Functionalized biocatalytic compositions

Abstract

The present invention relates to a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group.

Description

[Technical field]

[0001] The present invention relates to a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group. The present invention also relates to a method for producing said composition. [Background technology]

[0002] Proteins such as enzymes are frequently required, for example, in industrial, diagnostic or therapeutic applications. To stabilize proteins and / or render them resistant to various types of stress, it has been suggested in the prior art to immobilize proteins on the surface of a support and protect them with a layer of a protective material. Such an approach is described, for example, in WO 2015 / 014888, which discloses a biocatalytic composition comprising a solid support, a functional component such as an enzyme and a protective layer for protecting the functional component by embedding the functional component, and a method for producing such a biocatalytic composition. However, the biocatalytic compositions described, for example, in WO 2015 / 014888 cannot be used for therapeutic applications due to their lack of biocompatibility and bioavailability. Thus, there is a need to provide biocatalytic compositions that are compatible and useful for therapeutic applications. Summary of the Invention

[0003] The present invention provides a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group.

[0004] The present invention also provides a method for producing said composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group, the method comprising the steps of: (a) providing a solid support; (b) immobilizing the protein or a fragment thereof on a solid support; (c) forming a protective layer on the surface of the solid support to protect the enzyme or fragment thereof immobilized on the solid support; (d) immobilizing a functional component on the surface of the protective layer, the functional component immobilized on the surface of the protective layer being a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group; The present invention provides a method comprising:

[0005] It has surprisingly been found by the inventors of the present application that the compositions provided by the present invention, when applied in therapy, have an unexpectedly high biodistribution specificity, a surprisingly high mucoadhesion in vitro and in vivo, exhibit low cytotoxicity and do not disrupt the intestinal barrier even when localized in the gastrointestinal tract, thus making them highly promising for therapeutic applications, in particular for therapeutic applications in enzyme replacement therapy (ERT). [Brief description of the drawings]

[0006] [Figure 1] 1 shows a schematic diagram of a method for producing a composition of the invention: a) a protein or fragment is immobilized on a solid support; b) and c) a protective layer is grown around the immobilized protein or fragment thereof, embedding the immobilized protein or fragment thereof; and d) a functional component is immobilized on the surface of the protective layer. [Figure 2A-2B] Functionalization of nanoparticles with chitosan. (A) Shielded nanoparticles (NP-1) were reacted with FITC-chitosan for 30 min at 20 °C with stirring at 400 rpm. The histogram represents the fluorescence intensity (lex: 489 nm, lem: 515 nm) of shielded (NP-1) nanoparticles and reacted (NP-2) nanoparticles. (B) Kinetics of Cu(II)-catalyzed azide-alkyne cycloaddition reaction between ethynyl-modified silica nanoparticles (SNP) and 3-azido-7-hydroxycoumarin. [Figure 2C] (C) Kinetics of the copper-free azide-alkyne cycloaddition reaction between dibenzocyclooctyne-maleimide and 3-azido-7-hydroxycoumarin. [Diagram 3] Figure 1 shows the ex vivo interaction of chitosan-functionalized shielded nanoparticles with mucus layers depending on the surface functionalization level of the nanoparticles. Fluorescent shielded nanoparticles (NP-1) and fluorescent partially (NP-3) or fully (NP-2) chitosan-functionalized shielded nanoparticles (500 μg / mL) were added to porcine mucus layers for 1 h. Nanoparticle binding to mucus was assessed by measuring fluorescence (lex: 489 nm, lem: 515 nm). The histogram represents the percentage of nanoparticles bound to mucus. [Figure 4]Figure 1 shows the ex vivo interaction of chitosan-functionalized shielded nanoparticles with the mucus layer depending on the molecular weight (MW) of chitosan immobilized on the surface of the nanoparticles. Fluorescent chitosan-functionalized shielded nanoparticles (500 μg / mL) were added to porcine mucus layer for 1 h. (A) The image shows the nanoparticles bound to the mucus layer after washing (dark area within the circle). (B) Quantification of nanoparticles bound to the mucus layer is shown in the table. Different formulations of nanoparticles evaluated are: NP-4: fluorescent human recombinant lipase shielded nanoparticles; NP-6: fluorescent human recombinant lipase shielded nanoparticles partially functionalized with medium MW chitosan by electrostatic interactions; NP-7: fluorescent human recombinant lipase shielded nanoparticles partially functionalized with medium MW chitosan by click chemistry, NP-8: fluorescent human recombinant lipase shielded nanoparticles partially functionalized with low MW chitosan by click chemistry. [Diagram 5] Figure 1 shows the ex vivo interaction of nanoparticles functionalized with different polymers containing amino groups with the mucus layer. Fluorescence shielded nanoparticles (500 μg / mL) were added to porcine mucus layer for 1 h. (A) The image shows the nanoparticles bound to the mucus layer after washing (dark areas within the circle). (B) Quantification of nanoparticles bound to the mucus layer is shown in the table. The different nanoparticle formulations are: NP-4: fluorescent human recombinant lipase shielded nanoparticles; NP-9: fluorescent human recombinant lipase shielded nanoparticles whose surface is functionalized with an additional layer of polymerized APTES; NP-5: fluorescent human recombinant lipase shielded nanoparticles fully functionalized with medium MW chitosan by electrostatic interactions, and NP-10: fluorescent human recombinant lipase shielded nanoparticles whose surface is functionalized with polymerized silane-PEG-NH2. [Figure 6] Interaction of chitosan-functionalized shielding nanoparticles with the intestinal barrier model. (A) Differentiated Caco-2 and differentiated Caco-2 / HT29-MTX-E12 co-cultures were stained with Alcian Blue and imaged with a ZEISS light microscope. The dark areas are mucus stained with Alcian Blue. Representative images are shown. (B) Fluorescent fully chitosan-functionalized shielding nanoparticles (NP-2) were added to the apical side of the differentiated intestinal barrier model for 24 hours. Images show nanoparticles bound to mucus (dark areas) after washing enterocytes. [Figure 7A]Figure 1 shows the biodistribution of fully chitosan-functionalized shielded nanoparticles in mice. Radioactive fully chitosan-functionalized shielded nanoparticles (NP-2) were administered orally to mice by gavage. At the end of the day, the digestive tract (A) and blood and organs (B) were harvested and counted in a gamma counter. Data are expressed as percentage of injected dose per gram of organ (%ID / organ). [Figure 7B] (the above) [Figure 8] Figure 1 shows an in vitro cytotoxicity evaluation of nanoparticles. Caco-2 (AB) and HT29-MTX-E12 (CD) cells were exposed to increasing concentrations of nanoparticles (NP-1 or NP-2) for 24 hours (AC) and 48 hours (BD). Cell damage was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay. Cell viability is expressed as a percentage of control cells (untreated cells). Triton (1 mg / mL) is used as a negative control. Error bars indicate standard deviation. [Figure 9] Figure 1 shows the effect of nanoparticle exposure on transepithelial electrical resistance (TEER). Differentiated Caco-2 (AB) and differentiated Caco-2 / HT29-MTX-E12 co-cultures (CD) were exposed to nanoparticles (NP-1 or NP-2) for 24 h (AC) and 48 h (BD), covering 100% of the cell surface. Barrier integrity was assessed by measuring TEER. "NP-1 w / o cells" and "NP-2 w / o cells" refer to the addition of nanoparticles to the Transwell system without cells to assess the effect of the presence of nanoparticles on TEER measurements (background signal). EGTA (2.5 mM) is a calcium chelator that disrupts tight junctions and is used as a control for loss of barrier integrity. Untreated condition ("Untreated") refers to cells seeded in the Transwell system without any treatment and is used as a reference for TERR values. Error bars indicate standard deviation. [Figure 10]Figure 1 shows the effect of nanoparticle exposure on Lucifer Yellow translocation across the intestinal barrier. Differentiated Caco-2 (AB) and differentiated Caco-2 / HT29-MTX-E12 co-cultures (CD) were exposed to nanoparticles (NP-1 or NP-2) for 24 h (AC) and 48 h (BD), covering 100% of the cell surface. Barrier integrity was assessed by measuring the translocation of Lucifer Yellow (LY) from the donor compartment to the acceptor compartment for 90 min. Histograms represent the percentage of cumulative Lucifer Yellow (LY) permeability. EGTA (2.5 mM), a calcium chelator that disrupts tight junctions, is used as a control for loss of barrier integrity. The untreated condition ("Untreated") refers to cells seeded in the Transwell system without any treatment and is used as a reference for LY diffusion. Error bars indicate standard deviation. [Figure 11A] Evaluation of cellular uptake of nanoparticles in an intestinal barrier model. (A) Differentiated Caco-2 / HT29-MTX-E12 co-cultures were incubated with fluorescent fully chitosan-functionalized shielded nanoparticles (NP-2) for 24 h. Cells were stained with phalloidin-TRITC (cell membrane) and DAPI (nuclei). Nanoparticle localization in the intestinal barrier was evaluated by confocal microscopy. Cross lines in the XY images indicate the position of the XZ (bottom) and YZ (right) lateral views. [Figure 11B] (B) Differentiated Caco-2 / HT29-MTX-E12 co-cultures and THP-1 differentiated into M0 macrophages were treated with NP-2 for 24 h and analyzed by flow cytometry. The histograms represent the fluorescence of untreated cells (dashed line) and NP-2 treated cells (solid line). THP-1 are used as a positive control for nanoparticle internalization. [Figure 12]Figure 1 shows the activity of immobilized and shielded pancreatin on fully chitosan-functionalized shielded nanoparticles depending on the shielding composition. The biocatalytic activity of fully chitosan-functionalized shielded nanoparticles (NP-12, NP-14, and NP-16) containing immobilized and shielded pancreatin was evaluated at 37°C for 24 hours and compared to free pancreatin. The activity of each enzyme contained in the pancreatin was evaluated, namely protease (A), lipase (B), and amylase (C). The curves represent the percentage of remaining enzyme activity compared to the initial activity. The nanoparticles used were as follows: NP-14: partially shielded pancreatin nanoparticles with protective layers consisting of APTES and TEOS and functionalized with chitosan (A); NP-12: completely shielded pancreatin nanoparticles with protective layers consisting of APTES, TEOS, and benzyltriethoxysilane and functionalized with chitosan (B); NP-16: completely shielded pancreatin nanoparticles with protective layers consisting of APTES and TEOS and functionalized with chitosan (C). [Figure 13] The protective effect of chitosan on protease activity is shown. The protease activity of immobilized and shielded pancreatin nanoparticles was evaluated under acidic conditions (acetic acid solution, pH: 4) at 37°C for 1 h and compared with the activity of immobilized and shielded pancreatin nanoparticles under basic conditions (pH 8). The histogram represents the percentage of remaining activity compared to the initial activity. The nanoparticles used were: NP-13: partially shielded pancreatin nanoparticles with a protective layer consisting of APTES and TEOS; NP-14: partially shielded pancreatin nanoparticles with a protective layer consisting of APTES and TEOS and functionalized with chitosan. [Figure 14A]Figure 1 shows the ex vivo interaction of thiol-functionalized shielded nanoparticles with the mucus layer, depending on the percentage of thiol functionalization on the surface of the nanoparticles. Fluorescent thiol-functionalized shielded nanoparticles (500 μg / mL) were added to porcine mucus layer for 1 h. (A) The image shows the nanoparticles bound to the mucus layer after washing (dark areas within the circle). Quantification of nanoparticles bound to the mucus layer is shown in the table (B) and histogram (C). The different formulations of nanoparticles evaluated were: NP-17: fluorescent BSA nanoparticles partially (5%) functionalized with MPTS; NP-18: fluorescent BSA nanoparticles partially (10%) functionalized with MPTS; NP-19: fluorescent BSA nanoparticles partially (20%) functionalized with MPTS; NP-20: fluorescent BSA nanoparticles partially (50%) functionalized with MPTS; NP-21: fluorescent BSA nanoparticles fully (100%) functionalized with MPTS. [Figure 14B-14C] (the above) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] The present invention relates to a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group.

[0008] For purposes of interpreting this specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0009] It is to be understood that features, integers, properties, compounds described in connection with a particular aspect, embodiment or example of the invention are applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. All of the features disclosed in this specification (including the accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments.

[0010] The term "comprise" and its variants, such as "comprises" and "comprising", are generally used in the sense of include, i.e. "including but not limited to", i.e. allowing for the presence of one or more features or components.

[0011] The singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0012] The term "about" refers to a range of values ​​of ±10% of the specified value. For example, the phrase "about 200" includes ±10% of 200, or from 180 to 220.

[0013] The term "solid support" as used herein generally refers to particles. Preferably, the solid support is a monodisperse or polydisperse particle, more preferably a monodisperse particle. The solid support generally includes organic particles, inorganic particles, organic-inorganic particles, self-assembling organic particles, silica particles, gold particles, titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size of the solid support is generally between 1 nm and 1000 μm, preferably between 10 nm and 100 μm, in particular about 50 nm.

[0014] The terms "linker" or "crosslinker", used interchangeably herein, refer to any linking reagent that contains a group capable of binding to a specific functional group (e.g., primary amine, sulfhydryl, etc.). A linker in the context of the present invention typically links the surface of a solid support to an enzyme. For example, a linker may be immobilized on the surface of a solid support, e.g., a silica surface as a support material, and then an enzyme may be bound to an unoccupied binding site of the linker. Alternatively, a linker may be first bound to an enzyme, and then the linker bound to the enzyme may be bound to a solid support at its unoccupied binding site. Various types of linkers are known in the art, including, but not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as tags.

[0015] The term "protective layer" as used herein refers to a layer for protecting the functional properties of a protein or a fragment thereof, such as an enzyme or a fragment thereof, immobilized on the surface of a solid support. The protective layer of the present invention is usually constructed using components, at least a part of which are monomers that can interact with each other, usually by covalent bonds, and with the immobilized enzyme, usually by non-covalent bonds. The protective layer is formed on the surface of the solid support to protect the protein or a fragment thereof immobilized on the solid support. The protective layer is usually a homogeneous layer in which at least 50%, preferably at least 70%, more preferably at least 90% of the protein or a fragment thereof, such as an enzyme or a fragment thereof, is embedded in the protective layer.

[0016] The term "protein or fragment thereof" as used herein includes proteins that typically contain between 100 and 1500 amino acids, preferably between 100 and 800 amino acids, more preferably between 100 and 500 amino acids. A fragment of a protein described herein typically has the same functional properties as the protein from which it is derived. Preferred proteins or fragments thereof are enzymes or fragments thereof.

[0017] The term "enzyme or fragment thereof" includes naturally occurring enzymes or fragments thereof, and also includes artificially engineered enzymes or fragments thereof. An artificially engineered enzyme or fragment thereof is, for example, a variant or functionally active fragment of the enzyme. With respect to an enzyme of the present invention, "variant or functionally active fragment thereof" means that the fragment or variant (such as an analog, derivative or mutant) is capable of performing the same physiological function as the enzyme. Such variants include naturally occurring allelic variants and variants that do not occur naturally. Addition, deletion, substitution and derivatization of one or more of the amino acids are contemplated, so long as the modification does not result in loss of functional activity of the fragment or variant. Preferably, a functionally active fragment or variant has at least about 80% sequence identity to the relevant portion of the enzyme, more preferably at least about 90% sequence identity, even more preferably at least about 95% sequence identity, and most preferably at least about 98% sequence identity. A fragment of an enzyme described herein will usually have the same functional properties as the enzyme from which it is derived.

[0018] As used herein, "partially embedded protein" shall mean that the protein is not completely covered by the protective layer, and thus the protein is not completely embedded in the protective layer. In one embodiment, less than 50% of the protein of interest is covered by the protective layer, but typically at least 70% or more, thus improving the protection of the protein. In a preferred embodiment, at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the protein of interest is covered by the protective layer. In another preferred embodiment, about 70% to about 95%, more preferably about 80% to about 95%, even more preferably about 90% to about 95%, and most preferably about 90% to about 95, 96, 97, 98, or 99% of the protein of interest is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the protein of interest is covered by the protective layer. In a more particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the protein of interest is covered by the protective layer, leaving the active site uncovered.

[0019] The term "fully embedded protein" as used herein is intended to mean that the protein of interest according to the invention is completely, i.e. 100%, covered by the protective layer, i.e. also the active site is covered.

[0020] The term "at least partially embedded protein" as used herein is intended to mean that the protein is at least partially embedded and may be completely embedded by the protective layer. Thus, "at least partially embedded protein" means that the protective layer covers about 30% to 100%, preferably about 50% to about 100%, more preferably about 80% to about 100%, even more preferably about 90% to about 100%, and most preferably about 95% to about 100% of the protein or fragment thereof, and the active site is preferably covered.

[0021] As used herein, "partially embedded enzyme" is intended to mean that the enzyme is not completely covered by the protective layer, and thus the enzyme is not completely embedded in the protective layer. In one embodiment, less than 50% of the enzyme of interest is covered by the protective layer, but typically at least 70% or more is covered, thus improving the protection of the enzyme. In a preferred embodiment, at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the enzyme of interest is covered by the protective layer. In another preferred embodiment, about 70% to about 95%, more preferably about 80% to about 95%, even more preferably about 90% to about 95%, and most preferably about 90% to about 95, 96, 97, 98, or 99% of the enzyme of interest is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the enzyme of interest is covered by the protective layer. In a more particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the enzyme of interest is covered by the protective layer, leaving the active site uncovered.

[0022] The term "fully embedded enzyme" as used herein is intended to mean that the enzyme of interest according to the invention is completely, i.e. 100%, covered by the protective layer, i.e. also the active site is covered.

[0023] The term "at least partially embedded enzyme" as used herein is intended to mean that the enzyme is at least partially embedded and may be completely embedded by the protective layer. Thus, "at least partially embedded enzyme" means that the protective layer covers about 30% to 100%, preferably about 50% to about 100%, more preferably about 80% to about 100%, even more preferably about 90% to about 100%, and most preferably about 95% to about 100% of the enzyme or a fragment thereof, and the active site is preferably covered.

[0024] The term "functional component" as used herein refers to a component that retains its characteristics, functional properties, after being immobilized on the surface of the protective layer. A functional component in the sense of the present invention is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group.

[0025] As used herein, the term "a polymer comprising repeating units, each repeating unit comprising at least one amino group" refers to a polymer comprising multiple repeating units (monomers), each repeating unit comprising at least one amino group. Preferred polymers comprise multiple repeating units (monomers), each repeating unit comprising one amino group, particularly one primary amino group.

[0026] As used herein, the term "a polymer comprising repeating units, each repeating unit comprising at least one thiol group" refers to a polymer comprising multiple repeating units (monomers), each repeating unit comprising at least one thiol. A preferred polymer comprises multiple repeating units (monomers), each repeating unit comprising one thiol group.

[0027] As used herein, "polycarbophil-cysteine ​​conjugate" refers to a conjugate that contains cysteine ​​covalently bound to polycarbophil. Such conjugates can be prepared, for example, as described in Bernkop-Schnurch and Thaler, 2000, Journal of Pharmaceutical Sciences 89(7):901-9.

[0028] The term "polylysine" as used herein refers to α-polylysine and / or ε-polylysine (ε-poly-L-lysine, EPL), preferably ε-polylysine. α-Polylysine is a synthetic polymer and can be composed of either L-lysine or D-lysine. ε-Polylysine (ε-poly-L-lysine, EPL) is typically produced as a homopolypeptide of about 25-30 L-lysine residues.

[0029] As used herein, the term "polycysteine" can be composed of either L-cysteine ​​or D-cysteine, but preferably is composed of L-cysteine, and preferably contains from 2 to 30 cysteine ​​residues, more preferably from 2 to 5 cysteine ​​residues.

[0030] The term "polyglucosamine" as used herein refers to a linear aminopolysaccharide consisting of D-glucosamine and N-acetyl-D-glucosamine units linked by (1-4) glycosidic bonds. Polyglucosamine contains free amine (-NH2) groups and can be characterized by the ratio of N-acetyl-D-glucosamine units and D-glucosamine units, which is expressed as the degree of deacetylation (DDA) of the fully acetylated polymer chitin. The preferred polyglucosamine of the present invention is selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan and dermatan or derivatives thereof. Most preferred is chitosan or a derivative thereof.

[0031] The term "chitosan or its derivatives" as used herein refers to chitosan or its chitosan derivatives including chitosan having a molecular weight of preferably 2000 Da or more, preferably in the range of 25000-2000000 Da, more preferably about 50000-350000 Da, most preferably about 50000-190000 Da or 190000-310000 Da. The term chitosan derivatives includes esters, ethers or other derivatives formed by reaction of acyl or alkyl groups with OH groups. Examples are O-alkyl ethers of chitosan, O-acyl esters of chitosan. Suitable derivatives are given, for example, in GA E Roberts, Chitin Chemistry, MacMillan Press Ltd, London, 1992. Suitable salts of chitosan include nitrates, phosphates, sulfates, xanthates, hydrochlorides, glutamates, lactates and acetates.

[0032] In a first aspect, the present invention provides a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeat units, each repeat unit containing at least one amino group and / or at least one thiol group.

[0033] The protein or fragment thereof, such as an enzyme or fragment thereof, can be immobilized on the surface of the solid support by non-covalent or covalent bonds. Non-covalent bonds include pp (aromatic) interactions, van der Waals interactions, H-bond interactions, and electrostatic interactions, such as ionic interactions. Preferably, the protein or fragment thereof, such as an enzyme or fragment thereof, is immobilized on the surface of the solid support by covalent bonds or covalent bonds via linkers.

[0034] In one embodiment, the solid carrier is selected from the group of organic particles, inorganic particles, organic-inorganic particles, self-assembling organic particles, silica particles, gold particles, titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size is usually measured by measuring the diameter of the particle, and is usually between 1 nm and 1000 nm, preferably between 10 nm and 100 nm, particularly about 50 nm. When the solid carrier is a monodisperse particle, the particle size is usually between 1 nm and 1000 nm, preferably between 10 nm and 100 nm, particularly about 50 nm. When the solid carrier is a polydisperse particle, the particle size is usually between 1 nm and 1000 μm, preferably between 10 nm and 100 μm, particularly between 50 nm and 50 μm.

[0035] Generally, monodisperse particles or polydisperse particles, preferably monodisperse particles, are used as the solid carrier in the present invention. In a preferred embodiment, the monodisperse particles are spherical monodisperse particles. In a more preferred embodiment, the polydisperse particles are non-spherical polydisperse particles.

[0036] The solid support is usually provided in a suspension. The suspension of the solid support can be, for example, water, a buffer or a non-ionic surfactant or a mixture thereof, preferably a mixture of water and a non-ionic surfactant. Buffers that can be used in the method of the present invention include phosphate, piperazine-N,N'-bis(2-ethanesulfonic acid), 2-hydroxy-3-morpholinopropanesulfonic acid, N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), (3-(N-morpholino)propanesulfonic acid), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3-(N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid, ... N,N-bis(2-hydroxyethyl)amino)-2-hydroxypropanesulfonic acid, N,N-bis(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, diglycine, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, N,N-bis(2-hydroxyethyl)glycine, N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0037] In one embodiment, the surface of the solid support is modified to introduce molecules or functional chemical groups as anchor points, i.e. as anchor points of proteins, e.g. enzymes or linkers connecting proteins, e.g. enzymes, to the solid support. Preferably, said anchor points are amine functional chemical groups or moieties. As a non-limiting example, an amino-modified surface of a solid support, e.g. an amino-modified silica surface, may be used as the modified solid support. Such an amino-modified surface of a solid support may be obtained by reacting a solid support having a silica surface with an amino silane, e.g. APTES. Thus, in a preferred embodiment, the solid support is a solid support having a silica surface with an amino-modified surface, more preferably a solid support obtained by reacting a solid support having a silica surface with an amino silane, e.g. APTES. Such modified supports may form amide bonds between proteins, e.g. enzymes, and amine groups on the surface of the support material, or may form amide bonds between linkers and amine groups on the surface of the support material. In one embodiment, the molecules or functional chemical groups introduced as anchor points are uniformly distributed on the surface of the solid support.

[0038] In some embodiments, the protective layer has a defined thickness of about 1 to about 200 nm, usually 1 to about 100 nm, preferably about 1 to about 50 nm, more preferably about 1 to about 25 nm, even more preferably about 1 to about 20 nm, especially about 1 to about 15 nm. The most preferred defined thickness is about 1 to about 15 nm. In some embodiments, the layer has a defined thickness of about 5 to about 100 nm, preferably about 5 to about 50 nm, more preferably about 5 to about 25 nm, even more preferably about 5 to about 20 nm, especially about 5 to about 15 nm. The most preferred defined thickness is about 5 to about 15 nm. The protective layer is usually porous, with pore sizes between 1 nm and 100 nm, preferably between 1 nm and 20 nm.

[0039] In one embodiment, the protein or fragment thereof, e.g., the enzyme or fragment thereof, is partially embedded by the protective layer. In a preferred embodiment, the protein or fragment thereof, e.g., the enzyme or fragment thereof, is at least partially embedded by the protective layer. In a more preferred embodiment, the protein or fragment thereof, e.g., the enzyme or fragment thereof, is completely embedded by the protective layer.

[0040] In one embodiment, the protective layer embeds the solid support and embeds the protein or fragment thereof, such as an enzyme or fragment thereof, immobilized on the surface of the solid support. In one embodiment, the functional component immobilized on the surface of the protective layer is not embedded in the protective layer. Preferably, the protective layer completely embeds the solid support and completely embeds the protein or fragment thereof, such as an enzyme or fragment thereof, immobilized on the surface of the solid support. More preferably, the protective layer completely embeds the solid support and completely embeds the protein or fragment thereof, such as an enzyme or fragment thereof, immobilized on the surface of the solid support, and the functional component immobilized on the surface of the protective layer is not embedded in the protective layer. When the protective layer completely embeds the solid support and completely embeds the protein or fragment thereof, such as an enzyme or fragment thereof, immobilized on the surface of the solid support, the protein or fragment thereof, such as an enzyme or fragment thereof, is completely, i.e. 100%, covered by the protective layer, i.e. the active site is also covered and the solid support is completely, i.e. 100%, covered by the protective layer.

[0041] In a preferred embodiment, the protein or fragment thereof is an enzyme or fragment thereof.

[0042] In a more preferred embodiment, the protein or fragment thereof is selected from the group consisting of serum albumin or a fragment thereof, lipase or a fragment thereof, pancreatin, and a protein or a fragment thereof contained in pancreatin. The protein or a fragment thereof contained in pancreatin is typically a protein or a fragment thereof selected from the group consisting of proteases, amylases, and lipases.

[0043] In an even more preferred embodiment, the protein or fragment thereof is selected from the group consisting of serum albumin or a fragment thereof, lipase or a fragment thereof, and pancreatin.

[0044] In a particularly preferred embodiment, the protein or fragment thereof is a lipase or fragment thereof.

[0045] In one embodiment, the enzyme or fragment thereof is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, transpeptidases, or ligases, or fragments thereof and mixtures thereof. Particularly preferred are hydrolases or fragments thereof, more particularly lipases or fragments thereof.

[0046] The thickness of the protective layer can be measured by using a microscope such as a scanning electron microscope (SEM), a transmission electron microscope (TEM), a scanning probe microscope (SPM), light scattering methods, or by ellipsometry.

[0047] The composition of the present invention is usually produced in a reaction vessel such as a reactor. The formation of the protective layer is usually carried out by forming each protective layer by components, which build up the protective layer in a polycondensation reaction. The polycondensation can be carried out in different solvents, preferably in aqueous solutions. The polycondensation can be easily controlled and stopped as necessary, allowing the achievement of a defined thickness of the protective layer. The selection of components that can be used to build the protective layer can depend on the known structure of proteins, for example enzymes, to adapt the affinity of the protective layer according to optimal and / or desired parameters. As components of the protective layer, usually a structural component and a protective component are used to build the protective layer. A structural component that can be used is, for example, tetraethyl orthosilicate (herein named "TEOS" or "T"). Protective components that can be used are, for example, 3-aminopropyltriethoxysilane (herein designated as "APTES" or "A"), propyltriethoxysilane (herein designated as "PTES" or "P"), isobutyltriethoxysilane (herein designated as "IBTES"), hydroxymethyltriethoxysilane (herein designated as "HTMEOS" or H), benzyltriethoxysilane (herein designated as "BTES"), ureidopropyltriethoxysilane (herein designated as "UPTES"), or carboxyethyltriethoxysilane (herein designated as "CETES"). The structural component is usually a precursor of inorganic silica capable of forming four covalent bonds in the formed layer. The protective component is usually an organosilane with an organic moiety with the ability to interact with proteins, such as enzymes (e.g. enzymes). Preferred structural components are tetravalent silanes, in particular tetraalkoxysilanes. Preferred protective components are trivalent silanes, in particular trialkoxysilanes. More preferred structural components are mixtures of tetravalent and trivalent silanes, in particular mixtures of tetraalkoxysilanes and trialkoxysilanes. Even more preferred structural components are selected from the group consisting of tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate.Even more preferred protective components are selected from the group consisting of carboxyethylsilanetriol, benzylsilane, propylsilane, isobutylsilane, n-octylsilane, hydroxysilane, bis(2-hydroxyethyl)-3-aminopropylsilane, aminopropylsilane, ureidopropylsilane, (N-acetylglycyl)-3-aminopropylsilane, hydroxy(polyethyleneoxy)propyl]triethoxysilane, in particular benzyltriethoxysilane, propyltriethoxysilane, isobutyltriethoxysilane, n-acetylglycyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimethoxysilane, isobutylmethoxysilane, n-octyltrimethoxysilane, hydroxylethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureidopropyltrimethoxysilane (N-acetylglycyl)-3-aminopropyltrimethoxysilane, or benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymefylyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, ureidopropyltrihydroxyethoxysilane (N-acetylglycyl)-3-aminopropyltrihydroxymethoxysilane.

[0048] Particularly preferred components are TEOS as a structural component and APTES, PTES and / or HTMEOS as a protective component, preferably APTES. In particular, TEOS as a structural component and APTES as a protective component are used to build the protective layer.

[0049] The reaction time of the components with the solid support depends on the length of the linker, if one is used, and on the size of the protein, e.g., the enzyme. The reaction is usually carried out for between 0.5 and 10 hours, preferably between 1 and 5 hours, more preferably between 1 and 4 hours, even more preferably between 2 and 4 hours, preferably in an aqueous solution, preferably at room temperature of about 5 to about 25° C. or about 20° C. The formation of the protective layer can be stopped by actively stopping the polycondensation reaction, e.g. by removing unreacted components, e.g., by a washing step, or by self-termination of the polycondensation reaction caused by a limited amount of components.

[0050] In an even more preferred embodiment, the protein, e.g., enzyme, is immobilized on the solid support by at least partially modifying the surface of the solid support by introducing a molecule as an anchor point as described above for the protein, e.g., enzyme, and by using a linker, preferably a crosslinker, which binds the anchor point and the protein, e.g., enzyme.

[0051] In one embodiment, the molecules and / or linkers introduced as anchor points are evenly distributed on the surface of the solid support.

[0052] In a preferred embodiment, the crosslinking agent is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]suberate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryls, sulfhydryl-reactive 2-pyridyldithiols, BSOCOES (bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate]), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionate), or the like. Succinimidyl 6-(3-[2-pyridyldithio]propionate·2HCl), DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamido]hexanoate), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]propionamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamido]butane), DTME (dithio-bismaleimidylethane), BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinking agent is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]suberate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryls (e.g., sulfhydryl-reactive 2-pyridyldithio).Most preferred is glutaraldehyde.

[0053] After the protective layer is formed, the solid support comprising the protein, e.g., the enzyme, and the protective layer can be stored. Storage is usually achieved, for example, by washing the formed composition, e.g., with a buffer, and storing it suspended or dissolved in the buffer for a desired period of time. In a preferred embodiment, the solid support comprising the protein, e.g., the enzyme, and the protective layer is stored at a constant temperature of 2 to 25°C. In a further preferred embodiment, the solid support comprising the enzyme and the protective layer is stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid support comprising the protein, e.g., the enzyme, and the protective layer is stored for 10 to 30 hours at a constant temperature of 2 to 25°C, preferably at room temperature.

[0054] In one embodiment, the functional component binds to mucus.

[0055] In one embodiment, the polymer comprising repeat units, each repeat unit comprising at least one amino group and / or at least one thiol group, is a polymer comprising repeat units, each repeat unit comprising at least one amino group.

[0056] In one embodiment, the polymer comprising repeat units, each repeat unit comprising at least one amino group and / or at least one thiol group, is a polymer comprising repeat units, each repeat unit comprising at least one thiol group.

[0057] In one embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2 and polymerized silane comprising amino groups. In a preferred embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2 and polymerized APTES.

[0058] In a more preferred embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is selected from the group consisting of polyglucosamines selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; and polymerized silanes comprising amino groups, preferably polymerized APTES. In an even more preferred embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is a polyglucosamine, preferably a polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof, more preferably chitosan or derivatives thereof. The preferred polyglucosamines of the present invention are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof. Most preferred is chitosan or a derivative thereof. Preferred polymerized silane-PEG-NH2 is selected from the group consisting of silane-PEG4-NH2, silane-PEG2000-NH2, and silane-PEG5000-NH2. Preferred polymerized silanes containing amino groups are selected from the group consisting of APTES, amino-butyl-TES, amino-pentyl-TES, amino-hexyl-TES, amino-heptyl-TES, and amino-octyl-TES, in particular APTES.

[0059] In a further embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized silanes comprising amino groups, polymerized silanes comprising thiol groups, polycarbophil-cysteine ​​conjugates, polymerized silane-PEG-thiol and polycysteine.In an even more preferred embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is selected from the group consisting of polyglucosamines selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; polymerized silanes comprising thiol groups, preferably polymerized MPTS; polycarbophil-cysteine ​​conjugates; polymerized silane-PEG-thiol; and polycysteine. In an even more preferred embodiment, the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is a polyglucosamine or a polymeric silane comprising a thiol group, preferably a polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan and dermatan or derivatives thereof, more preferably chitosan or a derivative thereof or a polymeric silane comprising a thiol group, polycarbophil-cysteine ​​conjugate, and polymeric silane-PEG-thiol, preferably a polymeric silane comprising a thiol group.

[0060] In a particular embodiment, the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, dermatan or derivatives thereof, in particular chitosan or derivatives thereof, polymerized silane-PEG-NH2 selected from the group consisting of polymerized silane-PEG4-NH2, polymerized silane-PEG2000-NH2, polymerized silane-PEG5000-NH2, polymerized silanes comprising amino groups, preferably polymerized APTES, and polymerized silanes comprising thiol groups, preferably polymerized MPTS.

[0061] In one embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized silanes comprising amino groups, and polymerized silanes comprising thiol groups. In a preferred embodiment, the polymer comprising repeating units each of which comprises at least one amino group and / or at least one thiol group is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized APTES, and polymerized MPTS.

[0062] In a more preferred embodiment, the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamines selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymeric silane-PEG-NH2; polymeric silanes comprising amino groups, preferably polymeric APTES; and polymeric silanes comprising thiol groups, preferably polymeric MPTS. In a particular embodiment, the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, dermatan or derivatives thereof, in particular chitosan or derivatives thereof, polymeric silane-PEG-NH2 selected from the group consisting of polymeric silane-PEG4-NH2, polymeric silane-PEG2000-NH2, polymeric silane-PEG5000-NH2, polymeric silane comprising an amino group which is APTES, and polymeric silane comprising a thiol group which is MPTS.

[0063] In one embodiment, the polymer comprising repeating units, each repeating unit comprising at least one thiol group, is selected from the group consisting of polymerized silanes comprising thiol groups, polycarbophil-cysteine ​​conjugates, polymerized silane-PEG-thiol and polycysteine, preferably polymerized silanes comprising thiol groups, polycarbophil-cysteine ​​conjugates and polymerized silane-PEG-thiol, more preferably polymerized silanes comprising thiol groups, most preferably polymerized MPTS. In one embodiment, the polymerized silanes comprising thiol groups are preferably polymerized MPTS.

[0064] In one embodiment, 5% to 100%, preferably 10% to 100%, more preferably 50% to 100% of the surface of the protective layer is covered with a polymer comprising repeat units, each repeat unit containing at least one amino group and / or at least one thiol group.

[0065] In one embodiment, the functional component is immobilized on the surface of the protective layer by a bond, preferably a covalent bond. In a preferred embodiment, the functional component is immobilized on the surface of the protective layer by a non-covalent bond, preferably an electrostatic interaction. In a more preferred embodiment, a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is immobilized on the surface of the protective layer by a covalent bond.

[0066] In one embodiment, the functional component is immobilized on the surface of the protective layer using a spacer that binds to the surface of the protective layer and to the functional component. Thus, in one embodiment, the present invention includes a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or fragment thereof by embedding the protein or fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer that includes repeating units, each repeating unit containing at least one amino group and / or at least one thiol group, and the functional component is immobilized on the surface of the protective layer by a spacer. Examples of such spacers include polyethylenes such as PEG4, PEG2000, and PEG5000. The functional component immobilized on the surface of the protective layer by a spacer is usually produced by first reacting the spacer with the functional component to bind the spacer to the functional component, and then reacting the functional component bound to the spacer with the surface of the protective layer.

[0067] The immobilization of the functional component on the surface of the protective layer is usually carried out in a reaction vessel such as a reactor, for example by suspending the solid support carrying the protein, e.g., enzyme, embedded in said protective layer in water, a buffer or a non-ionic surfactant or a mixture thereof, preferably in a mixture of water and a non-ionic surfactant. The functional component is then added to the suspension and reacted with the surface of the protective layer, usually under stirring, to immobilize the functional component on the surface of the protective layer. Such a composition obtained is usually washed and resuspended in water, a buffer or a non-ionic surfactant or a mixture thereof. The immobilization occurs by non-covalent, e.g., electrostatic or covalent, bonding of the functional component. The functional component can be immobilized by chemically modifying the surface of the protective layer and the functional component using "click chemistry" such as, for example, copper-catalyzed click chemistry (copper-catalyzed azide-alkyne cycloaddition, see, for example, Kolb et al. (2001) Angew. Chem. 40(11) 2004-2021) or copper-free click chemistry (Wittig G, A Chem Ber, 1961, 94, 3260), for example, a solid support carrying a protein, e.g., an enzyme, embedded in the protective layer as described above, is first reacted with a reactive compound such as an ethynyl compound, the functional component is modified by adding a reactive compound, e.g., an azide residue, and then both components are reacted to immobilize the functional component on the surface of the protective layer.

[0068] In a further embodiment, the composition further comprises a chelating agent, which optionally comprises a radioactive or luminescent label. Preferably, the chelating agent is selected from the group consisting of DOTA, DTPA, NOTA, TETA, AAZTA, TRAP, NOPO and HEHA. More preferably, DOTA or HEHA is used. Even more preferably, a chelating agent comprising a radioactive or luminescent label is used, in particular p-SCN-Bn-DOTA or lutetium-177-radiolabeled-DOTA. When the composition further comprises a chelating agent, the solid support carrying the protein, e.g. the enzyme, embedded in the protective layer is usually pretreated with the chelating agent, and a different chelating agent comprising a radioactive or luminescent label is added to such a pretreated composition.

[0069] Preferably, a radioactive label is used, more preferably a compound of the lanthanide family, even more preferably gadolinium, lutetium, or europium.

[0070] In a further aspect, the present invention provides a composition as described above for use as a medicament.

[0071] In a further aspect, the present invention provides a composition for use in a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in a method of prevention, delay of progression or treatment of exocrine pancreatic insufficiency (EPI), lactase deficiency, sucrase-isomaltase deficiency, disaccharide intolerance, peptide allergy, inflammatory bowel disease (IBD), cystic fibrosis, and / or a pulmonary disease or disorder selected from the group consisting of Gaucher disease, Fabry disease, and mucopolysaccharidoses (MPS).

[0072] Also provided is the use of a composition described herein in the manufacture of a medicament for preventing, delaying progression or treating a pulmonary disease or disorder selected from the group consisting of exocrine pancreatic insufficiency (EPI), lactase deficiency, sucrase-isomaltase deficiency, disaccharide intolerance, peptide allergy, inflammatory bowel disease (IBD), cystic fibrosis, and / or Gaucher disease, Fabry disease, and mucopolysaccharidoses (MPS) in a subject.Also provided is the use of a composition described herein for preventing, delaying progression or treating a pulmonary disease or disorder selected from the group consisting of exocrine pancreatic insufficiency (EPI), lactase deficiency, sucrase-isomaltase deficiency, disaccharide intolerance, peptide allergy, inflammatory bowel disease (IBD), cystic fibrosis, and / or Gaucher disease, Fabry disease, and mucopolysaccharidoses (MPS) in a subject. Also provided is a method for preventing, delaying progression or treating a pulmonary disease or disorder selected from the group consisting of exocrine pancreatic insufficiency (EPI), lactase deficiency, sucrase-isomaltase deficiency, disaccharide intolerance, peptide allergy, inflammatory bowel disease (IBD), cystic fibrosis, and / or Gaucher disease, Fabry disease and mucopolysaccharidoses (MPS) in a subject, comprising administering to the subject a therapeutically effective amount of a composition described herein. Also provided is a use of a composition described herein in the manufacture of a medicament for a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy. Also provided is a use of a composition described herein in a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject. Also provided is a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject, comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0073] The phrases "effective amount" or "therapeutically effective amount" as used herein refer to an amount capable of producing one or more desired effects in a subject to which the composition of the present invention is administered. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0074] In a preferred embodiment, the present invention provides a composition for use in a method for the prevention, delay of progression or treatment of exocrine pancreatic insufficiency (EPI), lactase deficiency, sucrase-isomaltase deficiency, disaccharide intolerance, peptide allergy, inflammatory bowel disease (IBD) and cystic fibrosis, more preferably a composition for use in a method for the prevention, delay of progression or treatment of exocrine pancreatic insufficiency (EPI), lactase deficiency, sucrase-isomaltase deficiency, disaccharide intolerance, inflammatory bowel disease (IBD) and cystic fibrosis.

[0075] In a further preferred embodiment, the present invention provides compositions for use in methods of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy.

[0076] In a further preferred embodiment, the present invention provides a composition for use in a method for the prevention, delay of progression or treatment of a pulmonary disease or disorder selected from the group consisting of Gaucher disease, Fabry disease and mucopolysaccharidoses (MPS).

[0077] The term "treatment" / "treating" as used herein includes (1) delaying the appearance of clinical symptoms of a disease, disorder or condition that develops in an animal, particularly a mammal, especially a human, that is afflicted with or susceptible to a disease, disorder or condition, but has not yet experienced or exhibited clinical or asymptomatic symptoms of the disease, disorder or condition; (2) inhibiting the disease, disorder or condition (e.g., arresting, reducing, or delaying the onset of the disease, or in the case of maintenance treatment, the recurrence of at least one of its clinical or asymptomatic symptoms); and / or (3) alleviating the condition (i.e., causing regression of the disease, disorder or condition, or at least one of its clinical or asymptomatic symptoms). The benefit to the treated patient is statistically significant or at least perceptible to the patient or physician. However, it will be understood that when a pharmaceutical agent is administered to a patient to treat a disease, the result is not necessarily an effective treatment.

[0078] As used herein, "delayed progression" refers to increasing the time to onset of symptoms, e.g., of a pulmonary disease or disorder or cystic fibrosis, or symptoms associated with, e.g., a pulmonary disease or disorder or cystic fibrosis, or slowing the increase in severity of symptoms, e.g., of a pulmonary disease or disorder or cystic fibrosis. Additionally, as used herein, "delayed progression" includes reversing or inhibiting disease progression. "Inhibiting" disease progression or disease complications in a subject refers to preventing or reducing disease progression and / or disease complications in a subject.

[0079] Preventive treatment includes prophylactic treatment. In preventive applications, the pharmaceutical combination of the present invention is administered to a subject suspected of having or at risk of developing the above-mentioned disease or disorder, such as a pulmonary disease or disorder or cystic fibrosis. In therapeutic applications, the pharmaceutical combination is administered to a subject, such as a patient already suffering from the above-mentioned disease or disorder, such as a pulmonary disease or disorder or cystic fibrosis, in an amount sufficient to cure or at least partially arrest the symptoms of the disease. Amounts effective for this application will depend on the severity and course of the disease, previous therapy, the subject's health status and response to the drug, and the judgment of the treating physician.

[0080] If the subject's condition does not improve, the pharmaceutical combination of the present invention may be administered chronically over an extended period of time, including throughout the subject's lifespan, to ameliorate or otherwise control or limit the symptoms of the subject's disease or condition.

[0081] If the subject's condition improves, the pharmaceutical combination may be administered continuously; alternatively, the dose of the administered drug may be temporarily reduced or temporarily suspended for a predetermined length of time (i.e., a "drug holiday"). Once improvement of the patient's condition occurs, a maintenance dose of the pharmaceutical combination of the present invention is administered as needed. Thereafter, the dosage or frequency of administration, or both, is optionally reduced as a function of symptoms to a level at which the improved disease is maintained.

[0082] In a further aspect, the present invention relates to a method for producing a composition as described above, e.g. a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeat units, each repeat unit comprising at least one amino group and / or at least one thiol group, comprising: Next step: (a) providing a solid support; (b) immobilizing the protein or a fragment thereof on a solid support; (c) forming a protective layer on the surface of the solid support to protect the protein or a fragment thereof immobilized on the solid support; (d) immobilizing a functional component on the surface of the protective layer, the functional component immobilized on the surface of the protective layer being a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group; The present invention provides a method comprising:

[0083] Step (a) is usually carried out by providing the solid support suspended in water or a buffer. The suspension can be stirred, for example, at 400 rpm and 20° C. for 30 minutes. The immobilization of the protein, for example the enzyme, on the solid support in step (b) of the method is usually carried out by adding a solution of the protein, for example the enzyme, to the suspension of the solid support. In a preferred embodiment, the immobilization of the protein, for example the enzyme, on the solid support is carried out by providing a suspension of the solid support, adding a solution of the protein, for example the enzyme, and incubating the suspension with the added solution of the protein, for example the enzyme, so that the enzyme can bind to the surface of the solid support. In a preferred embodiment, the surface of the solid support is at least partially modified to improve the immobilization of the protein, for example the enzyme, on the solid support. In particular, the surface of the solid support is at least partially modified before the protein, for example the enzyme, is immobilized. The surface of the solid support can be at least partially modified by introducing molecules as anchor points for the protein, for example the enzyme, on the surface of the solid support, as described above. The formation of the protective layer according to step (c) of the present method is usually carried out by forming each protective layer with components, which build up the protective layer in a polycondensation reaction, as described above. The immobilization of the functional component on the surface of the protective layer according to step (d) of the present method is usually carried out as described above.

[0084] In one embodiment, the protective layer is formed by components, the structural component and the protective component being used to form the protective layer, the structural component being a precursor of inorganic silica capable of forming four covalent bonds in the formed layer, and the protective component being an organosilane.

[0085] In one embodiment, the protective layer encapsulates about 30% to about 100% of the protein, eg, enzyme.

[0086] In one embodiment, the solid support is selected from the group of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, magnetic particles and titanium particles. EXAMPLES

[0087] Materials and Methods: reagent: - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), Ammonium hydroxide (ACS grade, 28-30%), Low molecular weight chitosan (50000-190000 Da), Medium molecular weight chitosan (190000-310000 Da), Ethanol (ACS grade, anhydrous), Glutaraldehyde (Grade I, 25% in water), Copper sulfate, Sodium ascorbate, Chelex (registered trademark) ) 100 Sodium Form, Polysorbate 80, Sodium Chloride, Recombinant Human Pancreatic Lipase (HRL, Certified Reference Material), Pancreatin (4x USP Specification), Bovine Serum Albumin (BSA), Dibenzocyclooctyne-Maleimide (DBCO-mal, for Copper-Free Click Chemistry), 1,2-Di-O-Lauryl-rac-Glycero-3-(Glutaric Acid 6-Methylresorufin Ester), Copper Sulfate Pentahydrate, Sodium Ascorbate Acetic acid (glacial acetic acid, ACS reagent, ≥99.7%), Tris base, casein (from cow's milk), potassium dihydrogen phosphate, potassium monohydrogen phosphate, trichloroacetic acid, sodium carbonate, Folin-Ciocalteu reagent, amylase activity assay kit, dimethyl sulfoxide (DMSO), MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide), Triton X, Alcian Blue 8GX, acetic acid, Hanks' balanced salt solution (HBSS), Lucifer Yellow CH dilithium salt, EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid), PMA (phorbol 12-myristate 13-acetate), phalloidin-tetramethylrhodamine B isothiocyanate, and (3-mercaptopropyl)triethoxysilane (MPTS, ≥80% GC, technical) were purchased from Sigma-Aldrich.

[0088] -p-SCN-Bn-DOTA was purchased from Macrocyclics. - Benzyltriethoxysilane (B, 96%) was purchased from abcr GmbH. - 3-Azido-7-hydroxycoumarin was purchased from Biosynth Carbosynth. - Triethoxyethynylsilane (ETES) was purchased from Toronto Research Chemicals. - 1-Isothiocyanato-PEG3-azide was purchased from BroadPharm. -Silane-PEG4-NH2 was purchased from Nanocs. - Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were purchased from the European Collection of Certified Cell Cultures (ECACC). - THP-1 (human acute monocytic leukemia cell line) was purchased from LGC.

[0089] - Fetal bovine serum, penicillin / streptomycin (10'000U / ml penicillin / 10'000μg / ml streptomycin), MEM non-essential amino acids (100x), L-glutamine 200mM (100x), Dulbecco's phosphate buffered saline (DPBS) (1X), 0.25% trypsin-EDTA (1X), RPMI1640 medium, DMEM, HEPES, sodium pyruvate, D-glucose, β-mercaptoethanol were purchased from Gibco. - PierceTM BCA Protein Assay Kit was purchased from ThermoFisher Scientific. - VECTASHIELD antifade mounting medium with DAPI was purchased from Vector Laboratories. - ThinCert TM Cell culture insert plates (1.0 μm membrane) were purchased from Greiner bio-one.

[0090] Synthetic Silica Nanoparticles: Silica nanoparticles (50 nm) were synthesized according to the original Stober method described in WO 2015 / 014888 A1. Briefly, ethanol, distilled water (6 M) and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 h. TEOS (0.28 M) was added and the solution was stirred at 400 rpm for 22 h at 20° C. The solution was then centrifuged at 20000 g for 20 min and washed sequentially with ethanol and water. Particle size measurements were performed on SEM micrographs acquired at a magnification of 150000× using the image analysis software Olympus stream motion.

[0091] Enzyme and protein shielding: Silica nanoparticles in water-polysorbate 80 (8 mg / L) were reacted with APTES (2.75 mM) under stirring (400 rpm) at 20° C. for 30 min. Unreacted reagents were removed from the nanoparticle suspension using an Amicon stirred cell with a NMWL of 300 kDa, Biomax polyethersulfone ultrafiltration disk (hereafter referred to as the "washing step"). These nanoparticles are further referred to as amino-modified nanoparticles. The amino-modified nanoparticles were then incubated with 0.1% (v / v) aqueous glutaraldehyde solution under stirring (400 rpm) at 20° C. for 30 min. After the washing step, the nanoparticles were resuspended in MES buffer (10 mM, pH 6.2) containing polysorbate 80 (8 mg / L) and reacted with enzymes (recombinant human lipase) (348 μg / mL) or pancreatin (276 μg / mL) or proteins (bovine serum albumin, BSA) (374 μg / mL) for 1 h at 20° C. under stirring (400 rpm), further called immobilized enzyme / protein-nanoparticles. Before the shielding step consisting of polycondensation of silanes on the surface of the immobilized enzyme / protein, the nanoparticles were washed and resuspended in a HO-polysorbate 80 (8 mg / L) solution. The different shielding compositions, further called here "protein-shielded and / or enzyme-shielded nanoparticles", are as follows: - Shielding consisting of APTES and TEOS (AT): TEOS (7.75 mM) was added to the immobilized enzyme / protein nanoparticles and reacted for 1 h at 20° C. under stirring (400 rpm). After that, APTES (0.74 mM) was added to the reaction mixture. To obtain a complete protective layer, the silane polycondensation was stopped after 21 h by washing the nanoparticle suspension.

[0092] The silica nanoparticles obtained after silane polycondensation comprising bovine serum albumin protein immobilized on the surface of silica particles fully embedded by a protective layer comprising polycondensed silane were prepared as described in WO 2015 / 014888 A1 and are further referred to herein as "fully shielded nanoparticles", "fully protein shielded nanoparticles", "nanoparticle 1" or "NP-1".

[0093] The silica nanoparticles obtained after silane polycondensation, comprising pancreatin immobilized on the surface of silica particles fully embedded by a protective layer comprising polycondensed silane, were prepared as described in WO 2015 / 014888 A1 and are further referred to herein as "fully shielded nanoparticles", "enzyme fully shielded nanoparticles", "nanoparticles 15" or "NP-15".

[0094] The silica nanoparticles obtained after silane polycondensation, comprising pancreatin immobilized on the surface of silica particles partially embedded by a protective layer comprising polycondensed silane, were prepared as described in WO 2015 / 014888 A1 and are further referred to herein as "partially shielded nanoparticles", "enzyme partially shielded nanoparticles", "nanoparticles 13" or "NP-13". The silane polycondensation was stopped after 5 hours by washing the nanoparticle suspension in order to obtain a partial protective layer.

[0095] - Shielding made of APTES, TEOS, and benzyltriethoxysilane (ATB): TEOS (3,87 mM) was added to the immobilized enzyme / protein nanoparticles and reacted for 1 h at 20° C. under stirring (400 rpm). Then, APTES (0,74 mM) and benzyltriethoxysilane (3,34 mM) were added to the reaction mixture. After 21 h, the nanoparticle suspension was washed to stop the silane polycondensation.

[0096] The silica nanoparticles obtained after silane polycondensation comprising recombinant human lipase immobilized on the surface of silica particles fully embedded by a protective layer comprising polycondensed silane were prepared as described in WO 2015 / 014888 A1 and are further referred to herein as "fully shielded nanoparticles", "enzyme fully shielded nanoparticles", "nanoparticles 4" or "NP-4".

[0097] The silica nanoparticles obtained after silane polycondensation, comprising pancreatin immobilized on the surface of silica particles fully embedded by a protective layer comprising polycondensed silane, were prepared as described in WO 2015 / 014888 A1 and are further referred to herein as "fully shielded nanoparticles", "enzyme fully shielded nanoparticles", "nanoparticles 11" or "NP-11".

[0098] Particle size measurements were performed on SEM micrographs acquired at 150000× magnification using the image analysis software Olympus stream motion.

[0099] The different nanoparticles obtained after enzyme / protein shielding are summarized in Table 1 below.

[0100] Nanoparticle labeling: For nanoparticle labeling, an additional step was added to the nanoparticle process described in the "Enzyme and Protein Shielding" section.

[0101] - Lutetium 177( 177 Radiolabeling with Lu: All buffers used were pretreated with Chelex®. The amino-modified nanoparticles produced according to the "Enzyme and Protein Shielding" section above were resuspended in phosphate buffer (0.1 M, pH 7.4) containing polysorbate 80 (8 mg / L), and p-SCN-Bn-DOTA (1 mg / mL) was added and reacted at 20° C. for 1 h under stirring (400 rpm). After a washing step, the DOTA-labeled nanoparticles were resuspended in MES buffer (10 mM, pH 6.2) containing polysorbate 80 (8 mg / L), and enzymes were immobilized and shielded on these DOTA-labeled nanoparticles as described in the "Enzyme and Protein Shielding" section above. The nanoparticles were then resuspended in MES buffer (10 mM, pH 6.2) containing polysorbate 80 (8 mg / L), and enzymes were immobilized and shielded on these DOTA-labeled nanoparticles as described in the "Enzyme and Protein Shielding" section above. 177 The nanoparticles were incubated with Lu (2500 μCi) and sodium acetate (250 mM, pH 5.4) for 12 h at 45° C. After a washing step with sodium acetate (20 mM, pH 5.0) containing polysorbate 80 (8 mg / L), the nanoparticles were resuspended in EDTA (1 mM) and incubated overnight at room temperature (RT). The nanoparticles were then washed and resuspended in 0.9% sodium chloride containing polysorbate 80 (8 mg / L).

[0102] - Labeling with FITC: The amino-modified nanoparticles produced according to the above section "Enzyme and Protein Shielding" were resuspended in borate buffer (50 mM, pH 8.5) containing polysorbate 80 (8 mg / L), and FITC (50 μg / mL) was added and reacted under stirring (400 rpm) for 1 h at 20° C. After a washing step, the FITC-labeled nanoparticles were resuspended in MES buffer (10 mM, pH 6.2) containing polysorbate 80 (8 mg / L), and enzymes were immobilized and shielded on these DOTA-labeled nanoparticles as described above in the section "Enzyme and Protein Shielding".

[0103] Surface functionalization of nanoparticles: - Electrostatic binding of chitosan to protein-shielded and / or enzyme-shielded nanoparticles (chitosan-functionalized shielded nanoparticles) Protein-shielded and / or enzyme-shielded nanoparticles produced according to the above section "Enzyme and Protein Shielding" in HO containing polysorbate 80 (8 mg / L) were reacted with a solution of chitosan (500 μg / mL) in 0.1 M acetic acid for 30 minutes at 20° C. under stirring (400 rpm). After a washing step, the nanoparticles were resuspended in HO containing polysorbate 80 (8 mg / L). Complete functionalization with chitosan ("full functionalization") was achieved by applying a chitosan concentration based on theoretical calculations of the number of anchor points. Partial functionalization with chitosan ("partial functionalization") was achieved by applying a fraction of this number of anchor points (between 10% and 80%).

[0104] The silica nanoparticles obtained after full functionalization by electrostatic bonding of medium MW chitosan with BSA immobilized on the surface of the silica particles, fully embedded by a protective layer comprising polycondensed silane (AT) and further comprising medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e., fully functionalized with medium MW chitosan, are further referred to herein as "nanoparticle 2" or "NP-2".

[0105] The silica nanoparticles obtained after partial functionalization by electrostatic bonding of medium MW chitosan containing BSA immobilized on the surface of the silica particles, completely embedded by a protective layer containing polycondensed silane (AT) and further containing partial medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e. partially functionalized with medium MW chitosan, are further referred to herein as "nanoparticle 3" or "NP-3".

[0106] The silica nanoparticles obtained after full functionalization by electrostatic bonding of medium MW chitosan containing recombinant human lipase immobilized on the surface of the silica particles, fully embedded by a protective layer containing polycondensed silane (ATB) and further containing medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e., fully functionalized with medium MW chitosan, are further referred to herein as "nanoparticle 5" or "NP-5".

[0107] The silica nanoparticles obtained after partial functionalization by electrostatic bonding of medium MW chitosan containing recombinant human lipase immobilized on the surface of the silica particles, completely embedded by a protective layer containing polycondensed silane (ATB) and further containing medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e. partially functionalized with medium MW chitosan, are further referred to herein as "nanoparticles 6" or "NP-6".

[0108] The silica nanoparticles obtained after full functionalization by electrostatic bonding of medium MW chitosan containing pancreatin immobilized on the surface of the silica particles, fully embedded by a protective layer containing polycondensed silane (ATB) and further containing medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e., fully functionalized with medium MW chitosan, are further referred to herein as "nanoparticle 12" or "NP-12".

[0109] The silica nanoparticles obtained after full functionalization by electrostatic bonding of medium MW chitosan comprising pancreatin immobilized on the surface of the silica particles, partially embedded by a protective layer comprising polycondensed silane (AT) and further comprising medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e. fully functionalized with medium MW chitosan, are further referred to herein as "nanoparticle 14" or "NP-14".

[0110] The silica nanoparticles obtained after full functionalization by electrostatic bonding of medium MW chitosan containing pancreatin immobilized on the surface of the silica particles, fully embedded by a protective layer containing polycondensed silane (AT) and further containing medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e., fully functionalized with medium MW chitosan, are further referred to herein as "nanoparticle 16" or "NP-16".

[0111] - Click Chemistry Cu++ Ethyltriethoxysilane (ETES) (0.8 μmol) was added to a solution of protein-shielded and / or enzyme-shielded nanoparticles (250 μL, 10 mg / mL) produced according to the above section "Enzyme and Protein Shielding". The resulting mixture was stirred at 400 rpm at 20° C. for 15 min to obtain ethynyl-modified nanoparticles. Then, solutions of 3-azido-7-hydroxycoumarin (0.8 μmol) in acetic acid (0.1 M), CuSO4 (83 μL, 20 mM) in H2O containing polysorbate 80 (8 mg / L) and sodium ascorbate (208 μL, 100 mM) in H2O / PS80 were added sequentially to the nanoparticle suspension. The resulting mixture was stirred at 400 rpm at 20° C. for 22 h. Samples were collected at different reaction times and fluorescence (λ ex : 404 nm, λ em The cycloaddition reaction rate was monitored by measuring the optical density (A / D) of the reaction mixture at 477 nm.

[0112] - Click chemistry Cu++ free The pH of protein-shielded and / or enzyme-shielded nanoparticles (1 mL, 10 mg / mL) produced according to the "Enzyme and Protein Shielding" section above was adjusted to pH 9 by adding NaOH (5 M, 1 μL). A solution of dibenzocyclooctyne-maleimide (238 μL, 1 mg / mL) in DMSO was then added to the nanoparticle suspension. The resulting mixture was stirred at 400 rpm for 30 min at 20° C. The nanoparticles were washed three times with HO (1 mL) containing polysorbate 80 (8 mg / L) and resuspended in 1 mL of HO containing polysorbate 80 (8 mg / L) to obtain dibenzocyclooctyne-modified nanoparticles (nanoparticle-DBCO).

[0113] To nanoparticle-DBCO (100 μL, 10 mg / mL) was added a solution of 3-azido-7-hydroxycoumarin (4.75 μL, 1 mg / mL) in acetic acid (0.1 M). The resulting mixture was stirred at 400 rpm for 6 h at 20 °C. The nanoparticles were then washed three times with HO / PS80 (100 μL) and resuspended in 100 μL of HO containing polysorbate 80 (8 mg / L). Steady-state fluorescence measurements were performed (λ) using 100 μL of the 2 mg / mL nanoparticle suspension. ex : 404 nm, λ em :477nm).

[0114] - Functionalization of protein-shielded and / or enzyme-shielded nanoparticles with chitosan via click chemistry (chitosan-functionalized shielded nanoparticles) The pH of protein-shielded and / or enzyme-shielded nanoparticles (200 μL, 10 mg / mL) produced according to the "Enzyme and Protein Shielding" section above was adjusted to pH 9 by adding NaOH (5 M, 1 μL). A solution of dibenzocyclooctyne-maleimide in DMSO was then added to the nanoparticle suspension. The resulting mixture was stirred at 400 rpm for 30 min at 20° C. The nanoparticles were washed three times with HO (200 μL) containing polysorbate 80 (8 mg / L) and resuspended in 200 μL of HO containing polysorbate 80 (8 mg / L) to obtain dibenzocyclooctyne-modified nanoparticles (nanoparticle-DBCO). Azide-modified chitosan in acetic acid (0.1 M) was then added to the nanoparticle-DBCO suspension. The resulting mixture was stirred at 400 rpm for 6 h at 20° C. The nanoparticles were then washed three times with H2O containing polysorbate 80 (200 μL) and resuspended in 200 μL of H2O containing polysorbate 80 (8 mg / L).

[0115] The silica nanoparticles obtained after partial functionalization with medium MW chitosan by click chemistry comprising recombinant human lipase immobilized on the surface of the silica particles, completely embedded by a protective layer comprising polycondensed silane (ATB) and further comprising medium MW chitosan as a functional component immobilized on the surface of the protective layer, i.e. partially functionalized with medium MW chitosan, are further referred to herein as "nanoparticle 7" or "NP-7".

[0116] The silica nanoparticles obtained after partial functionalization with low MW chitosan by click chemistry comprising the enzyme recombinant human lipase immobilized on the surface of the silica particles, completely embedded by a protective layer comprising polycondensed silane (ATB) and further comprising low MW chitosan as a functional component immobilized on the surface of the protective layer, i.e. partially functionalized with low MW chitosan, are further referred to herein as "nanoparticle 8" or "NP-8".

[0117] - Functionalization of protein-shielded and / or enzyme-shielded nanoparticles with silane-PEG-NH2 (polymerized silane-PEG-NH2 functionalized shielded nanoparticles) The pH of a solution of protein-shielded and / or enzyme-shielded nanoparticles (2 mL, 10 mg / mL) produced according to the "Enzyme and Protein Shielding" section above was adjusted to pH 9 by adding NaOH (5 M). Silane-PEG-NH2 (567 μL, 10 mg / mL) was then added to the nanoparticle suspension. The resulting mixture was stirred at 400 rpm for 30 min at 20° C. The nanoparticles were washed three times with HO / PS80 (2 mL) and resuspended in 2 mL of HO containing polysorbate 80 (8 mg / L).

[0118] The silica nanoparticles obtained after functionalization with silane-PEG4-NH2 containing the enzyme recombinant human lipase immobilized on the surface of the silica particles, fully embedded by a protective layer containing polycondensed silane (ATB) and further containing polymerized silane-PEG4-NH2 as a functional component immobilized on the surface of the protective layer, i.e., fully functionalized with silane-PEG4-NH2, are further referred to herein as "nanoparticle 10" or "NP-10".

[0119] - Functionalization of protein-shielded and / or enzyme-shielded nanoparticles with APTES (polymerized APTES-functionalized shielded nanoparticles) The pH of a solution of protein-shielded and / or enzyme-shielded nanoparticles (2 mL, 10 mg / mL) produced according to the "Enzyme and Protein Shielding" section above was adjusted to pH 9 by adding NaOH (5 M). APTES (2.21 μL) was then added to the nanoparticle suspension. The resulting mixture was stirred at 400 rpm for 30 min at 20° C. The nanoparticles were washed three times with HO (2 mL) containing polysorbate 80 (8 mg / L) and resuspended in 2 mL of HO containing polysorbate 80 (8 mg / L).

[0120] The silica nanoparticles obtained after functionalization with APTES, comprising recombinant human lipase immobilized on the surface of the silica particles, completely embedded by a protective layer comprising polycondensed silane (ATB) and further comprising polymerized APTES as a functional component immobilized on the surface of the protective layer, i.e., functionalized with polymerized APTES, are further referred to herein as "nanoparticles 9" or "NP-9".

[0121] - Functionalization of protein-shielded and / or enzyme-shielded nanoparticles with MPTS (polymerized MPTS-functionalized shielded nanoparticles) To a solution (1 mL, 10 mg / mL) of protein-shielded and / or enzyme-shielded nanoparticles produced according to the above section "Enzyme and Protein Shielding" in phosphate buffer (10 mM, pH 8), different amounts of MPTS were added to functionalize the nanoparticle surface at different ratios: 0.23 μmol, 0.46 μmol, 0.91 μmol, 2.3 μmol, and 4.6 μmol to produce NP-17, NP-18, NP-19, NP-20, and NP-21, respectively. The resulting mixture was stirred at 400 rpm for 90 min at 20° C. The nanoparticles were washed three times with HO (1 mL) containing polysorbate 80 (8 mg / L) and resuspended in 1 mL of HO containing polysorbate 80 (8 mg / L). The partial / full functionalization was theoretically determined using a stoichiometric model calculation that works as follows: The unit surface area corresponding to a single silanol (R-Si-OH) functional group on the surface of a pure silica nanoparticle was calculated based on the silica molecular structure. The formula for calculating the stoichiometric number of functional groups is as follows: TIFF2025504835000001.tif14170n(-OH): number of functional groups on the nanoparticle surface expressed as moles of silanols; m: mass of the sample in milligrams; R: radius of a single nanoparticle expressed in nm; N A :mol -1 Avogadro's number, represented by d:mg.nm -3 The density of silica nanoparticles is expressed as: V:nm 3 The volume of a single nanoparticle, represented by: V U :nm 2 The unit surface area of ​​a single silanol functional group on the nanoparticle surface is represented by

[0122] Measuring the nanoparticle size allows the calculation of the surface area of ​​a single nanoparticle. Combining the unit surface area of ​​a functional group with the surface area of ​​the nanoparticle gives the total number of available functional groups on a single nanoparticle. This result is the basis for all stoichiometric calculations for nanoparticle functionalization.

[0123] The silica nanoparticles obtained after 5% partial functionalization with MPTS comprising BSA immobilized on the surface of the silica particles, which are completely embedded by a protective layer comprising polycondensed silane (AT) and further comprise polymerized MPTS as a functional component immobilized on the surface of the protective layer, i.e., partially functionalized (5% of the surface of the protein-shielded nanoparticles) with polymerized MPTS, are further referred to herein as "nanoparticle 17" or "NP-17".

[0124] The silica nanoparticles obtained after 10% partial functionalization with MPTS, comprising BSA immobilized on the surface of the silica nanoparticles, completely embedded by a protective layer comprising polycondensed silane (AT) and further comprising polymerized MPTS as a functional component immobilized on the surface of the protective layer, i.e., partially functionalized (10% of the surface of the protein-shielded nanoparticles) with polymerized MPTS, are further referred to herein as "nanoparticle-18" or "NP-18".

[0125] The silica nanoparticles obtained after 20% partial functionalization with MPTS comprising BSA immobilized on the surface of the silica particles, which are completely embedded by a protective layer comprising polycondensed silane (AT) and further comprise polymerized MPTS as a functional component immobilized on the surface of the protective layer, i.e., partially functionalized (20% of the surface of the protein-shielded nanoparticles) with polymerized MPTS, are further referred to herein as "nanoparticle-19" or "NP-19".

[0126] The silica nanoparticles obtained after 50% partial functionalization with MPTS comprising BSA immobilized on the surface of the silica particles, which are completely embedded by a protective layer comprising polycondensed silane (AT) and further comprise polymerized MPTS as a functional component immobilized on the surface of the protective layer, i.e., partially functionalized (50% of the surface of the protein-shielded nanoparticles) with polymerized MPTS, are further referred to herein as "nanoparticle-20" or "NP-20".

[0127] The silica nanoparticles obtained after full functionalization (100%) with MPTS, comprising BSA immobilized on the surface of the silica particles, fully embedded by a protective layer comprising polycondensed silane (AT) and further comprising polymerized MPTS as a functional component immobilized on the surface of the protective layer, i.e., fully functionalized (100% of the surface of the protein-shielded nanoparticles) with polymerized MPTS, are further referred to herein as "nanoparticle 21" or "NP-21".

[0128] The different nanoparticles obtained and used in the experiments outlined below are summarized in Table 1. JPEG2025504835000002.jpg95170

[0129] Protein quantification: The enzyme immobilization yield was quantified using the indirect Lowry protein quantification method. A standard regression curve with known protein concentrations was constructed using bovine serum albumin standard. The supernatant of the nanoparticles after enzyme immobilization was collected and centrifuged at 20 krcf for 3 min. Then, 1 mL of Lowry solution was added to 200 μL of samples and standards, vortexed, and incubated at room temperature for 15 min. Then, 100 μL of Folin's reagent 1N was added while vortexing, and incubated at room temperature for 30 min. Finally, the absorbance was read at 750 nm using a Biotek Synergy H1 reader.

[0130] Cell culture: For all experiments, cells were cultured at 37°C and 5% CO2. Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin.

[0131] For the development of the intestinal barrier model, cells were cultured on Transwell PET inserts (pore size 1 μm) at 2.6 × 10 5 cells / cm 2All cell models were used for experiments on day 21. For monoculture, Caco-2 cells were used. For coculture, Caco-2 cells and HT-29-MTX-E12 cells were used at a ratio of 75% to 25%.

[0132] THP-1 (human monocytic leukemia cell line) cells were maintained in culture in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, and 100 U / mL penicillin / streptomycin.

[0133] To differentiate THP-1 into macrophages, THP-1 cells were cultured in differentiation medium, i.e., RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2.5 g / L glucose, 50 pM β-mercaptoethanol. THP-1 were differentiated into M0 macrophages by incubation with 150 nM phorbol 12-myristate 13-acetate (PMA) for 24 h followed by incubation in differentiation medium for 24 h.

[0134] Mucus staining: The differentiated cell monolayer was fixed with 4% formalin for 15 min at room temperature and then washed with PBS. A 1% Alcian Blue solution in 3% acetic acid (pH 2.5) was added. After 30 min of incubation at room temperature, the cells were washed extensively with PBS and the insert membrane containing the cells was cut from the plastic insert holder and mounted on a glass slide.

[0135] Nanoparticle binding to mucus: For the ex vivo evaluation of the interaction of functionalized shielded nanoparticles with mucus, intestines of freshly slaughtered pigs were collected from a local slaughterhouse. The small intestine was cut longitudinally and the mucus was collected by scraping with a glass slide. 5 mL of 0.1 M sodium chloride was added to 1 g of mucus and stirred at 40 rpm for 1 h. The suspension was then centrifuged at 13125 g for 2 h. A clean part of the pellet was kept and the process was repeated once more. 33.6 mm 2 Transwell inserts with a surface area of ​​100 μL were covered with 50 mg of porcine mucus. The acceptor chamber was filled with 500 μL of HBSS pH 7.4. The donor chamber was filled with 250 μL of FITC-labeled nanoparticles diluted in HBSS pH 6.4. The plate was then incubated for 1 h at 37° C. with shaking (300 rpm). After incubation, sequential washing steps were performed with HO, 0.9% sodium chloride, 0.01% Triton X100. The percentage of nanoparticles bound to the mucus was determined by measuring the fluorescence (λ) in each compartment. ex : 489 nm, λ em The optical density was evaluated by measuring the optical density at 515 nm.

[0136] For in vitro evaluation of the interaction of functionalized shielding nanoparticles with mucus, differentiated cell cultures were exposed to FITC-labeled nanoparticles in DMEM for 24 h with shaking (300 rpm). After washing with PBS, binding of functionalized shielding nanoparticles to mucus was assessed optically.

[0137] Biodistribution: CD-1 mice were orally administered 100 mg / kg of radioactive chitosan-functionalized shielded nanoparticles by gavage after an overnight fast. At the end of the period, the animals were anesthetized by intraperitoneal injection of a mixture of ketamine hydrochloride (50 mg / kg) and xylazine hydrochloride (10 mg / kg) and then rapidly sacrificed by exsanguination via intracardiac puncture. Organs of interest were removed and weighed using a precision balance. Urine (including urine in the bladder) and feces were collected from mice individually housed in metabolic cages 24 hours after administration and analyzed for radioactivity. Radioactivity counting of samples was performed in an automatic gamma counter (Wallace Wizard 2470-Perkin Elmer) calibrated for lutetium-177 radionuclide (efficiency: 13.8%, LLOQ: 500 cpm). Radioactivity in sampled tissues was expressed as the percentage of ID per gram of tissue (%ID / g).

[0138] Viability assay: Cells were plated in 96-well flat-bottom cell culture plates at 2 x 10 4 Cells were seeded at a density of 1000 cells / well. After 24 h, the culture medium was changed and the cells were treated with increasing concentrations of shielded or functionalized shielded nanoparticles (0-1000 μg / mL) for 24 and 48 h. The cell monolayer was rinsed with medium and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) solution (1 mg / mL) was added to each well. The cell cultures were incubated at 37 °C for 2 h. Formazan crystals formed during the incubation period were dissolved in DMSO. After complete solubilization, the absorbance was measured at 570 nm and 680 nm (reference wavelength) using a Synergy H1 multimode microplate reader (Bio-Tek Instruments). Finally, the MTT results were expressed as a percentage of viability: [(sample absorbance - cell-free sample blank) * 100 / (untreated sample absorbance - cell-free sample blank)]. The viability of untreated control cells was arbitrarily defined as 100%.

[0139] Barrier Integrity: - TEER The integrity of the cell barrier was assessed by measuring the transepithelial electrical resistance (TEER) using a CellZscope system (NanoAnalytics). After refreshing the cell culture medium and treatment with nanoparticles, cells were equilibrated overnight at 37°C before TEER measurements. Inserts without cells and cell monolayers treated with 2.5 mM EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) were used as negative controls.

[0140] - Lucifer Yellow Transposition After nanoparticle exposure, the integrity of the cell layer was also assessed using Lucifer Yellow (LY). The medium of Caco-2 monocultures and Caco-2 / HT-29-MTX-E12 cocultures was removed and replaced with HBBS. LY (100 μM) was added to the apical side of the cells for 90 min at 37°C, and the basolateral medium was harvested and fluorescence (λ) was measured using a Synergy H1 multimode microplate reader (Bio-Tek Instruments). ex :428nm;λ em The fluorescence was analyzed at 540 nm.

[0141] Internal Migration: - confocal After incubation with FITC-labeled chitosan-functionalized shielding nanoparticles, cell monolayers grown on filter inserts were washed with PBS and fixed with 4% formalin for 15 min at room temperature. Cells were washed, permeabilized with Triton 0.1% for 15 min at room temperature, washed three times with PBS, and blocked with BSA 1% for 30 min at room temperature. Cells were then incubated with phalloidin-TRITC (tetramethyl-rhodamine B-isothiocyanate) for 1 h at room temperature. After the final washing step, cells were mounted on microscope glass slides with a drop of antifade mounting medium containing DAPI. Fluorescence microscopy analysis was performed with a confocal laser scanning microscope (Fluoview FV3000, Olympus).

[0142] - FACS For flow cytometry analysis, differentiated cell cultures pretreated with or without FITC-labeled chitosan-functionalized shielding nanoparticles were detached with trypsin and washed with PBS. Cells were then processed and data were acquired using a flow cytometer Cell Sorter SH800Z (Sony). Data were analyzed using Flowjo10 software.

[0143] Human Recombinant Lipase Activity Assay: To a solution of lipase-containing shielded nanoparticles and lipase-containing chitosan-functionalized shielded nanoparticles (60 μL, 3.3 mg / mL) in Tris buffer (0.1 M, pH 8.4), 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorufin ester) (60 μL, 100 μM) was added. Lipase activity kinetics was monitored by steady-state fluorescence measurements (λex / λem=529 / 600 nm) in a dark 96-well plate at 37°C for 30 min.

[0144] Activity assay of lipase from pancreatin: To a solution of shielded nanoparticles containing pancreatin and chitosan-functionalized shielded nanoparticles containing pancreatin (125 μL, 3.33 mg / mL) in Tris buffer (0.1 M, pH 8.4), 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorufin ester) (125 μL, 50 μM) was added. The resulting mixture was stirred at 750 rpm for 30 min at 37 °C in the dark. The nanoparticles were centrifuged and the supernatant containing the reaction product was collected. Steady-state fluorescence measurements (λex / λem=529 / 600 nm) were taken on 100 μL of the resulting solution in a dark 96-well plate.

[0145] Protease activity assay: To a solution of shielded nanoparticles containing pancreatin and chitosan-functionalized shielded nanoparticles containing pancreatin (50 μL, 10 mg / mL) in phosphate buffer (50 mM, pH 7) was added a solution of casein (250 μL, 0.65% w / v) in phosphate buffer (50 mM, pH 7). The resulting mixture was stirred at 750 rpm for 30 min at 37° C. The nanoparticles were centrifuged and the supernatant containing the reaction product was collected. To the supernatant (200 μL) was added a solution of TCA (110 mM, 167 μL) in H2O to precipitate the undigested casein. The resulting mixture was stirred at 750 rpm for 30 min at 37° C. The resulting solution was centrifuged and the supernatant containing the digested fragments of casein was collected. To this supernatant (200 μL) was added sodium carbonate solution (500 mM, 500 μL) followed by Folin-Ciocalteu reagent (0.5 M, 100 μL). The resulting mixture was stirred at 750 rpm for 30 min at 37 °C. The absorbance of 200 μL of the resulting solution was measured at 660 nm.

[0146] Protease activity assay under acidic conditions: Chitosan was coated on the surface of shielded nanoparticles containing pancreatin in acetic acid (0.1 M, pH 4). To evaluate the effect of chitosan on protease stability, the shielded nanoparticles containing pancreatin were incubated in acetic acid (0.1 M, pH 4) for 30 min. The activity of both the shielded nanoparticles containing pancreatin and the chitosan-functionalized shielded nanoparticles containing pancreatin after incubation in acetic acid (0.1 M, pH 4) was compared to the shielded nanoparticles containing pancreatin activity at basic pH (pH 8).

[0147] Amylase activity assay: To a solution of chitosan-functionalized shielded nanoparticles (2 μL, 10 mg / mL) containing pancreatin in activity buffer (51 μL) was added a solution of 4-nitrophenyl-4,6-ethylidene-aD-maltoheptaoside in activity buffer (107 μL). The resulting mixture was stirred at 750 rpm for 15 min at 25° C. The nanoparticles were centrifuged and the supernatant containing the reaction product was collected. The absorbance was measured at 405 nm for 150 μL of the resulting solution.

[0148] result: Example 1: Surface functionalization of nanoparticles with chitosan (comparative experiment) To generate mucoadhesive nanoparticles, the surface of the protective layer of shielded nanoparticles produced as described in WO 2015 / 014888 A1 was functionalized with chitosan. Different surface modification strategies were evaluated: non-covalent and covalent attachment. The electrostatic attachment of chitosan to shielded nanoparticles (NP-1) was evaluated using FITC-labeled chitosan. The increase in fluorescence after conjugation to reacted nanoparticles (NP-2) compared to shielded nanoparticles (NP-1) (6632 vs. 2556, respectively) proves the attachment of FITC-chitosan at the surface of the nanoparticles (Figure 2A). The covalent attachment of chitosan to NP-1 was performed by copper II-free and copper II-dependent click chemistry. To validate the attachment strategy, the azido fluorescent dye 3-azido-7-hydroxycoumarin was used. The increase in fluorescence on reacted nanoparticles proves the functionalization of shielded nanoparticles using both click reactions (Figure 2B-C). Azido-chitosan (N3-chitosan) was synthesized to facilitate its covalent attachment on the surface of shielding nanoparticles. These results validate the strategy of functionalizing nanoparticles with chitosan.

[0149] Example 2: Ex vivo interaction of chitosan-functionalized nanoparticles with mucus layers: Effect of the level of surface functionalization of nanoparticles (comparative experiment) Since functionalized nanoparticles need to interact with mucus, an ex vivo test model was set up by adding a layer of porcine intestinal mucus to a Transwell insert. Mucus-binding studies were then performed with fluorescent nanoparticles exhibiting different functionalization levels, either full coverage of the nanoparticle surface with chitosan (NP-2) or 10% coverage (NP-3) (partial functionalization). Compared to non-functionalized nanoparticles (NP-1), the presence of chitosan on the surface of the nanoparticles increases the interaction with mucus. Furthermore, Figure 3 shows that nanoparticles fully functionalized with chitosan (NP-2) interact to a higher degree than nanoparticles with only 10% surface functionalization (NP-3) (93% vs. 49% of the applied dose, respectively). Non-functionalized nanoparticles (NP-1) only show a weak interaction. These results demonstrate the mucoadhesive properties of chitosan-functionalized nanoparticles and highlight that the level of nanoparticle-mucus interaction can be modulated by adjusting the degree of surface functionalization.

[0150] Example 3: Ex vivo interaction of chitosan-functionalized nanoparticles with mucus layers: Immobilization process and chitosan size (comparative experiment) Chitosan on the surface of the nanoparticles promotes their interaction with mucus, and the effect of the chitosan immobilization process and sugar size on their interaction with mucus was evaluated.

[0151] Different strategies of chitosan immobilization were evaluated, namely electrostatic interactions (non-covalent binding) and click chemistry (covalent binding). Figure 4 shows that nanoparticles modified by electrostatic binding of chitosan (NP-6) and by click chemistry (NP-7 and NP-8) interact with the mucus layer, whereas non-functionalized nanoparticles (NP-1) show only weak interactions. This indicates that both strategies can be used to functionalize nanoparticles without affecting their biological behavior.

[0152] Furthermore, chitosans of various molecular weights (low molecular weight: 50,000-190,000 Da (NP-8) or medium molecular weight: 190,000-310,000 Da (NP-7)) were immobilized on the surface of nanoparticles by click chemistry. The results show that the nanoparticle-mucus interaction is maintained in both cases. The difference in nanoparticle retention on mucus for NP-7 and NP-8 can be explained by the amount of sugar residues immobilized on the surface (23 chitosan for NP-7 vs. 47 chitosan for NP-8).

[0153] Example 4: Ex vivo interaction of nanoparticles modified with different polymers containing amino groups with the mucus layer. We hypothesized that the interaction of functionalized nanoparticles with mucus is not only due to chitosan but more generally due to polymers containing amino groups. To evaluate this hypothesis, shielding nanoparticles with different functional groups were created. Ex vivo mucus binding studies were performed with shielding nanoparticles exhibiting various polymers containing amino groups and the interaction with the mucus layer was quantified with ImageJ software. Figure 5 shows that the surface functionalization of shielding nanoparticles with all the used polymers containing amino groups (NP-9: surface functionalized with a polymerized APTES layer; NP-5: surface functionalized with chitosan, NP-10: surface functionalized with silane-PEG4-NH2) surprisingly increases the retention of nanoparticles in the mucus layer.

[0154] Overall, these results highlight the important role of amino group-containing polymers in the nanoparticle–mucus interaction process.

[0155] Example 5: In vitro interactions of chitosan-functionalized nanoparticles with an intestinal barrier model To confirm the interaction of chitosan-functionalized nanoparticles with intestinal mucus, we developed in vitro models of intestinal barriers, namely, a mucus-nonproducing barrier (Caco-2 monoculture) and a mucus-producing barrier (Caco-2 / HT29-MTX-E12 coculture). To visualize the mucus on top of the cell layer surface, differentiated cells were stained with Alcian blue. The presence of blue mucus was detected in the coculture cell layer but not in the monoculture (Figure 6A). The apical end of both intestinal barrier models was exposed to fluorescent chitosan-functionalized shielding nanoparticles (NP-2) for 24 h, and the interaction of nanoparticles with the barrier was observed optically. Figure 6B shows that the presence of mucus increases the retention of NP-2 at the surface of the coculture barrier compared to the monoculture. These results demonstrate the specific mucus targeting of chitosan-functionalized nanoparticles and confirm the mucoadhesive properties of these nanoparticles.

[0156] Example 6: Biodistribution of chitosan-functionalized nanoparticles in mice (comparative experiment) To evaluate the ability of nanoparticles to interact with gastrointestinal mucus in vivo, radiolabeled chitosan-functionalized shielded nanoparticles (NP-2) were orally administered to CD-1 mice by gavage. At different time points (from 1 to 24 h), radioactivity was measured in tissues, blood, feces, and urine. The distribution of nanoparticles (NP-2) in the digestive system indicates that NP-2 is passing through the gastrointestinal tract. Compared with the residence time of the food bolus in the mouse stomach (1 h), the residence time of chitosan-functionalized nanoparticles is increased, with 60% of the initial amount of NP-2 remaining in the stomach 3 h after gavage (Figure 7A). Furthermore, urine and feces of mice housed in metabolic cages were collected to track the excretion balance. After 24 h, 95% of the initial amount of nanoparticles administered was recovered, which corresponds to the turnover time of intestinal mucus in mice (Figure 7A). These results demonstrate the remarkable mucoadhesive and specific distribution properties of chitosan-functionalized shielded nanoparticles in vivo.

[0157] To ensure specific localization of NP-2 in the digestive system, whole-organism biodistribution was performed at different time points (from 1 to 24 h). The results show that orally administered NP-2 remained in the gastrointestinal tract and did not cross the intestinal barrier, as no radioactive signal was detected at systemic levels (Figure 7B). This result indicates an unexpected high degree of safety of NP-2, suggesting that the nanoparticles do not disrupt the integrity of the intestinal barrier.

[0158] Taken together, these in vivo data reveal that surface functionalization of shielded nanoparticles with chitosan enables specific targeting to the intestinal mucus and suggests that the nanoparticles may be transiently implanted on the intestinal wall without any toxicity.

[0159] Example 7: In vitro cytotoxicity evaluation of nanoparticles (comparative experiment) To evaluate the safety of the nanoparticles, human colon adenocarcinoma cells Caco-2 (Figure 8A-B) and HT-29-MTX-E12 (Figure 8C-D) were treated with increasing concentrations of non-functionalized (NP-1) and chitosan-functionalized (NP-2) shielded nanoparticles (5 to 1000 mg / mL) for 24 h (Figure 8A-C) and 48 h (Figure 8B-D). Cell viability was assessed by measuring the activity of mitochondrial dehydrogenase by MTT assay. The results show that both nanoparticles do not significantly affect cell viability, supporting the biocompatibility of the nanoparticles with intestinal epithelial cells.

[0160] Example 8: Effect of nanoparticle exposure on transepithelial electrical resistance (TEER) (comparative experiment) Transepithelial electrical resistance (TEER) values ​​are a commonly used parameter to monitor the integrity and viability of cell monolayers. At day 21, the apical end of differentiated Caco-2- (Figure 8A-B) and Caco-2 / HT-29-MTX-E12- (Figure 8C-D) monolayers were exposed to non-functionalized (NP-1) and chitosan-functionalized shielding nanoparticles (NP-2) for 24 h (Figure 9A-C) and 48 h (Figure 9B-D), and TEER values ​​were recorded using the CellZscope system. Opening of tight junctions by EGTA induced a decrease in TEER values ​​compared to untreated monolayers. Figure 6 shows that exposure to nanoparticles does not affect the TEER values ​​of monocultures or cocultures compared to untreated monolayers. These results demonstrate the safety and biocompatibility of chitosan-functionalized shielding nanoparticles using a model of the intestinal barrier.

[0161] Example 9: Effect of nanoparticle exposure on Lucifer Yellow translocation across the intestinal barrier To ensure no disruption of the intestinal barrier by the nanoparticles, translocation studies were assessed using Lucifer Yellow (LY), a marker of barrier integrity. At day 21, differentiated Caco-2- (Figure 10A-B) and Caco-2 / HT-29-MTX-E12- (Figure 10C-D) monolayers were apically exposed to non-functionalized (NP-1) and chitosan-functionalized shielded nanoparticles (NP-2) for 24 h (Figure 10A-C) and 48 h (Figure 10B-D). LY leakage into the basolateral compartment was then measured over a 90 min period. Results show that LY permeability increases due to tight junction opening by EGTA, while fluorescent dye permeability remains constant upon exposure to nanoparticles in both models. This translocation study validates the safety and biocompatibility of chitosan-functionalized shielded nanoparticles using a model of the intestinal barrier.

[0162] Example 10: Evaluation of cellular uptake of nanoparticles in an intestinal barrier model To further evaluate potential toxicity and risk assessment, cellular uptake of nanoparticles was evaluated by confocal microscopy and flow cytometry after 24 h exposure of coculture monolayers with fluorescent NP-2. As observed in three-dimensional (3D) reconstructed z-slides obtained by confocal microscopy, nanoparticles were found only on the surface of Caco-2 / HT-29-MTX-E12 cocultures (Figure 11A). Chitosan-functionalized shielding nanoparticles form a coating on the cells, which can be explained by the interaction of NP-2 with mucus. The absence of cellular uptake was confirmed by flow cytometry. The fluorescence of enterocytes exposed to fluorescent NP-2 did not increase compared to untreated cells, in contrast to the fluorescence of THP-1 differentiated into macrophages used as a positive control (Figure 11B). Overall, these data demonstrate the safety of chitosan-functionalized shielding nanoparticles on the intestinal barrier.

[0163] Example 11: Stabilization of the activity of pancreatin immobilized and protected on nanoparticles The biocatalytic activity of the immobilized and protected pancreatin was evaluated and compared to free pancreatin at 37 °C for 24 h. The results shown in Figure 12 indicate an increase in the half-life of protease (A), lipase (B), and amylase (c) activity of NP-14, NP-12, and NP-16, respectively, compared to free pancreatin. These results highlight the benefit of the organosilane protective layer for stabilizing the biocatalytic activity of NP-12, NP-14, and NP-16.

[0164] Example 12: Protective effect of chitosan against protease activity To evaluate the lipase activity of pancreatin shielded nanoparticles functionalized or not with chitosan in a gastrointestinal-like environment, we evaluated the lipase activity assay in acidic conditions (pH 4). Unexpectedly, Figure 13 shows that in acidic conditions, the lipase activity of chitosan-functionalized shielded pancreatin nanoparticles (NP-14) is higher than that of non-functionalized shielded pancreatin nanoparticles (NP-13) (82% and 58% of remaining activity, respectively). This result demonstrates that in addition to its mucoadhesive properties, chitosan exerts an unexpected protective effect on the immobilized enzyme.

[0165] Example 13: Ex vivo interactions of nanoparticles functionalized with various ratios of thiol-containing compounds with mucus layers We hypothesized that the interaction of functionalized nanoparticles with mucus could also be caused by thiol functional groups. To evaluate this hypothesis, we created shielded nanoparticles containing increasing ratios of thiol functional groups on the surface of the nanoparticles, following the section "Functionalization of protein- and / or enzyme-shielded nanoparticles with MPTS (polymerized MPTS-functionalized shielded nanoparticles)" above. Ex vivo mucus binding studies were performed with shielded nanoparticles exhibiting various ratios of thiol functional groups on their surface, and their interaction with the mucus layer was quantified with ImageJ software. Figure 14 shows that the level of interaction of nanoparticles with the mucus layer increases depending on the amount of thiol functional groups on the surface of the nanoparticles.

[0166] These results demonstrate that nanoparticles functionalized with various ratios of thiol-functionalized polymers exhibit concentration-dependent binding to mucus, thus making the functionalized nanoparticles suitable for therapeutic applications such as ERT.

Claims

1. A composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group.

2. 2. The composition of claim 1, wherein the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymeric silane-PEG-NH2, and polymeric silane comprising an amino group.

3. 2. The composition of claim 1, wherein the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, and polymerized APTES.

4. 4. The composition according to claim 1, wherein the polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group, is a polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan and dermatan or derivatives thereof.

5. 2. The composition of claim 1, wherein the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is selected from the group consisting of polymerized silanes comprising thiol groups, polycarbophil-cysteine ​​conjugates, polymerized silane-PEG-thiols, and polycysteines.

6. 2. The composition of claim 1, wherein the polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group, is polymerized MPTS.

7. The composition of claim 1 , wherein the functional component is immobilized on the surface of the protective layer by non-covalent or covalent bonding.

8. 7. The composition of any one of claims 1 to 3, 5 and 6, wherein the protein or fragment thereof is an enzyme or fragment thereof.

9. 4. The composition according to claim 1, wherein the protein or fragment thereof is selected from the group consisting of serum albumin or a fragment thereof, lipase or a fragment thereof, pancreatin, and a protein or a fragment thereof contained in pancreatin.

10. 7. The composition of any one of claims 1 to 3, 5, and 6, wherein the protein or fragment thereof is selected from the group consisting of serum albumin or a fragment thereof, lipase or a fragment thereof, and pancreatin.

11. 7. The composition of claim 1, wherein the protein or fragment thereof is a lipase or fragment thereof.

12. 7. The composition of any one of claims 1 to 3, 5 and 6, wherein the protective layer embeds the solid support and embeds the protein or fragment thereof immobilized on the surface of the solid support.

13. The composition according to any one of claims 1 to 3, 5 and 6, wherein the functional component immobilized on the surface of the protective layer is not embedded by the protective layer.

14. 7. The composition of any one of claims 1 to 3, 5, and 6, further comprising a chelating agent, which optionally comprises a radioactive or luminescent label.

15. 10. A composition according to any one of claims 1 to 3, 5 and 6 for use as a medicament.

16. 7. The composition of any one of claims 1 to 3, 5 and 6 for use in a method for enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in a method for the prevention, delay of progression or treatment of exocrine pancreatic insufficiency (EPI), lactase deficiency, sucrase-isomaltase deficiency, disaccharide intolerance, peptide allergy, inflammatory bowel disease (IBD), cystic fibrosis, and lung diseases or disorders selected from the group consisting of Gaucher disease, Fabry disease and mucopolysaccharidoses (MPS).

17. 1. A method for producing a composition comprising a solid support, a protein or a fragment thereof immobilized on the surface of the solid support, a protective layer that protects the protein or the fragment thereof by embedding the protein or the fragment thereof, and a functional component immobilized on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit containing at least one amino group and / or at least one thiol group, the method comprising the steps of: (a) providing a solid support; (b) immobilizing the protein or a fragment thereof on a solid support; (c) forming a protective layer on the surface of the solid support to protect the protein or fragment thereof immobilized on the solid support; (d) immobilizing a functional component on the surface of the protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each repeating unit comprising at least one amino group and / or at least one thiol group; A method comprising:

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