protein cages, which are assembled into a defined crystalline material with uniformly distributed solvent channels. This is not only advantageous for achieving high material purity, but also for handling when used as a sorption material. For example, the apoferritin nanoparticles cross-linked with one another to form a crystalline structure can be handled as a whole and separated from blood, for example. The shape of the crystal can be adapted, for example, to the shape of the cavity of a sorption cartridge.In the composition according to the invention, the plurality of apoferritin nanoparticles according to the invention for use as toxin binders in hemodialysis can be present, for example, in water or an aqueous solution, for example a suitable aqueous buffer solution. The aqueous solution is preferably a biocompatible or pharmaceutically acceptable solution in which the nanoparticles are stable. In a third aspect, the present invention also relates to a sorption cartridge comprising, within its interior, a plurality of apoferritin nanoparticles for use as toxin binders in hemodialysis according to the first aspect of the invention or a composition for use as a toxin binder in hemodialysis according to the second aspect of the invention. A sorption cartridge according to the invention can, for example, be designed such that it can be integrated into a dialysis system.For this purpose, the sorption cartridge preferably has an inlet for the supply of blood and an outlet for the export of the blood.

[0066] Preferably, the sorption cartridge is designed such that the plurality of apoferritin nanoparticles, or the composition comprising a plurality of apoferritin nanoparticles, are retained in the sorption cartridge by suitable means. For example, the plurality of apoferritin nanoparticles or the composition can be contained in a first compartment within the sorption cartridge, which is separated from a second compartment within the sorption cartridge by one or more membranes, wherein the membrane or membranes are permeable to unbound toxin molecules and / or to proteins with bound toxin molecules, but not to the apoferritin nanoparticles. Blood to be purified of toxin molecules, e.g., PBUTs, can, for example, be passed through the second compartment.Toxin molecules present in the blood can, in free form or optionally bound to protein, pass across the membrane(s) into the first compartment, e.g. by diffusion, and be bound there by the apoferritin nanoparticles according to the invention. In a particularly preferred embodiment of the sorption cartridge according to the invention, the plurality of apoferritin nanoparticles or the composition are arranged in the sorption cartridge in such a way that during operation, e.g. in a dialysis system, direct contact occurs with the blood to be purified. In this preferred embodiment, a crystalline or non-crystalline (amorphous) composition of the apoferritin nanoparticles is located within the sorption cartridge or at least within a compartment within the sorption cartridge, whereby blood can come into direct contact with the composition within the sorption cartridge.In this form, the sorption material according to the invention forms an insoluble (amorphous or crystalline) solid that is significantly larger than the components of blood (e.g., erythrocytes with a size of approximately 2.5 pm). This size difference makes separation easy. The sorption material can be retained by relatively coarse filters while the blood flows through the sorption cartridge. The biocompatibility of the material according to the invention proves particularly advantageous here, as it eliminates the need to separate the sorption material from the blood, which not only enables closer contact between the sorption material and the toxins to be separated, but also facilitates integration into existing dialysis systems.

[0067] In a fourth aspect, the invention relates to a dialysis system comprising a dialyzer and at least one sorption cartridge according to the third aspect of the invention.

[0068] The at least one sorption cartridge can be arranged in the flow direction of the blood to be dialyzed a) upstream of the dialyzer, b) downstream of it or c) in a secondary circuit to the dialysis circuit.

[0069] In a fifth aspect, the invention also relates to a method for dialysis of blood, preferably human blood, comprising the step of contacting the blood with an apoferritin nanoparticle according to the invention according to the first aspect of the invention or with a composition according to the second aspect of the invention. The contacting step preferably takes place over a time and under conditions sufficient to bind a toxin contained in the blood, for example a PBUT or a heavy metal, to the apoferritin nanoparticles according to the invention or the composition. The composition can comprise the apoferritin nanoparticles in amorphous or cross-linked (crystalline) form. The term “contacting” here particularly encompasses direct contacting of blood with the apoferritin nanoparticles or the composition, i.e. without separation by, for example, a semipermeable membrane.The dialysis method according to the invention is particularly advantageous for the treatment of chronic kidney disease (CKD) and / or for alleviating the consequences of chronic kidney disease (CKD). In a further aspect, the invention therefore also relates to a method for the therapeutic treatment of a patient with chronic kidney disease (CKD) and / or for the therapeutic alleviation of the consequences of chronic kidney disease (CKD), comprising a method for dialysis of blood according to the above fifth aspect of the invention.

[0070] The invention is explained in more detail below with reference to the attached drawings and embodiments purely for illustrative purposes.

[0071] Figure 1. Schematic representation of embodiments of the apoferritin nanoparticle according to the invention. A. Schematic representation of an apoferritin nanoparticle according to the invention with a change in the amino acid sequence in the region of the inner cavity; B. Schematic representation of an apoferritin nanoparticle with a change in the amino acid sequence in the region of the inner cavity with functionalized cysteine ​​residues; C. Schematic representation of an apoferritin nanoparticle with a change in the amino acid sequence in the region of a triple channel; D. Schematic representation of an apoferritin nanoparticle with a change in the amino acid sequence in the region of the inner cavity (with functionalization of a cysteine ​​residue), and with an additional change in the amino acid sequence in the region of a triple channel.

[0072] Figure 2. Schematic representation of a crystalline arrangement (lower part of the figure) of several apoferritin nanoparticles according to the invention, composed of apoferritin nanoparticles from Figure 1 (upper part of the figure).

[0073] Figure 3. Schematic representation of embodiments of a dialysis system according to the invention. A. Dialysis system with an upstream sorption cartridge according to the invention; B. Dialysis system with a downstream sorption cartridge according to the invention; C. Dialysis system with an upstream sorption cartridge according to the invention in a secondary circuit.

[0074] Figure 4. Results of adsorption experiments with functionalized apoferritin nanoparticles for binding PBUTs. a), b), c) Adsorption capacity of untreated and functionalized ferritin towards representative PBUTs at concentrations expected in CNK patient blood

[0026] , d) Light microscopic image of the Ftn-Phe crystals used as adsorbents. e) Ratio of phosphorylated AKT to AKT as a measure of platelet activation and associated blood coagulation. f) Relative concentration of TMF a mRNA in endothelial cells. Ftn (neg) -Phe: Ftn functionalized with 2-iodo-N-phenylacetamide (Phe) (neg Apoferritin nanoparticles; Ftn (neg) -C10: Ftn functionalized with 2-bromo-N-decylacetamide (CIO) (neg) - Apoferritin nanoparticles.

[0075] Figure 5. a) Adsorption capacity of genetically modified ferritin variants towards indoxyl sulfate. Ftn (neg)-Dock: Variant with toxin binding site (sequence: Ftn (neg) -dock03, SEQ ID NO: 21); Ftn (neg) -Ap: Variant with reduced negative surface charge on the inner surface (sequence: Ftn (neg) -Ap4, SEQ ID NO: 17); Ftn (neg) -Ap channel: variant with reduced negative surface charge on the inner surface and in the region of the triple channel (sequence: Ftn (neg) -Ap4-3A, SEQ ID NO: 18). b) Indoxyl sulfate and its interactions with three side chains in a designed toxin binding site (computer model).

[0076] Figure 6. Adsorption capacity of crystalline (left) and non-crystalline (right) genetically modified ferritin variants according to the invention towards indoxyl sulfate (IS). Ftn (neg) : Apoferritin nanoparticle variant with increased negative charge on the outer surface (SEQ ID NO: 12); Ftn (neg) -Ap4: Variant with 4 mutations compared to Ftn (neg)and thus altered reduced negative surface charge on the inner surface (SEQ ID NO: 17); Ftn(neg)-Apl6: variant with 16 mutations compared to Ftn (neg) and thus altered reduced negative surface charge on the inner surface (SEQ ID NO: 20).

[0077] Figure 7. Adsorption capacity of crystalline genetically modified ferritin variants according to the invention with specially developed binding sites towards indoxyl sulfate (IS) compared to Ftn (neg) . Ftn (neg) -03 (= Ftn (neg) -dock03, SEQ ID NO: 21); Ftn (neg) -23 (= Ftn (neg) - dock23, SEQ ID NO: 22); Ftn (neg) -43 (= Ftn (neg) -dock43, SEQ ID NO: 22). Figure 8. Adsorption capacity of crystalline genetically modified ferritin variants according to the invention with specially developed binding sites towards para-cresyl sulfate (pCS) and phenyl acetate (PhAc) compared to Ftn (neg) . Ftn (neg) -03 (= Ftn (neg)-dock03, SEQ ID NO:

[0078] 21); Ftn (neg) -23 (= Ftn (neg) -dock23, SEQ ID NO: 22); Ftn (neg) -43 (= Ftn (neg) -dock43, SEQ ID NO:

[0079] 22).

[0080] Figure 9. Adsorption capacity of a crystalline genetically modified ferritin variant according to the invention (Ftn (neg) -Ap4-3A-dock43; SEQ ID NO: 24) with combined sequence modifications to indoxyl sulfate (IS) compared to Ftn (neg) and the variants Ftn (neg) -Ap4 (SEQ ID NO: 17), Ftn (neg) -Ap4-3A (SEQ ID NO: 18) and Ftn (neg) - dock43 (SEQ ID NO: 23). Ftn (neg) -43 = Ftn (neg) -dock43; Ftn (neg) -Ap4-3A-43 = Ftn (neg) -Ap4-3A- dock43.

[0081] Figure 1 shows a highly simplified and schematic representation of embodiments of apoferritin nanoparticles 1 according to the invention for use as toxin binding agents in hemodialysis. As can be seen from Figure 1, the apoferritin nanoparticles 1 have a generally spherical shape, with a protein shell formed by apoferritin subunits 2, which encloses an inner cavity 4 and in which channels 3 are formed at the interfaces between the apoferritin subunits 2, through which toxins from the external environment can pass, for example by diffusion, into the inner cavity 4 and be bound there, for example by hydrophobic interaction. Figure 1 schematically shows four individual apoferritin nanoparticles 1, wherein Figure 1A shows an embodiment in which sequence modification sites 5 are arranged in the region of the inner cavity 4, i.e.Sites where modifications to the amino acid sequence of the apoferritin subunits 2 have been made, i.e., individual amino acids or entire amino acid sequence regions have been exchanged for other amino acids or amino acid sequence regions compared to the wild-type sequence of the respective apoferritin subunit 2. Functionalization of amino acids, e.g., cysteine ​​residues, has not been performed here. Binding of toxins can occur, for example, through hydrophobic interaction with hydrophobic amino acid residues provided at the sequence modification sites 5.In the embodiment of an apoferritin nanoparticle 1 for use as a toxin binding agent in hemodialysis shown in Figure 1B, any existing native cysteine ​​residues were first replaced with alanine and then four amino acids at four sequence modification sites 5 were replaced with cysteine, which was functionalized at its SH group with a covalently bound organic compound residue (here, for example, an N-phenylacetamidyl residue). In the example, four cysteine ​​residues are functionalized with a hydrophobic organic compound residue for the binding of uremic toxins. Figure 1C schematically shows an embodiment of an apoferritin nanoparticle 1 for use as a toxin binding agent in hemodialysis in which only sequence modification sites 5 are provided in the region of a triple channel, i.e., an amino acid sequence that is modified compared to the wild-type sequence is present only in the region of a triple channel.Figure 1C schematically shows an embodiment of an apoferritin nanoparticle 1 for use as a toxin binding agent in hemodialysis, in which, in addition to sequence modification sites 5 in the region of a triple channel, sequence modification sites 5 are also present in the region of the inner cavity 4, wherein one of the sequence modification sites 5 is shown here as a functionalized sequence modification site 5 by way of example.

[0082] Figure 2 schematically shows the assembly of apoferritin nanoparticles 1 according to the invention (upper part of the figure) into a crystalline arrangement 100 (lower part of the figure). Under suitable conditions, the apoferritin nanoparticles 1 can be assembled into a well-defined macroscopic crystalline material with uniformly distributed solvent channels. This ensures high material purity and ease of handling, and is also helpful for material characterization. To fix the arrangement, the apoferritin nanoparticles 1 can optionally be cross-linked with one another.

[0083] Figure 3 schematically illustrates a dialysis system 200 according to the invention in various embodiments. Figure 2A shows an embodiment in which a sorption cartridge 201 is arranged upstream of a dialyzer 202, here a hollow fiber membrane module, in the direction of blood flow indicated by the arrows. The flow direction is determined by a corresponding pump, which is not shown here. The body boundary is schematically indicated here by the dashed line 203. Blood is conveyed to the sorption cartridge 201 via a line 205, e.g., a hose. The sorption cartridge 201 contains an adsorption material according to the invention, e.g., a composition 100 in the form of a crystalline arrangement according to the second aspect of the invention. The blood can be brought into direct contact with the composition 100 containing the apoferritin nanoparticles 1 according to the invention. Any substances contained in the blood, e.g.,Uremia toxins bound to proteins can be bound by the adsorption material. The blood, freed of toxins or at least depleted of them, is returned to the dialyzer 202 and then to the patient's body via a line 204 leading to the body. A dialysate circuit 206 can ensure an exchange or counterflow of dialysate.

[0084] Figure 3B shows a dialysis system 200 according to the invention, in which the sorption cartridge 201 according to the invention is arranged downstream of the dialyzer 202 in the direction of blood flow. Otherwise, the dialysis system 200 in this embodiment corresponds to the embodiment shown in Figure 3A, so reference is made to the explanations therein for further details. Here, too, the blood to be freed of toxins or to be removed comes into direct contact with the apoferritin nanoparticles 1 or a composition 100 comprising the apoferritin nanoparticles 1 in the sorption cartridge 201.

[0085] Figure 3C shows a dialysis system 200 according to the invention, in which the sorption cartridge 201 according to the invention with apoferritin nanoparticles 1 according to the invention is arranged in a secondary circuit 207 to the bloodstream. Instead of or in addition to a dialysate circuit (not shown here), adsorption material can be introduced into the dialyzer 202 to bind toxins passing from the blood through the membranes of the hollow fibers. In this embodiment, the blood to be purified does not come into direct contact with the apoferritin nanoparticles 1 or a composition 100 comprising the apoferritin nanoparticles 1. Examples

[0086] 1. Chemical modification (functionalization) of the inner surface of apoferritin nanoparticles

[0087] Apoferritin nanoparticles according to one embodiment of the invention were produced by chemically modifying the inner surface with small hydrophobic molecules. For this purpose, the protein was genetically modified so that the amino acid cysteine ​​is present only at certain exposed sites on the inner surface. This amino acid is the only amino acid in the inner cavity with a thiol group in its side chain, which was used as an anchor point for the reaction with hydrophobic molecules. The protein container can be fragmented into its subunits under acidic conditions (pH 2). The internal cysteines can then be functionalized with the hydrophobic molecules. The molecules used were 2-iodo-N-phenylacetamide and 2-bromo-N-decylacetamide, which can bind to the thiol group as a result of a substitution reaction.Since a thiol group is exclusively found in the cysteine ​​residue, and the modified protein contains this group only within the cavity, only the inner surface is selectively modified with hydrophobic groups. Complete functionalization can be verified by a significant increase in the protein mass using ESI-MS measurements. This method also allows for the determination of whether each cysteine ​​residue has been functionalized. Currently, up to 96 hydrophobic molecules can be incorporated per nanoparticle. After functionalization, an increased adsorption capacity for the IS and pCS, as well as for phenylacetate, can be measured, as shown in Fig. 3a.

[0088] Materials and methods

[0089] General

[0090] All chemicals were obtained from commercial sources and used without further purification. Where possible, all solutions were prepared using ultrapure water (prepared using a Purelab Flex 2 system, resistivity 18.2 Mfi cm) and analytical-grade reagents, unless otherwise stated.

[0091] Mutagenesis

[0092] The introduction of cysteine ​​anchor sites was performed by several cycles of QuikChange™ site-directed mutagenesis using a two-step polymerase chain reaction (PCR) protocol

[0030] . The primers used for the different mutation sites are given in Table 1.

[0093] Table 1. Primer sequences (in 5 '-3 ' direction) for PCR protocol for mutagenesis

[0094] A mixture of 2.9 pL pET-22b(+) plasmid containing the gene of interest (7 ng pL x), 1 pL 10 mM dNTP-Mix, 5 pL Reaktionspuffer lOx (100 mM KCl, 100 mM (NH4)2SO4, 200 mM Tris-HCl pH 8,8, 20 mM MgSO4, 1 % Triton® X-100, 1 mg mL 1nuclease-free bovine serum albumin (BSA)), 1 pL Pfu DNA polymerase (2.5 U pL '), and 38.1 pL ultrapure water were prepared. The mixture was halved, and 1 pL of forward or reverse primer (10 pmol pL ') was added to each tube. A PCR thermocycler (Eppendorf Mastercycler Nexus PCR Cycler) was prepared with an initial heating step for 30 s at 95 °C. The first step of the PCR protocol consisted of 3 cycles of 30 s denaturation at 95 °C, annealing for 1 min at 61 °C, followed by elongation for 6 min at 68 °C. After the first 3 cycles, the separated mixtures with the forward and reverse primers were combined, and the PCR was continued for 16 more cycles using the same parameters as for the first 3 cycles, followed by a final elongation phase for 10 min at 68 °C to complete the PCR. Digestion of the parental plasmid was performed by adding 1 pL Dpnl (10 U pL 1) and incubation overnight at 37°C. DpnI is inactivated by heating at 80°C for 20 min, and the mixture is purified using the NucleoSpin® Gel and PCR Clean-Up Kit according to the manufacturer's instructions. Calcium-competent E. coli DH5a cells were incubated with 200 ng of the purified plasmid for 30 min on ice, followed by a heat shock for 45 s at 42°C. Next, the cells were incubated in Super Optimal Broth (SOB) medium for 1 h, centrifuged at 1000 g, resuspended in 100 μL medium, plated on an LB agar plate, and incubated for 16 h at 37°C. A single colony was picked and incubated overnight at 37 °C and 250 rpm in 5 mL of sterile LB medium supplemented with 150 pg mL 1 Ampicillin. The next day, the plasmids were extracted using the NucleoSpin® Plasmid Miniprep Kit according to the manufacturer's instructions.

[0095] The sequence was confirmed by mixing 500 ng of plasmid with 25 pmol of T7 forward or reverse primer in 10 pL of solution and submitting it for DNA sequencing (Eurofins Genomics). Plasmids with the desired mutations were then selected as parent plasmids for further mutagenesis until all 5 mutations were present.

[0096] Production and purification of ferritin cysteine ​​variants

[0097] The production of negatively charged ferritin variants (Ftn (neg) ) and cysteine-bearing variants was performed as previously published

[0031] . First, calcium-competent E. coli BL21-Gold(DE3) cells were thawed on ice for 10 min. Then, 1 pL of 40 ng pL' 1Plasmid solution was added to the cells, and the mixture was incubated on ice for 30 min. Heat shock was performed by incubating the mixture at 42°C for 45 s, followed by 2 min incubation on ice. The cells were suspended in 1 ml of SOB medium and incubated for 1 h at 37°C. The cells were centrifuged at 1000 g, and 1 ml of the mixture was removed. The cell pellet was resuspended in the remaining solution and plated on an LB agar plate containing 150 pg ml-1 ampicillin and incubated overnight at 37°C. To prepare precultures, colonies of transformed E. coli BL21-Gold (DE3) cells (Agilent) were incubated overnight in 5 ml of sterile LB-Miller medium supplemented with 150 pg ml-1 1 Sodium ampicillin, at 37°C and 180 rpm. Then, 400 ml of Terrific Broth (TB) medium containing 150 pg ml' 1Sodium ampicillin was inoculated with 4 ml of the preculture. The cells were grown at 37°C and 180 rpm until an ODeoo of 0.6 was reached. Protein overexpression was induced by the addition of isopropyl-ß-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.25 mM, and the cells were incubated for a further 48 h at 18°C. The cells were harvested by centrifugation at 4000 g. Pellets were stored at -20°C until further use.

[0098] Cells from 400 ml of culture were resuspended in 20 ml of buffer (50 mM Tris, pH 7.5, 0.3 M NaCl). Cell lysis was achieved by sonication (60% amplitude) for six 1-min cycles on ice with 1-min intervals using a Vibra-Cell VCX-130 ultrasonic processor (Sonics). The resulting suspension was centrifuged at 14,000 g for 20 min to separate cellular debris from soluble proteins. Denaturation of most E. coli proteins was achieved by heating the supernatant to 65 °C for 10 min in a water bath. The denatured proteins were separated by centrifugation at 14,000 g for 15 min. Remaining proteins in the solution were precipitated with ammonium sulfate at a final concentration of 70% of its saturation concentration, followed by centrifugation at 14,000 g for 20 minutes. After resuspension of the pellet in 10 ml of buffer (50 mM Tris, pH 7.5, 0.15 M NaCl), the ammonium sulfate precipitation was repeated.The resulting pellet was dissolved in 50 ml of IEC loading buffer (50 mM Tris, pH 7.5, 0.15 M NaCl) and purified by ion exchange chromatography (IEC) with a linear gradient from 0.15 to 1 M NaCl using a 5 ml HiTrap™ Q HP anion exchange column (Cytiva). All FTn. (neg) Fractions containing 4xCys were collected and concentrated to a final volume of 2 ml using a Sartorius Vivaspin® Turbo 15 (MWCO 30,000) filter unit. Finally, the sample was purified by gel filtration using a HiLoad 16 / 600 Superdex™ 200 pg column. All chromatography steps were performed on an Äkta pure system from Cytiva. All Ftn (neg) Fractions containing -4xCys were collected and stored at 4°C until further use.

[0099] Functionalization with 2-iodo-N-phenylacetamide

[0100] 5 mg Ftn (neg) -3xCys or Ftn (neg)-4xCys were incubated for 4 h in decomposition buffer (10 mM phosphate; 50 mM NaCl, pH 2). After 3 h, 10 equivalents (eq.) (based on each cysteine) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Iris Biotech GmbH) were added from a 10 mg mL solution. 1Stock solution was added to the solution. The solution was then made up to 15 mL with reassembly buffer (50 M Tris, 50 mM NaCl, pH 7.6) and concentrated to a final volume of 200 μL using a membrane filter (Sartorius Vivaspin Turbo 15; 30 kDa MWCO). Another 10 eq. of TCEP was added, and the solution was made up to a volume of 2 mL. The pH was adjusted to 7.6 with 1M NaOH or HCl. Subsequently, 2 mL of ethanol containing 20 eq. of 2-iodo-N-phenylacetamide (but GmbH) was added to the solution, and the mixture was stirred for 1 h at 300 rpm in the dark. The solution was then made up to a total volume of 30 mL with reassembly buffer. The protein reassembled overnight. Finally, the solution was concentrated to 2 ml and purified by gel filtration on a HiLoad 16 / 600 Superdex™ 200 pg column. Protein-containing fractions were collected and stored at 4°C for further use.

[0101] Functionalization with 2-bromo-N-decylacetamide

[0102] The functionalization followed exactly the protocol for functionalization using 2-iodo-N-phenylacetamide. However, during the functionalization reaction, the protein / TCEP solution was made up to 800 pL instead of 2 mL, and then 3.2 mL of ethanol containing 40 eq. of 2-bromo-N-decylacetamide (Sigma-Aldrich) was added to the solution. All other steps were performed according to the protocol for 2-iodo-N-phenylacetamide. Hanging-drop crystallization

[0103] Crystallization of small amounts of protein or functionalized protein variants was performed using the hanging-drop vapor diffusion technique. Reservoir solution (100 mM Tris, 500 mM MgOAc, pH 8.5) was prepared in a manual 24-well plate set. Droplets were prepared on siliconized coverslips (Jena Bioscience) by mixing 2 pL of reservoir solution with 1 pL of 50 mM Tris, 1 M NaCl, pH 7.5 buffer, and 1 pL of the respective ferritin variant. The plates were incubated at 25°C. After one day, the first crystals were visible.

[0104] Batch crystallization

[0105] For the crystallization of larger quantities of Ftn (neg) and functionalized variants of Ftn (neg) Batch crystallization approach based on a protocol by Rayment was used

[0032] . For a standard experiment, 250 pL of a 50 mM Tris 1 M NaCl pH 7.5 buffer was carefully mixed with equal amounts of Ftn (neg)-Stock solution with a concentration of 12 mg mL' 1 in 50 mM Tris 0.3 M NaCl pH 7.5 buffer. Then, 500 pL of the precipitant solution (133 mM Tris, 333 mM MgOAc, pH 8.5) was added dropwise with continuous shaking. The mixture was stored at ambient temperature of 20 °C for 7 days before the crystals were fixed.

[0106] Crystal fixation

[0107] For the adsorption experiments, the stability of the crystals was increased. They were fixed with the crosslinker Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate, Sigma-Aldrich). The crystals were centrifuged for 2 min at 1000 g. In a standard experiment with a total mass of 3 mg of crystals, the crystallization solution was removed until 246 pL remained. Subsequently, 64 pL of a freshly prepared aqueous Sulfo-SMCC solution containing 4.8 mg mL 1added, resulting in a final concentration of 1 mg mL 1 The mixture was allowed to stand at room temperature for 4 h and then made up to 1 mL with ultrapure water. The crystals were collected by centrifugation at 1500 g for 2 min. The supernatant was removed, and the crystals were then resuspended in ultrapure water. This procedure was repeated up to three times to wash the crystals free of residual cross-linking agent. The material was stored at an ambient temperature of 20°C until further use. Crystals were photographed under a Leica S9D microscope using a FlexaCamCl.

[0108] For fixation with glutaraldehyde (Merck), 50 μl of a 2.5% aqueous glutaraldehyde solution was added to 1 ml of crystal solution containing 3 mg of crystals, resulting in a final concentration of 0.00119%. The crystals were incubated for 4 h and then washed three times with ultrapure water. The crystals were stored at 20°C until further use.

[0109] After glutaraldehyde crosslinking, the crystals dissolve in a 60 mg mL' 1 BSA solution. Stability was increased by an additional fixation step. The process was repeated, but the crystals were only incubated for 10 minutes before washing. Toxin adsorption assays, however, showed a significantly reduced adsorption capacity. It is assumed that the polymerization of the glutaraldehyde leads to pore blockage.

[0110] Production of non-crystalline adsorbent

[0111] 500 pL of a 6 mg mL' 125 pL of 2.5% glutaraldehyde solution was added to the protein solution. Complete mixing was ensured by gently pipetting the solution up and down three times. The mixture was kept overnight at an ambient temperature of 20°C. The next day, a white precipitate formed. The material was washed three times with ultrapure water and stored at 20°C until further use. ESI-MS measurements

[0112] The protein sample was buffered to ultrapure water using an Amicon® Ultra 0.5 ml (MWCO 30,000 Da) centrifugal filter. The protein sample was made up to a volume of 500 pL with ultrapure water, concentrated to approximately 20 pL, and then topped up to 500 pL. The buffering was repeated 5 times, and the concentration was adjusted between 0.15 and 0.2 mg ml. 1The protein mass was determined using electron spray ionization time-of-flight mass spectrometry (Agilent 6224 ESI-TOF). The measurement was performed in positive mode.

[0113] Toxin assay

[0114] Adsorption experiments were conducted to determine the uremic toxin adsorption capacity of the ferritin variants. All solutions and samples were handled in glassware (Macherey-NAGEL Vials N9), as adsorption of the toxins to the polypropylene walls of the reaction tubes was observed in initial experiments.

[0115] First, a stock solution of the desired toxin with a concentration of 50 pg mL' 1 for pCS and IS and 500 pg mL' 1 for PheAc. From the initial stock solutions, a calibration series with concentrations of 0.01, 0.05, 0.1, 0.2, 0.5, 0.7 and 1 pg mL' 1 for later determination of the absolute toxin concentrations in the samples.

[0116] The stock solutions were further diluted to achieve the final uremic toxin concentrations expected in a stage 5 CNK patient (41 mg L' 1 for pCS,

[0033] 44 mg L' 1 for IS

[0026] and 474 mg L' 1 for PheAc

[0034] ). The adsorbent was centrifuged for 2 min at 1500 g, and the complete supernatant was removed from the sample. 150 pL of the respective toxin solution was added, and the crystals were incubated for 3 h at room temperature. Additionally, 150 pL of the toxin solution was incubated as a control. Three aliquots of 10 pL were taken from each sample and diluted 100-fold in ultrapure water. Finally, the crystals were washed with water, dried under vacuum, and weighed.

[0117] Uremia toxin concentration was quantified using a reversed-phase high-performance liquid chromatography (RP-HPLC) system with a C18 column (Zorbax Extend-C18, Agilent) coupled to an electron spray ionization quadrupole liner ion trap mass spectrometer (ESI-QTRAP). A mixture of HPLC-grade water (LiChrosolv® Merck) and acetonitrile (LiChrosolv® Merck), both with added 0.1% formic acid (Honeywell Fluka), was used as the solvent. The specific compositions at each step during the 15-minute chromatography program are summarized in Table 2.

[0118] Table 2. Sequence of the HPLC program

[0119] The control was measured before each sample incubated with the crystals.

[0120] Chromatograms were analyzed using Analyst® Instrument Control and Data Processing Software. Peaks were integrated, and the toxin concentration was determined from the calibration series. The amount of adsorbed uremic toxin was determined from the concentration difference between the control and the samples. The adsorption capacity was then determined by dividing the mass of adsorbed toxins by the mass of the crystals.

[0121] Quantitative polymerase chain reaction (qPCR) analysis of mRNA expression in human aortic endothelial cells. Human aortic endothelial cells (hAoECs) (Promocell) were cultured in Endothelial Cell Growth Medium MV (Promocell). The cells were plated in 24-well plates (15 x 10 4 cells / well) at 80% confluence and incubated for 6 h with 100 ng mb 1Lipopolysaccharides (LPS) or functionalized or unfunctionalized protein crystals were incubated. After the incubation period, total RNA was extracted using the RNAeasy mini kit (Qiagen). Reverse transcription was performed using 1 pg of total RNA (600 ng), random hexamers, and Verso reverse transcriptase (Thermo Scientific) according to the manufacturer's instructions. For real-time PCR, gene expression levels were quantified using SYBR Green I dye chemistry on a LightCycler 480 system (Roche Applied Sciences). The following primers were used for the relative quantification of targeted gene expression - for human TNF-alpha: forward primer 5'-GCCCAGGCAGTCAGATCATCT-3' (SEQ ID NO: 8), reverse primer 5'-TTGAGGTTTGCTACAACATGG-3' (SEQ ID NO: 9) and for human beta-actin: forward primer 5'-CAACCGCGAGAAGATGAC-3' (SEQ ID NO: 10), reverse primer 5'-GTCCATCACGATGCCAGT-3' (SEQ ID NO: 11).Data were expressed as mean levels of gene expression relative to the expression of the reference gene (ß-actin).

[0122] Platelet activation assay

[0123] Platelets from three donors were isolated by centrifugation at 260 g for 15 minutes. After a second centrifugation step, platelets were resuspended in HEPES buffer pH 6.6 (10 mM HEPES, 136 mM NaCl, 2.7 mM KCl, 2 mM MgCl, and 5 mM glucose). Platelet suspensions were prepared in the presence of 1:15 acid citrate dextrose (ACD) and 1 U mL' 1 Apyrase was centrifuged again and then resuspended in Hepes buffer pH 7.45 (10 mM Hepes, 136 mM NaCl, 2.7 mM KCl, 2 mM MgCl, 5 mM glucose, and 0.1% BSA). 15 x 10 6 Platelets were incubated for 15 min with 4 nmol L' 1 Thrombin in the presence of 2 mmol L' 1 CaCh or various protein crystals. The platelets were incubated with 4% SDS lysis buffer (200 mmol L' 1Tris, 600 mmol L' 1 NaCl, 4% SDS) including EDTA-free Halt protease inhibitor cocktail (1:10; Sigma-Aldrich) and Halt phosphatase inhibitor cocktail (1:10; Sigma-Aldrich). The protein amount was quantified according to the protocol for the DC protein assay (Bio-Rad). An equal amount of protein from each sample was separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. Anti-p-Akt antibodies (1:1000;

[0124] Cell Signaling) and anti-tubulin (1:1000; Cell Signaling) were used. The blots were incubated overnight at 4°C. A second anti-rabbit antibody (1:1000;

[0125] Cell Signaling) was used and incubated for 1 h at room temperature. Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using Quantity One Software (Bio-Rad Laboratories).

[0126] sequences

[0127] The amino acid sequences of Ftn (neg) -nanoparticle subunits and the cysteine-containing variants Ftn (neg) -3xCys (with 3 cysteine ​​residues) and Ftn (neg) 4xCys (with 4 cysteine ​​residues) are listed below. Variants with only one (Ftn (neg) -lxCys) and two cysteine ​​residues (Ftn (neg) -2xCys) are also shown. For Ftn (neg) The mutations compared to the wild-type H chain (SEQ ID NO: 1) are shown. Mutations compared to Ftn (neg)(SEQ ID NO: 12), including originally present cysteine ​​residues that were altered to alanine residues, are indicated. The cysteine ​​residues in the cysteine-containing variants are underlined.

[0128] Ftn (neg) , (SEQ ID NO: 12): Variant with increased negative charge on the outer surface; A18E, C90E, C102E, K86Q, H105E TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQ SHEEREHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKH TLGDSDNES

[0129] Ftn (neg) -3xCys (SEQ ID NO: 13): Variant with 3 cysteine ​​residues on the inner surface; K53C, E64C, C130A, K143C

[0130] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFACYFLHQ SHEERCHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPHLADFIETHYLNEQVCAIKELGDHVTNLRKMGAPESGLAEYLFDKH TLGDSDNES

[0131] Ftn (neg)-4xCys (SEQ ID NO: 14): variant with 4 cysteine ​​residues on the inner surface;

[0132] K53C, E64C, C130A, K143C, S178C

[0133] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFACYFLHQ SHEERCHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPHLADFIETHYLNEQVCAIKELGDHVTNLRKMGAPESGLAEYLFDKH TLGDCDNES

[0134] Ftn (neg) -lxCys (SEQ ID NO: 15): variant with 1 cysteine ​​residue on the inner surface;

[0135] K53C, C130A

[0136] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFACYFLHQ SHEEREHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKH TLGDSDNES

[0137] Ftn (neg) -2xCys (SEQ ID NO: 16): variant with 2 cysteine ​​residues on the inner surface;

[0138] K53C, E64C, C130A

[0139] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFACYFLHQ SHEERCHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKH TLGDSDNES

[0140] Sequences of other variants examined are shown below. Mutations compared to Ftn (neg) (SEQ ID NO: 12) are indicated. The term "binding site" refers to toxin binding sites formed by simple amino acid exchange (without additional functionalization).

[0141] Ftn (neg) -Ap4 (SEQ ID NO: 17): Variant with reduced negative surface charge in the region of the inner cavity; E61V, E62A, D131F, E140W

[0142] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQ SHVAREHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPHLCFFIETHYLNWQVKAIKELGDHVTNLRKMGAPESGLAEYLFDK HTLGDSDNES

[0143] Ftn (neg)-Ap4-3A (SEQ ID NO: 18): Variant with reduced negative surface charge in

[0144] Area of ​​the inner cavity and pores, as well as increased pore size; E61 V,

[0145] E62A, T122A, D123A, N125A D13 IF, E140W

[0146] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQ SHVAREHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLAAAKADPHLCFFIETHYLNWQVKAIKELGDHVTNLRKMGAPESGLAEYLFDK HTLGDSDNES

[0147] Ftn (neg) -Ap7 (SEQ ID NO: 19): Variant with 7 hydrophobic amino acids on the inner surface; E61V, E62A, H128F, D131W, N139V, E140W, K143V

[0148] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQ SHVAREHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPFLCWFIETHYLVWQVVAIKELGDHVTNLRKMGAPESGLAEYLFDK HTLGDSDNES

[0149] Ftn (neg)-Apl6 (SEQ ID NO: 20): Variant with 16 hydrophobic amino acids on the inner surface; K49V, Y54F, H57W, Q58L, E61V, E62A, H65L, H128F, H136Y, D131W, H136Y N139V, E140W, K143V, E147L, H151Y, N154A

[0150] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALVNFAKFFLWL SHVARELAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLATDKNDPFLCWFIETYYLVWQVVAIKLLGDYVTALRKMGAPESGLAEYLFDK HTLGDSDNES

[0151] Ftn (neg) -dock03 (SEQ ID NO: 21): Variant with a binding site on the inner surface near the triple channel; E64R, H65K, K68E, K71R, Q75D, R76K, H128E, D131E, F132H, T135K, H136R, Y137H, N139R, E140R

[0152] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQ SHEERRKAEELMRLQNDKGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLE LHKLATDKNDPELCEHIEKRHLRRQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHT LGDSDNES

[0153] Ftn (neg)-dock23 (SEQ ID NO: 22): Variant with a binding site on the inner surface in the middle of the subunit; H57E, Q58R, H60I, E61G, E64G, K68D, T135D, H136K, Y137H, N139E, E140K, A144N

[0154] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLER SIGERGHAEDLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLE LHKLATDKNDPHLCDFIEDKHLEKQVKNIKELGDHVTNLRKMGAPESGLAEYLFDKH TLGDSDNES

[0155] Ftn (neg) -dock43 (SEQ ID NO: 23): Variant with a binding site on the inner surface near the E-helix; H57E, Q58R, E64R, H65K, K68E, T135D, H136R, Y137H, N139R, E140R, E147Y

[0156] TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLER SHEERRKAEELMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLE LHKLATDKNDPHLCDFIEDRHLRRQVKAIKYLGDHVTNLRKMGAPESGLAEYLFDKH TLGDSDNES

[0157] Ftn (neg)-Ap4-3A-dock43 (SEQ ID NO: 24): variant with reduced negative surface charge in the region of the inner cavity and at the pores, as well as increased pore size, and also with a binding site on the inner surface near the E-helix; E61V, E62A, T122A, D123A, N125A D131F, H57E, Q58R, E64R, H65K, K68E, T135D, H136R, Y137H, N139R, E140R, E147Y TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLER SHVARRKAEELMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLL ELHKLAAAKADPHLCFFIEDRHLRRQVKAIKYLGDHVTNLRKMGAPESGLAEYLFDK HTLGDSDNES

[0158] Results:

[0159] The assembly into a macroscopic material with the apoferritin nanoparticle of the invention as a building block was achieved by batch crystallization techniques (see above). A protein solution was gently mixed with constant shaking while the precipitant solution was added. After about 24 h, the first crystals could be observed. The size of the crystals can be adjusted by changing the protein and precipitant concentration. To increase stability, the crystals were fixed with a cross-linking agent. Initial experiments were carried out with glutaraldehyde. In stability tests in a 60 mg mL 1 However, in BSA solution, which was chosen to simulate the high protein content of blood, the crystals dissolve. Therefore, stabilization was achieved using a sulfo-SMCC crosslinker, which enabled stable fixation by maintaining the adsorption capacity.

[0160] To improve the adsorption of hydrophobic and partially negatively charged PBUTs, hydrophobic ligands were introduced into the inner cavity of the apoferritin nanoparticles according to the invention. The invention provides a modular material that can be adapted to the type of ligand. To this end, a generic modification site was achieved by incorporating cysteine ​​residues that can be modified by their thiol group as an anchor site for chemical derivatization. On the other hand, native cysteine ​​residues were exchanged for alanine residues to prevent modification at undesired positions. Important design criteria for the introduction of cysteine ​​residues on the cavity surface were the distance between the sites and the solvent-accessible surface area (SASA). A high SASA value should correspond to high reactivity.For this purpose, ferritin variants with three and four introduced cysteines per subunit, designated Ftn. (neg) -3xCys and Ftn (neg) - 4xCys. The total number of anchor sites per assembled protein cage is 72 or 96 with 24 subunits and 3 or 4 cysteine ​​residues. The mutations were introduced into the Ftn( neg) gene was introduced and the variants were overexpressed in E. coli bacteria. Purification followed the published protocol for Ftn (neg)

[0031] In ion exchange chromatography (IEC) and size exclusion chromatography (SEC), no significant change in elution behavior was observed compared to the parent protein. The introduction of the desired mutations was verified by electrospray ionization mass spectrometry (ESI-MS), with the detected mass matching the calculated mass (not shown).

[0161] For the chemical modification of cysteine ​​thiol groups, the a-halogenocarbonyls 2-iodo-N-phenylacetamide (Phe) used. Apoferritin nanoparticles functionalized with the above compounds are designated Ftn(neg)-Phe and Ftn(neg)-C10. The overall strategy for the chemical modification of the internal surface and subsequent assembly into a heterogeneous material is basically as follows: First, the protein cage is disassembled into its subunits under acidic conditions. In the disassembled state, the thiol groups are readily accessible to the haloacetamide derivatives. The molecules to be bound themselves are not soluble in an aqueous solution. Therefore, the functionalization is carried out in a solution containing high proportions of ethanol to facilitate the solubility of the hydrophobic molecules. After incubation of the reactants, the mixture is diluted, and the protein cage is allowed to reassemble.After SEC purification, the resulting chromatogram shows a similar elution volume to the unfunctionalized cage (not shown), indicating complete reassembly of the protein cage. This finding is confirmed in negatively stained TEM images, which show intact cage structures (not shown). SEC of functionalized Ftn. (neg) -4xCys shows a slight shift to a higher elution volume in conjunction with a slightly increased size, which is in good agreement with the results of dynamic light scattering (DLS) measurements.

[0162] To verify the successful and complete functionalization of the cysteine ​​anchor sites, ESLMS measurements were performed. Initial experiments revealed multiple mass peaks per charged species (not shown). Each of these could be assigned to the ferritin subunit containing one to four of the desired molecules, indicating that a mixture of varying degrees of functionalization was present in the sample. Fine-tuning of the reaction conditions, particularly the ethanol content in the mixture, the ratio of reactive molecules to cysteines, and the addition of a reducing agent (TCEP), enabled reproducible, complete functionalization of all sites, as demonstrated by ESI-MS (not shown). Derivatization was also demonstrated by X-ray crystallography of the functionalized ferritin (not shown).Finally, macroscopic crystalline materials were prepared from the functionalized variants under the same conditions described above, indicating that functionalization did not affect the external surface. Subsequently, PBUT adsorption assays were performed with the unfunctionalized protein-based material and the material functionalized with aliphatic or phenyl molecules. For this purpose, the respective sample was incubated in solutions of three different uremic toxins: indoxyl sulfate (IS), p-cresyl sulfate (pCS), and phenylacetic acid (PheAc) at concentrations expected in an end-stage CKD patient [26, 33, 34]. After incubation for 3 h, the PBUT concentration in the supernatant and the respective control samples was determined by HPLC-MS / MS techniques.The absolute values ​​are determined by comparison with a calibration experiment conducted at the beginning of each experimental setup and every 20 samples. In addition, control measurements were taken shortly before each sample to ensure direct comparability. One challenge was the nonspecific adsorption of the IS to the polymer vials of the reaction vessels, which was overcome by switching to glass vials for sample incubation and storage. Finally, the protein-based material was vacuum-dried and weighed to determine its mass. The adsorption capacity, i.e., the ratio of the adsorbed PBUT mass to the total mass of the material, was calculated and is shown in Figure 4a-c.

[0163] Crystals of the unfunctionalized protein Ftn (neg) adsorbed all three tested toxins with a capacity between 247 and 283 pg g 1 for pCS and IS and 2710 pg g 1for PheAc. The reason for the significantly higher PheAc adsorption capacity is most likely the 10-fold higher concentration of PBUT in the assay. For IS and pCS, functionalization with phenyl molecules (Phe) leads to an increase in adsorption capacity to 458 and 372 pg g ' , respectively. For functionalization with the aliphatic molecules (CIO), enhanced adsorption is only observable for IS (Fig. 4a). For the toxin PheAc, no significant enhancement of adsorption could be observed after incorporation of the hydrophobic molecules. To test whether a highly ordered material with a uniform distribution of solvent channels and pores is advantageous for adsorption, the material was compared with samples of non-crystalline protein material prepared by adding a cross-linker to a protein solution and incubating overnight (not shown). The resulting material was tested for its adsorption capacity towards IS.No significant difference was found between the two types of materials, suggesting that the macroscopic shape of the material does not affect the adsorption of the IS. The capacities of the protein-based adsorbent according to the invention are in a similar range, but are smaller than the values ​​of other published materials, for example, P87 zeolites with capacities of up to 1000 pg mL-1

[0035] or carbon-based adsorbents with capacities of up to 3200 pg mL-1

[0036] with respect to IS. To the best of the inventors' knowledge, the adsorbent with the highest capacity published to date is a zirconium-based MOF with a capacity of up to 156 mg g'. 1

[0024] Nevertheless, these materials are inherently good adsorption materials, and efforts must be made to improve their biocompatibility. In contrast, the protein-based material of the present invention exhibits intrinsic biocompatibility. The adsorption capacity can be adjusted and further improved.

[0164] To demonstrate the biocompatibility of the material according to the invention, endothelial cells and isolated platelets were incubated with the crystalline material. Endothelial cells showed no expression of tumor necrosis factor-α (TNF-α), indicating no endotoxin contamination from the bacterial origin of the material (Fig. 4d). Platelets showed no activation, indicating that the material did not induce blood coagulation (Fig. 4c). Similar results were obtained for crystals cross-linked with glutaraldehyde (not shown). Furthermore, no significant difference was observed between the chemically modified and unmodified protein materials, suggesting that functionalization did not affect biocompatibility.

[0165] Overall, the synthesized material according to the invention, based on the bottom-up assembly of protein cages, demonstrates its suitability for blood purification applications. The resulting material exhibits stability in blood-like systems and good adsorption of three PBUTs. Crystalline and non-crystalline adsorption materials exhibit similar behavior. Furthermore, it was shown that by introducing anchor sites, up to 96 water-insoluble aliphatic and phenyl molecules could be incorporated into the cavity of the ferritin protein cage. No decrease in biocompatibility was observed after the modification. An increase in adsorption capacity due to the chemical modifications was observed.By providing ligands other than exclusively hydrophobic ones, such as amphiphilic ligands, a mixture of hydrophobic and hydrophilic ligands, or by modifying the amino acid residues surrounding the ligands, the adsorption capacity can very likely be further increased, since PBUTs exhibit both hydrophobic and hydrophilic properties. The modular nature of the material according to the invention allows adaptation for other applications, such as treatment strategies for heavy metal poisoning through the incorporation of chelating agents. Furthermore, genetic modifications can be used to introduce positively charged amino acids around the anchor sites to satisfy the negative charge of the PBUTs, while binding the hydrophobic part of the toxins to the inserted molecules. All these modifications to the inner surface have no influence on the assembly of the material.

[0166] 2. Modification of the inner surface and channel region of apoferritin nanoparticles according to the invention (without additional chemical functionalization).

[0167] Efficient adsorption to the apoferritin nanoparticles according to the invention can also be achieved by a suitable combination of amino acids with hydrophobic, aromatic, polar, or charged side chains, thereby creating binding sites inside the protein container even in the unfunctionalized protein. This is shown as an example in Figure 5b. Here, the delocalized and hydrophobic ring system of the IS is bound by the ring system of the amino acid tyrosine, while the polar, negatively charged sulfate group is stabilized by partially positively charged nitrogen atoms in the amino acid histidine. To design these protein variants, the binding affinity of the proteins to the toxins was determined using "ligand docking" protocols from the Rosetta software package. Subsequently, various amino acids in the protein were randomly altered in the computer model, and the affinity was determined again.This process was repeated until no further increase in affinity was observed. The protein containers designed in this way were produced in the laboratory, remained soluble, and assembled into complete containers. The introduced mutations were confirmed by ESI-MS measurements. Variants with increased affinity for IS were designed and successfully produced. Adsorbents made from these variants sometimes showed a significantly increased adsorption capacity for the corresponding toxin, as shown in Fig. 5a (Fig. 5b). (neg) - Dock = Ftn (neg) -dock03, SEQ ID NO: 21).

[0168] Furthermore, amino acids of the apoferritin nanoparticle can be specifically exchanged to alter the properties of the apoferritin nanoparticle. For example, by exchanging negatively charged amino acids with hydrophobic amino acids, the negative surface potential in the inner cavity could be reduced. This resulted in a significant increase in the adsorption capacity for the PBUTs, which are also negatively charged under physiological conditions (Figure 5a, Ftn). (neg) -Ap = Ftn (neg) -Ap4, SEQ ID NO: 17). Since the modified region is located near the triple channel, it can be assumed that the increased adsorption is due to improved mass transport into the inner region of the apoferritin nanoparticle. Mass transport into the inner cavity region can be further enhanced by modifications in the region of the

[0169] triple channel. For example, the exchange of amino acids with charged or sterically demanding residues with the amino acid alanine led to a significant increase in the adsorption capacity (Figure 5a, Ftn (neg) -Ap+Channel = Ftn (neg) -Ap4-3 A, SEQ ID NO: 18). Binding sites created by amino acid exchange can be combined with modifications that enhance mass transport to further improve adsorption. Variants with more than one binding site per apoferritin subunit are also possible.

[0170] In the variant Ftn mentioned above (neg)-Ap4 (SEQ ID NO: 17), amino acids with negatively charged side chains, such as glutamic and aspartic acid, were replaced at four positions (E61, E62, D131, and E140) per subunit in the inner cavity with nonpolar or aromatic derivatives (E61V, E62A, D131F, and E140W). Based on the surface potential, a shift in polarity from negative to uncharged was observed (not shown). However, a negatively charged region remained on the inner surface because the responsible amino acids are partially buried in the protein backbone, and mutations at these sites were omitted to maintain protein stability. Since the introduced amino acids carry hydrophobic or even aromatic side chains, it is possible that these residues can also act as adsorption sites for the partially hydrophobic PBUTs.In order to distinguish whether possible effects on adsorption capacities are due to a general reduction of the negative surface charge or to specific adsorption to hydrophobic residues, another variant called Ftn was used. (neg) -Apl6 was developed (SEQ ID NO: 20). As with the Ap4 variant, the four charged amino acids at positions 61, 62, 131, and 140 were replaced by nonpolar derivatives (D at position 131 was replaced by W instead of F), and twelve additional nonpolar amino acids were introduced at surface-exposed positions.

[0171] The adsorption capacities of Ftn (neg) -Ap4 and Ftn (neg) -Apl6 were compared to Ftn (neg)determined for both crystalline and non-crystalline forms (see Fig. 6; crystalline forms left bars; non-crystalline forms right bars). When comparing the adsorption capacities of the crystalline materials towards indoxyl sulfate (IS), no significant difference could be found between unmodified (Ftn (neg ) and modified ferritin (Ftn (neg) -Ap4, Ftn (neg) -Apl6) was observed (see Fig. 6). However, for the non-crystalline material, a significant increase in adsorption capacity was observed for both Ap variants. Despite the 12 additional hydrophobic amino acids, the adsorption capacity of Ftn (neg) -Apl6 similar to that of Ftn (neg)-Ap4. The reduction of the negative surface charge therefore appears to be the main reason for the increased PBUT adsorption. This is consistent with the assumption that the introduction of hydrophobic molecules results in almost no significant improvement in adsorption capacity, and the change in absorption capacity is due to a general reduction of negative charges. In addition to reducing the negative charge in the inner cavity and pores of the protein cage, another strategy was pursued to increase the adsorption of PBUTs. In order to stabilize both the polar and the hydrophobic part of the PBUTs, the creation of a unique binding site by introducing suitable amino acids was sought. For this purpose, ligand docking protocols from the Rosetta software suite (see

[0040] ) were used. IS was used as the ligand. Three other possible ferritin variants were identified: Ftn (neg) -dock03 (SEQ ID NO: 21), Ftn(neg) -dock23 (SEQ ID NO: 22) and Ftn (neg) -dock43 (SEQ ID NO: 22). The binding sites are located near the triple channel (Ftn (neg) -dock03), in the center of the subunit (Ftn (neg) -dock23) and near the 4-way channel (Ftn (neg) -dock43).

[0172] The adsorption capacities of the new variants with respect to IS were determined for crystalline arrangements and are shown in Figure 7. As can be seen from Figure 7, the adsorption capacity for all three variants compared to unmodified Ftn (neg) be significantly improved (Ftn (neg) -03 = Ftn (neg) -dock03, Ftn (neg) -23 = Ftn (neg) -dock23, Ftn (neg) -43 = Ftn (neg) -dock43). The variant Ftn(neg)-03 showed the highest adsorption capacity with a value almost doubled.

[0173] The three variants Ftn developed specifically for IS (neg) -dock03, Ftn (neg) -dock23 and Ftn (neg)- dock43 were also determined with regard to their adsorption capacity towards pCS and PheAc, as shown in Figure 8. Here, an interesting trend could be observed: While for IS, the variants Ftn (neg) -03 and -43 both performed better than Ftn (neg) - 23, cut the variant Ftn (neg) -23 performed better than the other variants in terms of pCS and PheAc adsorption. These results suggest that some selectivity in the binding sites can be introduced by targeting unique features of the toxins.

[0174] Furthermore, it was tested whether the different features could be combined in one structure to increase the binding capacity of the proteins. The mutations that change the binding site in Ftn (neg) -43 were combined with the mutations of Ftn (neg)-Ap4-3 A combined to reduce the surface charge on the inner cavity and the triple channel, resulting in the variant Ftn (neg) -Ap4-3A-43.

[0175] The adsorption capacity of this variant was determined for the adsorbent material with non-crystalline morphology and compared with the results for unmodified Ftn (neg) and the previously mentioned variants were compared (Figure 9). The measured values ​​indicate that individual positive effects due to different modifications can be added together by combining the mutations in one structure. The variant Ftn (neg) -Ap4-3 A-43 showed the highest adsorption capacities of all variants investigated in this study.

[0176] The advantage of simply changing the properties of the apoferritin nanoparticles according to the invention through amino acid exchange is that the proteins can be used without further chemical modification. This reduces the likelihood of allergic reactions or other side effects and makes them more cost-effective to produce than functionalized variants. Stronger binding sites can be designed. Furthermore, the present invention opens up the possibility of providing further apoferritin nanoparticles or apoferritin nanoparticle compositions with further improved affinity, optionally adapted to specific toxins.

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Claims

PATENT CLAIMS 1. Apoferritin nanoparticles for use as a toxin binder in hemodialysis, wherein the apoferritin nanoparticle comprises apoferritin subunits which, when assembled into the apoferritin nanoparticle, form an internal cavity, and wherein the apoferritin nanoparticle does not contain any nanoparticles in its internal cavity.

2. Apoferritin nanoparticles for use as toxin binders in hemodialysis according to claim 1, wherein at least one of the apoferritin subunits in the region of the inner cavity and / or in the region of a triple channel and / or in the region of a quadruple channel has an amino acid sequence altered compared to the wild-type sequence.

3. Apoferritin nanoparticles for use as toxin binders in hemodialysis according to claim 2, wherein the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence has, in the region of the inner cavity, at least one cysteine ​​residue additional to the wild-type sequence or arranged at a different position in the sequence, and wherein the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence is functionalized or functionalizable by covalent bonding of an organic compound residue to the cysteine ​​residue.

4. Apoferritin nanoparticles for use as toxin binders in hemodialysis according to claim 3, wherein the at least one apoferritin subunit with an amino acid sequence modified from the wild-type sequence has, in the region of the inner cavity, two, three or four cysteine ​​residues to each of which an organic compound residue is covalently bound.

5. Apoferritin nanoparticles for use as toxin binders in hemodialysis according to claim 3 or 4, wherein the organic ligand covalently bound to the at least one additional cysteine ​​residue or to another position in the sequence Verbindungsrest ausgewählt ist aus der Gruppe bestehend aus N-phenylacetamidyl, N- decylacetamidyl, N-(l-amino-2-phenylethyl)acetamidyl, N-(l-amino-2- phenylpropyl)acetamidyl, N-(l-amino-2-phenylbutyll)acetamidyl, N-(l, l-diamino-2- phenylethyl)acetamidyl, N-(l, l-diamino-2-phenylpropyl)acetamidyl, N-(l, l-diamino-2- phenylbutyll)acetamidyl, N-(3,4,5-tris(trifluoromethyl)phenyl)acetamidyl, N-(3,4,5- trinitrophenyl)acetamidyl, N-(l-amino-2-(3,4,5-trinitrophenyl)ethyl)acetamidyl und N-(2- (heptylamino)ethyl)acetamidyl.

6. Apoferritin nanoparticles for use as toxin binding agents in hemodialysis according to one of the preceding claims, wherein the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence is modified in the region of the cavity in its amino acid sequence compared to the wild-type sequence such that the apoferritin nanoparticle has a higher binding affinity to a toxin compared to an apoferritin nanoparticle composed of apoferritin subunits with an amino acid sequence unchanged compared to the wild-type sequence.

7. Apoferritin nanoparticles for use as toxin binding agents in hemodialysis according to one of the preceding claims, wherein the at least one apoferritin subunit with an amino acid sequence modified compared to the wild-type sequence is modified in the region of a triple channel in its amino acid sequence compared to the wild-type sequence such that the transport of a toxin into the cavity of the apoferritin nanoparticle is facilitated compared to an apoferritin nanoparticle with an unchanged amino acid sequence.

8. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of the preceding claims, wherein the toxin is a protein-bound uremic toxin, preferably selected from the group consisting of indoxyl sulfate, para-cresyl sulfate, phenyl acetate, and p-hydroxyhippuric acid.

9. Apoferritin nanoparticles for use as toxin binders in hemodialysis according to one of the preceding claims, wherein the apoferritin nanoparticle is composed of 24 apoferritin subunits, and at least two, preferably at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10, more preferably at least 12, at least 14, at least 16, at least 18, at least 20 or at least 22, particularly preferably all 24 subunits have an amino acid sequence which is altered compared to the wild-type sequence.

10. Apoferritin nanoparticles for use as toxin binders in hemodialysis according to claim 9, wherein the subunits have an amino acid sequence modified from the wild-type sequence, which is selected from one of the amino acid sequences according to SEQ ID NO: 13-24, preferably from one of the amino acid sequences according to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, or SEQ ID NO:

24.

11. A composition for use as a toxin binder in hemodialysis, comprising a plurality of apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of claims 1 to 10.

12. A composition for use as a toxin binder in hemodialysis according to claim 11, wherein the plurality of apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of claims 1 to 10 are cross-linked to one another.

13. A sorption cartridge comprising in its interior a plurality of apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of claims 1 to 10 or a composition for use as a toxin binder in hemodialysis according to any one of claims 11 or 12.

14. Sorption cartridge according to claim 13, wherein the plurality of apoferritin nanoparticles or the composition are in a first compartment within the sorption cartridge which is separated from a second compartment within the sorption cartridge by one or more membranes, wherein the membrane or membranes are permeable to unbound toxin molecules and / or to proteins with toxin molecules bound thereto, but not to the apoferritin nanoparticles.

15. Dialysis system comprising a dialyzer and at least one sorption cartridge according to one of claims 13 or 14.

16. Dialysis system according to claim 15, wherein the at least one sorption cartridge is arranged in the flow direction of the blood to be dialyzed a) upstream of the dialyzer, b) downstream of the dialyzer or c) in a secondary circuit to the dialysis circuit.

Images