Novel toxin-binding agents for hemodialysis.

Abstract

The present invention relates to a novel toxin binder for hemodialysis. The present invention relates to apoferritin nanoparticles for use as a toxin binder in hemodialysis. The apoferritin nanoparticles contain apoferritin subunits that, when assembled into apoferritin nanoparticles, form an internal space, but do not contain nanoparticles. The present invention also relates to a composition, a sorbent cartridge, and a dialysis system.

Description

[Technical Field]

[0001] The present invention relates to novel toxin-binding agents for hemodialysis. [Background technology]

[0002] Approximately 9% of the world's population suffers from chronic kidney disease (CKD) of varying severity [Non-Patent Document 1]. As the disease progresses, the likelihood of developing cardiovascular disease increases significantly [Non-Patent Document 2]. As a result, 1.2 million people died directly from CKD in 2017, and an additional 1.4 million died from cardiovascular disease attributable to CKD [Non-Patent Document 1]. High cardiovascular risk is attributed to various uremic toxins, such as indoxyl sulfate (IS) and paracresyl sulfate (pCS) [Non-Patent Documents 3-10]. Conventional hemodialysis methods only partially separate these toxins from plasma, even with prolonged or frequent treatments [Non-Patent Document 11]. Because the toxins are partially hydrophobic, they bind to plasma proteins (e.g., serum albumin), preventing their efficient diffusion through the pores of the dialysis membrane [Non-Patent Documents 12, 13]. These toxins are therefore known as protein-bound uremic toxins (PBUT). On average, 95% of IS and pCS are bound to proteins

[14] , which reduces the separation efficiency during hemodialysis.

[0003] One approach to improving the separation of PBUT is the administration of oral or intravenous adjuvants. Intravenous adjuvants aim to remove toxins from plasma protein binding sites, thereby increasing the soluble fraction and the fraction that can be separated by dialysis. This can be achieved, for example, with ibuprofen ((RS)-2-(4-isobutylphenyl)propanoic acid) [Non-Patent Document 15], mesna (sodium 2-sulfanylethanesulfonate) [Non-Patent Document 16], or acetylcysteine ​​[Non-Patent Document 17]. However, effective separation of pCS and IS has only been demonstrated with ibuprofen. Oral adjuvants, such as the carbon-based AST-120 (Kremezin®), adsorb toxins generated by microbial activity in the gut before they enter the bloodstream [Non-Patent Document 18] or, like the synbiotic NatuREN G, inhibit the production of these toxins [Non-Patent Document 19]. In a systematic comparison of all studies on the reduction of protein-bound uremic toxins up to January 2, 2020 [Non-Patent Document 20], the aforementioned methods demonstrated a maximum 44% reduction in the toxins IS and pCS [Non-Patent Document 18]. However, fractional plasma separation, adsorption, and dialysis (FPAD) has achieved significantly higher reduction rates of 71–78% [Non-Patent Documents 20, 21]. This indicates that direct adsorption of protein-bound toxins is one of the most effective methods for reducing toxins in a patient's plasma. However, the drawback of this system is the complex plasma separation and filtration required to separate albumin from the remaining blood components, which must not come into contact with the adsorbent material [Non-Patent Document 22], making it very expensive for routine use.

[0004] There remains a lack of effective adsorbent materials for PBUT that are sufficiently biocompatible and hemocompatible to allow direct contact of PBUT with blood and thus facilitate its integration into conventional hemodialysis treatment. Current PBUT adsorption methods do not combine a) high affinity for toxins with b) favorable hemocompatibility and biocompatibility. The prior art contains only studies on conventional adsorbent materials, such as activated carbon [Non-Patent Document 23], metal-organic frameworks [Non-Patent Document 24], or zeolites [Non-Patent Document 25], but these studies mainly concern the affinity of these materials for uremic toxins. Only a few studies have considered the biocompatibility of the materials [Non-Patent Document 23].

[0005] Patent Document 1 describes charged protein containers for constructing nanostructured materials, constructed from ferritin complexes capable of encapsulating nanoparticles within. Such ferritin complexes containing nanoparticles are also intended for use in dialysis, particularly for removing hydrophobic toxins. However, no relevant research has been described. Patent Document 2 describes nanoparticles containing protein cages, such as bacterial ferritin-like protein cages, which contain at least one guest material, such as a metal, within their interior. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] DE 10 2016 108 017 [Patent Document 2] International Publication No. 2004 / 001019 [Non-patent literature]

[0007] [Non-Patent Document 1] Bikbov,B.et al.,2020,Global,regional,and national burden of chronic kidney disease,1990-2017:a systematic analysis for the Global Burden of Disease Study 2017,The Lancet 395,709-733 [Non-patent document 2] Schlieper, G., Hess, K, Floege J., Marx, N., 2016, The vulnerable patient with chronic kidney disease, Nephrology Dialysis Transplantation 31, 382-390, doi.org / 10.1093 / ndt / gfv041 [Non-patent document 3] Ito, S.; Yoshida, M. Protein-Bound Uremic Toxins: New Culprits of Cardiovascular Events in Chronic Kidney Disease Patients.Toxins 2014,6,665-678.doi.org / 10.3390 / toxins6020665 [Non-patent document 4] Karbowska,M.;Kaminski,TW;Marcinczyk,N.;Misztal,T.;Rusak,T.;Smyk,L.;Pawlak,D.The Uremic Toxin Indoxyl Sulfate Accelerates Thrombotic Response after Vascular Injury in Animal Models.Toxins 2017,9,229.doi.org / 10.3390 / toxins9070229 [Non-Patent Document 5] Dou L.,Bertrand,E.,Cerini,C.,Faure,V.,Sampol,J.,Vanholder,R.,Berland,Y,,Brunet,P.,2004,The uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation and wound repair,Kidney International 65,442–451,doi.org / 10.1111 / j.1523–1755.2004.00399.x

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[0008] The object of the present invention is to provide a sorbent for separating protein-bound uremic toxins (PBUT) during dialysis, which has as high an affinity as possible for PBUT, but is sufficiently biocompatible and hemocompatible to be able to come into direct contact with blood. [Means for solving the problem]

[0009] In a first aspect, the present invention achieves this object by providing apoferritin nanoparticles for use as a toxin binder in hemodialysis, the apoferritin nanoparticles comprising apoferritin subunits that form an internal space when assembled into apoferritin nanoparticles, and that do not contain nanoparticles in the internal space.

[0010] Surprisingly, apoferritin nanoparticles have been shown to be highly suitable for use as adsorbents for toxins in blood, for example, for separating protein-bound uremic toxins (PBUT). The apoferritin nanoparticles according to the present invention have internal spaces (hereinafter also referred to as cavities) in which toxins can be bound, and furthermore, have high biocompatibility and hemocompatibility. They are therefore highly suitable for removing toxins, such as PBUT, or other toxins, such as heavy metals, from blood during dialysis, e.g., hemodialysis. They can thus be advantageously used, for example, in the treatment and / or alleviation of the consequences of chronic kidney disease (CKD). The protein shell of the apoferritin nanoparticles according to the present invention has channels or pores that allow small molecules, such as PBUT, to enter the interior of the particle but prevent macromolecules, such as proteins and cells, from entering. The affinity of ferritin nanoparticles of the present invention for toxins can be specifically tailored and optimized, for example, by altering the amino acid sequence of at least one subunit within the interior space, e.g., to ensure a suitable number of hydrophobic amino acid residues or a suitable mixture of hydrophobic and hydrophilic amino acid residues, or by functionalizing one or more amino acid residues with chemical groups. Furthermore, transport of substances into ferritin nanoparticles can be improved by enlarging or modifying the surface charge in the pore region, e.g., by specifically exchanging sterically hindered or charged amino acids with smaller hydrophobic amino acids. Thus, the interior surface and pore region of the nanoparticle (protein cage) can be altered by sequence modification or chemical functionalization to increase affinity for toxins and / or facilitate toxin transport into the nanoparticle without compromising assembly or biocompatibility. Furthermore, protein cages can also be constructed into well-defined crystalline materials with uniformly distributed solvent channels. This ensures high material purity, facilitates handling and use as adsorbents, and aids in material characterization.

[0011] The term "ferritin" (abbreviated as "Ftn") refers to a granular protein complex (globular protein) composed of 24 protein subunits that form a hollow nanocage and naturally bind iron. The term "apoferritin" or "apoferritin nanoparticle" refers to the protein complex without iron binding. This protein complex has an internal cavity with a diameter of approximately 6-8 nm and an external diameter of approximately 12-13 nm (see, for example, [Non-Patent Documents 28, 29]). Apoferritin can be composed of identical subunits, such as heavy chains (H chains), or various ratios of light chains (L chains) and heavy chains (H chains). The human ferritin heavy chain (hFTN-H, HFt, or FTH) is encoded by the FTH1 gene and is a naturally occurring protein of approximately 21 kDa containing 183 amino acids (UniProtKB No. P02794, 2007-01-23 v2; see NCBI NP_002023.2). The human ferritin light light chain (hFTN-L, LFt, or FTL) is encoded by the FTL gene and is a naturally occurring protein of approximately 19.5 kDa containing 175 amino acids (UniProtKB No. P02792, 2007-01-23 v2; see NCBI NP_000137.2). Both the light and heavy chains have a tertiary structure consisting of four bundled α-helices (A, B, C, and D), with the B and C helices connected via a non-helical loop (BC loop) consisting of 18 amino acids. Furthermore, a fifth α-helix (E) is present at the C-terminus, protruding into space. The N-terminus and the A and C helices are located on the outside of ferritin, while the B and D helices are oriented on the inside of ferritin. Ferritin has channels or pores in its protein shell that connect the internal space with the external environment ([Non-Patent Document 28]). In this regard, due to the spatial arrangement of apoferritin subunits, triple channels (triaxial channels) are formed at the contact sites between three apoferritin subunits, and quadruple channels (tetraaxial channels) are formed at the contact sites between four apoferritin subunits (see, for example, [Non-Patent Document 28]).Apoferritin nanoparticles, which are typically constructed from 24 subunits, contain eight triple channels and six quadruple channels (see, for example, [Non-Patent Document 28]).

[0012] The term "human H-chain ferritin" or "human heavy-chain ferritin" (abbreviated as HuHF) refers to human ferritin whose subunits are composed only of human heavy chains, rather than a mixture of heavy (H) and light (L) chains. This term also encompasses H-chain variants whose amino acid sequences have been altered relative 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 heavy chain of human ferritin).

[0013] The term "toxin-binding agent" in relation to apoferritin nanoparticles means that apoferritin nanoparticles bind to toxins in their space, i.e., internal cavity, so that the toxins can be removed from body fluids, such as blood, while bound to the apoferritin nanoparticles. In this regard, the bond between the toxin and the apoferritin nanoparticles is preferably a non-covalent bond, and can be formed, for example, by hydrophobic interaction, electrostatic interaction, or hydrogen bond. Examples of possible toxins include protein-bound uremic toxins such as indoxyl sulfate (IS) and paracresyl sulfate (pCS), organic heavy metal compounds, heavy metal ions, and other chemicals and compounds, which preferably have the property of being able to bind by non-covalent bonds. However, covalent binding is not excluded.

[0014] In principle, the term "protein-bound uremic toxins" or "PBUTs" refers to compounds that are poorly soluble in water (hydrophobic) and present in serum bound to proteins. Examples of PBUTs include indoxyl sulfate (IS), paracresyl sulfate (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-furanpropanoic acid (CMPF), and p-cresyl glucuronide (see, for example, [Non-Patent Document 37], while [Non-Patent Document 41] lists 33 PBUTs).

[0015] The term "hemodialysis" refers to an extracorporeal blood treatment that removes metabolic waste products (e.g., urea, uric acid, creatinine, excess phosphate, etc.) and / or excess fluid from the blood. Hemodialysis is used regularly in people whose kidneys no longer function properly, for example, in chronic kidney disease (CKD). The phrase "for use in hemodialysis" encompasses use in the context of hemeapheresis, particularly whole blood apheresis. The term "hemeapheresis" refers to a method for the extracorporeal removal of specific blood components, or pathogenic substances, microorganisms, etc., from blood (see, for example, [Non-Patent Document 42]).

[0016] The term "chronic kidney disease" (CKD) refers to a chronic disease of the kidneys that involves a gradual and long-term (3 months or longer) decline in kidney function leading to permanent kidney insufficiency or complete kidney failure (ICD-10 code: N18). In particular, as used herein, the term relates to stage 5 chronic kidney disease (ICD-10 code: N18.5), in which the glomerular filtration rate (GFR) is less than 15 mL / min and renal replacement therapy is required to purify the blood. As used herein, the term "chronic renal insufficiency" is synonymous with the term "chronic kidney disease."

[0017] The expression that an apoferritin nanoparticle "does not contain nanoparticles in its interior space" means that the interior space of the apoferritin nanoparticle is empty, i.e., does not contain separate nanoparticles designed to bind, for example, toxins.

[0018] As used herein, the term "within a region of the internal space" in reference to an apoferritin subunit refers to portions of the apoferritin subunit, particularly amino acid residues of this apoferritin subunit that are oriented in the internal space of the apoferritin nanoparticle, specifically the B helix (amino acids 50-77 of the heavy chain of human apoferritin), the D helix (amino acids 128-159 of the heavy chain of human apoferritin), the E helix (amino acids 165-174 of the heavy chain of human apoferritin), and the C-terminal region (175-183) following the E helix.

[0019] As used herein, the term "within the triple channel region" in relation to an apoferritin subunit refers to a portion of the apoferritin subunit, particularly an amino acid residue of this apoferritin subunit located in the triple channel of the constructed apoferritin nanoparticle, or an amino acid residue that directly affects toxin transport into the apoferritin nanoparticle; that is, replacing this residue leads to enhanced toxin transport into the apoferritin nanoparticle. For example, this may refer to the amino acids at the end of the C helix and the beginning of the D helix (e.g., amino acids 118-141, preferably amino acids 120-140, in the heavy chain of human apoferritin). This term also encompasses amino acids outside the apoferritin subunit. As used herein, "enhanced toxin transport" (or "improved toxin transport") means that, when incubated with a given toxin concentration under suitable conditions for a certain period of time, more toxin molecules reach the space in apoferritin nanoparticles modified within the triple channel region than in apoferritin nanoparticles not modified within the triple channel region. The term "pore" as sometimes used herein is synonymous with the term "triple channel."

[0020] As used herein, the term "within the quadruple channel region" in relation to an apoferritin subunit refers to a portion of the apoferritin subunit, particularly the amino acid residues of this apoferritin subunit located in the quadruple channel of an apoferritin nanoparticle. As an example, amino acids M158, L165, Y168, L169, and H173 are located within the quadruple channel region (see, for example, [Non-Patent Document 41]). This term encompasses amino acid residues of each subunit that are located in the quadruple channel of an apoferritin nanoparticle in an assembled apoferritin nanoparticle, for example, lining the inner surface of the quadruple channel of the assembled apoferritin nanoparticle or present at the opening of the channel, or amino acid residues that directly affect the shape, e.g., diameter, or physicochemical properties of the channel.

[0021] The term "redesigned apoferritin nanoparticle" or "redesigned apoferritin subunit" is used herein in reference to apoferritin nanoparticles or apoferritin subunits having an amino acid sequence that is modified relative to the wild-type sequence. This term encompasses apoferritin nanoparticles or apoferritin subunits having a modified amino acid sequence in which some or all of the replaced amino acid residues are additionally functionalized with chemical groups.

[0022] The term "functionalized" means that an additional chemical group or compound residue is covalently attached to a suitable amino acid residue, such as cysteine. An example is a cysteine ​​residue with an N-phenylacetamide residue attached to the thiol group (SH group).

[0023] The term "organic compound residue" refers to a carbon-containing chemical side group or molecule covalently attached to, for example, a cysteine ​​residue. It may be an aliphatic or aromatic compound residue; the term is not understood herein to be limited to purely hydrocarbon compounds, but also encompasses heteroaliphatic or heteroaromatic compound residues, i.e., compound residues with heteroatoms such as nitrogen, oxygen, phosphorus, or sulfur. The term "aromatic compound residue" refers to any compound residue with an aromatic or heteroaromatic group. The term "aliphatic compound residue" refers to any organic compound residue that is an aliphatic or heteroaliphatic compound residue, i.e., does not contain an aromatic group.

[0024] The term "aliphatic residue" includes cyclic or acyclic, linear (straight-chain) or branched, saturated or unsaturated carbon residues that are not aromatic. The term "heteroaliphatic residue" refers to an aliphatic residue in which one or more C atoms of the carbon skeleton are replaced by a heteroatom, such as oxygen, sulfur, nitrogen, or phosphorus.

[0025] The term "aromatic residue" (also called "aryl") refers to a chemical group having aromatic character, including polycyclic aromatic groups with many members and polycyclic ring systems with at least one aromatic ring. Examples of aromatic residues (aryl groups) are benzyl and phenyl. The term "heteroaromatic residue" (also called heteroaryl) refers to an aromatic residue in which one or more C atoms of the carbon skeleton are replaced by a heteroatom, such as oxygen, sulfur, nitrogen, or phosphorus.

[0026] The term "dialyzer" refers to a replaceable blood purification unit of a dialysis system. As an example, the dialyzer may be a hollow fiber membrane module in which hollow fiber-shaped biocompatible semipermeable polymer membranes are arranged in a housing, so that, for example, blood flows through the hollow fibers, and dialysate flows through the inside of the housing along the outside of the hollow fibers (e.g., in the opposite direction to the blood flow), and preferably low-molecular-weight compounds such as salts, protein fragments, or nucleic acid fragments diffuse from the blood through the membrane into the dialysate and can be removed by the dialysate.

[0027] For example, reference to a range "1 to 10" means that all intermediate values ​​are also disclosed. Where a reference can only include integers, such as the number of atoms, it also means that only the integers are disclosed. Any narrower ranges that fall within a broader range are also disclosed by reference to the broader range, and such narrower ranges include ranges that do not include any of the limits of the broader range (e.g., a range of 2 to 5 within a range of 1 to 10).

[0028] In this specification, amino acids are represented by three-letter or one-letter codes as needed. The list of amino acids and their corresponding codes is as follows: Amino acid: 3-letter code: 1-letter code Alanine: Ala: A Arginine: Arg:R Asparagine:Asn:N Aspartic acid: Asp:D Cysteine:Cys:C Glutamine:Gln:Q Glutamic acid: Glu:E Glycine:Gly:G Histidine:His:H Isoleucine: Ile:I Leucine:Leu:L Lysine: Lys:K Methionine:Met:M Phenylalanine:Phe:F Pro Line: Pro:P Serine:Ser:S Threonine: Thr:T Tryptophan:Trp:W Tyrosine:Tyr:Y Valin:Val:V

[0029] Mutations in protein sequences in the form of amino acid exchanges are represented by the format X1PX2, where X1 denotes the original amino acid (single letter code) that is exchanged at position P, X2 denotes the amino acid (single letter code) that replaces the original amino acid at the same position after the exchange, and P denotes the position in the amino acid sequence where the exchange took place.

[0030] Apoferritin nanoparticles for use as toxin binders in hemodialysis may have an amino acid sequence modified relative to the wild-type sequence in the region of the internal space, for example, a cysteine ​​residue at a position where no cysteine ​​residue is present in the wild-type sequence. The cysteine ​​residue may be further functionalized. Alternatively or additionally, apoferritin nanoparticles for use as toxin binders in hemodialysis may have an amino acid sequence modified relative to the wild-type sequence in the region of the triple or quadruple channel, for example, by amino acid exchange, to facilitate toxin transport into the space of the apoferritin nanoparticles.

[0031] In a preferred embodiment, at least one of the apoferritin subunits of the apoferritin nanoparticle has an amino acid sequence within a region of the interior space that is modified relative to the wild-type sequence. n L mwhere 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 modified relative to the respective wild-type sequences. If at least one of the apoferritin subunits having a modified amino acid sequence is a heavy chain subunit, it is modified relative to the wild-type heavy chain sequence, and if at least one of the apoferritin subunits having a modified amino acid sequence is a light chain subunit, it is modified relative to the wild-type light chain sequence. For human apoferritin nanoparticles, i.e., apoferritin nanoparticles consisting of each human subunit, the relevant reference is the wild-type sequence of each human subunit (for the heavy chain, see UniProtKB No. P02794, 2007-01-23 v2 or NCBI NP_002023.2; for the light chain, see UniProtKB No. P02792, 2007-01-23 v2 or NCBI NP_000137.2). In the present application, the wild-type sequence of the human heavy chain is also referred to as the sequence having SEQ ID NO: 1, and the wild-type sequence of the human light chain is also referred to as the sequence having SEQ ID NO: 2, with the N-terminal methionine omitted to distinguish it from the aforementioned database sequences, since it is not part of the sequence of the completed protein.

[0032] SEQ ID NO: 1: Human ferritin heavy chain TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0033] SEQ ID NO: 2: Human ferritin light chain SSQIRQNYSTDVEAAVNSLVNLYLQASYTYLSLGFYFDRDDVALEGVSHFFRELAEEKREGYERLLKMQNQRGGRALFQDIKKPAEDEWGKTPDAMKAAMALEKKLNQALLDLHALGSARTDPHLCDFLETHFLDEEVKLIKKMGDHLTNLHRLGGPEAGLGEYLFERLTLKHD

[0034] In a preferred embodiment of the apoferritin nanoparticles according to the present invention for use as a toxin binder in hemodialysis, the amino acid sequence of at least one apoferritin subunit having an amino acid sequence modified relative to the wild-type sequence is modified relative to the wild-type sequence in a spatial region so that the apoferritin nanoparticles have a higher binding affinity for toxins compared to apoferritin nanoparticles made from apoferritin subunits having an amino acid sequence that is not modified relative to the wild-type sequence. In this embodiment of the apoferritin nanoparticles according to the present invention, the modification to the amino acid sequence of at least one apoferritin subunit in the internal spatial region is preferably designed so that the binding affinity of apoferritin nanoparticles having at least one modified subunit in its amino acid sequence for toxins to be removed from blood is higher than that of apoferritin nanoparticles having a subunit whose amino acid sequence is not modified relative to the wild-type sequence. As an example, the modification may be an increase in the number of hydrophobic amino acid residues. Examples of amino acids with hydrophobic amino acid residues include alanine, valine, methionine, leucine, isoleucine, proline, tryptophan, and phenylalanine. However, for binding to a specific toxin, it may be advantageous to have a higher proportion of polar / neutral, acidic, or basic amino acids in at least one apoferritin subunit. 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. For binding to a specific toxin, it may also be advantageous to provide a specific mixture and / or distribution of amino acids with hydrophobic and polar / neutral amino acid residues. The binding affinity of such modified apoferritin nanoparticles relative to unmodified apoferritin nanoparticles for toxins such as PBUT can be easily determined by those skilled in the art.

[0035] In a preferred embodiment of the apoferritin nanoparticles of the present invention for use as a toxin binder in hemodialysis, at least one apoferritin subunit in the region of the internal space has an amino acid sequence modified relative to the wild-type sequence, has at least one additional cysteine ​​residue or a cysteine ​​residue at a different position in the sequence compared to the wild-type sequence, and at least one apoferritin subunit having an amino acid sequence modified relative to the wild-type sequence is functionalized or can be functionalized by covalent bonding of an organic compound residue, for example, an aliphatic or aromatic compound residue, with the cysteine ​​residue.Functionalization preferably serves to increase the binding affinity of the apoferritin nanoparticles of the present invention for toxins to be removed from blood, for example, protein-bound uremic toxins.

[0036] Particularly preferably, in at least one apoferritin subunit having an amino acid sequence modified relative to wild-type sequence, the cysteine ​​residues that may naturally occur in the region of the internal space are replaced by other amino acid residues, for example, alanine residues.This allows the specific positioning of the introduced cysteine ​​residues, and thus the specific positioning of the site that can be functionalized in the apoferritin subunit according to the present invention.Preferably, this allows the cysteine ​​residues that may exist in the region of the internal space in at least one apoferritin subunit having an amino acid sequence modified relative to wild-type sequence to be replaced by other amino acid residues, for example, alanine residues, and at least one cysteine ​​residue is introduced into the position in the region of the internal space where no cysteine ​​residue exists in natural protein.

[0037] Particularly preferably, in apoferritin nanoparticles according to the present invention for use as toxin binders in hemodialysis, at least one apoferritin subunit having an amino acid sequence modified relative to the wild-type sequence has, compared to the wild-type sequence, two, three, or four cysteine ​​residues in the region of the internal space to which the respective organic compound residues are or can be covalently bound. Preferably, in this embodiment, there are three or four cysteine ​​residues in the region of the internal space, each of which can be functionalized in the same or different manner. If all 24 subunits of an apoferritin nanoparticle according to the present invention are or can be functionalized in this manner, this results in 72 to 96 functionalization sites, i.e., sites within the apoferritin nanoparticle that can or can be functionalized. As mentioned above, preferably, each cysteine ​​residue of a native protein that may be present in the region of the internal space is replaced with, for example, an alanine residue, 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 on the apoferritin nanoparticles according to the invention may be functionalized in the same or different ways, i.e. each with the same or different chemical groups.

[0038] The organic compound residue may be a hydrophobic, hydrophilic, or amphiphilic compound residue. Hydrophobic compound residues are preferred for binding, for example, to hydrophobic uremic toxins. However, for binding to hydrophobic uremic toxins, it may be advantageous to provide an amphiphilic compound residue for functionalization or to functionalize different cysteine ​​residues within the interior space with different compound residues, for example, a mixture of hydrophilic and hydrophobic compound residues. Adjusting the hydrophobicity or hydrophilicity of the compound residue is known to those skilled in the art and can be determined by routine experimentation, as needed. For example, the hydrophobicity of the compound residue may be influenced by the size or length of the hydrocarbon residue, and the hydrophilicity may be influenced by the presence of polar or charged residues.

[0039] The organic compound residue may be, for example, an acetamidyl compound having the general structure -CH2-C(O)-NH-R. The residue R may be an aliphatic, heteroaliphatic, aromatic, or heteroaromatic residue. For aliphatic and heteroaliphatic residues, the maximum chain length, i.e., the maximum number of atoms constituting the compound residue, is preferably 35 atoms, with a maximum chain length of 34, 33, 32, 31, or 30 atoms being particularly preferred. For aromatic and heteroaromatic residues, electron-deficient aromatic systems are preferred, allowing for effective π-π interactions with the electron-rich aromatic system of the toxin. Examples of electron-deficient aromatic residues are 3,4,5-trinitrophenyl, 3,4,5-trihalogenyl (e.g., 3,4,5-trifluorophenyl), or 3,4,5-tris(trihalogenomethyl)phenyl residues (e.g., 3,4,5-tris(trifluoromethyl)phenyl residues). Examples of electron-deficient heteroaromatic groups are pyridine, quinoline, or isoquinoline, and their electron-deficient nature can be further enhanced by electron-withdrawing substituents such as nitro groups, tertiary amine groups, positively charged groups, or halogen atoms. For aromatic and heteroaromatic residues, a maximum molecular size of 45 atoms is preferred, preferably 44, 43, 42, 41, or 40 atoms, i.e., residues consisting of up to 45 atoms, including heteroatoms, preferably 44, 43, 42, 41, or 40 atoms are preferred. For aromatic and heteroaromatic residues, a maximum molecular size of 39 atoms is preferred, with a maximum of 38, 37, 36, 35, 34, 33, 32, 31, or 30 atoms being particularly preferred. Because a very high proportion of PBUTs has a formal negative charge under physiological conditions, amphiphilic compound residues with a combination of hydrophobic and positively charged groups are preferred. Examples of groups that are positively charged under physiological conditions include primary, secondary, tertiary, and quaternary amines. For heteroaliphatic compounds, these groups are preferably contained in a chain. Depending on the shape of the toxin to be attached, these groups can be incorporated into alternating chains, preferably with a maximum chain length of no more than 35 atoms, particularly preferably 34, 33, 32, 31, or 30 atoms. For aromatic and heteroaromatic residues, the charged groups are preferably arranged with suitable symmetry about the aromatic or heteroaromatic system.Again, it is preferred that the maximum molecular size of the compound residue not be exceeded, preferably a molecular size of 45, preferably 44, 43, 42, 41, or 40 atoms, more preferably a molecular size of 39 atoms, and particularly preferably 38, 37, 36, 35, 34, 33, 32, 31, or 30 atoms. For toxins such as p-cresyl sulfate or phenylacetic acid, for example, 1-amino-2-phenylethyl or 1,1-diamino-2-phenylethyl residues are suitable. Preferred compound residues for conjugation with protein-bound uremic toxins that can be or are covalently bound to at least one additional cysteine ​​residue or to cysteine ​​residues located at different positions in the sequence are N-phenylacetamidyl, N-decylacetamidyl, N-(1-amino-2-phenylethyl)acetamidyl, N-(1-amino-2-phenylpropyl)acetamidyl, N-(1-amino-2-phenylbutyl)acetamidyl, N-(1,1-diamino-2-phenylbutyl)acetamidyl, N-(1,1-diamino-2-phenylethyl ...ethyl)acetamidyl, N-(1,1-diamino-2-phenylbutyl)acetamidyl The compounds listed are preferably covalently linked to cysteine ​​residues via the acetamide group. For this purpose, halogenated acetamide derivatives may be used, which react with the thiol group of cysteine. Furthermore, it is obviously possible to use other suitable coupling groups, such as haloalkyl or maleimide derivatives, for binding to the thiol group.

[0040] The organic compound residue may be a chelating agent that binds to heavy metals, such as lead, antimony, arsenic, cadmium, nickel, mercury, thallium, or uranium. Examples of chelating agents (complexing agents) include ethylenediaminetetraacetic acid (EDTA), dimercaptosuccinic acid (DMSA), or dimercaptopropanesulfonic acid (DMPS). The term "heavy metal" includes heavy metal ions.

[0041] In a preferred embodiment of the apoferritin nanoparticles according to the present invention for use as a toxin binder in hemodialysis, the amino acid sequence of at least one apoferritin subunit having an amino acid sequence modified relative to the wild-type sequence can be modified relative to the wild-type sequence in the triple channel region, instead of or in addition to modifying the amino acid sequence in the region of the internal space, so that toxin transport into the space of the apoferritin nanoparticle through the triple channel is promoted compared to apoferritin nanoparticles having an unmodified amino acid sequence. In this embodiment, the amino acid sequence of at least one apoferritin subunit can be modified relative to the wild-type sequence only in the triple channel region without modifying the amino acid sequence in the region of the internal space, for example, to promote transport into the apoferritin nanoparticles only, or the amino acid sequence of at least one apoferritin subunit can be modified relative to the wild-type sequence in the triple channel region in addition to modifying the amino acid sequence in the region of the internal space. Preferably, at least one modification to the amino acid sequence is present in the triple channel region of at least two, preferably three, apoferritin subunits forming the triple channel. For example, there can be 1, 2, 3 or more, for example, up to 12 amino acid modifications in 1, 2 or 3 of the apoferritin subunits that form the triple channel.When the modifications are made to 2 or 3 of the apoferritin subunits, they can be the same or different for each subunit, but preferably the same.Particularly preferred apoferritin nanoparticles for use as toxin binder in hemodialysis are made up of identical subunits, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 positions in the triple channel region, and have the same amino acid sequence modifications relative to the wild-type sequence in the triple channel region.As mentioned above, these modifications can exist alone or in combination with the modifications to the amino acid in the region of the internal space.

[0042] The toxin to be bound in hemodialysis using apoferritin nanoparticles according to the present invention is preferably a protein-bound uremic toxin selected from the group consisting of indoxyl sulfate, paracresyl sulfate, phenyl acetate, and p-hydroxyhippuric acid.

[0043] Apoferritin nanoparticles according to the present invention for use as toxin binders in hemodialysis preferably have modified amino acid sequences relative to the wild-type sequence in the internal space region and / or the triple channel region and / or the quadruple channel region in at least 2, more preferably at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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. Preferably, the subunits constituting the apoferritin nanoparticles according to the present invention are identical in terms of their amino acid sequences. More preferably, according to one embodiment, the apoferritin nanoparticles according to the present invention described above are modified by the introduction of cysteine ​​residues functionalized with suitable compound residues, preferably at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, preferably all 24 subunits are similarly modified and functionalized. In one embodiment in which the apoferritin nanoparticles have modified amino acid sequences within the triple channel region, preferably at least 2, more preferably at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, preferably all 24 subunits have modified amino acid sequences relative to the wild-type sequence within the triple channel region, either alone or in combination with modified amino acid sequences within the interior space region.

[0044] In a preferred embodiment of the apoferritin nanoparticles according to the invention for use as a toxin binder in hemodialysis, preferably at least 2, more preferably at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, preferably all 24 subunits have an amino acid sequence in the interior space region and / or in the triple channel region and / or in the quadruple channel region that is modified relative to the wild-type sequence and is selected from one of the amino acid sequences according to SEQ ID NO: 13 to 24. In this regard, preferably, the subunits that have an amino acid sequence that is modified relative to the wild-type sequence each have the same sequence. For example, if apoferritin nanoparticles according to the present invention for use as a toxin binder in hemodialysis have 24 subunits in the region of the internal space and / or in the region of the triple channel and / or in the region of the quadruple channel, which have amino acid sequences modified relative to the wild-type sequence, preferably, the 24 subunits 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 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.

[0045] Particularly preferably, the apoferritin nanoparticles according to the present invention are composed solely of subunits consisting of heavy chains, particularly preferably human heavy chains. With regard to the amino acid sequence, the chains may be modified relative to the wild-type sequence in the outer protein regions (e.g., the A and C helices), for example, to generate a specific external charge, such as a negative or positive charge. Furthermore, particularly preferably, the apoferritin nanoparticles according to the present invention do not have cysteine ​​residues on the exterior of the nanoparticles, and, if necessary, have cysteine ​​residues only in the interior space, so that functionalization with organic compound residues occurs only at the cysteine ​​residues in the interior space. Functionalization may be achieved by disassembling the apoferritin nanoparticles into subunits under suitable conditions and then functionalizing the individual subunits. When restored to the original conditions, the subunits spontaneously reform apoferritin nanoparticles. Suitable conditions are known to those skilled in the art and are described, for example, in [Non-Patent Documents 31, 38, 39] and below.

[0046] In a second aspect, the present invention also relates to a composition for use as a toxin binder in hemodialysis, comprising a plurality of apoferritin nanoparticles according to the present invention for use as a toxin binder in hemodialysis according to the first aspect of the present invention described above. Such a composition may advantageously be used as an adsorbent for binding toxins in the blood, for example for binding protein-bound uremic toxins (PBUT) or heavy metals.

[0047] In the composition according to the present invention for use as a toxin binder in hemodialysis, a plurality of apoferritin nanoparticles according to the present invention for use as a toxin binder in hemodialysis may be in amorphous form or in a specific arrangement, for example, in crystalline form. The term "amorphous apoferritin nanoparticle composition" herein refers to a composition of apoferritin nanoparticles in which the apoferritin nanoparticles are bonded to each other only by forming an irregular or unclear structure, particularly an amorphous structure. A specific arrangement, for example, a crystalline arrangement, of apoferritin nanoparticles according to the present invention can be achieved by methods known to those skilled in the art (see, for example, [Non-Patent Document 31]). In the case of an arrangement in crystalline form, for example, the apoferritin nanoparticles for use as a toxin binder in hemodialysis included in the arrangement are preferably crosslinked. This type of adsorbent has apoferritin nanoparticles according to the present invention as a protein cage, constructed into a specific crystalline material with uniformly distributed solvent channels. This is advantageous not only for achieving high purity of the material but also for handling when used as a sorption material. For example, apoferritin nanoparticles crosslinked into a crystalline structure can be handled as a whole and separated from blood. The morphology of the crystals can be adapted to the shape of the space in the adsorbent cartridge, for example. In the composition according to the present invention, a plurality of apoferritin nanoparticles according to the present invention for use as a toxin binder in hemodialysis can be present in, for example, 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.

[0048] In a third aspect, the present invention also relates to a sorbent cartridge comprising therein a plurality of apoferritin nanoparticles for use as a toxin binder 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. The sorbent cartridge according to the invention may be configured to be integrated into, for example, a dialysis system. To this end, the sorbent cartridge preferably has an inlet for supplying blood and an outlet for discharging blood.

[0049] Preferably, the sorbent cartridge is configured in such a way that a plurality of apoferritin nanoparticles or a composition comprising a plurality of apoferritin nanoparticles can be retained in the sorbent cartridge by a suitable means within the sorbent cartridge. For example, a plurality of apoferritin nanoparticles or a composition can be contained in a first compartment in the sorbent cartridge, separated from a second compartment in the sorbent cartridge by one or more membranes, which are permeable to unbound toxin molecules and / or proteins to which the toxin molecules are bound, but not permeable to apoferritin nanoparticles. Blood from which toxin molecules, e.g., PBUT, are removed can be passed through, for example, the second compartment. Toxin molecules present in the blood, either in free form or optionally bound to proteins, can pass through the membrane(s), for example, by diffusion, to the first compartment, where they can be bound by apoferritin nanoparticles according to the present invention. However, in a particularly preferred embodiment of the sorbent cartridge according to the present invention, a plurality of apoferritin nanoparticles or a composition are arranged in the sorbent cartridge in such a way that they come into direct contact with the blood to be purified, for example, during operation of a dialysis system. In this preferred embodiment, the crystalline or amorphous composition of apoferritin nanoparticles is located within the sorbent cartridge, or at least within a compartment within the sorbent cartridge, allowing direct contact of the blood with the composition within the sorbent cartridge. In this form, the sorbent material according to the present invention forms an insoluble (amorphous or crystalline) solid that is significantly larger than blood components (e.g., red blood cells, approximately 2.5 μm). This size difference facilitates separation. The sorbent material can be retained using a relatively coarse filter, while the blood flows through the sorbent cartridge. The biocompatibility of the material according to the present invention has been found to be particularly advantageous in this case, since it is not necessary to separate the sorbent material from the blood, thereby providing closer contact between the toxins to be separated and the sorbent material, as well as facilitating integration into existing dialysis systems.

[0050] In a fourth aspect, the present invention relates to a dialysis system comprising a dialyzer and at least one sorbent cartridge according to the third aspect of the invention.

[0051] The at least one sorbent cartridge may be positioned a) upstream, b) downstream, or c) in a sub-circuit of the dialysis circuit relative to the dialyzer in the direction of flow of blood to be dialyzed.

[0052] In a fifth aspect, the present invention also relates to a method for dialysis of blood, preferably human blood, comprising a step of contacting the blood with apoferritin nanoparticles according to the present invention according to the first aspect of the present invention or a composition according to the second aspect of the present invention. The contacting step is preferably carried out for a time and under conditions sufficient for toxins contained in the blood, such as PBUT or heavy metals, to bind to the apoferritin nanoparticles or the composition. The composition may contain apoferritin nanoparticles in amorphous or crosslinked (crystalline) form. In particular, the term "contacting" as used herein includes contacting blood directly with apoferritin nanoparticles or the composition, i.e., without a semipermeable membrane between them, for example. The dialysis method according to the present invention is particularly advantageous for treating chronic kidney disease (CKD) and / or mitigating its consequences.

[0053] Therefore, in a further aspect, the present invention also relates to a method for the therapeutic treatment of patients 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 present invention.

[0054] The invention will now be described in detail, purely by way of example, with the aid of the accompanying drawings and exemplary embodiments. [Brief explanation of the drawings]

[0055] [Figure 1]1A and 1B are schematic diagrams of embodiments of apoferritin nanoparticles according to the present invention. A is a schematic diagram of apoferritin nanoparticles according to the present invention in which the amino acid sequence in the region of the internal space has been modified. B is a schematic diagram of apoferritin nanoparticles according to the present invention in which the amino acid sequence in the region of the internal space has been modified and cysteine ​​residues have been functionalized. C is a schematic diagram of apoferritin nanoparticles according to the present invention in which the amino acid sequence in the region of the triple channel has been modified. D is a schematic diagram of apoferritin nanoparticles according to the present invention in which the amino acid sequence in the region of the internal space has been modified (cysteine ​​residues have been functionalized) and further in which the amino acid sequence in the region of the triple channel has been modified. [Figure 2] FIG. 2 is a schematic diagram of a crystalline arrangement (bottom) of a plurality of apoferritin nanoparticles according to the present invention constructed from the apoferritin nanoparticles of FIG. 1 (top). [Figure 3] 1A-C are schematic diagrams of embodiments of dialysis systems according to the present invention: (A) a dialysis system with an upstream sorbent cartridge according to the present invention; (B) a dialysis system with a downstream sorbent cartridge according to the present invention; and (C) a dialysis system with a sorbent cartridge according to the present invention in the auxiliary circuit. [Figure 4] These figures show the results of adsorption experiments using functionalized apoferritin nanoparticles for binding with PBUT. Figures a), b), and c) show the adsorption amounts of untreated and functionalized ferritin for each concentration of PBUT expected in the blood of CKD patients [Non-Patent Document 26]. Figure d) is an optical microscope image of crystals of Ftn-Phe acting as an adsorbent. Figure e) shows the relative concentration of TNF-α mRNA in endothelial cells. Figure f) shows the ratio of phosphorylated AKT to AKT as an indicator of platelet activation and the associated blood coagulation. Ftn(neg)-Phe: Ftn(neg)-apoferritin nanoparticles functionalized with 2-iodo-N-phenylacetamide (Phe); Ftn(neg)-C10: Ftn(neg)-apoferritin nanoparticles functionalized with 2-bromo-N-decylacetamide (C10). [Figure 5]Figure 1 shows the adsorption of indoxyl sulfate to recombinant ferritin mutants. Ftn(neg)-Dock: a mutant with a toxin-binding site (sequence: Ftn(neg)-dock03, SEQ ID NO: 21), Ftn(neg)-Ap: a mutant with reduced negative surface charge on the inner surface (sequence: Ftn(neg)-Ap4, SEQ ID NO: 17), and Ftn(neg)-Ap-Channel: a mutant with reduced negative surface charge on the inner surface and within the triple channel region (sequence: Ftn(neg)-Ap4-3A, SEQ ID NO: 18). b) Interactions between indoxyl sulfate and three side chains in the designed toxin-binding site (computer model). [Figure 6] Graphs showing the adsorption of indoxyl sulfate (IS) to crystalline (left) and amorphous (right) recombinant ferritin mutants according to the present invention: Ftn(neg): an apoferritin nanoparticle mutant with increased negative charge on the external surface (SEQ ID NO: 12); Ftn(neg)-Ap4: a mutant with four mutations relative to Ftn(neg) resulting in a corresponding decrease in negative surface charge on the internal surface (SEQ ID NO: 17); and Ftn(neg)-Ap16: a mutant with 16 mutations relative to Ftn(neg) resulting in a corresponding decrease in negative surface charge on the internal surface (SEQ ID NO: 20). [Figure 7] 1 is a graph showing the adsorption of crystalline recombinant ferritin variants according to the present invention with specifically engineered binding sites for 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), and Ftn(neg)-43 (=Ftn(neg)-dock43, SEQ ID NO:22). [Figure 8]1 is a graph showing the adsorption of crystalline recombinant ferritin variants according to the present invention with specifically engineered binding sites for paracresyl sulfate (pCS) and phenyl acetate (PhAc) compared to Ftn(neg): Ftn(neg)-03 (=Ftn(neg)-dock03, SEQ ID NO:21), Ftn(neg)-23 (=Ftn(neg)-dock23, SEQ ID NO:22), and Ftn(neg)-43 (=Ftn(neg)-dock43, SEQ ID NO:22). [Figure 9] 1 is a graph showing the adsorption amounts of a crystalline recombinant ferritin mutant according to the present invention (Ftn(neg)-Ap4-3A-dock43; SEQ ID NO:24) with sequence modifications to indoxyl sulfate (IS) compared to Ftn(neg), as well as the mutants 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. DETAILED DESCRIPTION OF THE INVENTION

[0056] FIG. 1 shows a highly simplified schematic representation of an embodiment of an apoferritin nanoparticle 1 according to the present invention for use as a toxin-binding agent in hemodialysis. As can be seen from FIG. 1 , the apoferritin nanoparticle 1 has a roughly spherical shape with a protein shell formed from apoferritin subunits 2 surrounding an internal space 4. Channels 3 are formed at the interfaces between the apoferritin subunits 2, through which toxins can enter the internal space 4 from the external environment, e.g., by diffusion, and bind thereto, e.g., by hydrophobic interactions. FIG. 1A shows an embodiment in which sequence modification sites 5, i.e., sites in which modifications have been made 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 replaced with other amino acids or amino acid sequence regions, relative to the wild-type sequence of each apoferritin subunit 2. Functionalization of amino acids, e.g., cysteine ​​residues, has not been performed here. Toxin binding can occur, for example, through hydrophobic interactions with hydrophobic amino acid residues present at sequence modification site 5. In the embodiment of apoferritin nanoparticles 1 for use as a toxin binder in hemodialysis shown in FIG. 1B, first, any naturally occurring cysteine ​​residues present are replaced with alanine, and then, at the four sequence modification sites 5, four amino acids are replaced with cysteines whose SH groups are functionalized with covalently bound organic compound residues (here, for example, N-phenylacetamidyl residues). In this example, the four cysteine ​​residues are functionalized with one hydrophobic organic compound residue to bind uremic toxins. In the embodiment of apoferritin nanoparticles 1 for use as a toxin binder in hemodialysis shown schematically in FIG. 1C, only sequence modification sites 5 within the triple channel region are provided, i.e., the amino acid sequence modified relative to the wild-type sequence is present only within the triple channel region.In the embodiment of apoferritin nanoparticles 1 for use as a toxin binder in hemodialysis shown schematically in Figure 1D, in addition to sequence modification sites 5 within the region of the triple channel, sequence modification sites 5 are also present within the region of the internal space 4, and one of the sequence modification sites 5 is shown here as a functionalized sequence modification site 5 by way of example.

[0057] Figure 2 shows a schematic representation of apoferritin nanoparticles 1 (top) and their aggregates, crystalline compositions 100 (bottom). Under suitable conditions, apoferritin nanoparticles 1 can be assembled into well-defined macroscopic crystalline materials with uniformly distributed solvent channels. This ensures high material purity and ease of handling, and also aids in characterization. To stabilize the alignment, apoferritin nanoparticles 1 may be crosslinked to each other, if necessary.

[0058] FIG. 3 shows various embodiments of a dialysis system 200 according to the present invention. FIG. 3A shows an embodiment in which a sorbent cartridge 201 is connected upstream of a dialyzer 202 (in this case, a hollow fiber membrane module) in the direction of blood flow indicated by the arrow. The flow direction is determined by a suitable pump (not shown here). The external body surface is shown here diagrammatically by a dashed line 203. Blood is delivered to the sorbent cartridge 201 via a line 205, e.g., a tube. A sorbent according to the present invention, e.g., a composition 100 in the form of a crystalline array according to the second aspect of the present invention, is located within the sorbent cartridge 201. The blood can be brought into direct contact with the composition 100 comprising apoferritin nanoparticles 1 according to the present invention. Uremic toxins, e.g., protein-bound uremic toxins, that may be present in the blood can be bound to the sorbent. The toxin-free, or at least toxin-depleted, blood is returned to the dialyzer 202 and then to the patient's body via a line 204 leading to the body. The dialysate circuit 206 may be used for dialysate exchange or for dialysate counterflow.

[0059] 3B shows a dialysis system 200 according to the present invention in which a sorbent cartridge 201 according to the present invention is downstream of a dialyzer 202 in the direction of blood flow. In this embodiment, the dialysis system 200 is otherwise the same as the embodiment of FIG. 3A, so see the above description for details. Again, the blood to be cleared or depleted of toxins comes into direct contact with apoferritin nanoparticles 1 or a composition 100 comprising apoferritin nanoparticles 1 within the sorbent cartridge 201.

[0060] 3C shows a dialysis system 200 according to the present invention in which a sorbent cartridge 201 according to the present invention having apoferritin nanoparticles 1 according to the present invention is placed in an auxiliary circuit 207 to the blood circulation. Instead of or in addition to a dialysate circuit (not shown here), the sorbent can be sent to the dialyzer 202 to bind to toxins in the blood passing through the hollow fibers via the membrane. In this embodiment, the blood to be purified does not come into direct contact with the apoferritin nanoparticles 1 or the composition 100 comprising the apoferritin nanoparticles 1. [Example]

[0061] Chemical modification (functionalization) of the inner surface of apoferritin nanoparticles Apoferritin nanoparticles according to one embodiment of the present invention were fabricated by chemically modifying the inner surface with hydrophobic small molecules. This involved genetically modifying the protein so that the amino acid cysteine ​​was present only at specific exposed sites on the inner surface. This amino acid, the only one in the inner cavity with a thiol group on its side chain, served as an anchor site for reaction with hydrophobic molecules. The protein container could be separated into subunits under acidic conditions (pH 2). The inner cysteines could then be functionalized with hydrophobic molecules. 2-iodo-N-phenylacetamide and 2-bromo-N-decylacetamide molecules were used, which can be attached to the thiol groups after the substitution reaction. Because thiol groups are only present on cysteines and the modified protein contains them only inside the cavity, only the inner surface is selectively modified with hydrophobic groups. Complete functionalization can be confirmed by a significant increase in protein mass using ESI-MS, which can also be used to confirm that all cysteines have been functionalized. Previously, up to 96 hydrophobic molecules could be incorporated per nanoparticle. After functionalization, improved adsorption capacity for IS, pCS, and phenyl acetate was measured, as can be seen from Fig. 4a–c.

[0062] Materials and Methods general All chemicals were obtained commercially and used without further purification. Wherever possible and unless otherwise stated, all solutions were made up with ultrapure water (produced with a Purelab-Flex-2-System, resistivity 18.2 MΩ·cm) and analytical-quality reagents.

[0063] Mutation introduction Introduction of cysteine ​​anchor sites was achieved by several cycles of site-directed QuikChange® mutagenesis using a two-step polymerase chain reaction (PCR) protocol

[30] . The primers used for the various mutation sites are listed in Table 1.

[0064] [Table 1]

[0065] 2.9 µL of pET-22b(+) plasmid containing the gene of interest (7 ng µL -1 ), 1 μL of 10 mM dNTP-Mix, 5 μL of reaction buffer 10x (100 mM KCl, 100 mM (NH4)2SO4, 200 mM Tris-HCl (pH 8.8), 20 mM MgSO4, 1% Triton® X-100, 1 mg mL -1 1 μL of nuclease-free bovine serum albumin (BSA), 1 μL of Pfu-DNA polymerase (2.5 U / μL) -1 ), and 38.1 μL of ultrapure water. The mixture was divided into halves and 1 μL of forward primer or reverse primer (10 pmol μL) was added. -1 ) was added to each tube. A PCR thermal cycler (Eppendorf Mastercycler Nexus PCR Cycler) was primed with an initial heating step at 95°C for 30 seconds. The first step of the PCR protocol consisted of three cycles of denaturation at 95°C for 30 seconds, a 1-minute incubation step at 61°C, followed by a 6-minute extension at 68°C. After the first three cycles, the separated mixture was combined with the forward and reverse primers, and PCR was continued for 16 additional cycles with the same parameters as the first three cycles, followed by a final 10-minute extension step at 68°C to complete the PCR. Digestion of the parental plasmid was performed with 1 μL of DpnI (10 U μL -1 ) and incubated overnight at 37°C. DpnI was heat-inactivated at 80°C for 20 minutes, and the mixture was purified using NucleoSpin® Gel and PCR Clean-Up Kit according to the manufacturer's instructions. Calcium-competent E. coli DH5α cells were incubated with 200 ng of purified plasmid on ice for 30 minutes and then heat-shocked at 42°C for 45 seconds. The cells were then incubated in "Super Optimal Broth" (SOB) medium for 1 hour, centrifuged at 1000 g, resuspended in 100 μL of medium, plated on LB agar plates, and incubated at 37°C for 16 hours. Individual colonies were picked and diluted with 150 μg mL -1The cells were incubated overnight at 37° C. and 250 rpm in 5 mL of sterile LB medium supplemented with 100 μL of ampicillin. The next day, the plasmids were extracted with a NucleoSpin® Plasmid Miniprep Kit.

[0066] The sequence was confirmed by mixing 500 ng of the plasmid with 25 pmol of T7 forward or reverse primer in 10 μL of solution and sending it for DNA sequencing (Eurofins Genomics). Plasmids with the desired mutations were then selected as plasmid stocks and further mutagenesis was performed until all five mutations were obtained.

[0067] Preparation and purification of ferritin-cysteine ​​mutants Negatively charged ferritin mutant (Ftn (neg) The construction of mutants with cysteine ​​and cysteine ​​was performed as previously published

[31] . First, calcium-competent E. coli BL21-Gold(DE3) cells were thawed on ice for 10 min. Next, 1 μL of 40 ng / μL of ATP was added. -1 The plasmid solution was added to the cells, and the mixture was incubated on ice for 30 minutes. The mixture was incubated at 42°C for 45 seconds, followed by a heat shock by incubating on ice for 2 minutes. The cells were suspended in 1 mL of SOB medium and incubated at 37°C for 1 hour. The cells were centrifuged at 1000 g, and 1 mL of the mixture was removed. The cell pellet was suspended in the remaining solution and diluted to 150 μg mL. -1 The resulting mixture was plated onto an LB agar plate supplemented with 150 μg mL of ampicillin and incubated overnight at 37°C. To prepare the preculture, a colony of transformed E. coli BL21-Gold(DE3) cells (Agilent) was added to 150 μg mL -1 The cells were incubated overnight at 37°C and 180 rpm in 5 mL of sterile LB-Miller medium supplemented with 150 μg mL of sodium ampicillin. -1 4 mL of the preculture was inoculated into 400 mL of Terrific Broth (TB) medium supplemented with 100 mL of sodium ampicillin. 600The cells were incubated at 37°C and 180 rpm until the RI reached 0.6. Protein overexpression was induced by adding isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.25 mM, and the cells were incubated at 18°C ​​for an additional 48 h. Cells were harvested by centrifugation at 4000 g. The pellet was stored at -20°C until further use.

[0068] Cells from a 400 mL culture were resuspended in 20 mL of buffer (50 mM Tris, pH 7.5, 0.3 M NaCl). Cells were lysed on ice by sonication (60% amplitude) six times, with 1-minute intervals between each interval, using a Vibra-Cell VCX-130 sonicator (Sonics). The resulting suspension was centrifuged at 14,000 g for 20 minutes to separate cellular debris from soluble proteins. The supernatant was heated to 65°C in a water bath for 10 minutes to denature most E. coli proteins. The denatured proteins were separated by centrifugation at 14,000 g for 15 minutes. Proteins remaining in solution were precipitated with ammonium sulfate to a final concentration of 70% of saturation, followed by centrifugation at 14,000 g for 20 minutes. The pellet was rebuffered with 10 mL of buffer (50 mM Tris, pH 7.5, 0.15 M NaCl), after which 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) using a 5 mL HiTrap® Q HP anion exchange column (Cytiva) with a linear gradient of 0.15 to 1 M NaCl. (neg) The -4xCys-containing fractions were collected and concentrated to a final volume of 2 mL using a Sartorius Vivaspin® Turbo 15 (MWCO 30000) filtration device. Finally, the sample was purified by gel filtration using a HiLoad 16 / 600 Superdex® 200 pg column. All chromatographic steps were performed on an Äkta pure system from Cytiva. (neg) All fractions containing -4xCys were collected and stored at 4°C until further use.

[0069] Functionalization with 2-iodo-N-phenylacetamide 5mg of Ftn (neg) -3xCys or Ftn (neg) -4xCys was incubated in digestion buffer (10 mM phosphate, 50 mM NaCl, pH 2) for 4 hours. After 3.5 hours, 10 mg mL -1 Ten equivalents (eq.) (for each cysteine) of tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Iris Biotech GmbH) from a stock solution of 10 sachets were added to the solution. The solution was then brought to 15 mL with reconstitution 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). An additional 10 eq. of TCEP was added, bringing the solution volume to 2 mL. The pH was adjusted to 7.6 with 1 M NaOH or HCl. Next, 2 mL of ethanol containing 20 eq. of 2-iodo-N-phenylacetamide (abcr GmbH) was added to the solution, and the mixture was stirred at 300 rpm in the dark for 1 h. The solution was then brought to a total volume of 30 mL with reconstitution buffer. The protein was reconstituted 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 until further use.

[0070] Functionalization with 2-bromo-N-decylacetamide Functionalization followed the exact same protocol as for functionalization with 2-iodo-N-phenylacetamide, except that during the functionalization reaction, the protein / TCEP solution was reduced to 800 μL 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 carried out according to the protocol for 2-iodo-N-phenylacetamide.

[0071] Hanging drop crystallization Crystallization of small amounts of protein or functionalized protein variants was performed using the hanging drop vapor diffusion method. Reservoir solution (100 mM Tris, 500 mM MgOAc, pH 8.5) was prepared in a manual 24-well plate. 2 μL of reservoir solution was mixed with 1 μL of 50 mM Tris, 1 M NaCl (pH 7.5) buffer and 1 μL of each ferritin variant and dropped onto a siliconized cover slide (Jena Bioscience). The plate was incubated at 25°C. The first crystals were visible after 1 day.

[0072] Batch Crystallization A large amount of Ftn (neg) and Ftn (neg) To crystallize functionalized variants of α-glucan, a batch crystallization technique based on the protocol by Rayment was used

[32] . In a typical experiment, 250 μL of 50 mM Tris 1 M NaCl (pH 7.5) buffer was added to a solution of α-glucan at a concentration of 12 mg mL in 50 mM Tris 0.3 M NaCl (pH 7.5) buffer. -1 The same amount of Ftn (neg) The stock solution was carefully mixed. Next, 500 μL of precipitating solution (133 mM Tris, 333 mM MgOAc, pH 8.5) was added dropwise with constant shaking. The mixture was stored at ambient temperature of 20 °C without stirring for 7 days before the crystals were allowed to settle.

[0073] Fixation of crystals For adsorption experiments, it was necessary to increase the stability of the crystals. To this end, the crystals were fixed with the cross-linker Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, Sigma-Aldrich). The crystals were centrifuged at 1000 g for 2 min. For a standard test with a total mass of 3 mg of crystals, the crystallization solution was removed until 246 μL remained. Next, 64 μL of freshly prepared 4.8 mg mL solution was added. -1 Add an aqueous solution of Sulfo-SMCC to a final concentration of 1 mg mL -1The mixture was left at ambient temperature for 4 hours and then made up to 1 mL with ultrapure water. The crystals were separated by centrifugation at 1500 g for 2 minutes. The supernatant was removed, and the crystals were then resuspended in ultrapure water. This process was repeated up to three times to wash away any remaining crosslinker from the crystals. The material was stored at ambient temperature at 20°C until further use. The crystals were photographed using a FlexaCam C1 under a Leica S9D microscope.

[0074] To fix the crystals with glutaraldehyde (Merck), 50 μL of 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 hours and then washed three times with ultrapure water. The crystals were stored at 20°C until further use.

[0075] After glutaraldehyde cross-linking, the crystals remained at 60 mg mL -1 The crystals were dissolved in a BSA solution of 1000 mg / ml. This stability could be improved by an additional fixation step. This procedure was repeated, but the crystals were incubated for only 10 minutes before washing. However, toxin adsorption assays showed a significant decrease in the amount of adsorbed toxin. It was assumed that the pores were blocked by polymerization of glutaraldehyde.

[0076] Preparation of amorphous adsorbents 500 μL of 6 mg mL -1 To the protein solution, 25 μL of 2.5% glutaraldehyde solution was added. The solution was carefully pipetted forward and reverse three times to ensure thorough mixing. The mixture was kept overnight at ambient temperature of 20°C. The next day, a white precipitate formed. This material was washed three times with ultrapure water and stored at 20°C until further use.

[0077] ESI-MS measurement The protein sample was rebuffered with ultrapure water using an Amicon® Ultra 0.5 mL (MWCO 30,000 Da) centrifugal filter. The protein sample was brought to a volume of 500 μL with ultrapure water, concentrated to approximately 20 μL, and then re-buffered to 500 μL. This rebuffering process was repeated five times to achieve concentrations ranging from 0.15 to 0.2 mg mL.-1 The mass of the protein was measured using an electrospray ionization time-of-flight mass spectrometer (Agilent 6224 ESI-TOF) in the positive mode.

[0078] Toxin assay To measure the amount of uremic toxins adsorbed by the ferritin mutants, we performed adsorption experiments. In the initial experiments, we observed adsorption of the toxins onto the polypropylene walls of the reaction tubes. Therefore, all solutions and samples were handled in glassware (Macherey-NAGEL vial N9).

[0079] First, 50 μg mL for pCS and IS -1 , 500 μg mL for PheAc -1 Stock solutions of the toxin of interest were prepared at concentrations of 0.01, 0.05, 0.1, 0.2, 0.5, 0.7, and 1 μg mL for later determination of absolute toxin concentrations in samples. -1 A set of standard solutions for the calibration curve was prepared with concentrations of

[0080] The stock solution was further diluted to achieve the final uremic toxin concentration expected in a stage 5 CKD patient (41 mg L for pCS). -1 [Non-patent document 33], and 44 mg L -1 [Non-patent Document 26], and 474 mg L -1 The adsorbent was centrifuged at 1500 g for 2 minutes, and all the supernatant was removed from the sample. 150 μL of each toxin solution was added, and the crystals were incubated at ambient temperature for 3 hours. Additionally, 150 μL of the toxin solution was incubated as a control. Three 10 μL aliquots were taken from all samples and diluted 100-fold with ultrapure water. Finally, the crystals were washed with water, vacuum dried, and weighed.

[0081] Uremic toxin concentrations were quantified using a reversed-phase high-performance liquid chromatography (RP-HPLC) system using a C18 column (Zorbax Extend-C18, Agilent) coupled with an electrospray ionization quadrupole linear ion trap mass spectrometer (ESI-QTRAP). The solvent used was a mixture of HPLC-quality water (LiChrosolv®, Merck) and acetonitrile (LiChrosolv®, Merck), both supplemented with 0.1% formic acid (Honeywell Fluka). The specific composition of each step of the 15-minute chromatography program is summarized in Table 2.

[0082] [Table 2]

[0083] A control substance was measured before incubating each sample with the crystals. Chromatograms were analyzed using Analyst® Instrument Control and Data Processing software. Peaks were integrated, and toxin concentrations were determined from calibration standards. The amount of adsorbed uremic toxin was determined from the difference in concentration between the control substance and the sample. The adsorption amount was then determined by dividing the mass of adsorbed toxin by the mass of the crystals.

[0084] 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). Cells were seeded at 80% confluence into 24-well plates (15 × 10 4 cells / well), 100ng mL -1The cells were incubated with lipopolysaccharide (LPS) or functionalized or non-functionalized protein crystals for 6 hours. After incubation, total RNA was extracted using the RNAeasy Minikit (Qiagen). Reverse transcription was performed using 1 μg of total RNA (600 ng), random hexamer primers, and Verso-Reverse-Transcriptase (Thermo Scientific) according to the manufacturer's instructions. Real-time PCR quantified gene expression levels using SYBR Green I dye on a LightCycler 480 system (Roche Applied Sciences). For relative quantification of target gene expression, the primers used were: forward primer 5'-GCCCAGGCAGTCAGATCATCT-3' (SEQ ID NO:8) and reverse primer 5'-TTGAGGGTTTGCTACAACATGG-3' (SEQ ID NO:9) for human TNFα; forward primer 5'-CAACCGCGAGAAGATGAC-3' (SEQ ID NO:10) and reverse primer 5'-GTCCATCACGATGCCAGT-3' (SEQ ID NO:11) for human β-actin. Data are expressed as the average level of gene expression relative to the expression of the reference gene (β-actin).

[0085] Platelet activation assay Platelets from three donors were isolated by centrifugation at 260 g for 15 min. 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, 5 mM glucose). The platelet suspension was diluted with 1:15 acid citrate dextrose (ACD) and 1 U mL -1 The cells were centrifuged again in the presence of 15 x 10 apyrase 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). 6 2mmol L -14 nmol L in the presence of CaCl2 or various protein crystals -1 Platelets were incubated with 100 μL of thrombin for 15 min. Platelets were lysed in 4% SDS lysis buffer (200 mmol L−1) containing EDTA-free Halt Protease Inhibitor Cocktail (1:10, Sigma-Aldrich) and Halt Phosphatase Inhibitor Cocktail (1:10, Sigma-Aldrich). -1 Tris, 600 mmol L -1 The blots were dissolved in 10% NaCl, 4% SDS (NaCl, 4% SDS). Protein content was quantified according to the protocol of the DC protein assay (Bio-Rad). Equal amounts of protein from each sample were separated by 10% SDS polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and blocked with 5% bovine serum albumin (BSA) for 1 hour at ambient temperature. Anti-p-Akt antibody (1:1000, Cell Signaling) and anti-tubulin antibody (1:1000, Cell Signaling) were used as primary antibodies. The blots were incubated overnight at 4°C. A second anti-rabbit antibody (1:1000, Cell Signaling) was used for 1 hour at ambient temperature. Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using Quantity One Software (Bio-Rad Laboratories).

[0086] array Ftn (neg) Nanoparticle subunit, cysteine-containing mutant Ftn (neg) -3xCys (having three cysteine ​​residues), and Ftn (neg) The amino acid sequence of 4xCys (having four cysteine ​​residues) is shown below. (neg) -1xCys) and 2 (Ftn (neg) The mutant with Ftn (-2xCys) is also shown. (neg) For Ftn, mutations relative to the wild-type H chain (SEQ ID NO: 1) are indicated. (neg)Mutations compared to (SEQ ID NO:12) are shown with the naturally occurring cysteine ​​residue converted to an alanine residue. The cysteine ​​residue in the cysteine-containing mutants is underlined.

[0087] Ftn (neg) , (SEQ ID NO: 12): Mutants with increased negative charges on the outer surface; A18E, C90E, C102E, K86Q, H105E TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0088] Ftn (neg) -3xCys (SEQ ID NO: 13): a mutant with three cysteine ​​residues on the internal surface; K53C, E64C, C130A, K143C TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFA C YFLHQSHEER C HAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLADFIETHYLNEQV C AIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0089] Ftn (neg) -4xCys (SEQ ID NO: 14): a mutant with four cysteine ​​residues on the internal surface; K53C, E64C, C130A, K143C, S178C TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFA C YFLHQSHEER CHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLADFIETHYLNEQV C AIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGD C DNES

[0090] Ftn (neg) -1xCys (SEQ ID NO: 15): a variant with one cysteine ​​residue on the internal surface; K53C, C130A TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFA C YFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0091] Ftn (neg) -2xCys (SEQ ID NO: 16): a mutant with two cysteine ​​residues on the internal surface; K53C, E64C, C130A TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVALKNFA C YFLHQSHEER C HAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLADFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0092] The sequences of other mutants investigated are as follows: Ftn (neg) (SEQ ID NO: 12). The term "binding site" as used herein refers to a toxin binding site formed by a simple amino acid exchange (without additional functionalization).

[0093] Ftn(neg) -Ap4 (SEQ ID NO: 17): mutants with reduced negative surface charge within the region of the inner space; E61V, E62A, D131F, E140W TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDDRDDVALKNFAKYFLHQSH VA REHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLC F FIETHYLN W QVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0094] Ftn (neg) - Ap4-3A (SEQ ID NO: 18): Mutants with reduced negative surface charge in the interior space and pore and enlarged pore size; E61V, E62A, T122A, D123A, N125A, D131F, E140W TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDDRDDVALKNFAKYFLHQSH VA REHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLA AA K A DPHLC F FIETHYLN W QVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0095] Ftn (neg) Ap7 (SEQ ID NO: 19): a variant with 7 hydrophobic amino acids on the inner surface; E61V, E62A, H128F, D131W, N139V, E140W, K143V TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDDRDDVALKNFAKYFLHQSH VA REHAEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPF LC W FIETHYL VW QV V AIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0096] Ftn (neg) - Ap16 (SEQ ID NO: 20): a 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 TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDRDDVAL V NFAK F FL WL SH VA RE L AEKLMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDP F LC W FIET Y YL VW QV V AIK L LGD Y VT A LRKMGAPESGLAEYLFDKHTLGDSDNES

[0097] Ftn (neg) dock03 (SEQ ID NO: 21): A mutant 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 TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDDRDDVALKNFAKYFLHQSHEER RK AE E LM R LQN DKGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDP E LC EH IE K RHLRRQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0098] Ftn (neg) dock23 (SEQ ID NO: 22): a mutant with a binding site on the inner surface of the central subunit; H57E, Q58R, H60I, E61G, E64G, K68D, T135D, H136K, Y137H, N139E, E140K, A144N TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDDRDDVALKNFAKYFL ER S IG ER G HAE D LMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLCDFIE DKH L EK QVK N IKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0099] Ftn (neg) dock43 (SEQ ID NO: 23): mutants with a binding site on the internal surface near the E helix; H57E, Q58R, E64R, H65K, K68E, T135D, H136R, Y137H, N139R, E140R, E147Y TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDDRDDVALKNFAKYFL ER SHEER RK AE E LMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLATDKNDPHLCDFIE DRH L RR QVKAIK YLGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0100] Ftn (neg) - Ap4-3A-dock43 (SEQ ID NO: 24): Mutants with reduced negative surface charge in the interior space and pore, enlarged pore size, and additional binding sites on the interior surface near the E helix; E61V, E62A, T122A, D123A, N125A, D131F, H57E, Q58R, E64R, H65K, K68E, T135D, H136R, Y137H, N139R, E140R, E147Y TTASTSQVRQNYHQDSEEAINRQINLELYASYVYLSMSYYFDDRDDVALKNFAKYFL ER SH VA R RK AE E LMKLQNQRGGRIFLQDIQKPDEDDWESGLNAMEEALELEKNVNQSLLELHKLA AA K A DPHLC F FIE DRH L RR QVKAIK Y LGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0101] result Macroscopic materials were constructed using the batch crystallization technique (see above) with apoferritin nanoparticles as building blocks. The protein solution was carefully mixed by constant shaking while the precipitant solution was added. The first crystals were visible after approximately 24 hours. The size of the crystals could be adjusted by varying the protein and precipitant concentrations. To increase stability, the crystals were fixed using a cross-linking agent. Initial experiments were performed with glutaraldehyde. However, a 60 mg mL solution was chosen to mimic the high protein content of blood. -1 In a stability test using a BSA solution, the crystals dissolved. Therefore, we stabilized them with a Sulfo-SMCC crosslinker to maintain the adsorption amount and ensure stable immobilization.

[0102] To enhance the adsorption of hydrophobic and partially negatively charged PBUT, hydrophobic ligands were introduced into the interior space of apoferritin nanoparticles according to the present invention. The present invention provides a modular material adaptable to the nature of the ligand. To this end, cysteine ​​residues, which can be modified by their thiol groups serving as anchor sites for chemical derivatization, were incorporated to provide a general site for modification. Meanwhile, to avoid modification at unwanted positions, the native cysteine ​​residues were replaced with alanine residues. The key design criteria for introducing cysteine ​​residues into the cavity surface were the distance between sites and the solvent-accessible surface area (SASA). A higher SASA value should result in higher reactivity. To this end, ferritin mutants were designed with three and four cysteines introduced per subunit, respectively, and were characterized as Ftn. (neg) -3xCys and Ftn (neg) Therefore, the total number of anchor sites per constructed protein cage was 72 or 96, with 24 subunits and 3 or 4 cysteine ​​residues. (neg) Mutations were introduced into the gene and the mutants were overexpressed in E. coli. (neg) The protocol published in [Non-Patent Document 31] was followed. Ion exchange chromatography (IEC) and size exclusion chromatography (SEC) revealed no significant changes in elution behavior compared to the starting protein. Introduction of the desired mutations was confirmed by electrospray ionization mass spectrometry (ESI-MS), and the detected masses were consistent with the calculated masses (not shown).

[0103] Chemical modification of cysteine ​​thiol groups involves the use of α-halogenocarbonyl 2-iodo-N-phenylacetamide (Phe).

[0104] [ka]

[0105] and 2-bromo-N-decylacetamide (C10) were used.

[0106] [ka]

[0107] Apoferritin nanoparticles functionalized with the above compound were treated with Ftn (neg) -Phe and Ftn (neg) This is referred to as -C10. The overall approach for chemically modifying the internal surface and subsequent assembly to form heterogeneous genetic material is essentially as follows: First, the protein cage is decomposed into subunits under acidic conditions. In the decomposed state, the thiol groups are readily accessible to halogenoacetamide derivatives. The molecule to be conjugated itself is not soluble in aqueous solution. Therefore, functionalization is carried out in a solution with a high ethanol content to facilitate the dissolution of hydrophobic molecules. After incubation of the reaction partners, the protein cage can be reassembled by diluting the mixture. After purification by SEC, the resulting chromatogram shows the same elution volume as the unfunctionalized cage (not shown), indicating complete reassembly of the protein cage. This result is also confirmed by negative stain TEM images showing the intact cage structure (not shown). Functionalized Ftn (neg) The SEC of -4xCys shows a slight shift towards higher elution volumes, associated with a slightly larger size, which is in good agreement with the dynamic light scattering (DLS) results.

[0108] To confirm successful and complete functionalization of the cysteine ​​anchor sites, ESI-MS measurements were performed. In initial experiments, multiple mass peaks were observed for each charged species (not shown). These could be assigned to ferritin subunits bearing one to four desired molecules, indicating that a mixture of functionalized sites with varying degrees of functionalization was present in the sample. Fine-tuning the reaction conditions, particularly the percentage of ethanol in the mixture, the ratio of reactive molecules to cysteines, and the addition of a reducing agent (TCEP), enabled reproducible and complete functionalization of all sites, as confirmed by ESI-MS (not shown). Derivatization was also confirmed by X-ray crystallography of the functionalized ferritin (not shown). Finally, macroscopic crystals of the functionalized variant were prepared under the same conditions as described above, indicating that the functionalization did not affect the external surface.

[0109] Next, we performed PBUT adsorption assays using unfunctionalized protein-based materials and materials functionalized with aliphatic or phenyl molecules. To do this, each sample was incubated in a solution of three uremic toxins, indoxyl sulfate (IS), p-cresyl sulfate (pCS), and phenylacetic acid (PheAc), at concentrations expected in end-stage CKD patients [26, 33, 34]. After 3 hours of incubation, the PBUT concentrations in the supernatant and each control sample were measured using HPLC-MS / MS. Absolute values ​​were determined by comparison with a calibration curve experiment, which was performed before the start of each experiment and every 20 samples. In addition, controls were measured immediately before each sample to allow for direct comparison. Nonspecific adsorption of IS to the polymer reaction vessel flask was an issue, but this was overcome by switching to glass flasks for sample incubation and storage. Finally, the protein-based materials were vacuum-dried and weighed to determine their mass. The adsorption amount, i.e., the ratio of the mass of adsorbed PBUT to the total mass of the material, was calculated and is shown in Figures 4a–c.

[0110] Unfunctionalized protein Ftn (neg) The crystals adsorbed all three tested toxins, with adsorption amounts of 247–283 μg g−1 for pCS and IS. -1, PheAc 2710 μg g -1 The significantly higher adsorption of PheAc is likely due to the 10-fold higher concentration of PBUT in the assay. For IS and pCS, functionalization with a phenyl moiety (Phe) resulted in adsorption of 458 or 372 μg g -1 The adsorption capacity increased to 1000 μg mL. Functionalization with aliphatic molecules (C10) only observed an increase in adsorption capacity for IS (Figure 4a). For PheAc toxin, no significant increase in adsorption capacity was observed after incorporation of hydrophobic molecules. To investigate whether a highly ordered material with a uniform distribution of solvent channels and pores would be advantageous for adsorption, this material was compared with a sample of amorphous protein material prepared by adding a crosslinker to the protein solution and incubating overnight (not shown). The resulting material was tested for adsorption capacity toward IS. No significant difference was observed between the two materials, indicating that the macroscopic shape of the material does not affect IS adsorption. The adsorption capacity of our protein-based adsorbent is in a similar range, but is higher than that of other published materials, e.g., up to 1000 μg mL for IS. -1 P87 zeolite [Non-Patent Document 35] with an adsorption capacity of up to 3200 μg mL -1 This is smaller than the value of a carbon-based adsorbent with an adsorption capacity of 156 mg g [Non-Patent Document 36]. To the best of our knowledge, the highest adsorbent with an adsorption capacity of 156 mg g has been published to date. -1 Zirconium-based MOFs have adsorption capacities of 1000 kJ / kg [Non-Patent Document 24]. However, these materials are excellent adsorbents in themselves, and efforts to improve their biocompatibility are needed. In contrast, the protein-based materials of the present invention have inherent biocompatibility. Adsorption capacities can be tuned and further improved.

[0111] To demonstrate the biocompatibility of the material, endothelial cells and isolated platelets were incubated with the crystalline material. Endothelial cells showed no expression of tumor necrosis factor alpha (TNF-α), indicating the absence of endotoxin contamination due to the bacterial origin of the material (Figure 4d). Platelets were not activated, indicating that this material does not induce blood clotting (Figure 4c). Similar results were obtained with crystals cross-linked with glutaraldehyde (not shown). Furthermore, no significant differences were observed between chemically modified and unmodified protein materials, indicating that functionalization does not affect biocompatibility.

[0112] Overall, the materials synthesized according to the present invention, based on the bottom-up construction of protein cages, have been shown to be highly suitable for blood purification applications. The resulting materials exhibit stability in organ systems such as blood and exhibit good adsorption of three types of PBUT. Crystalline and amorphous adsorbents exhibit similar behavior. Furthermore, it has been shown that the introduction of anchoring moieties allows the incorporation of up to 96 water-insoluble aliphatic and phenyl molecules into the cavity of the ferritin protein cage. No decrease in biocompatibility was observed after modification. Chemical modification is expected to increase the adsorption capacity. Because PBUT possesses both hydrophobic and hydrophilic properties, it is highly likely that the adsorption capacity will be further enhanced by the addition of ligands other than those exhibiting only hydrophobicity, such as amphiphilic ligands, a mixture of hydrophobic and hydrophilic ligands, or by modifying the amino acid residues surrounding the ligands. The modular nature of the materials according to the present invention allows for other applications, such as the treatment of heavy metal contamination by incorporating chelating agents. Furthermore, genetic engineering allows the introduction of positively charged amino acids around the anchoring moieties to counteract the negative charge of PBUT while simultaneously binding the hydrophobic moiety of the toxin to the molecule being incorporated. All these modifications to the internal surface do not affect the construction of the material. [Example]

[0113] Modification of the inner surface and channel region of apoferritin nanoparticles according to the present invention (without additional chemical functionalization) Efficient adsorption to apoferritin nanoparticles according to the present invention can also be achieved by using a suitable combination of amino acids with hydrophobic, aromatic, polar, or charged side chains, allowing binding sites within the protein container to be created even in unfunctionalized proteins. An example is shown in Figure 5b. Here, the delocalized hydrophobic ring system of the IS is attached to the ring system of the amino acid tyrosine, and the negatively charged polar sulfate group is stabilized by the partially positively charged nitrogen atom of the amino acid histidine. To design these protein variants, the "ligand docking" protocol in the Rosetta software package was used to determine the binding affinity of the protein for the toxin. Next, various amino acids in the protein were randomly varied in the computer model, and the affinity was determined again. This procedure was repeated until no increase in affinity was observed.

[0114] The protein containers designed in this way were fabricated in the laboratory. The containers remained soluble and assembled into complete containers. The introduced mutations were confirmed by ESI-MS measurements. Mutants with enhanced affinity for IS were designed and successfully fabricated. As can be seen in Figure 5a, these mutant adsorbents showed, to some extent, significantly increased adsorption of the appropriate toxin (Ftn). (neg) -Dock=Ftn (neg) -dock03, SEQ ID NO:21).

[0115] Furthermore, the properties of apoferritin nanoparticles can be altered by specifically exchanging amino acids. For example, by exchanging negatively charged amino acids with hydrophobic amino acids, the negative surface potential can be reduced within the region of the internal cavity. In this way, the adsorption amount can be significantly increased for PBUT, which is negatively charged even 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, the increased adsorption is likely due to improved transport of material into the inner region of the apoferritin nanoparticle. And, material transport into the inner cavity region can be improved by modifications within the triple channel region. For example, replacing amino acids with charged or sterically hindered residues with the amino acid alanine significantly increased the amount of adsorption (Figure 5a, Ftn (neg) -Ap+Channel=Ftn (neg) -Ap4-3A, SEQ ID NO:18). Binding sites created by amino acid exchange can be combined with modifications that improve mass transport to further improve adsorption. Furthermore, variants with multiple binding sites per apoferritin subunit are possible.

[0116] The mutant Ftn already mentioned above (neg) In -Ap4 (SEQ ID NO:17), amino acids with negatively charged side chains, such as glutamine and aspartic acid, were substituted with nonpolar or aromatic derivatives (E61V, E62A, D131F, and E140W) at four positions per subunit in the interior space (E61, E62, D131, and E140). The polarity changed from negative to uncharged due to the surface potential (not shown). However, negatively charged regions remained on the interior surface. This is because the responsible amino acids were somewhat hidden in the protein backbone, and these sites were not mutated to maintain protein stability. Because the introduced amino acids have hydrophobic or aromatic side chains, these residues may further serve as adsorption sites for the partially hydrophobic PBUT. To distinguish whether the effect on adsorption, if any, was due to a general decrease in negative surface charge or specific adsorption of hydrophobic residues, Ftn was analyzed. (neg) A further mutant, designated -Ap16, was generated (SEQ ID NO:20). Similar to the Ap4 mutant, four charged amino acids at positions 61, 62, 131, and 140 were substituted with nonpolar derivatives (W instead of D at position 131), and 12 additional nonpolar amino acids were introduced at surface-exposed positions.

[0117] Ftn(neg) -Ap4 and Ftn (neg) The amount of adsorption of -Ap16 was measured for both crystalline and amorphous forms of Ftn (neg) The adsorption of indoxyl sulfate (IS) from the crystalline material was compared with that of unmodified ferritin (Ftn). (neg) ) and modified ferritin (Ftn (neg) -Ap4, Ftn (neg) No significant difference was observed between the Ap variants (Ap16) and Ftn (see Figure 6). However, on amorphous materials, a significant increase in adsorption was observed for both Ap variants. Despite the addition of 12 hydrophobic amino acids, Ftn (neg) The amount of adsorption of -Ap16 is Ftn (neg) The adsorption of PBUT was similar to that of -Ap4. Therefore, the decrease in negative surface charge appears to be the main reason for the increased adsorption of PBUT. This is consistent with the assumption that the introduction of hydrophobic molecules does not significantly improve the adsorption, and that the change in adsorption is due to a general decrease in negative charge.

[0118] In addition to reducing the negative charges in the interior space and pores of the protein cage, we pursued further approaches to increase the adsorption of PBUT. To stabilize both the polar and hydrophobic parts of PBUT, we envisioned introducing suitable amino acids to create unique binding sites. For this purpose, we used the ligand docking protocol of the Rosetta software suite (see [Non-Patent Document 40]). We used IS as the ligand. Three further potential ferritin mutants, Ftn (neg) -dock03 (SEQ ID NO: 21), Ftn (neg) -dock23 (SEQ ID NO: 22), and Ftn (neg) The binding site was determined to be near the triple channel (Ftn (neg) -dock03), subunit center (Ftn (neg) -dock23), and near the quadruple channel (Ftn (neg) -dock43).

[0119] The adsorption of the new mutants to IS was measured for the crystal assemblies and is shown in Figure 7. As can be seen from Figure 7, the unmodified Ftn (neg) The adsorption capacity of all three variants is significantly improved compared to Ftn (neg) -03=Ftn (neg) -dock03, Ftn (neg) -23=Ftn (neg) -dock23, Ftn (neg) -43=Ftn (neg) -dock43). (neg) -03 showed the greatest adsorption amount.

[0120] Three mutant Ftn specifically engineered for IS (neg) -dock03, Ftn (neg) -dock23, and Ftn (neg) As shown in Figure 8, the adsorption of the mutant Ftn-dock43 to pCS and PheAc was also measured. An interesting trend was observed. (neg) -03 and Ftn (neg) -43 are both Ftn (neg) Better than -23, but mutant Ftn (neg) -23 was superior to other mutants in adsorbing pCS and PheAc. These results indicate that it is possible to introduce a degree of selectivity into the binding site by targeting specific features of the toxin.

[0121] Furthermore, we tested whether we could combine various features that improve protein binding in a single structure. (neg) Mutations that create the binding site for -43 were identified as Ftn (neg) Combined with the -Ap4-3A mutation, which reduces the internal space and surface charge of the triple channel, mutant Ftn (neg) -Ap4-3A-43 was prepared.

[0122] The adsorption capacity of this mutant was measured on the amorphous form of the adsorbent and compared with that of the unmodified Ftn (neg)The results were compared with those of the mutants mentioned above (Figure 9). The measurements show that by combining mutations into one structure, the individual positive effects of the different modifications can be added together. (neg) -Ap4-3A-43 shows the highest adsorption amount among all the mutants examined in this study. [Industrial Applicability]

[0123] The advantage of simply changing the properties of apoferritin nanoparticles according to the present invention by amino acid exchange is that the protein can be used without further chemical modification. This reduces the possibility of allergic reactions and other side effects, and makes them cheaper to produce than functionalized variants. It also allows for the design of stronger binding sites. Furthermore, the present invention opens the possibility of providing other apoferritin nanoparticles or apoferritin nanoparticle compositions with even improved affinity, tailored to specific toxins as needed.

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Claims

1. Apoferritin nanoparticles for use as a toxin binder in hemodialysis, comprising: The apoferritin nanoparticles contain apoferritin subunits that form an internal space when assembled, and the internal space does not contain nanoparticles. Apoferritin nanoparticles for use as a toxin binder in hemodialysis, characterized in that

2. At least one of the apoferritin subunits within the region of the internal space and / or within the region of the triple channel and / or within the region of the quadruple channel has an amino acid sequence that is modified relative to the wild-type sequence. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to claim 1.

3. The at least one apoferritin subunit having an amino acid sequence within the region of the internal space that is modified relative to the wild-type sequence has at least one additional cysteine ​​residue or a cysteine ​​residue at a different position in the sequence compared to the wild-type sequence, and the at least one apoferritin subunit having an amino acid sequence that is modified relative to the wild-type sequence is functionalized or can be functionalized by covalent bonding of an organic compound residue to the cysteine ​​residue. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to claim 2.

4. The at least one apoferritin subunit having an amino acid sequence modified relative to the wild-type sequence has two, three, or four cysteine ​​residues within the region of the internal space to which each organic compound residue is covalently bound, compared to the wild-type sequence. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to claim 3.

5. The covalently bonded organic compound residue located at the at least one additional cysteine ​​residue or at a cysteine ​​residue at a different position in the sequence may be N-phenylacetamidyl, N-decylacetamidyl, N-(1-amino-2-phenylethyl)acetamidyl, N-(1-amino-2-phenylpropyl)acetamidyl, N-(1-amino-2-phenylbutyl)acetamidyl, N-(1,1-diamino-2-phenylethyl)acetamidyl , N-(1,1-diamino-2-phenylpropyl)acetamidyl, N-(1,1-diamino-2-phenylbutyl)acetamidyl, N-(3,4,5-tris(trifluoromethyl)phenyl)acetamidyl, N-(3,4,5-trinitrophenyl)acetamidyl, N-(1-amino-2-(3,4,5-trinitrophenyl)ethyl)acetamidyl, and N-(2-(heptylamino)ethyl)acetamidyl. Apoferritin nanoparticles for use as toxin binders in hemodialysis according to claim 3 or 4.

6. The amino acid sequence of the at least one apoferritin subunit having an amino acid sequence that is modified relative to the wild-type sequence is modified relative to the wild-type sequence within the region of space such that the apoferritin nanoparticles have a higher binding affinity for a toxin compared to apoferritin nanoparticles made from apoferritin subunits having an amino acid sequence that is not modified relative to the wild-type sequence. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of claims 1 to 5.

7. The amino acid sequence of the at least one apoferritin subunit having an amino acid sequence modified compared to the wild-type sequence is modified compared to the wild-type sequence within the region of the triple channel such that toxin transport into the space of the apoferritin nanoparticle is enhanced compared to apoferritin nanoparticles having an unmodified amino acid sequence. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of claims 1 to 6.

8. The toxin is preferably a protein-bound uremic toxin selected from the group consisting of indoxyl sulfate, paracresyl sulfate, phenyl acetate, and p-hydroxyhippuric acid. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of claims 1 to 7.

9. The apoferritin nanoparticles are composed of 24 apoferritin subunits, and at least 2, preferably at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, and particularly preferably all 24 subunits have an amino acid sequence that is modified relative to the wild-type sequence. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to any one of claims 1 to 8.

10. The subunit has an amino acid sequence that is modified relative to the wild-type sequence, selected from one of the amino acid sequences according to SEQ ID NO: 13 to 24, preferably 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. Apoferritin nanoparticles for use as a toxin binder in hemodialysis according to claim 9.

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

10. A composition for use as a toxin binder in hemodialysis, comprising:

12. A plurality of apoferritin nanoparticles cross-linked to each other for use as a toxin binder in hemodialysis according to any one of claims 1 to 10.

12. The composition of claim 11 for use as a toxin binding agent in hemodialysis.

13. 13. The method of claim 12, further comprising administering to said patient a drug containing 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 claim 11 or 12. An adsorbent cartridge characterized by:

14. The plurality of apoferritin nanoparticles or compositions are contained in a first compartment within the sorbent cartridge separated from a second compartment within the sorbent cartridge by one or more membranes, the one or more membranes being permeable to unbound toxin molecules and / or proteins bound to toxin molecules, but not to the apoferritin nanoparticles. The sorbent cartridge of claim 13.

15. A dialyzer comprising at least one sorbent cartridge according to claim 13 or 14. A dialysis system characterized by:

16. The at least one sorbent cartridge is arranged a) upstream, b) downstream, or c) in an auxiliary circuit of the dialysis circuit in the direction of blood flow to be dialyzed.

16. The dialysis system of claim 15.

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