Methods for protein immobilization

Abstract

The present invention relates to a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally, 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 containing at least one amino group and / or at least one thiol group. The present invention also relates to a composition obtained by this method.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally, 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 containing at least one amino group and / or at least one thiol group. The present invention also relates to a composition obtained by this method. [Background technology]

[0002] Proteins, such as enzymes, are frequently required for industrial, diagnostic, or therapeutic applications. In the prior art, to stabilize proteins and / or confer resistance to various types of stress, it has been proposed to immobilize proteins on the surface of a support and protect it with a layer of protective material. Such a method is described, for example, in International Publication 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 at least partially embedding it, as well as a process for producing such a biocatalytic composition. However, the method described in International Publication 2015 / 014888 is difficult to use on a large scale due to the low protein loading per dry weight of the particles. Therefore, there is a need to provide an improved immobilization method to obtain a high protein loading per dry weight of the particles, enabling the resulting particles to have high enzymatic activity. [Overview of the Initiative]

[0003] The present invention provides a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally 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 containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspension; (b) A step of immobilizing a protein or fragment thereof on a solid carrier, comprising: i) adding a linker to a suspension of the solid carrier; and ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, wherein the linker connects the solid carrier to the protein or fragment thereof; (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker or part thereof that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment; and optionally (d) A step of immobilizing a functional component on the surface of a 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.

[0004] The present invention also provides a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally 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 comprising at least one amino group and / or at least one thiol group, and the composition can be obtained by the method described herein.

[0005] Surprisingly, the present inventors of this application have found that the methods provided herein and described herein can be used for large-scale protein immobilization to provide a high loading of immobilized protein per unit dry weight of particles that significantly increases particle activity. [Brief explanation of the drawing]

[0006] [Figure 1] A schematic representation of a method for producing the composition of the present invention is provided: a) immobilizing the processed PAL or fragment thereof onto a solid carrier; b) and c) growing a protective layer around the immobilized processed PAL or fragment thereof to embed it; and d) immobilizing the functional component onto the surface of the protective layer. [Figure 2] This shows the added value of the covalent bond between the PAL surface and the protective layer. (A) Quantification of PAL performed on PAL-based silica nanoparticles NP-1(1), NP-1(2), and the reaction supernatant of NP-1. (B) PAL loading per dry weight of SNPs. (C) PAL activity of SNPs expressed as U / g SNPs. (D) PAL specific activity expressed as U / g PAL. [Figure 3] This shows the phenylalanine ammonia lyase (PAL) activity of nanoparticles. The biocatalytic activity of engineered PAL immobilized and protected on nanoparticles was quantified after exposure to phenylalanine (unit: U / g). [Figure 4A-B] This demonstrates the resistance of PAL to external stress. PAL-based silica nanoparticles NP-1 and modified PAL were exposed to acidic conditions (pH 4), and their stability was evaluated by measuring PAL enzyme activity at different time points. [Figure 4C] This demonstrates the resistance of PAL to external stress. PAL-based silica nanoparticles NP-1 and engineered PAL were exposed to proteases, and their stability was evaluated by measuring PAL enzyme activity at different time points. [Figure 5]This study demonstrates the in vitro biocompatibility and efficacy of PAL-based silica nanoparticles NP-1 in a model of the intestinal barrier. (A) In vitro evaluation of intestinal barrier integrity by measurement of transepithelial electrical resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to PAL-based silica nanoparticles NP-1 (9.7 mU) for 6 hours in the presence or absence of pancreatin (30 mU), or in pancreatin (30 mU) alone. The graph shows the time course profile of averaged, normalized TEER data over 6 hours. The dashed line represents the untreated state. (B) In vitro metabolism of phenylalanine (Phe) in a model of the intestinal barrier. Differentiated Caco-2 / HT29-MTX-E12 cocultures cultured in cell medium containing 0.4 mM Phe were exposed to NP-1 (9.7 mU) or engineered PAL (9.7 mU) with or without pancreatin (30 mU) at the apical end of the barrier for 6 hours. Phe metabolism was assessed by quantification of trans-cinnamic acid (TCA) at the basal end of the barrier. The graph shows the time course profile of TCA accumulation over 6 hours. [Figure 6] This shows the quantification of trans-cinnamic acid (TCA) in rat urine. Wistar rats were administered either PAL-based silica nanoparticles NP-1 (n=5) or inactive nanoparticles NP-2 (n=5) intraduodenally, and d5-Phe was administered simultaneously via enteral nutrition. Urine was collected over 24 hours after administration and analyzed by LC-MS. The graph shows the d5-hypric acid concentration in the urine. **p<0.01 (by t-test). [Figure 7]Shows the plasma concentration of Phe in BTBR-Pahenu2 / J mice. BTBR-Pahenu2 / J mice with free access to drinking water containing L-Phe were administered PAL-based silica nanoparticles NP-1 (0.581 U; 7 mg), inert nanoparticles NP-2 (7 mg), or engineered PAL (0.581 U) twice a day for 12 days via duodenal administration. Blood samples were collected on days 0, 4, 6, 8, 10, and 12 to extract plasma and analyzed by LC-MS. (A) The graph shows the plasma concentration of Phe in BTBR-Pahenu2 / J mice. (B) The graph shows the normalized plasma concentration of Phe in BTBR-Pahenu2 / J mice. [Figure 8] Shows the absorbance at a wavelength of 460 nm of PAL-based silica nanoparticles NP-1, NP-1(1), and NP-1(2). [Figure 9] Schematically shows a method for generating the composition of the present invention: a) immobilizing a disaccharidase or a fragment thereof (denoted as "protein") on a solid support; b) and c) growing a protective layer around the immobilized disaccharidase or a fragment thereof to embed the immobilized disaccharidase or a fragment thereof; d) immobilizing a functional component on the surface of the protective layer. [Figure 10] Shows the added value of the covalent bond between the lactase surface and the protective layer. (A) Quantification of lactase performed on the reaction supernatants of lactase-based silica nanoparticles NP-2(1), NP-2(2), and NP-2. (B) Lactase loading per dry weight of SNP. [Figure 11] Shows the disaccharidase activity of the nanoparticles. (A) Lactase activity (unit: U / g) of lactase-based silica nanoparticles NP-2 after exposure to lactose. (B) Invertase activity (unit: U / mg) of invertase-based silica nanoparticles NP-3 after exposure to sucrose. (C) Isomaltase activity (unit: U / g) of isomaltase-based silica nanoparticles NP-4 after exposure to isomaltose. (D) Isomaltase activity and invertase activity (unit: U / g) of invertase / isomaltase-based silica nanoparticles NP-5 after exposure to isomaltose and sucrose, respectively. [Figure 12] It shows the in vitro biocompatibility of a representative nanoparticle model for the intestinal barrier. (A) In vitro evaluation of the integrity of the intestinal barrier by measuring transepithelial electrical resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 co-cultures were exposed to inert nanoparticles NP-1 (0.5 mg / mL and 1 mg / mL) for 16 hours. The TEER data were normalized to the control point consisting of the equilibrium value before the addition of inert nanoparticles NP-1 (defined as the control) and set as 100%. The graph represents the transition of the time-course profile of the averaged normalized TEER data over 16 hours. The dashed line represents the untreated state. (B) In vitro evaluation of the inflammatory effect on the intestinal epithelial barrier. Differentiated Caco2-HT29-MTX-E12 and M0-differentiated THP-1 were co-cultured and exposed to inert nanoparticles NP-1 (1 mg / mL) at 37 °C for 16 hours. Lipopolysaccharide (LPS) (50 μg / mL and 100 μg / mL) was used as a positive control to induce an inflammatory response. The TEER data were normalized with the equilibrium value before the addition of inert nanoparticles NP-1 or LPS set as 100%. The graph represents the transition of the time-course profile of the averaged normalized TEER data over 16 hours in the presence of inert nanoparticles NP-1. [Figure 13] It shows the difference in the size of the rat cecum. Wistar rats were administered lactase-based silica nanoparticles NP-2, inert silica nanoparticles NP-1, or vehicle intraduodenally daily for 15 days, and lactose was immediately administered by tube feeding. The size of the cecum was evaluated at the end. (A) It is a photograph of the digestive tract of a rat. The circle indicates the cecum. (B) It is an MRI image of the digestive tract of a rat. The arrow indicates the cecum. (C) The histogram shows the size of the cecum evaluated by MRI imaging (unit: cm3). *p < 0.05, **p < 0.01, one-way ANOVA test. [Figure 14]This study demonstrates the in vitro digestion of sucrose in a model of the intestinal barrier. Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to different amounts of invertase-based silica nanoparticles NP-3 (0.5 mU or 1 mU) at the barrier tip for 4 hours in the presence of sucrose. Sucrose hydrolysis was evaluated by quantifying glucose at the barrier base. The graph shows the time course profile of glucose accumulation over 4 hours. [Figure 15] The absorbance of lactase-based silica nanoparticles NP-2, NP-2(1), and NP-2(2) at a wavelength of 460 nm is shown. [Figure 16] The process for producing the compositions of the present invention is schematically shown: a) A solid carrier is supplied with lipases, proteases, amylases having closed lids, and substances that interact with the lid domains of lipases (shown as full circles) to immobilize the lipases, proteases, and amylases having open lids onto the solid carrier; b) and c) A protective layer is grown around the immobilized lipases, proteases, and amylases having open lids to embed all three enzymes; d) Functional components are immobilized on the surface of the protective layer. [Figure 17A-B] The in vivo distribution of chitosan-functionalized inert DOTA-labeled silica nanoparticles 111In-NP-1 (n=5) and unfunctionalized inert DOTA-labeled silica nanoparticles 111In-NP-2 (n=4) in miniature pigs is shown (unit: %ID). (A) SPECT / CT images were acquired 0.25, 3, 8, and 24 hours after intraduodenal administration of 111In-NP-1 and 111In-NP-2. The graph shows the relative quantification of nanoparticles at imaging time points in the small intestinal compartment (unit: %). (B) The histogram shows the area under the curve (AUC) of residence time in the small intestine for 111In-NP-1 and 111In-NP-2. [Figure 17C]This shows the in vivo distribution (in %ID) of chitosan-functionalized inert DOTA-labeled silica nanoparticles 111In-NP-1 (n=5) and unfunctionalized inert DOTA-labeled silica nanoparticles 111In-NP-2 (n=4) in miniature pigs. Blood samples were collected 0.25, 3, 8, and 24 hours after intraduodenal administration of 111In-NP-1 or free 111In. The histogram shows the relative quantification of radioactivity in the blood compartment. [Figure 18] The activity of lipase and / or fragments thereof, protease and / or fragments thereof, and amylase and / or fragments thereof, composed of pancreatin immobilized and protected on nanoparticles, is shown. (A) Lipase activity of pancreatin-based silica nanoparticle NP-3 after exposure to olive oil (unit: U / g). (B) Protease activity of pancreatin-based silica nanoparticle NP-5 after exposure to casein (unit: U / mg). (C) Amylase activity of pancreatin-based silica nanoparticle NP-3 after exposure to amylase substrate solution (unit: U / g). [Figure 19] This shows the relative quantification of plasma triglycerides (TG) in pancreatic duct ligation (PDL) rats. PDL rats were administered either pancreatin-based silica nanoparticles NP-3 (n=1) or inactive nanoparticles NP-4 (n=1) via duodenum, followed by enteral nutrition of triolein 5 minutes later. Blood samples were collected before the procedure and at 0.25, 0.5, 1, 1.5, 2, 4, and 6 hours later. Plasma extraction and analysis by LC-MS were performed. The graph shows the peak area of ​​triolein (TG(54:3)). [Figure 20]This study compares plasma triglyceride (TG) concentrations in healthy miniature pigs treated with pancreatin-based silica nanoparticles NP-3 and miniature pigs that underwent pancreatic duct ligation (PDL). Olive oil was administered to both a healthy miniature pig (n=1) and a PDL-ligated miniature pig (n=1), followed by administration of pancreatin-based silica nanoparticles NP-3 to the PDL-ligated miniature pig. Blood samples were collected before administration and at 0.083, 0.25, 0.5, 1, 2, 3, 4, and 6 hours after administration, and TG analysis was performed. (A) The graph shows the plasma TG concentrations in miniature pigs determined using a Konelab analyzer. (B) The histogram shows the area under the curve (AUC) for plasma TG concentrations in miniature pigs. [Figure 21] This shows the fecal fat content of miniature pigs on a high-fat diet. Healthy miniature pigs and PDL miniature pigs were fed a high-fat diet for 25 days. PDL miniature pigs were administered pancreatin-based silica nanoparticles NP-3 twice daily for 10 days. Feces were collected on days 8, 9, and 10, and fecal homogenates were analyzed by near-infrared spectroscopy to quantify the fat content in the feces. (A) The histogram shows the absolute values ​​of fecal fat. (B) The graph shows the normalized fecal fat homogenate fat content for healthy miniature pigs and untreated PDL miniature pigs. [Figure 22]This demonstrates the in vitro biocompatibility of pancreatin-based silica nanoparticles NP-5 with respect to the intestinal barrier. (A) In vitro evaluation of intestinal barrier integrity by measurement of transepithelial electrical resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to gradually increasing doses of pancreatin-based silica nanoparticles NP-5 or pancreatin (32.9~263.6 U / m2) for 20 hours. TEER data were normalized with the equilibrium value before the addition of pancreatin-based silica nanoparticles NP-5 or pancreatin set to 100%. The graph shows the time course profile of averaged and normalized TEER data over 20 hours. The dashed line represents the untreated state. (B) Confocal image of tight junctions in a model of the intestinal barrier. Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to pancreatin-based silica nanoparticles NP-5 or pancreatin (263.6 U / m2) for 20 hours. Cells were stained with zonula occludens 1 (ZO-1) and evaluated using a confocal microscope (white signals in the image). [Figure 23] This shows the added value of the pancreatin surface, protective layer, and covalent bonding. (A) Quantification of proteins performed on the reaction supernatant of pancreatin-based silica nanoparticles NP-3 and NP-3(1). (B) Pancreatin load per dry weight of SNP. (C) Lipase activity of SNP expressed in μmol / min. (D) Pancreatin specific activity of pancreatin expressed in U / g. [Figure 24] This shows the absorbance of pancreatin-based silica nanoparticles NP-3 and NP-3(1) at a wavelength of 460 nm. [Figure 25] The process for producing the composition of the present invention is schematically shown: a) A lipase or fragment thereof having a closed lid and a substance (shown as a full circle) that interacts with the lid domain of the lipase or fragment thereof are supplied to a solid carrier to immobilize the lipase or fragment thereof having an open lid onto the solid carrier; b) and c) A protective layer is grown around the immobilized lipase or fragment thereof having an open lid to embed the immobilized lipase or fragment thereof. [Figure 26]Figure 26 shows the 3D structure of pancreatic lipase in two states: a) the inactive conformation (lid closed) and b) the active conformation (lid open). The active site of pancreatic lipase is covered by a lid that prevents substrates from reaching the enzyme's active site (Figure 26a). The opening of the lipase lid is induced by interaction with substances that interact with the lipase lid domain, such as bile salts and / or protein cofactors called collipases, which allows for the stabilization of the active conformation of pancreatic lipase (Figure 26b). [Figure 27] This shows the rate of hydrolysis of lipase substrates by recombinant human pancreatic lipase (HRL) with or without colipase (CLPS) immobilized on the surface of silica nanoparticles (SNPs) and protected by an organosilica layer consisting of APTES, TEOS, and benzyltriethoxysilane (ATB), a) in its free form; and b) with or without colipase (CLPS). [Figure 28] This shows the rate of hydrolysis of lipase substrates by porcine pancreatic lipase (PL) with and without colipase (CLPS) immobilized on the surface of silica nanoparticles (SNPs) and protected by an organic silica layer consisting of APTES, TEOS, and benzyltriethoxysilane (ATB). [Figure 29] The rate of hydrolysis of lipase substrates by free human recombinant lipase (HRL) is shown using gradually increasing concentrations of sodium taurocholate (NaTc). [Figure 30] This shows the rate of hydrolysis of lipase substrates by porcine pancreatic lipase (PL), immobilized on the surface of silica nanoparticles (SNPs) and protected by an organosilica layer consisting of APTES, TEOS, and benzyltriethoxysilane (ATB), with or without sodium taurocholate (NaTc). [Figure 31] This shows the 3D structure of pancreatic lipase activated by a peptide that mimics colipase. [Figure 32]This demonstrates the added value of the covalent bond between the HRL surface and the protective layer. (A) Quantification of proteins performed on the reaction supernatant of HRL-based silica nanoparticles NP-1, NP-1(1), and NP-1(2). (B) HRL loading per dry weight of SNPs. (C) Lipase activity of nanoparticles of SNPs expressed in UμM / min / g. (D) HRL specific activity of HRL expressed in UμM / min / g. [Figure 33] The absorbance of HRL-based silica nanoparticles NP-1, NP-1(1), and NP-1(2) at a wavelength of 460 nm is shown. [Modes for carrying out the invention]

[0007] The present invention relates to a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally 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 containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspension; (b) A step of immobilizing a protein or fragment thereof on a solid carrier, comprising: i) adding a linker to a suspension of the solid carrier; and ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, wherein the linker connects the solid carrier to the protein or fragment thereof; (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker or part thereof that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment; and optionally (d) A step of immobilizing a functional component on the surface of a 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.

[0008] The present invention also provides a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally 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 comprising at least one amino group and / or at least one thiol group, and the composition can be obtained by the method described herein.

[0009] For the purposes of interpreting this specification, the following definitions apply, and wherever used in the singular, the plural form is also included, and vice versa. It should be understood that the terms used herein are intended solely to describe and not to limit a particular embodiment.

[0010] Features, integers, properties, and compounds described in relation to specific aspects, embodiments, or examples of the present invention should be understood to be applicable to any other aspects, embodiments, or examples described herein, insofar as they do not contradict each other. All features and / or steps of methods or processes disclosed herein (including the claims, abstract, and drawings) can be combined in any combination, except for any combination in which at least some of such features and / or steps are mutually exclusive. The present invention is not limited to the details of the embodiments described above.

[0011] The term "comprise," and its variations such as "comprises" and "comprising," are generally used to mean "include," that is, "to include, but not limited to," meaning to allow the presence of one or more features or components.

[0012] The singular forms "a," "an," and "the" include references to the plural form unless otherwise explicitly stated.

[0013] The term "approximately" refers to a range of values ​​that is ±10% of a given value. For example, the expression "approximately 200" includes ±10% of 200, i.e., from 180 to 220.

[0014] As used herein, the term “solid carrier” usually refers to particles. Preferably, the solid carrier is monodisperse or polydisperse particles, more preferably monodisperse particles. The solid carrier typically includes organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, and titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size of the solid carrier is typically 1 nm to 1000 μm, preferably 10 nm to 100 μm, and particularly about 50 nm.

[0015] In this specification, the terms “linker” or “crosslinker” as used synonymously refer to any linking reagent containing a group capable of binding to a specific functional group (e.g., primary amine, sulfhydryl, etc.). In the context of the present invention, a linker typically connects the surface of a solid carrier to a protein or fragment thereof, i.e., an enzyme, e.g., lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof. For example, a linker can be immobilized on the surface of a solid carrier, e.g., a silica surface as the carrier material, and then a protein or fragment thereof, i.e., an enzyme, e.g., lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, can be bound to the unoccupied binding sites of the linker. Alternatively, the linker may first bind to a protein or fragment thereof, i.e., an enzyme, e.g., lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, and then the linker bound to the enzyme, e.g., lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, may be bound to the solid carrier at its unoccupied binding sites. 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 tag linkers known in the art.

[0016] As used herein, the term “protective layer” refers to a layer for protecting the functional properties of proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, immobilized on the surface of a solid carrier. The protective layer of the present invention is typically constructed of building blocks, at least a portion of which are monomers capable of interacting with each other, usually by covalent bonds, and with proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, usually immobilized by non-covalent bonds. The protective layer is formed on the surface of a solid carrier to protect the proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, immobilized on the solid carrier. The protective layer is typically a homogeneous layer in which at least 50%, preferably at least 70%, more preferably at least 90%, of the proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are embedded within the protective layer.

[0017] The term “protein or fragment thereof” includes both naturally occurring proteins or fragments thereof and artificially engineered proteins or fragments thereof. Artificially engineered proteins or fragments thereof are, for example, protein variants or functionally active fragments. Accordingly, in this specification, the terms “protein fragment,” “fragment thereof” in relation to a protein, and “functionally active fragment of a protein” are used synonymously. In relation to a protein in this 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 functions as the protein. Such variants include naturally occurring allelic variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that such modifications do not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the protein, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98%. The protein fragments defined herein typically have the same functional properties as the protein, i.e., the full-length protein from which the fragment is derived. The protein fragments typically contain 100 to 1000 amino acids, preferably 150 to 500 amino acids, and more preferably 300 to 450 amino acids. Preferred proteins or fragments thereof of the present invention are enzymes or fragments thereof, more preferably enzymes or fragments thereof selected from the group consisting of hydrolases and lyases, or fragments thereof. Even more preferred proteins or fragments thereof of the present invention are selected from the group consisting of lipases or fragments thereof, proteases or fragments thereof, amylases or fragments thereof, pancreatin or proteins or fragments thereof composed of pancreatin, engineered phenylalanine ammonia lyase (PAL) or fragments thereof, and disaccharide-degrading enzymes or fragments thereof.

[0018] The term “lipase or its fragment” includes naturally occurring lipases or their fragments, as well as artificially engineered lipases or their fragments. Artificially engineered lipases or their fragments are, for example, lipase variants or functionally active fragments. Therefore, in this specification, the terms “lipase fragment,” “its fragment” in relation to lipase, and “functionally active fragment of lipase” are used synonymously. In relation to the lipases of this invention, “variant or functionally active fragment” means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as lipase. Such variants include naturally occurring allelic variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the lipase, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98%. The lipase fragments as defined herein typically have the same functional properties as the lipase, i.e., the full-length enzyme from which it is derived, and include at least a lid domain and a substrate-binding region. The lipase fragments typically contain 100 to 450 amino acids, preferably 150 to 400 amino acids, and more preferably 200 to 350 amino acids. Preferred lipases or fragments thereof are lipases or fragments thereof extracted from the pancreas, more preferably lipases or fragments thereof extracted from the pancreas of a pig, and even more preferably lipases or fragments thereof composed of pancreatin. In preferred embodiments, the lipase or fragment thereof is recombinant human pancreatic lipase (HRL) or fragment thereof, or porcine pancreatic lipase or fragment thereof, preferably recombinant human pancreatic lipase (HRL) or fragment thereof, more preferably full-length recombinant human pancreatic lipase (HRL).

[0019] The term “protease or fragment thereof” includes naturally occurring proteases or fragments thereof, as well as artificially engineered proteases or fragments thereof. Artificially engineered proteases or fragments thereof are, for example, protease variants or functionally active fragments. Accordingly, in this specification, the terms “protease fragment,” “its fragment” in relation to a protease, and “functionally active fragment of a protease” are used synonymously. In this invention, “variant or functionally active fragment thereof” in relation to a protease means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the protease. Such variants include naturally occurring allelic variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the protease, 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. The protease fragments defined herein typically have the same functional properties as the protease from which they are derived. The protease fragments typically contain 50 to 200 amino acids, preferably 75 to 175 amino acids, and more preferably 100 to 150 amino acids. Preferred proteases or fragments thereof are proteases or fragments thereof extracted from the pancreas, more preferably lipases or fragments thereof extracted from the pancreas of a pig, and even more preferably proteases or fragments thereof composed of pancreatin.

[0020] The term “amylase or its fragment” includes naturally occurring amylase or its fragment, as well as artificially engineered amylase or its fragment. Artificially engineered amylase or its fragment is, for example, an amylase variant or a functionally active fragment. Therefore, in this specification, the terms “amylase fragment,” “its fragment” in relation to amylase, and “functionally active fragment of amylase” are used synonymously. In this invention, “variant or functionally active fragment” of amylase means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as amylase. Such variants include naturally occurring allele variants and variants that do not exist in nature. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the amylase, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least about 98%. The amylase fragments defined herein typically have the same functional properties as the amylase from which they are derived. The amylase fragments typically contain 100 to 550 amino acids, preferably 200 to 500 amino acids, and more preferably 300 to 450 amino acids. Preferred amylases or fragments thereof are amylases or fragments thereof extracted from the pancreas, more preferably amylases or fragments thereof extracted from the pancreas of a pig, and even more preferably amylases or fragments thereof composed of pancreatin.

[0021] The term "pancreatin," as used herein and also known as "pancreatic enzymes," refers to pancreatic enzyme preparations derived from the pancreatic glands of pigs, including lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof. The term "pancreatin" as used herein also includes formulated pancreatic enzymes, such as capsules containing pancreatic enzymes, for example, Zenpep®.

[0022] As used herein, the terms “phenylalanine ammonia lyase or fragment thereof” or “PAL or fragment thereof” refer to a class of enzymes within the aromatic amino acid lyase family (EC 4.3.1.23, EC 4.3.1.24, and EC 4.3.1.25), which also includes histidine ammonia lyase and tyrosine ammonia lyase. PAL is sometimes called phenylalanine / tyrosine ammonia lyase because it can use tyrosine as well as phenylalanine as a substrate. PAL catalyzes the conversion of L-phenylalanine to trans-cinnamic acid and ammonia. PAL activity refers to the enzymatic activity of the PAL polypeptide. PAL may also include the cofactor 3,5-dihydro-5-methylidene-4H-imidazole-4-one (MIO). This cofactor is required for catalytic activity and is formed by cyclization and dehydration of the conserved active site Alal67-Serl68-Glyl69 tripeptide segment.

[0023] When used herein in reference to phenylalanine ammonia lyase or its fragments, the terms “manipulated” and “not naturally occurring” refer to phenylalanine ammonia lyase or its fragments that have been modified in a manner otherwise not found in nature, corresponding to the natural or native form of phenylalanine ammonia lyase or its fragments. The term “manipulated phenylalanine ammonia lyase or its fragments” does not include, nor does it encompass, “wild-type” and “naturally occurring” phenylalanine ammonia lyase or its fragments. As used herein, “wild-type” and “naturally occurring” refer to forms of phenylalanine ammonia lyase or its fragments that exist in nature. For example, wild-type phenylalanine ammonia lyase or its fragments are polypeptides present in living organisms that can be isolated from natural sources and have not been intentionally modified by artificial manipulation. Manipulated PAL or its fragments are, for example, variants or functionally active fragments of manipulated phenylalanine ammonia lyase. Accordingly, the terms “engineered phenylalanine ammonia lyase fragment,” “the fragment” in relation to the engineered phenylalanine ammonia lyase, and “functionally active fragment of the engineered phenylalanine ammonia lyase” are used synonymously herein. “Variant or functionally active fragment” in relation to the engineered phenylalanine ammonia lyase of the present invention means that the fragment or variant (such as an analog, derivative, or variant not found in nature) can exhibit the same or improved physiological function as wild-type phenylalanine ammonia lyase. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that such modification does not result in a loss of functional activity of the fragment or variant. The PAL fragment comprises a homotetrameric enzyme, where at least one monomer, preferably all four monomers, of the homotetrameric enzyme typically contains 100 to 550 amino acids, preferably 200 to 500 amino acids, more preferably 300 to 450 amino acids."Improved physiological function" or "improved enzymatic properties" refers to an engineered PAL that exhibits improvement in any enzymatic property compared to a reference PAL polypeptide, such as a wild-type PAL polypeptide. Improved properties include, but are not limited to, increased protein expression, increased thermal activity, increased heat tolerance, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity and / or affinity, increased specific activity, increased resistance to inhibition of substrates and / or end products, increased chemical stability, improved chemoselectivity, improved solvent stability, increased resistance to acidic pH, increased resistance to proteolytic activity (i.e., decreased sensitivity to proteolysis), reduced aggregation, increased solubility, decreased immunogenicity, and altered temperature profiles. A preferred engineered phenylalanine ammonia lyase or fragment thereof of the present invention is the engineered phenylalanine ammonia lyase described in International Publication No. 2018 / 148633. In a preferred embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, or at least 97% sequence identity with respect to the sequence of SEQ ID NO: 1. In one embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof is SEQ ID NO: 2, 3, 4, or 5. In a particular preferred embodiment, the manipulated phenylalanine ammonia lyase or fragment thereof comprises the polypeptide shown in SEQ ID NO: 5.

[0024] As used herein, the term “partially embedded engineered phenylalanine ammonia lyase” means that the engineered phenylalanine ammonia lyase is not completely covered by the protective layer, and therefore, the engineered phenylalanine ammonia lyase is not completely embedded in the protective layer. In one embodiment, less than 50% of the engineered phenylalanine ammonia lyase of interest is covered by the protective layer, but typically at least 70% is covered, thereby improving the protection of the engineered phenylalanine ammonia lyase. 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 engineered phenylalanine ammonia lyase 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 engineered phenylalanine ammonia lyase of interest is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most specifically about 95% of the manipulated phenylalanine ammonia lyase of the target is covered by a protective layer. In a particularly more preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most specifically about 95% of the manipulated phenylalanine ammonia lyase of the target is covered by a protective layer, and the active site is not covered.

[0025] As used herein, the term “fully embedded engineered phenylalanine ammonia lyase” means that the engineered phenylalanine ammonia lyase of the present invention is completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered. Preferably, the engineered phenylalanine ammonia lyase or fragment thereof according to the present invention is completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered.

[0026] As used herein, the term “at least partially embedded engineered phenylalanine ammonia lyase” means that the engineered phenylalanine ammonia lyase is at least partially embedded and may be completely embedded by a protective layer. Thus, “at least partially embedded engineered phenylalanine ammonia lyase” means that the protective layer covers about 30% to 100%, preferably about 50% to 100%, more preferably about 80% to 100%, even more preferably about 90% to 100%, and most preferably about 95% to 100% of the engineered phenylalanine ammonia lyase or fragment thereof, and the active site is preferably covered.

[0027] The term “disacase or fragment thereof” includes naturally occurring disacases or fragments thereof, as well as artificially engineered disacases or fragments thereof. Disacases are glycoside hydrolases, enzymes that break down certain sugars called disaccharides into simpler sugars called monosaccharides. In the human body, disacases are mostly produced in an area of ​​the small intestine wall called the brush border. Examples of disacases include lactase, maltase, isomaltase, trehalase, and sucrase (also called invertase). Artificially engineered disacases or fragments thereof are, for example, variants or functionally active fragments of disacases. Therefore, in this specification, the terms “fragment of a disacase,” “fragment thereof” and “functionally active fragment of a disacase” are used synonymously. In relation to the disaccharide-degrading 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 exhibiting the same physiological function as the disaccharide-degrading enzyme. Such variants include naturally occurring allelic variants and variants that do not exist naturally. Addition, deletion, substitution, and derivatization of one or more amino acids are attempted, provided that such modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the disaccharide-degrading 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. The disaccharide-degrading enzyme fragments as defined herein typically have the same functional properties as the disaccharide-degrading enzyme from which they are derived. The disaccharide-degrading enzyme fragments typically contain 100 to 1000 amino acids, preferably 300 to 800 amino acids, and more preferably 500 to 700 amino acids.

[0028] As used herein, the term “partially embedded disaccharidase” means that the disaccharidase is not completely covered by the protective layer, and therefore not completely embedded in the protective layer. In one embodiment, less than 50% of the disaccharidase of interest is covered by the protective layer, but typically at least 70% is covered, thereby improving the protection of the disaccharidase. 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 disaccharidase 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 disaccharidase is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more specifically about 90%, and most specifically about 95% of the disaccharidase of interest is covered by the protective layer. In a particularly more preferred embodiment, about 70%, especially about 80%, more specifically about 90%, and most specifically about 95% of the target disaccharide-degrading enzyme is covered by a protective layer, while the active site is left uncovered.

[0029] As used herein, the term “fully embedded disaccharidase” means that the disaccharidase of interest according to the present invention is completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered.

[0030] As used herein, the term “at least partially embedded disaccharidase” means that the disaccharidase is at least partially embedded and may be completely embedded by a protective layer. Thus, “at least partially embedded disaccharidase” means that the protective layer covers about 30% to 100%, preferably about 50% to 100%, more preferably about 80% to 100%, even more preferably about 90% to 100%, and most preferably about 95% to 100% of the disaccharidase or fragment thereof, and the active site is preferably covered.

[0031] As used herein, the term “partially embedded protein” means that a protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is not completely covered by the protective layer; and therefore, a protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is not completely embedded in the protective layer. In one embodiment, less than 50% of the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, is covered by the protective layer, but typically at least 70% is covered, thereby 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, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, 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%, most preferably about 90% to about 95%, 96%, 97%, 98%, or 99% of the protein, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, is covered by a protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more specifically about 90%, and most specifically about 95% of the protein, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, is covered by a protective layer. In a particularly more preferred embodiment, about 70%, particularly about 80%, more specifically about 90%, and most specifically about 95% of the protein, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, is covered by a protective layer, and the active site is not covered.

[0032] As used herein, the term “fully embedded protein” means that the proteins according to the present invention, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, are completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered. Preferably, the lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof according to the present invention are completely, i.e., 100%, covered by a protective layer, i.e., the active site is also covered.

[0033] As used herein, the term “at least partially embedded protein” means that a protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, is at least partially embedded by a protective layer, and may be fully embedded. 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 a fragment thereof, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, and the active site is preferably covered.

[0034] As used herein, “substances that interact with the lid domain of lipase or its fragments” typically refers to substances that bind to and / or to the region surrounding the lid domain of lipase or its fragments, thereby causing the lid domain to transition the lipase or its fragments to an open structure and / or to maintain the open structure of the lipase or its fragments. The lid domain of lipase is usually amphiphilic; in the closed structure, their hydrophilic side faces the solvent and their hydrophobic side faces directly toward the catalyst pocket (Brocca S., Secundo F., Ossola M., Alberghina L., Carrea G., Lotti M. (2003). Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes. Protein Sci. 12, 2312-2319.10.1110 / ps.0304003). When lipase transitions to an open structure, its hydrophobic surface is exposed and contributes to the substrate-binding region. Preferably, a substance that interacts with the lid domain of the lipase or its fragment locks the lipase or fragment into its active conformation. Once locked into its active conformation, the lipase is usually fully activated. Substances that interact with the lid domain of the lipase or its fragment to cause it to transition to an open structure include collipase or its fragment, collipase-mimicking peptides, and amphiphilic molecules.

[0035] As used herein, the term “amphiphilic molecule” refers to molecules such as chemical compounds that have both a polar (water-soluble) and a nonpolar (water-insoluble) moiety in their structure. The term may also refer to molecules such as chemical compounds that have both hydrophobic and hydrophilic regions. Examples of amphiphilic molecules include bile salts, phospholipids, and nonionic detergents.

[0036] The terms “open structure” or “open structure of lipase or its fragment” as used interchangeably herein refer to a structure of lipase or its fragment in which a substrate can enter and be converted into the active site of the lipase. In a closed structure, the entry of a substrate into the active site of the lipase or its fragment and its conversion are restricted or impossible. The structure of lipase or its fragment, i.e., whether the lipase is in an open or closed structure, can be determined by X-ray crystallography, enzyme activity testing, or site-directed spin labeling (SDSL) method. It can also be determined from electron paramagnetic resonance (EPR).

[0037] As used herein, the term “colipase or fragment thereof” includes naturally occurring colipases or fragments thereof, as well as artificially engineered colipases or fragments thereof. Artificially engineered colipases or fragments thereof are, for example, lipase variants or functionally active fragments. In relation to the colipases of this 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 colipase. Such variants include naturally occurring allelic variants and variants that do not exist naturally. Addition, deletion, substitution, and derivatization of one or more amino acids are intended, provided that the modification does not result in a loss of functional activity of the fragment or variant. Preferably, the functionally active fragment or variant has at least about 80% sequence identity with the relevant portion of the lipase, 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. The collipase fragments defined herein have the same functional properties as the collipase from which they are derived. The preferred collipase is the collipase having uniplot number: P02703.

[0038] As used herein, the term “colipase-mimicking peptide” refers to a peptide consisting of 10 to 40 amino acids, which allows specific amino acid residues to be geometrically positioned in appropriate locations to interact with the amino acids of the pancreatic lipase structure, thereby inducing conformational elongation and lid opening of the lipase, and thereby possessing the same functional properties as a lipase. The lipase-mimicking peptide that can be used in the present invention is preferably the peptide shown in SEQ ID NO: 6.

[0039] As used herein, the term “bile salt” refers to bile acids conjugated with taurine or glycine, and includes sodium taurocholate, sodium glycocholate, sodium glycodeoxycholate, sodium taurodeoxycholate, sodium glycochenodeoxycholate, and sodium taurochenodeoxycholate.

[0040] As used herein, the term “nonionic detergent” refers to a surfactant, and includes tetraethylene glycol monooctyl ether, octyl-bD-glucopyranoside, N,N-dimethyldodecylamine-N-oxide, and b-octylglucomaltoside.

[0041] As used herein, the term “phospholipid” refers to a class of lipids whose molecules have a hydrophilic “head” containing a phosphate group, linked by an alcohol residue (usually a glycerol molecule), and two hydrophobic “tails” derived from fatty acids. Phospholipids include lecithins and lysolecithins.

[0042] As used herein, the term “functional component” refers to a component that, after being immobilized on the surface of a protective layer, retains its characteristic functional properties. In the sense of the present invention, a functional component is a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0043] As used herein, the term “polymer comprising repeating units, each containing at least one amino group” refers to a polymer comprising a large number of repeating units (monomers), each of which contains at least one amino group. Preferred polymers comprise a large number of repeating units (monomers), each of which contains one amino group, in particular one primary amino group.

[0044] As used herein, the term “polymer comprising repeating units, each containing at least one thiol group” refers to a polymer comprising a large number of repeating units (monomers), each of which contains at least one thiol group. A preferred polymer comprises a large number of repeating units (monomers), each of which contains one thiol group.

[0045] As used herein, the term “polycarbophil-cysteine ​​conjugate” refers to a conjugate containing cysteine ​​covalently bonded to polycarbophil. Such conjugates can be produced, for example, as described in Bernkop-Schnurch and Thaler, 2000, Journal of Pharmaceutical Sciences 89(7):901-9.

[0046] As used herein, the term “polylysine” refers to α-polylysine and / or ε-polylysine (ε-poly-L-lysine, EPL), preferably ε-polylysine. α-Polylysine is a synthetic polymer and may consist of L-lysine or D-lysine. ε-Polylysine (ε-poly-L-lysine, EPL) is typically produced as a homopolypeptide of approximately 25 to 30 L-lysine residues.

[0047] As used herein, the term "polycysteine" may consist of L-cysteine ​​or D-cysteine, preferably L-cysteine, and preferably containing 2 to 30 cysteine ​​residues, more preferably 2 to 5 cysteine ​​residues.

[0048] As used herein, the term “polyglucosamine” refers to a linear aminopolysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units linked by (1-4) glycosidic bonds. Polyglucosamine contains a free amine (-NH2) group and is characterized by the ratio of N-acetyl-D-glucosamine units to D-glucosamine units, which is expressed as the degree of deacetylation (DDA) of fully acetylated polymer chitin. Preferred polyglucosamines of the present invention are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, and dermatan or their derivatives. Most preferred are chitosan or its derivatives.

[0049] As used herein, the term “chitosan or its derivatives” refers to chitosan or chitosan derivatives, including salts, having a molecular weight preferably of 2000 Da or more, preferably in the range of 25000 to 2000000 Da, more preferably in the range of about 50000 to 350000 Da, and most preferably in the range of about 50000 to 190000 Da or 190000 or 310000 Da. The term chitosan derivatives include esters, ethers, or other derivatives formed by the reaction of an acyl group or alkyl group with an OH group. Examples of these are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Suitable derivatives are described, for example, in GAERoberts, Chitin Chemistry, MacMillan Press Ltd, London, 1992. Suitable salts of chitosan include nitrates, phosphates, sulfates, xanthogenic salts, hydrochlorides, glutamates, lactates, and acetates.

[0050] In a first embodiment, the present invention provides a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally 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 containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspension; (b) A step of immobilizing a protein or fragment thereof on a solid carrier, comprising: i) adding a linker to a suspension of the solid carrier; and ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, wherein the linker connects the solid carrier to the protein or fragment thereof; (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker or part thereof that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment; and optionally (d) A step of immobilizing a functional component on the surface of a 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.

[0051] In one embodiment, a method for producing a composition is provided, the composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and optionally, 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 containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspension; (b) A step of immobilizing a protein or fragment thereof on a solid carrier, comprising: i) adding a linker to a suspension of the solid carrier; and ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, wherein the linker connects the solid carrier to the protein or fragment thereof; (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker or a part thereof that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment thereof.

[0052] In a further embodiment, the present invention provides a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally, 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 containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspension; (b) A step of immobilizing a protein or fragment thereof on a solid carrier, comprising: i) adding a linker to a suspension of the solid carrier; and ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, wherein the linker connects the solid carrier to the protein or fragment thereof; (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker or part thereof that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment; and optionally (d) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer comprising repeating units, each comprising at least one amino group and / or at least one thiol group, provided that the protein or fragment thereof is not a lipase or fragment thereof, a protease or fragment thereof, an amylase or fragment thereof, pancreatin or a protein or fragment thereof composed of pancreatin, engineered phenylalanine ammonia lyase (PAL) or fragment thereof, and a disaccharide-degrading enzyme or fragment thereof, preferably, the protein or fragment thereof is not a lipase or fragment thereof, a protease or fragment thereof, an amylase or fragment thereof, pancreatin or a protein or fragment thereof composed of pancreatin, engineered phenylalanine ammonia lyase (PAL) or fragment thereof, and a disaccharide-degrading enzyme or fragment thereof as described herein.

[0053] In one embodiment, a method for producing a composition is provided, the composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and optionally, 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 containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspension; (b) A step of immobilizing a protein or fragment thereof on a solid carrier, comprising: i) adding a linker to a suspension of the solid carrier; and ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, wherein the linker connects the solid carrier to the protein or fragment thereof; (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker, or a part thereof, that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment thereof, provided that the protein or fragment thereof is not a lipase or fragment thereof, a protease or fragment thereof, an amylase or fragment thereof, pancreatin or a protein or fragment thereof composed of pancreatin, engineered phenylalanine ammonia lyase (PAL) or fragment thereof, and a disaccharide-degrading enzyme or fragment thereof, preferably, the protein or fragment thereof is not a lipase or fragment thereof, a protease or fragment thereof, an amylase or fragment thereof, pancreatin or a protein or fragment thereof composed of pancreatin, engineered phenylalanine ammonia lyase (PAL) or fragment thereof, and a disaccharide-degrading enzyme or fragment thereof as described herein.

[0054] Proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, can be immobilized on the surface of a solid support by non-covalent or covalent bonds. Non-covalent bonds include electrostatic interactions such as pp (aromatic) interactions, van der Waals interactions, H-bond interactions, and ionic interactions. Preferably, proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are immobilized on the surface of a solid support by covalent bonds or by linker-mediated covalent bonds.

[0055] Solutions of proteins or their fragments typically contain proteins or their fragments in a buffer. Commonly usable buffers include phosphates, chlorides, citrates, MES, MOPS, HEPES, PIPES, ACES, or mixtures thereof. In addition, the solution may contain sugar alcohols or nonionic surfactants, as described herein. Solutions of proteins or their fragments can be prepared, for example, by dissolving the protein or its fragments in water to reconstitute a stock buffer for the protein or its fragments.

[0056] In one embodiment, the solid support is selected from the group consisting of organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, and titanium particles, preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size is usually measured by measuring the diameter of the particles and is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is monodisperse particles, its size is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. If the solid support is polydisperse particles, its size is typically from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, and particularly about 50 nm to 50 μm. In one embodiment, the composition comprises a solid carrier, the solid carrier comprising at least 4%, preferably at least 10%, more preferably at least 20%, even more preferably 4% to 50%, particularly 10% to 40%, more specifically 25% to 35%, and even more specifically 15% to 25% of immobilized protein or fragment thereof, based on the dry weight of the solid carrier.

[0057] Typically, 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.

[0058] Solid carriers are usually provided in suspension. The suspension of solid carriers can be done, for example, in water, a buffer, or a nonionic surfactant, or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. Nonionic surfactants are typically ethoxylated sorbitan esters such as PEG-40 sorbitan diisostearate, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407; and fatty acid ethoxylated esters such as PEG-5 oleate, PEG-8 stearate, polyoxyl stearate 40, and polyoxyl hydroxystearate 15. The material is selected from the group consisting of silates, fatty alcohol ethoxylates such as steareth 40; fatty acid esters such as ascorbyl palmitate, beeswax, polyglyceryl-3 oleate, propylene glycol monocaprylate, and propylene glycol monolaurate; fatty alcohols such as cetostearyl alcohol, cetyl alcohol, myristic alcohol, and stearyl alcohol; glycerides; pegylated triglycerides; and sugar esters, preferably polysorbates, more preferably polysorbate 80 (PS80).The buffers that can be used in the method of the present invention include phosphates, 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)propane-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3-(N,N-bis[2-hydroxy These are ethyl]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.

[0059] In one embodiment, a substance that interacts with the lid domain of lipase or a fragment thereof is added to a suspension of a solid carrier, preferably together with the lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof, before immobilizing the lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof onto the solid carrier. The lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof may be added to the suspension of a solid carrier in the form of pancreatin before immobilizing the lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof onto the solid carrier.

[0060] The immobilization of proteins or their fragments, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, onto solid carriers is typically carried out by adding a solution containing proteins or their fragments, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, or pancreatin or a solution thereof, to a suspension on a solid carrier. In a preferred embodiment, the immobilization of proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof, onto a solid carrier is carried out by providing a suspension of the solid carrier and a solution containing proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof, or pancreatin or a solution thereof, wherein the suspension of the solid carrier is incubated with the solution containing proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof, or pancreatin or a solution thereof, so that the proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof, can bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of a protein or fragment thereof, such as lipase, protease or fragment thereof, and amylase or fragment thereof, or pancreatin or a solution thereof, on a solid carrier is carried out by providing a suspension of the solid carrier, providing a solution containing a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, or pancreatin or a solution thereof, and providing a solution of a substance that interacts with the lid domain of the lipase or fragment thereof, wherein the suspension of the solid carrier is incubated with the solution containing a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, or pancreatin or a solution thereof, and with the solution of a substance that interacts with the lid domain of the lipase or fragment thereof, so that the protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, can bind to the surface of the solid carrier.

[0061] In a further preferred embodiment, the protein or fragment thereof is immobilized on the solid carrier by linkers, preferably bifunctional crosslinkers, that bind to the protein or fragment thereof, or to the fragment thereof, and to the surface of the solid carrier, preferably bifunctional crosslinkers, which are covalently bound to the protein or fragment thereof and to the surface of the solid carrier.

[0062] In one embodiment, the surface of a solid support is modified to introduce molecules or functional chemical groups as anchor points, i.e., as anchor points for proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or as anchor points for linkers connecting proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, to the solid support. Preferably, the anchor points are amine functional chemical groups or parts. In a non-limiting example, an amino-modified surface of a solid support, such as an amino-modified silica surface, can be used as a modified solid support. Such an amino-modified surface of a solid support can be obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Therefore, in a preferred embodiment, the solid support is a solid support having a silica surface having an amino-modified surface, and more preferably a solid support obtained by reacting a solid support having a silica surface with an aminosilane, such as APTES. Such modified carriers can form amide bonds between proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylase or fragments thereof, and amine groups on the surface of the carrier material, or between a linker and amine groups on the surface of the carrier material. In one embodiment, the molecules or functional chemical groups introduced as anchor points are uniformly distributed on the surface of the solid carrier.

[0063] In one embodiment, the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, a colipase mimetic peptide, and an amphiphilic molecule. Preferably, the substance that interacts with the lid domain of lipase or a fragment thereof is selected from the group consisting of colipase or a fragment thereof, a colipase mimetic peptide, and a bile salt; more preferably, it is selected from the group consisting of colipase or a fragment thereof, a colipase mimetic peptide, and sodium taurocholate; and even more preferably, it is selected from the group consisting of colipase or a fragment thereof, the colipase mimetic peptide shown in SEQ ID NO: 6, and sodium taurocholate. Most preferably, the substance that interacts with the lid domain of lipase or a fragment thereof is a bile salt, particularly sodium taurocholate.

[0064] In one embodiment, a substance that interacts with the lid domain of the lipase or fragment thereof specifically interacts with the lid domain of the lipase or fragment thereof such that the lipase or fragment thereof transitions to an open structure and / or maintains an open structure.

[0065] In one embodiment, about 50% to 100%, preferably about 80% to 100%, more preferably about 90% to 100%, and even more preferably about 100% of the lipase or fragment thereof immobilized on the surface of the solid carrier is in an open structure.

[0066] In some embodiments, the protective layer has a specified thickness of about 1 to about 200 nm, typically about 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, and particularly about 1 to about 15 nm. The most preferred specified thickness is about 1 to about 10 nm. In some embodiments, the layer has a specified 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, and particularly about 5 to about 15 nm. The most preferred specified thickness is about 5 to about 10 nm. The protective layer is usually porous, with a pore size of 1 to 100 nm, preferably 1 to 20 nm.

[0067] In one embodiment, a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, is partially embedded by a protective layer. In a preferred embodiment, a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, is at least partially embedded by a protective layer. In a more preferred embodiment, a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, is completely embedded by a protective layer.

[0068] In one embodiment, a solid carrier is embedded in the protective layer, and proteins or fragments thereof, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, immobilized on the surface of the solid carrier are embedded. In one embodiment, functional components immobilized on the surface of the protective layer are not embedded by the protective layer. Preferably, the solid carrier is completely embedded in the protective layer, and proteins or fragments thereof, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, immobilized on the surface of the solid carrier are completely embedded. More preferably, the solid carrier is completely embedded in the protective layer, and proteins or fragments thereof, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, immobilized on the surface of the solid carrier are completely embedded, and functional components immobilized on the surface of the protective layer are not embedded by the protective layer. When the solid carrier is completely embedded in the protective layer, and the proteins or fragments thereof immobilized on the surface of the solid carrier, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, are completely embedded, i.e., 100%, by the protective layer, i.e., the active sites are also covered, and the solid carrier is completely, i.e., 100%, covered by the protective layer.

[0069] In preferred embodiments, the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof used in the present invention are composed of pancreatin. In more preferred embodiments, pancreatin is used to immobilize the lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof on the surface of a solid carrier.

[0070] The thickness of the protective layer can be measured using a microscope such as a scanning electron microscope (SEM), transmission electron microscope (TEM), or scanning probe microscope (SPM), or by light scattering or ellipsometry.

[0071] The compositions of the present invention are typically produced in a reaction vessel such as a reactor. The formation of the protective layer is usually carried out by forming each protective layer with building blocks, which construct the protective layer through polycondensation reactions. Polycondensation can be carried out in different solvents, preferably aqueous solutions. Polycondensation can be easily controlled and stopped as needed, making it possible to achieve a specified thickness of the protective layer. The selection of building blocks that can be used to construct the protective layer may depend on the known structures of proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof, in order to adapt the affinity of the protective layer according to optimal and / or desired parameters. As building blocks for the protective layer, structural building blocks and protective building blocks for constructing the protective layer are typically used. A structural building block that can be used is, for example, tetraethyl orthosilicate (referred to herein as "TEOS" or "T"). The protective building blocks that can be used may be, for example, 3-aminopropyltriethoxysilane (referred to herein as "APTES" or "A"), propyltriethoxysilane (referred to herein as "PTES" or "P"), isobutyltriethoxysilane (referred to as "IBTES"), hydroxymethyltriethoxysilane (referred to herein as "HTMEOS" or "H"), benzyltriethoxysilane (referred to herein as "BTES"), ureidopropyltriethoxysilane (referred to as "UPTES"), or carboxyethyltriethoxysilane (referred to herein as "CETES"). The structural building blocks are typically precursors of inorganic silica and can form four covalent bonds in the resulting layer. The protective building blocks are typically organosilanes and have an organic moiety capable of interacting with proteins, such as lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof. Preferred structural building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes. More preferred structural building blocks areA mixture of tetravalent and trivalent silanes, particularly a mixture of tetra-alkoxysilane and tri-alkoxysilane. More preferred structural building blocks are selected from the group consisting of tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate. A more preferred protective building block is selected from the group consisting of carboxyethylsilanetriol, benzylsilane, propylsilane, isobutylsilane, n-octylsilane, hydroxysilane, bis(2-hydroxyethyl)-3-aminopropylsilane, aminopropylsilane, ureidopropylsilane, (N-acetylglycyl)-3-aminopropylsilane, and hydroxy(polyethyleneoxy)propyl]triethoxysilane, particularly benzyltriethoxysilane (BTES), propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, and (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimeth Selected from xysilane, isobutyltrimethoxysilane, n-octyltrimethoxysilane, hydroxyrunethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, ureidopropyltriethoxysilane, (N-acetylglycyl)-3-aminopropyltriethoxysilane, or benzyltrimethoxysilane, propyltrimethoxysilane, isobutyltrimethoxysilane, n-octyltrimethoxysilane, hydroxymethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureidopropyltrimethoxysilane, (N-acetylglycyl)-3-aminopropyltrimethoxysilane, or benzyltrihydroxyethoxysilane,Selected from propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane (hydroxymefilyltrihydroxyethoxysilane), bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, and ureidopropyltrihydroxyethoxysilane (N-acetylglycyl)-3-aminopropyltrihydroxymethoxysilane.

[0072] Particularly preferred building blocks are TEOS as structural building blocks and APTES, BTES, and / or HTMEOS as protective building blocks, preferably APTES and / or BTES. In particular, TEOS as structural building blocks and APTES and / or BTES as protective building blocks are used to construct the protective layer.

[0073] The reaction time between the building blocks and the solid support bearing the immobilized enzymes may depend on the length of the linker if one is used, and the size of the protein, such as lipase or its fragments, protease or its fragments, and amylase or its fragments. The reaction is usually carried out over a period of 0.5 to 10 hours, preferably 1 to 5 hours, more preferably 1 to 4 hours, and even more preferably 2 to 4 hours, preferably in an aqueous solution, preferably at room temperature of about 5 to about th°C or about 20°C. The formation of the protective layer can be stopped by actively halting the polycondensation reaction, for example, by removing unreacted building blocks through a washing step, or by the self-termination of the polycondensation reaction caused by a limited amount of building blocks.

[0074] In a more preferred embodiment, proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are immobilized on a solid carrier by at least partially modifying the surface of the solid carrier by introducing molecules as anchor points for proteins or fragments thereof, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, using linkers, preferably crosslinkers, that bind to anchor points and proteins, such as lipase or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, as described above.

[0075] In one embodiment, the molecules and / or linkers introduced as anchor points are uniformly distributed on the surface of the solid support.

[0076] Step (a) of this method is typically carried out by providing a solid carrier suspended in water, a buffer, a nonionic surfactant, or a mixture thereof, preferably suspended in water and / or a nonionic surfactant, more preferably suspended in water and / or a nonionic surfactant without a buffer in the suspension, even more preferably suspended in a mixture of water and a nonionic surfactant, particularly suspended in a mixture of water and a nonionic surfactant without a buffer in the suspension. Immobilization of a protein or fragment thereof onto the solid carrier in step (b) of this method is typically carried out by adding a solution of the protein or fragment thereof to the suspension of the solid carrier. Preferably, a linker connecting the solid carrier and the protein or fragment thereof is added to the suspension of the solid carrier before adding the solution of the protein or fragment thereof to the suspension of the solid carrier. In a preferred embodiment, immobilization of a protein or fragment thereof onto the solid carrier is carried out by providing a suspension of the solid carrier and adding a solution of the protein or fragment thereof, and the suspension to which the solution of the protein or fragment thereof has been added is incubated so that the protein can bind to the surface of the solid carrier. In a more preferred embodiment, the immobilization of the protein or fragment thereof to the solid carrier in step b) is carried out by i) adding a linker to a suspension containing the solid carrier provided in step (a), and ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, the linker connecting the solid carrier to the protein or fragment thereof, and preferably the suspension to which the solution of the protein or fragment thereof has been added is incubated so that the protein can bind to the surface of the solid carrier. In one embodiment, a building block for the protective layer, preferably a monomer of the building block for the protective layer, more preferably an organosilane, even more preferably a triethoxysilane, particularly APTES, is added to the suspension containing the solid carrier and the linker before the solution of the protein or fragment thereof is added. In a preferred embodiment, the surface of the solid carrier is at least partially modified to improve the immobilization of the protein or fragment thereof to the solid carrier. In particular, the surface of the solid carrier is at least partially modified before the protein or fragment thereof is immobilized.As described above, the surface of a solid support can be modified, at least partially, by adding molecules that serve as anchor points for proteins or their fragments to the surface of the solid support.

[0077] The suspension containing the solid support is typically incubated after each of the above addition steps so that the protein or fragment thereof is attached to the solid support, preferably the surface of the solid support, via a linker, preferably by covalent bond, thereby immobilizing the protein or fragment thereof on the solid support, for example, to allow reactions between the solid support and / or the molecule as an anchor point, the solid support and the linker, and the solid support containing the linker and the protein or fragment thereof.

[0078] In one embodiment of step (b), the protein or fragment thereof is immobilized on a solid carrier by linking the solid carrier to the protein or fragment thereof via a linker, and the solid carrier is connected to the protein or fragment thereof by covalent bonds between the linker and the solid carrier, and between the linker and the protein or fragment thereof. The linker preferably connects the surface of the solid carrier to the protein or fragment thereof by covalent bonds. More preferably, the linker is added to the suspension of the solid carrier in step (b) i) in a molar excess relative to the protein or fragment thereof added to the suspension containing the solid carrier and linker in step (b) ii), preferably, the linker is added to the suspension of the solid carrier in step (b) in a molar excess of 1 to 1000 times relative to the protein or fragment thereof added to the suspension containing the solid carrier and linker in step (b) ii), more preferably, the linker is added to the suspension of the solid carrier in step (b) in a molar excess of 2 to 300 times relative to the protein or fragment thereof added to the suspension containing the solid carrier and linker in step (b) ii), and even more preferably, the linker is added to the suspension of the solid carrier in step (b) in a molar excess of 4 to 250 times relative to the protein or fragment thereof added to the suspension containing the solid carrier and linker in step (b) ii).

[0079] In a preferred embodiment, linkers that did not connect the solid carrier to the protein or its fragment in step (b) are present while a protective layer is formed on the surface of the solid carrier in step (c). In a more preferred embodiment, linkers that did not connect the solid carrier to the protein or its fragment in step (b) are not removed in step (b) or step (c), or between steps (b) and (c). In a particular embodiment, linkers that did not connect the solid carrier to the protein or its fragment in step (b) are not removed in step (b) or step (c), or between steps (b) and (c), and the linkers or a portion thereof that did not connect the solid carrier to the protein or its fragment in step (b) covalently bond the protective layer to the protein or its fragment in step (c). After adding the protein in ii), the amount of linkers that did not connect the solid carrier to the protein or its fragment in step (b) is usually 30% to 70%, preferably 40% to 60%, and more preferably about 50% of the amount of linkers added to the solid carrier in step (b). In one embodiment, there is no washing step between step (b)(i), where the linker is added to the suspension of the solid carrier provided in step (a), and step (b)ii), where a solution of protein or its fragments is added to the suspension containing the solid carrier and the linker. In one embodiment, there is no washing step between any of steps (a) to (c). In one embodiment, there is no washing step between step (b)(i), where the linker is added to the suspension of the solid carrier provided in step (a), and step (b)ii), where a solution of protein or its fragments is added to the suspension containing the solid carrier and the linker, and there is no washing step between any of steps (a) to (c).

[0080] In preferred embodiments, the crosslinker may be glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl, sulfhydryl-reactive 2-pyridyldithiol, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) Selected from the group consisting of HCl), DST (disuccinimidyl tartrate), sulfo-LC-SMPT (4-sulfosuccinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoic acid), SPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl 6-(3-[2-pyridyldithio]-propionamide)hexanoic acid), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-(2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimerimidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryl (e.g., sulfhydryl-reactive 2-pyridyldithio), and a colipase-mimicking peptide, the colipase-mimicking peptide may be functionalized with a chemical group that enables covalent bonding to the surface of a solid support.In a more preferred embodiment, the crosslinker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2HCl), DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane). More preferably, the crosslinker is selected from glutaraldehyde, disuccinimidyl tartrate, disuccinimidyl suberate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated sulfhydrils (e.g., sulfurhydryl-reactive 2-pyridyldithio). Most preferably, glutaraldehyde is selected.

[0081] In one embodiment, after the protective layer is formed, a solid carrier containing proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, and the protective layer can be stored. Storage is usually achieved, for example, by washing the formed composition with a buffer and storing it suspended or dissolved in the buffer for a desired period of time. In a preferred embodiment, the solid carrier containing proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, and the protective layer, is stored at a constant temperature between 2 and 25°C. In a more preferred embodiment, the solid carrier containing proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, and the protective layer, is stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier containing proteins, such as lipase or its fragments, protease or its fragments, and amylase or its fragments, and the protective layer, is stored at a constant temperature between 2 and 25°C, preferably at room temperature, for 10 to 30 hours.

[0082] A preferred method of the present invention is a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally 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 containing at least one amino group and / or at least one thiol group, and the method comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspended state, preferably in a suspended state in water and / or a nonionic surfactant, and more preferably in a suspended state in a mixture of water and a nonionic surfactant. (b) A step of immobilizing a protein or fragment thereof onto a solid carrier, preferably, the surface of the solid carrier is at least partially modified before the protein or fragment thereof is immobilized onto the solid carrier, i) a linker is added to a suspension of the solid carrier, or i) a linker is added to a suspension of the solid carrier after at least partially modification of the surface of the solid carrier, and ii) a solution of the protein or fragment thereof is added to a suspension containing the solid carrier and the linker, the linker connecting the solid carrier and the protein or fragment thereof, (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker, or a part thereof, that did not connect the solid carrier and the protein or fragment thereof in step (b), covalently bonds the protective layer to the protein or fragment thereof, and optionally, (d) A step of immobilizing a functional component on the surface of a 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.

[0083] A more preferred method of the present invention is a method for producing a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment by embedding it, and optionally 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 comprising at least one amino group and / or at least one thiol group, and comprises the following steps: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspended state, preferably in a suspended state in water and / or a nonionic surfactant, and more preferably in a suspended state in a mixture of water and a nonionic surfactant. (b) A step of immobilizing a protein or fragment thereof onto a solid carrier, preferably, before the protein or fragment thereof is immobilized onto the solid carrier, the surface of the solid carrier is at least partially modified, i) a linker is added to a suspension of the solid carrier, or i) a linker is added to a suspension of the solid carrier after at least partially modification of the surface of the solid carrier, and ii) a solution of the protein or fragment thereof is added to a suspension containing the solid carrier and the linker, the linker connecting the solid carrier and the protein or fragment thereof, (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker or a part thereof that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment thereof.

[0084] In one embodiment, an optional functional component binds to mucus.

[0085] In one embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a polymer comprising repeating units, each containing at least one amino group.

[0086] In one embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a polymer comprising repeating units, each containing at least one thiol group.

[0087] In one embodiment, the polymer comprising repeating units, each containing 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 containing an amino group. In a preferred embodiment, the polymer comprising repeating units, each containing 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.

[0088] In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; and polymerized silanes containing an amino group, preferably polymerized APTES. In an even more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is polyglucosamine, preferably polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof, and more preferably chitosan or a derivative thereof.

[0089] The preferred polyglucosamine of the present invention is selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan, and dermatan or their derivatives. The most preferred is chitosan or its derivatives. The preferred polymerized silane-PEG-NH2 is selected from the group consisting of silane-PEG4-NH2, silane-PEG2000-NH2, and silane-PEG5000-NH2. The preferred polymerized silane containing an amino group is selected from the group consisting of APTES, amino-butyl-TES, amino-pentyl-TES, amino-hexyl-TES, amino-heptyl-TES, and amino-octyl-TES, with APTES being particularly preferred.

[0090] In further embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of polyglucosamine, polymerized silane-PEG-NH2, polymerized silane containing an amino group, polymerized silane containing a thiol group, polycarbophil-cysteine ​​conjugate, polymerized silane-PEG-thiol, and polycysteine. In more preferred further embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratan, and dermatan or derivatives thereof, and are selected from the group consisting of polyglucosamine; polymerized silane-PEG-NH2; polymerized silane containing a thiol group, preferably polymerized MPTS; polycarbophil-cysteine ​​conjugate; polymerized silane-PEG-thiol; and polycysteine. In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is a thiol-containing polyglucosamine or polymerized silane, 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 thiol-containing polymerized silane, polycarbophil-cysteine ​​conjugate, and polymerized silane-PEG-thiol, preferably a thiol-containing polymerized silane.

[0091] In certain embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polymerized silane-PEG-NH2, polymerized silane-PEG2000-NH2, polymerized silane-PEG5000-NH2, polymerized silanes containing amino groups, preferably polymerized APTES, and polymerized silanes containing thiol groups, preferably polymerized MPTS.

[0092] In one embodiment, the polymer comprising repeating units, each containing 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 silane containing an amino group, and polymerized silane containing a thiol group. In a preferred embodiment, the polymer comprising repeating units, each containing 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.

[0093] In a more preferred embodiment, the polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is selected from the group consisting of polyglucosamine selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratan and dermatan or derivatives thereof; polymerized silane-PEG-NH2; polymerized silane containing an amino group, preferably polymerized APTES; and polymerized silane containing a thiol group, preferably polymerized MPTS. In certain embodiments, polymers comprising repeating units, each containing at least one amino group and / or at least one thiol group, are selected from the group consisting of chitin, chitosan, polyglucosaminoglycans, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polymerized silane-PEG-NH2, polymerized silane-PEG2000-NH2, polymerized silane-PEG5000-NH2, polymerized silanes containing amino groups that are APTES, and polymerized silanes containing thiol groups that are MPTS.

[0094] In one embodiment, the polymer comprising repeating units, each containing at least one thiol group, is selected from the group consisting of polymerized silanes containing thiol groups, polycarbophil-cysteine ​​conjugates, polymerized silane-PEG-thiols, and polycysteine, preferably selected from the group consisting of polymerized silanes containing thiol groups, polycarbophil-cysteine ​​conjugates, and polymerized silane-PEG-thiols, more preferably polymerized silanes containing thiol groups, and most preferably polymerized MPTS. In one embodiment, the polymerized silane containing thiol groups is preferably polymerized MPTS.

[0095] In one embodiment, 5% to 100%, preferably 10% to 100%, and more preferably 50% to 100% of the surface of the protective layer is covered with a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group.

[0096] In one embodiment, an optional functional component is immobilized on the surface of the protective layer by bonding, preferably covalent bonding. In a preferred embodiment, an optional functional component is immobilized on the surface of the protective layer by non-covalent bonding, preferably by electrostatic interaction. In a more preferred embodiment, a polymer comprising repeating units, each containing at least one amino group and / or at least one thiol group, is immobilized on the surface of the protective layer by covalent bonding.

[0097] In one embodiment, an optional functional component is immobilized on the surface of a protective layer using a spacer that binds to the surface of the protective layer and the functional component. Thus, in one embodiment, the present invention comprises a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and optionally 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 comprising 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 polyethylene such as PEG4, PEG2000, and PEG5000. Functional components immobilized on the surface of the protective layer by a spacer are typically produced by first reacting the spacer and the functional component such that the spacer binds to the functional component, and then the functional component bound to the spacer reacts with the surface of the protective layer.

[0098] The selective immobilization of functional components onto the surface of the protective layer is typically carried out by suspending a solid support carrying a protein, such as an enzyme, embedded in the protective layer, in a reaction vessel such as a reactor, for example, in water, a buffer, or a nonionic surfactant or a mixture thereof, preferably in a mixture of water and a nonionic surfactant, as described above. Nonionic surfactants are typically ethoxylated sorbitan esters such as EG-40 diisostearate P-sorbitan, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 407; and fatty acid ethoxylated surfactants such as PEG-5 oleate, PEG-8 stearate, polyoxyl stearate 40, and polyoxyl hydroxystearate 15. The functional component is selected from the group consisting of silates, fatty alcohol ethoxylates such as steareth 40; fatty acid esters such as ascorbyl palmitate, beeswax, polyglyceryl-3 oleate, propylene glycol monocaprylate, and propylene glycol monolaurate; fatty alcohols such as cetostearyl alcohol, cetyl alcohol, myristic alcohol, and stearyl alcohol; glycerides; pegylated triglycerides; and sugar esters, preferably polysorbates, more preferably polysorbate 80 (PS80). 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. Typically, such compositions obtained are washed and resuspended in water, buffer, or a nonionic surfactant or a mixture thereof. Immobilization is achieved by non-covalent bonds, such as electrostatic bonds, or by covalent bonds of the functional component.Functional components can be immobilized by chemically modifying the surface of the protective layer and the functional components using "click chemistry" such as copper-catalyzed click chemistry (Copper-catalyzed azide-alkyne cycloaddition, see, e.g., Kolb et al. (2001) Angew. Chem. 40(11) 2004-2021), or by using click chemistry without copper (Wittig G, A Chem Ber, 1961, 94, 3260), for example, by first reacting a solid support carrying a protein, such as an enzyme, embedded in the protective layer as described above with a reactive compound such as an ethynyl compound, modifying the functional components by adding a reactive compound, such as an azide residue, and then reacting the two components to immobilize the functional components on the surface of the protective layer.

[0099] In a further embodiment, the present invention provides compositions described herein for use as pharmaceuticals.

[0100] In further embodiments, the present invention provides compositions described herein for use in methods of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in methods for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI). In preferred embodiments, the present invention provides compositions for use in methods for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI). In further preferred embodiments, the present invention provides compositions for use in methods of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy.

[0101] Furthermore, the use of the composition described herein for the manufacture of a medicament for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided. Furthermore, the use of the composition described herein for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided. Furthermore, a method for the prevention, delay of progression, or treatment of exocrine pancreatic insufficiency (EPI) in a subject is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject. Furthermore, the use of the composition described herein for the manufacture of a medicament for a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, is provided. Furthermore, the use of the composition described herein in a method of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject is provided. Furthermore, a method for enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in a subject is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject.

[0102] In further embodiments, the present invention provides compositions described herein for use in methods for the prevention, delay of progression, or treatment of phenylketonuria (PKU). The use of compositions described herein for the manufacture of pharmaceuticals for the prevention, delay of progression, or treatment of phenylketonuria (PKU) in subjects is also provided. Furthermore, the use of compositions described herein for the prevention, delay of progression, or treatment of phenylketonuria (PKU) in subjects is also provided. Furthermore, a method for the prevention, delay of progression, or treatment of phenylketonuria (PKU) in subjects is provided, comprising administering a therapeutically effective amount of the composition described herein to the subject. Preferably, when administered to a subject in the method of the present invention, the composition breaks down phenylalanine in the intestines of the subject.

[0103] In further embodiments, the present invention provides compositions described herein for use in methods for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance. In one embodiment, the present invention provides compositions for use in methods for the prevention, delay of progression, or treatment of lactase deficiency or sucrase-isomaltase deficiency. Lactase deficiency includes primary (hereditary) lactase deficiency, secondary (acquired) lactase deficiency, and congenital lactase deficiency, preferably secondary lactase deficiency. Sucrase-isomaltase deficiency includes, and is preferred, congenital sucrase-isomaltase deficiency (CSID). The use of compositions described herein for the manufacture of pharmaceuticals for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in subjects is also provided. Furthermore, the use of the compositions described herein for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in subjects is provided. Also provided are methods for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in subjects, comprising administering a therapeutically effective amount of the compositions described herein to the subjects.

[0104] The compositions according to the present invention are preferably pharmaceutical compositions comprising a therapeutically effective amount of the composition described herein and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical compositions according to the present invention are suitable for oral administration to a subject. Unless otherwise indicated, the pharmaceutical compositions according to the present invention are prepared by known methods.

[0105] The compositions of the present invention, such as pharmaceutical compositions, may be administered over a continuous period of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks, followed by a suspension of one week or part thereof, two weeks, three weeks, four weeks, five weeks, or six weeks. The compositions of the present invention, such as pharmaceutical compositions, can be conveniently administered in unit dose form. The unit of enzyme activity ("U") can be expressed as the weight or mass of substrate hydrolyzed per unit time.

[0106] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount of the composition of the present invention that is capable of producing one or more desired effects in a subject to which it is administered. Determining the therapeutic effective dose is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein.

[0107] As used herein, the terms “treatment” and “to treat” include: (1) delaying the onset of clinical symptoms of a condition, disorder, or pathology in animals, particularly mammals, particularly humans, that are suffering from or susceptible to a condition, disorder, or pathology but have not yet experienced or manifested clinical or subclinical symptoms of the condition, disorder, or pathology; (2) inhibiting a condition, disorder, or pathology (e.g., stopping, reducing, or delaying the onset of the disease with respect to at least one clinical or subclinical symptom, or stopping, reducing, or delaying its recurrence in the case of maintenance treatment); and / or (3) reducing a pathology (i.e., causing a regression of the condition, disorder, or pathology, or at least one of its clinical or subclinical symptoms). The benefit to the patient being treated is statistically significant or at least perceptible to the patient or physician. However, it will be understood that when a patient is given medicine to treat a disease, the result is not always an effective treatment.

[0108] As used herein, “delay in progression” means extending the time to the onset of symptoms or delaying the increase in the severity of symptoms. Furthermore, as used herein, “delay in progression” includes setback or inhibition of disease progression. “Inhibition” of disease progression or disease complications in a subject means preventing or mitigating disease progression and / or disease complications in a subject.

[0109] Preventive measures include prophylactic treatment. For preventive use, the drug combination of the present invention is administered to subjects suspected of having or at risk of developing the above-mentioned disease or disorder. For therapeutic use, the drug combination is administered to subjects, for example, patients already suffering from the above-mentioned disease or disorder, in an amount sufficient to cure or at least partially cessate the symptoms of the disease. The effective dose for such use will depend on the severity and course of the disease, previous treatments, the subject's health status and response to the drug, and the judgment of the attending physician.

[0110] If the target condition does not improve, the pharmaceutical combination of the present invention may be administered chronically, i.e., over a long period including the subject's entire life, to improve, or otherwise control or limit the symptoms of the target disease or condition.

[0111] If the patient's condition improves, the drug combination can be administered continuously; alternatively, the dose of the administered drug may be temporarily reduced or temporarily stopped over a specific period (i.e., a “drug-free period”). Once the patient’s condition improves, a maintenance dose of the drug combination of the present invention may be administered as needed. Thereafter, the dose, or the frequency of administration, or both, may be reduced as a function of the symptoms to a level at which the improved disease is maintained.

[0112] In a further embodiment, the present invention provides a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and optionally 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 comprising at least one amino group and / or at least one thiol group, and the composition can be obtained by the method described above.

[0113] In a further embodiment, the present invention provides a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and an optional 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 comprising at least one amino group and / or at least one thiol group, and the composition is obtainable by the method described above, provided that the protein or fragment thereof is not a lipase or fragment thereof, a protease or fragment thereof, an amylase or fragment thereof, pancreatin or a protein or fragment thereof composed of pancreatin, engineered phenylalanine ammonia lyase (PAL) or fragment thereof, and a disaccharide-degrading enzyme or fragment thereof, preferably, the protein or fragment thereof is not a lipase or fragment thereof, a protease or its Dynepen, an amylase or fragment thereof, pancreatin or a protein or fragment thereof composed of pancreatin, engineered phenylalanine ammonia lyase (PAL) or fragment thereof, and a disaccharide-degrading enzyme or fragment thereof as described herein.

[0114] The composition comprises a solid carrier, a protein or fragment thereof, a protective layer protecting the protein or fragment thereof, and an optional functional component, as described above. [Examples]

[0115] Phenylalanine ammonia lyase Materials and methods reagent: - 99% tetraethyl orthosilicate (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), polysorbate 80, acetic acid, bovine serum albumin (BSA), Tris buffer, L-phenylalanine, pancreatin, and pronase were purchased from Sigma-Aldrich. BSA, pancreatin, and pronase were dissolved in water to reconstitute the stock buffer. - Chitosan 95 / 500 P was purchased from Heppe Medical Chitosan GmbH. - Manipulated PAL (SEQ ID NO: 5) was supplied by Nestle Health Science at a concentration of 90 mg / mL in 25 mM sodium phosphate, 250 mM sodium chloride, 5% D-mannitol, 0.2% poloxamer 188, pH 7.5. - Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were purchased from the European Collection of Authenticated Cell Cultures (ECACC). - ThinCert TM The cell culture insert plates (1.0 μm membrane) were purchased from Greiner bio-one. - Fetal bovine serum, penicillin / streptomycin (10,000 U / ml penicillin / 10,000 μg / ml streptomycin), L-glutamine 200 mM (100x), Dulbecco phosphate-buffered saline (DPBS) (1x), 0.25% trypsin-EDTA (1x), DMEM, and white DMEM were purchased from Gibco. - Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, was purchased from Corning. - The animal feed Altromin 1324 was purchased from Altromin International. - I purchased the phenylalanine-free 5LF2 animal feed from LabDiet. - The catheter was purchased from Instech Laboratories.

[0116] Synthesis of silica nanoparticles (SNPs): Silica nanoparticles (50 nm) were synthesized according to the original Stoeber process as described in International Publication No. 2015 / 014888. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 hour. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm at 20°C for 22 hours. The solution was then centrifuged at 20,000 g for 20 minutes and washed sequentially with ethanol and water. Particle size was measured using SEM micrographs acquired at a magnification of 150,000x with image analysis software Olympus Stream Motion.

[0117] Generation of PAL-based silica nanoparticles NP-1: SNPs (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.9 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.9 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Processed PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized processed PAL using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20°C.

[0118] Generation of inert nanoparticles NP-2: SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA solution was added to achieve a final BSA concentration of 1.42 mg / mL, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized BSA using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a water bath at 20°C.

[0119] Generation of PAL-based silica nanoparticle NP-1 variants: The following experiments investigated the effects of covalently bonding enzymes to a protective layer on enzyme stability and activity.

[0120] In the initial experiment, PAL-based silica nanoparticles (NP-1(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L) was mixed with APTES (3.9 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. / Washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). The manipulated PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized manipulated PAL using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then subjected to H2O / The particles were washed three times in PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). NP-1(1) was cured overnight in a water bath at 20°C.

[0121] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888, and PAL-based silica nanoparticles (NP-1(2)) were generated in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 59 nm) and PS80 (8 mg / L) were added to phosphate buffer (25 mM, pH 7.5), to which APTES (3.9 mM) was added. The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The manipulated PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized manipulated PAL using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-1(2) was cured overnight in a water bath at 20°C.

[0122] In the third experiment, PAL-based silica nanoparticles (NP-1) were generated in H2O / PS80 (8 mg / L) according to the section titled "Generation of NP-1" above. The nanoparticles were not washed between each chemical step in order to retain the excess glutaraldehyde in the reaction mixture, which had not bonded to the manipulated PAL. Therefore, glutaraldehyde remained present during layer growth, causing covalent bonding between the protective layer and the manipulated PAL. This covalent bonding between the protective layer and the manipulated PAL can be observed by the appearance of a yellow / orange color with a maximum absorbance at 460 nm. This color is due to the formation of imine bonds by the reaction of the aldehyde group of the glutaraldehyde linker with the primary amine amino acids in the manipulated PAL and organosilica layers. The absorbance at 460 nm of PAL-based silica nanoparticles NP-1(1), NP-1(2), and NP-1 was measured after the formation of the organic silica layer and after the final particle washing, i.e., after the organic silica layer was formed and the particles were washed three times in H2O / PS80 and resuspended in H2O / PS80, as described in the section titled "Production of NP-1" in "Example 1". The immobilization and protection of phenylalanine ammonia lyase described above resulted in a much higher absorbance at 460 nm for NP-1 than for NP-1(1) and NP-1(2) (see Figure 8). NP-1(1) and NP-1(2) still show some absorbance at this wavelength, because imine bonds are formed even during enzyme immobilization. However, the absorbance of NP-1 is significantly higher than the others, indicating that further imine bonds were formed by covalent bonding between the protective layer and the manipulated PAL.

[0123] Activity assay of PAL-based silica nanoparticles NP-1: In a 96-well plate, NP-1 (20 μL, 1.75 mg / mL) in Tris buffer (0.1 M, pH 7.5) was mixed with L-Phe solution (180 μL, 50 mM). The pharmacokinetics of PAL were monitored with a spectrophotometer at 37°C for 30 minutes at λ = 290 nm.

[0124] Resilience to external stress: - Tolerance to acidic conditions Manipulated PAL or PAL-based silica nanoparticles NP-1 were incubated at pH 4 for 24 hours. Enzyme activity was evaluated at 0, 1, 3, 6, and 24 hours, as described in "NP-1 Activity Assay". - Resistance to proteases Modified PAL or PAL-based silica nanoparticles NP-1 were treated with pancreatin (30 mU) or pronase (0.8 U) and incubated at 37°C for 4 hours with shaking at 300 rpm. Enzyme activity was evaluated at 0, 0.1, 0.25, 0.5, 1, 2, and 4 hours, as described in "NP-1 Activity Assay".

[0125] Cell culture: In all experiments, cells were cultured at 37°C and 5% CO2.

[0126] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) cells were cultured in DMEM supplemented with 10% thermo-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin.

[0127] For the development of an intestinal barrier model, a Transwell PET insert (pore size 1 μm) was used to collect 2.6 × 10⁶ of material. 5 cells / cm 2 Cells were seeded at the following density. All cell models were used in the experiment on day 21. Caco-2 cells and HT-29-MTX-E12 cells were used in co-culture at a ratio of 75% to 25%.

[0128] Transepithelial electrical resistance Cell barrier integrity was evaluated by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). After refreshing the cell medium and treating it with nanoparticles, TEER was automatically measured every 15 minutes for up to 24 hours in the range of 1 Hz to 100,000 Hz.

[0129] Metabolism of Phe in vitro Differentiated Caco-2 / HT29-MTX-E12 cells were co-cultured in white DMEM (hereinafter referred to as "cell medium") supplemented with 1% thermo-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin.

[0130] The intestinal barrier was exposed to PAL-based silica nanoparticles NP-1 (9.7 mU) or modified PAL (9.7 mU) for 6 hours at the apical end of the barrier in the presence of pancreatin (30 mU). At each time point, a 150 μL aliquot was withdrawn from the basal end of the intestinal barrier and replaced with an equal volume of pre-warmed cell medium. The barrier was further incubated at 37°C. The absorbance of the withdrawn aliquot samples was measured at 290 nm to quantify the level of trans-cinnamic acid (TCA).

[0131] animal: All animal experiments were conducted under a license approved by the National Animal Testing Service of the Danish Ministry of Food, Agriculture and Fisheries. - Wisteria rat: This study was conducted on male Wistar rats (8 weeks old) belonging to the Janvier stock in France. 〇Food and drinking water: The rats were given free access to a pellet-type complete diet called "Altromin 1324." The animals also had free access to drinking water. Duodenal catheterization The animals were anesthetized with isoflurane (2-4%) in the induction chamber and then transferred to a nose cone containing isoflurane for surgery. The catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the duodenum to the mesenteric region, near the opening of the biliary and pancreatic ducts. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's neck. The abdominal and cervical incisions were then closed with sutures. The animals were kept warm throughout the procedure and carefully monitored until they had fully recovered from anesthesia. Evaluation of the effectiveness of PAL-based silica nanoparticles NP-1 in rats Prior to drug administration and d5-L-Phe administration, the animals were starved for 4 hours. Then, rats were administered NP-1 (0.85 U) or NP-2 (8.5 mg) intraduodenally, followed immediately by enteral nutrition of 3.6 mg of d5-L-Phe. The rats were housed in metabolic cages for 24 hours. ○ Urine collection, metabolic cage Urine was collected over 24 hours in a metabolic cage. Total excretion volume was obtained, and the urine was collected in Eppendorf tubes and stored at -80°C until shipment for analysis. Measurement of d5-hypric acid in rat urine: Quantification of the target analyte was performed using an LC system: Thermo Vanquish Horizon Binary Pump and mass spectrometer: Thermo Q Exactive. Urine samples were prepared as follows: 50 μL of urine sample was spiked into 5 μL of ISTD (100 μM 13C6-HIP, final concentration 2 μM in each sample), 200 μL of 100% methanol was added, and the mixture was vortexed. After being placed on ice for 20 minutes, the samples were centrifuged at 16000 g at 4°C for 10 minutes. The supernatant was transferred to a fully recovered MS glass vial and used for analysis. The injection volume used was 2.5 μL, and the runtime was 4.8 minutes at a flow rate of 1 mL / min. Mobile phase A was H2O and formic acid (0.1%), and mobile phase B was methanol and formic acid (0.1%). Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) (gradient: 10% B to 100% B). Mass spectrometry (MS) was performed using a Thermo Q Exactive mass spectrometer in DDA top5 capture mode. The MS parameters were as follows: MS1 resolution: 70,000 and MS2 resolution: 17,500. HCD fragmentation was performed at normalized stepwise collision energies of 10, 20, and 30. Data analysis was performed using Thermo quan Browser software.

[0132] - BTBR-Pahenu2 / J: This experiment was conducted on male and female stock BTBR- Pahenu 2 / J mice (8 weeks old) from the Jackson Laboratory in the United States. 〇Diet and drinking water: The mice were allowed to freely consume a phenylalanine-free diet (5LF2, LabDiet). The animals could drink drinking water freely. 〇Duodenal catheterization The animals were anesthetized with isoflurane (2-4%). The abdominal cavity was incised through a white line, and a catheter (C19PB-MGI1923, Instech Laboratories) was placed into the duodenum from the opposite side of the mesentery. The tip of the catheter was advanced and positioned near the opening of the bile and pancreatic ducts. The catheter was ligated to the intestinal wall, a subcutaneous tunnel was dug and exposed at the neck of the animal, and then closed. Subsequently, the incisions in the abdominal wall and neck were sutured closed. The animals were kept on a warming bed throughout the procedure and carefully monitored until they fully recovered from anesthesia. 〇Evaluation of the effectiveness of PAL-based silica nanoparticles NP-1 in mice Before use in the test, the mice were maintained on a phenylalanine-free diet for at least 3 days, then the drinking water was supplemented with low-concentration (0.03 g / L) L-Phe for 3 days, and then the L-Phe concentration in the drinking water was increased to 0.5 g / L. The mice could freely drink the L-Phe-supplemented drinking water at night. NP-1 (0.581 U, 7 mg), NP-2 (7 mg), or engineered PAL (0.581 U) was administered intraduodenally to the mice twice a day for 12 days. Blood samples were collected with EDTA on days 0, 4, 6, 8, 10, and 12. The blood samples were then centrifuged (10 minutes, 4 °C, 2000 × g), and at least 20 μL of plasma was transferred to an Eppendorf tube and stored at -80 °C until analysis of the Phe content. 〇Measurement of plasma Phe in mice: Quantification of the analyte of interest was performed using an LC system: Thermo Vanquish Horizon Binary Pump and a mass spectrometer: Thermo TSQ Quantiva.

[0133] Plasma samples were prepared as follows: 20 μL of plasma sample was centrifuged at 13.2 krpm at 4°C for 10 minutes. 10 microliters of the supernatant was added to 10 μL of ISTD (1000 μM D5-Phe) and 80 μL of 100% MeOH. The sample was then vortexed and centrifuged at 13.2 krpm at 4°C for 10 minutes, after which 50 μL of the supernatant was dried at 30°C under a gentle flow of nitrogen. 500 microliters of 0.1% (v / v) formic acid in water were added, and the sample was shaken at 900 rpm for 10 minutes at 15°C before centrifugation (13.2 krpm, 10 minutes, 4°C). Finally, 350 μL of the supernatant was transferred to a total recovery glass vial for analysis.

[0134] The injection volume used was 2 μL, and the runtime was 5 minutes at a flow rate of 1 mL / min. Mobile phase A was H2O and formic acid (0.1%), and mobile phase B was methanol and formic acid (0.1%). Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) (gradient: 100% A to 100% B).

[0135] MS was performed using a Thermo TSQ Quantiva mass spectrometer in capture mode: selective reaction monitoring.

[0136] The MS parameters were as follows: Q§1 resolution: 0.7; Q3 resolution: 0.7; Fragmentation: CID fragmentation in argon (1.5 mTorr). The analyte concentration was calculated from the peak area ratio of Phe and the internal standard d5-Phe. Data analysis was performed using Thermo Quan Browser software.

[0137] result: Example 1: Improvement of PAL stability by covalent bonding to the protective layer A series of three experiments were conducted to demonstrate that covalent bonding of a protective layer to the PAL surface via glutaraldehyde crosslinking further improves enzyme stability compared to immobilized proteins protected by an organosilica layer that interacts only electrostatically.

[0138] In the first experiment, PAL-based silica nanoparticles NP-1(1) were produced under unbuffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, PAL-based silica nanoparticles NP-1(2) were produced under buffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the third experiment, PAL-based silica nanoparticles NP-1 were produced under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0139] To determine the PAL immobilization yield on the surfaces of NP-1(1), NP-1(2), and NP-1, protein quantification was performed on the reaction supernatant. Surprisingly, the results showed that enzyme immobilization yield doubled under conditions where the presence of glutaraldehyde was maintained (NP-1) (Figure 2A), and consequently, the enzyme load per dry weight of SNP doubled compared to buffer conditions where glutaraldehyde was removed by washing (NP-1(2)) (Figure 2B). Similarly, enzyme immobilization under conditions where the presence of glutaraldehyde was maintained (NP-1) resulted in a 1.5-fold increase in the enzyme load per dry weight of SNP compared to buffer conditions where glutaraldehyde was removed by washing (NP-1(2)) (Figure 2B).

[0140] The biocatalytic activity of NP-1(1), NP-1(2), and PAL immobilized and protected on NP-1 was evaluated. Even more surprising than the increased load due to enzyme immobilization while maintaining the presence of glutaraldehyde was the threefold increase in nanoparticle specific activity compared to buffered conditions where glutaraldehyde is removed by a washing step, and the twofold increase compared to unbuffered conditions where glutaraldehyde is removed by a washing step (Figure 2C). This extremely surprising fact of a threefold increase in nanoparticle specific activity is closely related to the specific activity (units per gram of PAL) of the enzyme immobilized under unbuffered conditions where the presence of glutaraldehyde is maintained, which is comparable to the specific activity of the enzyme immobilized under buffered conditions. This result is completely unexpected, as the enzyme is thought to have significantly higher activity in the presence of a buffer (Figure 2D). In summary, covalent bonding of the protective layer to the PAL surface unexpectedly improves its load and stability compared to PAL protected by an organic silica layer via electrostatic interactions alone.

[0141] Example 2: Phenylalanine ammonia lyase (PAL) activity of PAL-based silica nanoparticles NP-1 The biocatalytic activity of immobilized, protected, and manipulated PAL was evaluated. The results shown in Figure 3 report PAL activity at NP-1. This demonstrates the ability to access and convert L-Phe of shielded, functionalized SNPs despite shielding and functionalization. Verification of biocatalytic activity on NP-1 confirms the potential for using nanoparticles for therapeutic purposes.

[0142] Example 3: Resistance to external stress PAL-based silica nanoparticles NP-1 are nanoparticles developed for use in the gastrointestinal tract. Due to the physiological characteristics of the gastrointestinal tract, NP-1 is subjected to various stresses. To ensure the sustained activity of NP-1 in the gastrointestinal tract, the protection of immobilized and engineered PAL was evaluated. First, NP-1 or engineered PAL was placed under acidic conditions (pH 4). Monitoring of PAL activity over 24 hours revealed that enzyme activity was sustained on NP-1 (Figure 4B), while the free form of engineered PAL lost its activity over time (Figure 4A). These data demonstrate that engineered PAL immobilized and protected on NP-1 is protected in an acidic environment.

[0143] During digestion, pancreatic enzymes, particularly proteases that can affect therapeutic enzymes in the gastrointestinal tract, are released. Therefore, NP-1 and engineered PAL were exposed to various proteases at 37°C. First, to mimic physiological digestive conditions, NP-1 or engineered PAL was incubated with pancreatin (30 mU), a mixture of pancreatic enzymes extracted from porcine pancreas, and then PAL activity was evaluated. After 4 hours, both NP-1 and engineered PAL exhibited sustained PAL activity (Figure 4C). Next, to further evaluate the benefits of the protective shield under harsh conditions, NP-1 or engineered PAL was exposed to pronase (0.88 U), a cocktail of purified proteases. The results shown in Figure 4C indicate that 80% of the PAL activity on NP-1 was maintained after 4 hours of exposure, while the enzymatic activity of engineered PAL was lost after 4 hours. In summary, these data demonstrate the added value of immobilizing and protecting manipulated PALs on nanoparticles, highlighting the potential of NP-1 for therapeutic applications of the gastrointestinal tract.

[0144] Example 4: In vitro biocompatibility and efficacy of PAL-based silica nanoparticles NP-1 Maintaining the integrity of the intestinal barrier is fundamental to preventing unwanted lumen contents, such as pathogens or food allergens, from entering the body. To evaluate the biocompatibility of nanoparticles, a monolayer of Caco2-HT29-MTX-E12 cells was exposed to NP-1 in the presence or absence of pancreatin to mimic digestive conditions, and transepithelial electrical resistance (TEER) was measured. The data shown in Figure 5A report that the integrity of the intestinal epithelial barrier was maintained after 6 hours of contact with NP-1, with or without pancreatin. This result demonstrates the in vitro biocompatibility of NP-1 for gastrointestinal application.

[0145] NP-1 was developed to metabolize Phe in the intestinal lumen. To evaluate the in vitro efficacy of NP-1, a monolayer of Caco2-HT29-MTX-E12 cells cultured in cell medium containing 0.4 mM L-Phe was exposed at its apical end to NP-1 (9.7 mU) or engineered PAL (9.7 mU) for 6 hours in or without pancreatin (30 mU). Quantification of Phe metabolites at the basal side of the barrier is shown in Figure 5B. The graph reports that TCA accumulated at the basal extrabasal side of the barrier under all conditions. This result demonstrates the in vitro efficacy of NP-1 in the digestive environment and suggests its use for therapeutic purposes.

[0146] Example 5: In vivo activity of PAL-based silica nanoparticles NP-1 in rats To evaluate the transition from in vitro to in vivo applications, the efficacy of NP-1 was assessed in rats. Rats were administered either NP-1 (nanoparticles containing engineered PAL) or NP-2 (nanoparticles containing bovine serum albumin (BSA) for which Phe is not a substrate) intraduodenally before enteral nutrition of d5-Phe. In vivo, TCA, a metabolite of Phe, is rapidly metabolized to hypric acid. The in vivo activity of NP-1 was then evaluated by quantifying d5-hypric acid in rat urine collected over 24 hours post-administration. As shown in Figure 6, the significant increase in d5-hypric acid in rats administered with NP-1 compared to NP-2 indicates that NP-1 has the ability to digest Phe within the intestinal lumen, suggesting the use of NP-1 for therapeutic purposes.

[0147] Example 6: In vivo therapeutic efficacy of PAL-based silica nanoparticles NP-1 in mice Phenylketonuria (PKU) is characterized by a deficiency in the intracellular hepatic enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the essential amino acid phenylalanine to tyrosine. In PAH deficiency, the concentration of phenylalanine, which is toxic to the brain, becomes abnormally high. The basis of PKU treatment is a low-phenylalanine diet combined with phenylalanine-free L-amino acids. Currently, enzyme replacement therapy using recombinant phenylalanine ammonia lyase is available via subcutaneous injection, but this treatment can induce hypersensitivity reactions and immune-mediated acute hypersensitivity reactions.

[0148] NP-1 was developed to exhibit sustained PAL activity in a gastrointestinal environment (acidic and exposed to proteases). Our method for controlling patients' Phe levels involves breaking down Phe in the intestines (from food intake) to avoid its absorption and accumulation in the blood.

[0149] This treatment strategy is based on BTBR-, a representative animal model of the disease in which the gene encoding PAH is mutated. Pahenu The evaluation was performed using a 2 / J mouse.

[0150] Mice that could ingest L-Phe supplemented drinking water were administered NP-1, NP-2, or engineered PAL twice daily for 12 days. Impressively, mice administered NP-1 showed a steady decrease in plasma Phe concentration throughout the study period, while mice administered engineered PAL showed unstable plasma concentrations (Figure 7). As shown in Figure 7B, normalization of plasma Phe concentrations revealed a 30% decrease in plasma Phe concentration in mice at the end of NP-1 treatment.

[0151] The interaction between nanoparticles and intestinal mucus leads to the transient engraftment of NP-1 and sustained PAL activity in the intestinal wall, while the free form of the manipulated PAL is washed away in the intestine. These results confirm and establish a strategy involving the degradation of Phe in the intestine, supporting the remarkable therapeutic efficacy of NP-1 against PKU.

[0152] Disaccharide-degrading enzyme Materials and methods reagent: - 99% tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), polysorbate 80, acetic acid, lactase (USP standard), invertase, bovine serum albumin (BSA), invertase activity assay kit, lactose, lipopolysaccharide (LPS), and phorbol 12-myristo 13-acetate (PMA) were purchased from Sigma-Aldrich. BSA, lactase, and invertase were dissolved in water to reconstitute the stock buffer. - Chitosan 95 / 500 P was purchased from Heppe Medical Chitosan GmbH. Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were purchased from the European Collection of Authenticated Cell Cultures (ECACC). - THP-1 (human acute monocytic leukemia cell line) was purchased from LGC. - ThinCert TM The cell culture insert plates (1.0 μm membrane) were purchased from Greiner bio-one. - Fetal bovine serum, penicillin / streptomycin (10,000 U / ml penicillin / 10,000 μg / ml streptomycin), MEM non-essential amino acids (100x), L-glutamine 200 mM (100x), Dulbecco phosphate-buffered saline (DPBS) (1x), 0.25% Trypsin-EDTA (1x), RPMI 1640 Medium, DMEM, HEPES, sodium pyruvate, D-glucose, and s-mercaptoethanol were purchased from Gibco. - Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, LDEV-free, was purchased from Corning. - Altromin 1319 and AIN93G modified 200g polysaccharides for animal feed were purchased from Altromin International. - The catheter was purchased from Instech Laboratories. - I purchased the TB100 Holtex blood glucose meter kit from MediSafe. - Bifidobacterium adolescentis-derived oligo-α-1,6-glucosidase 13A recombinant (isomaltase) was purchased from Creative Enzymes at a concentration of 1 mg / mL in 35 mM NaHepes buffer, pH 7.5, 750 mM NaCl, 200 mM imidazole, 3.5 mM CaCl2, 0.02% sodium azide, and 25% (v / v) glycerol.

[0153] Synthesis of silica nanoparticles: Silica nanoparticles (50 nm) were synthesized according to the original Stoeber process as described in International Publication No. 2015 / 014888. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 hour. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm at 20°C for 22 hours. The solution was then centrifuged at 20,000 g for 20 minutes and washed sequentially with ethanol and water. Particle size was measured using SEM micrographs acquired at a magnification of 150,000x with image analysis software Olympus Stream Motion.

[0154] Enzyme shielding and surface functionalization - Generation of lactase-based silica nanoparticles NP-2: SNPs (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.9 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.9 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Lactase (7 mg / mL, 0.1 mM) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized lactase using APTES (8.4 mM) and TEOS (125.9 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were washed three times in H2O / PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a water bath at 20°C.

[0155] - Lactase-based silica nanoparticle NP-2 variant: The following experiments investigated the effects of covalently bonding enzymes to a protective layer on enzyme stability and activity.

[0156] In the initial experiment, lactase-based silica nanoparticles (NP-2(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L) was mixed with APTES (3.3 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Then, glutaraldehyde (3.3 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Priming was performed by adding APTES (3.3 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Lactase (5.2 mg / mL, 0.1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized lactase using APTES (6.5 mM) and TEOS (93 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then removed from H2O / The sample was washed three times in PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). NP-2(1) was cured overnight in a 20°C water bath.

[0157] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888, and lactase-based silica nanoparticles (NP-2(2)) were generated in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 69 nm) and PS80 (8 mg / L) were added to phosphate buffer (25 mM, pH 7.5), to which APTES (3.3 mM) was added. The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Then, glutaraldehyde (3.3 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Priming was performed by adding APTES (3.3 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Lactase (5.2 mg / mL, 0.1 mM) was added, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized lactase using APTES (6.5 mM) and TEOS (93 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-2(2) was cured overnight in a 20°C water bath.

[0158] In the third experiment, lactase-based silica nanoparticles (NP-2) were produced in H2O / PS80 (8 mg / L) according to the section titled "NP-2 Production" above. The nanoparticles were not washed between each chemical step in order to retain the excess glutaraldehyde in the reaction mixture, which had not bound the solid support to lactase. Therefore, glutaraldehyde remained present during layer growth, causing covalent bond formation between the protective layer and lactase. The covalent bond between the protective layer and lactase can be observed by the appearance of a yellow / orange color with a maximum absorbance at 460 nm. This color is due to the formation of imine bonds by the reaction of the aldehyde group of the glutaraldehyde linker with the primary amine amino acids of the lactase and organosilica layer. The absorbance at 460 nm of lactase-based silica nanoparticles NP-2(1), NP-2(2), and NP-2 was determined after the formation of the organic silica layer and after the final particle washing, i.e., after the organic silica layer was formed and the particles were heated to H2O as described in the section titled "Formation of NP-2" in "Example 2". / Measurements were taken after washing three times in PS80 and resuspending in H2O / PS80: The immobilization and protection of the disaccharide-degrading enzymes described above indicates that the absorbance of NP-2 at 460 nm is higher than that of NP-2(1) and NP-2(2) (see Figure 15). NP-2(1) and NP-2(2) still show some absorbance at this wavelength, which is due to the formation of imine bonds during enzyme immobilization. However, the absorbance of NP-2 is significantly higher than the others, indicating that further imine bonds were formed by covalent bonding between the protective layer and lactase.

[0159] - Generation of invertase-based silica nanoparticles NP-3: SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Inverdase (1.726 mg / mL, 0.03 mM) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized inverdase using APTES (5.4 mM) and TEOS (81.3 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were washed three times in H2O / PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 115 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-3 was cured overnight in a water bath at 20°C.

[0160] - Generation of isomaltase-based silica nanoparticles NP-4: SNPs (10 mg / mL, 59 nm) were added to H2O / PS80 (8 mg / L) with APTES (3.6 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.6 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.6 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Isomaltase (3.55 mg / mL, 0.05 mM) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized inverdase using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were washed three times in H2O / PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 82 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). NP-4 was cured overnight in a water bath at 20°C.

[0161] - Generation of invertase / isomaltase-based silica nanoparticles NP-5: APTES (3.6 mM) was added to an SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.6 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.6 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Isomaltase (1.77 mg / mL, 0.025 mM) and invertase (4.05 mg / mL, 0.07 mM) were added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized enzyme using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were washed three times in H2O / PS80 (8 mg / L) by centrifugation at 20,000 rcf for 5 minutes, and then resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 82 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). NP-5 was cured overnight in a water bath at 20°C.

[0162] - Generation of inert nanoparticles NP-1: SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA solution was added to achieve a final BSA concentration of 1.42 mg / mL, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized BSA using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) (by centrifugation at 20,000 rcf for 5 minutes) and resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20°C. SNP-BSA-AT was cured overnight at 20°C.

[0163] Disaccharide-degrading enzyme activity assay: - Lactase activity assay A suspension of lactase-based silica nanoparticles NP-2 (30 μL, 2.3 mg / mL) in phosphate buffer (100 mM, pH 6.5) / MgCl2 (5 mM) was mixed with lactose solution (100 μL, 50 mg / mL). The reaction mixture was incubated in a thermomixer at 37°C and 750 rpm for 20 minutes. Samples were taken every 2.5 minutes, and glucose production was monitored using a blood glucose meter.

[0164] - Invertase activity assay The activity of invertase-based silica nanoparticles NP-3 was evaluated using Sigma's invertase assay kit. 1X reaction buffer (94 μL) was added to NP-3 (94 μL, 147 μg / L). Next, 1X sucrose solution (11.76 μL) was added. The reaction mixture was incubated in a thermomixer at 37°C and 300 rpm for 20 minutes. The sample was centrifuged at m20000 rcf for 5 minutes. The supernatant was collected, and 85 μL was transferred to a 96-well plate. 90 μL of master mix (prepared by mixing enzyme mix, dye reagent, and assay buffer) was added to each well. The reaction mixture was incubated in the dark at room temperature for 20 minutes. Absorbance was measured at λ = 570 nm.

[0165] - Isomaltase activity assay The activity of isomaltase-based silica nanoparticles NP-4 was evaluated using a blood glucose meter. An isomaltose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 minutes. Then, NP-4 (25 μL, 67 μg) in 50 mM phosphate buffer, pH 6.8 was added to the isomaltose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 minutes. Samples were collected at 2 minutes, 5 minutes, and 10 minutes, and glucose concentrations were determined using a blood glucose meter.

[0166] - Co-immobilized isomaltase-invertase activity assay The isomaltase activity of invertase / isomaltase-based silica nanoparticles NP-5 was evaluated using a blood glucose meter. An isomaltose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 minutes. Then, NP-5 (25 μL, 67 μg) in 50 mM phosphate buffer, pH 6.8 was added to the isomaltose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 minutes. Samples were collected at 2 minutes, 5 minutes, and 10 minutes, and glucose concentrations were determined using a blood glucose meter.

[0167] The invertase activity of the invertase / isomaltase silica nanoparticle NP-5 was evaluated using a blood glucose meter. A sucrose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 minutes. Then, NP-5 (25 μL, 67 μg) in 50 mM phosphate buffer, pH 6.8 was added to the sucrose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 minutes. Samples were collected at 2 minutes, 5 minutes, and 10 minutes, and glucose concentrations were determined using a blood glucose meter.

[0168] Cell culture: In all experiments, cells were cultured at 37°C and 5% CO2.

[0169] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) cells were cultured in DMEM supplemented with 10% thermo-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin.

[0170] THP-1 (human monocytic leukemia cell line) cells were cultured and maintained in RPMI1640 supplemented with 10% thermoactivated fetal bovine serum, 2 mM L-glutamine, and 100 U / mL penicillin / streptomycin.

[0171] To differentiate THP-1 into macrophages, THP-1 cells were cultured in the following differentiation medium: RPMI1640 containing 10% thermoinactivated fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2.5 g / L glucose, and 50 pM β-mercaptoethanol. THP-1 cells were differentiated into M0 macrophages by incubation with 150 nM phorbol 12-myrisstart 13-acetate (PMA) for 24 hours, followed by incubation in differentiation medium for 24 hours.

[0172] - Intestinal barrier model For the development of an intestinal barrier model, a Transwell PET insert (pore size 1 μm) was used to collect 2.6 × 10⁶ of material. 5 cells / cm 2 Cells were seeded at the following density. All cell models were used in the experiment on day 21. Caco-2 cells and HT-29-MTX-E12 cells were used in co-culture at a ratio of 75% to 25%.

[0173] - Immunodeficient intestinal barrier model To develop an immunodeficient intestinal barrier model, M0 differentiated THP-1 cells were added to the intestinal barrier model on day 21. Immune cells were attached to the posterior surface of a Transwell membrane containing a co-culture of pre-differentiated cells with a 25% Matrigel solution via a dropper method.

[0174] Transepithelial electrical resistance Cell barrier integrity was evaluated by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). After refreshing the cell medium and treating it with nanoparticles, TEER was automatically measured every 15 minutes for up to 24 hours in the range of 1 Hz to 100,000 Hz.

[0175] Intensive digestion of sucrose Differentiated Caco-2 / HT29-MTX-E12 cocultures in PBS were exposed to NP-3 (0.5 mU and 1 mU) in the presence of sucrose for 4 hours at the leading edge of the barrier. At each time point, 150 μL aliquots were withdrawn from the basal end of the intestinal barrier and replaced with the same volume of pre-warmed PBS. The barrier was further incubated at 37°C. The absorbance of the withdrawn aliquot samples was measured at 570 nm to quantify the glucose level.

[0176] animal: All animal experiments were conducted under a license approved by the National Animal Testing Service of the Danish Ministry of Food, Agriculture and Fisheries.

[0177] This study was conducted on male Wistar rats (8 weeks old) from Janvier, France.

[0178] - Food and drinking water: As a maintenance diet, the rats were given free access to a pellet-type complete feed called "Altromin 1319." The animals were also free to drink water.

[0179] From one week before treatment through the experimental period, rats were given ad libitum access to a low-sugar diet (AIN 93G modified 200g polysaccharides).

[0180] - Duodenal catheterization The animals were anesthetized with isoflurane (2-4%) in the induction chamber and then transferred to a nose cone containing isoflurane for surgery. The catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the mesenteric duodenum, near the biliary and pancreatic duct opening. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's neck. The abdominal and cervical incisions were then closed with sutures. The animals were kept warm throughout the procedure and carefully monitored until they had fully recovered from anesthesia.

[0181] - Medication and lactose administration Prior to drug administration and lactose administration, the animals were starved for 4 hours. Then, rats were administered intraduodenally with lactase-based silica nanoparticles NP-2 (97U), inactive nanoparticles NP-1 (54mg), or vehicle (1.5mL of NaCl 0.9%-polysorbate 80 8mg / mL), followed immediately by enteral nutrition of 3g of lactose. The rats received daily drug and enteral nutrition for 15 days.

[0182] - Cecal analysis After the experiment, all animals were scanned under complete anesthesia (scan time approximately 5 minutes) using a Bruker Pharmascan 7 Tesla with a rat volume coil to assess cecal size. A region of interest in the cecum was depicted in all acquired slices. MRI images were used to determine cecal volume.

[0183] During the autopsy, photographs of the digestive tract were taken to visualize the dilation of the cecum.

[0184] result: Example 1: Improvement of lactase loading by covalent bonding to the protective layer In the first experiment, lactase-based silica nanoparticles NP-2(1) were produced under unbuffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, lactase-based silica nanoparticles NP-2(2) were produced under buffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the third experiment, lactase-based silica nanoparticles NP-2 were produced under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0185] To determine the lactase immobilization yield on lactase-based silica nanoparticles NP-2(1), NP-2(2), and the surface of NP-2, lactase was quantified in the reaction supernatant. Surprisingly, the results showed that under conditions where the presence of glutaraldehyde was maintained (NP-2), the enzyme immobilization yield increased 26-fold (Figure 10A), and consequently, the enzyme load per dry weight of SNPs increased 24-fold compared to under buffer conditions where glutaraldehyde was removed by the washing step (NP-2(2)) (Figure 10B). Similarly, under conditions where the presence of glutaraldehyde was maintained (NP-2), the enzyme load per dry weight of SNPs doubled compared to under buffer conditions where glutaraldehyde was removed by the washing step (NP-2(2)) (Figure 10B).

[0186] In summary, the covalent bonding of the protective layer to the lactase surface unexpectedly improved its loading compared to lactase protected by an organosilica layer via electrostatic interactions alone.

[0187] Example 2: Disaccharide-degrading enzyme activity of lactase-based silica nanoparticles NP-2, invertase-based silica nanoparticles NP-3, isomaltase-based silica nanoparticles NP-4, and invertase / isomaltase-based silica nanoparticles NP-5 The biocatalytic activity of four immobilized and protected disaccharide-degrading enzymes was evaluated. Figure 11 shows the results reporting the enzymatic activity of each nanoparticle: lactase activity of lactase-based silica nanoparticle NP-2 (Figure 11A), invertase activity of invertase-based silica nanoparticle NP-3 (Figure 11B), isomaltase activity of isomaltase-based silica nanoparticle NP-4 (Figure 11C), and dual enzymatic activity (isomaltase and invertase) of invertase / isomaltase-based silica nanoparticle NP-5 (Figure 11D). These data indicate that disaccharides reach the catalytic site of the enzymes and are cleaved with high enzymatic activity. The verification of biocatalytic activity in NP-2, NP-3, NP-4, and NP-5 confirms the potential application of immobilization and protection strategies to a wide range of disaccharide-degrading enzymes for therapeutic purposes.

[0188] Example 3: Biocompatibility of nanoparticles for application to the gastrointestinal tract The intestinal mucosa consists of a single layer of epithelial cells tightly bound together by intercellular tight junctions, adjacent to a subepithelial region containing the lamina propria. The intestinal mucosa functions as a barrier between the external environment and the internal environment. Its integrity is a crucial parameter for ensuring the body's protection against undesirable contaminants such as microorganisms. The lamina propria contains diffuse lymphoid tissue composed of immune cells that maintain homeostasis or respond to the breakdown of epithelial protection.

[0189] To evaluate the biocompatibility of nanoparticles, the inventors developed representative models of lactase-based silica nanoparticles NP-2 and invertase-based silica nanoparticles NP-3. This model consists of inactive nanoparticles NP-1, which have the same functionalized outer surface as NP-2 and NP-3 but lack enzymatic activity.

[0190] To evaluate the safety of the nanoparticles, the inventors first focused on maintaining the integrity of the intestinal barrier in the presence of inactive nanoparticles NP-1 (Figure 12A). Transepithelial electrical resistance (TEER) measurements across a monolayer of Caco2-HT29-MTX-E12 cells showed that the integrity of the intestinal epithelial barrier was maintained even after 24 hours of contact with NP-1. This result demonstrates the in vitro biocompatibility of NP-1.

[0191] To further characterize the effects of nanoparticles on the intestinal barrier, the inventors evaluated the ability of NP-1 to induce an inflammatory response. For this purpose, the inventors developed an immunodeficient intestinal barrier model and monitored its integrity. Figure 12B shows that barrier integrity decreases in a dose-dependent manner when treated with LPS, an inflammatory component. This loss of integrity reveals the recruitment of macrophages from the basal to the apical side of the barrier.

[0192] Most importantly, TEER measurements showed that the integrity of the epithelial barrier was not compromised even in contact with NP-1 (Figure 12B). NP-1 did not stimulate macrophage recruitment at the apical end of the intestinal barrier, leading to the conclusion that NP-1 does not induce inflammation.

[0193] In summary, these results demonstrate the in vitro safety of nanoparticles for gastrointestinal application.

[0194] Example 4: In vivo efficacy of lactase-based silica nanoparticles NP-2 Lactose malabsorption is caused by an imbalance between the amount of lactose ingested and the ability of lactase to hydrolyze disaccharides. Lactose digestion and absorption occur in the small intestine. In cases of lactose malabsorption, undigested lactose reaches the large intestine and comes into contact with the gut microbiota. Bacterial fermentation of lactose leads to the production of short-chain fatty acids and gases, resulting in enlargement of the cecum in particular.

[0195] Rats administered high doses of lactose daily via enteral nutrition for 15 days showed increased substrate availability for fermentation in the large intestine and cecal enlargement compared to rats fed a normal diet without additional lactose (referred to as "vehicle" and "lactose-free" conditions, respectively) (Figure 13). Importantly, a significant reduction in cecal size was reported in rats administered lactase-based silica nanoparticles NP-2, while administration of inactive nanoparticles (NP-1) had no effect on cecal size reduction. These results demonstrate the in vivo biocatalytic activity of lactase-based silica nanoparticles NP-2 for lactose digestion.

[0196] Enzyme replacement therapy using exogenous lactase derived from microorganisms is available, but results regarding the precise efficacy rate are inconsistent (Montalto et al., World J Gastroenterol 2006, Jan 14;12(2):187-91). The rule used to calculate the amount of lactase is 7500 units per 16 grams of lactose. In addition to this close relationship between the amount of lactose hydrolyzed and the required enzyme units, gastric pH and bile salt concentration affect not only the effectiveness of exogenous lactase but also the loss of specific localization of the enzyme in the intestines.

[0197] Figure 13 demonstrates the in vivo efficacy of lactase-based silica nanoparticles NP-2 at a dose of 97 units per 3 g of lactose. Compared to the dosages of currently available lactase preparations, this single dose is a remarkable 14.5% lower. In other words, this set of data demonstrates the high value of NP-2 for lactose digestion and highlights the therapeutic potential of lactase-based silica nanoparticles NP-2 for patients with disaccharide digestion disorders, such as those with lactose malabsorption.

[0198] Example 5: In vitro efficacy of invertase-based silica nanoparticles NP-3 Congenital sucrase-isomaltase deficiency (CSID) is characterized by complete or near-complete absence of sucrose activity and varying degrees of reduced isomaltase activity.

[0199] The efficacy of invertase-based silica nanoparticles NP-3 in sucrose digestion was evaluated in an intestinal barrier model. A monolayer of differentiated Caco2-HT29-MTX-E12 cells was exposed to NP-3 for 4 hours at its apical end in the presence of its substrate. Figure 14 shows the quantification of sucrose hydrolysis products in the basal compartment of the barrier. This graph reports that glucose accumulates in a dose-dependent manner at the basal side of the barrier as the amount of invertase-based silica nanoparticles NP-3 increases, but glucose is not detected in the untreated intestinal barrier. This result demonstrates the in vitro efficacy of invertase-based silica nanoparticles NP-3 and clearly indicates the use of NP-3 for therapeutic purposes.

[0200] Lipase, amylase, and protease Materials and methods reagent: - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), Chelex® 100 sodium form, polysorbate 80, pancreatin (4x USP specification), bovine serum albumin (BSA), monobasic potassium phosphate, dibasic potassium phosphate, sodium taurocholate hydrate, acetic acid, olive oil, acacia gum arabic, sodium chloride, sodium hydroxide, Trizma base, hydrogen chloride, ammonium acetate, sodium acetate, pancreatin, bile salts, butanol, methanol, isopropanol, acetonitrile, NH4 acetate, amylase activity assay kit, 4% buffered formalin, Triton X-100 were purchased from Sigma-Aldrich. BSA and pancreatin were dissolved in water to reconstitute the stock buffer. - The p-SCN-Bn-DOTA was purchased from Macrocyclics. - Benzyltriethoxysilane (B, 96%) was purchased from abcr GmbH. - Chitosan 95 / 500 P was purchased from Heppe Medical Chitosan GmbH. - Indium chloride ( 111 -In) was purchased from Curium. - Animal feeds Altromin 1324 and Altromin 9033 were purchased from Altromine International. - The catheter was purchased from Instech Laboratories. - Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were purchased from the European Collection of Authenticated Cell Cultures (ECACC). - ThinCert TM The cell culture insert plates (1.0 μm membrane) were purchased from Greiner bio-one. - Fetal bovine serum, penicillin / streptomycin (10,000 U / ml penicillin / 10,000 μg / ml streptomycin), MEM non-essential amino acids (100x), L-glutamine 200 mM (100x), Dulbecco phosphate-buffered saline (DPBS) (1x), 0.25% Trypsin-EDTA (1x), RPMI 1640 Medium, and DMEM were purchased from Gibco. - The mounting medium, containing anti-zonula occludens 1 (ZO-1) antibody, goat anti-rabbit IgG conjugated to Alexa Fluor 488, and DAPI, was purchased from ThermoFischer Scientific.

[0201] Synthesis of silica nanoparticles (SNPs): Silica nanoparticles (50 nm) were synthesized according to the original Stoeber process as described in International Publication No. 2015 / 014888. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 hour. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm at 20°C for 22 hours. The solution was then centrifuged at 20,000 g for 20 minutes and washed sequentially with ethanol and water. Particle size was measured using SEM micrographs acquired at a magnification of 150,000x with image analysis software Olympus Stream Motion.

[0202] Enzyme shielding and protein shielding, and surface functionalization - Production of inert DOTA-labeled silica nanoparticles NP-1 functionalized with chitosan: SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Then, DOTA (3.8 mM) was added and the mixture was stirred at 50°C for 1 hour. The DOTA-labeled particles were washed three times by centrifugation in H2O / PS80 (8 mg / L, Chelex) at 20000 rcf for 20 minutes, resuspended in H2O / PS80 (8 mg / L, Chelex), and the tips were sonicated. APTES (3.8 mM) was added to the DOTA-labeled particles. The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Then, glutaraldehyde (3.8 mM) was added and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA solution was added to achieve a final BSA concentration of 1.42 mg / mL, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized BSA using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then subjected to H2O / The particles were washed three times in PS80 (8 mg / L) (by centrifugation at 20,000 rcf for 5 minutes) and resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20°C. SNP-BSA-AT was cured overnight at 20°C.

[0203] - Generation of non-functionalized NP-2 in inert DOTA-labeled silica nanoparticles: Enzyme immobilization and shielding were performed as described in International Publication No. 2015 / 014888. APTES (3.8 mM) was added to SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. DOTA (3.8 mM) was then added, and the mixture was incubated at 50°C for 1 hour. The DOTA-labeled particles were washed three times by centrifugation in H2O / PS80 (8 mg / L, Chelex) (20000 rcf for 20 minutes), resuspended in H2O / PS80 (8 mg / L, Chelex), and the tips were sonicated. APTES (3.8 mM) was added to the DOTA-labeled particles. The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Next, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA solution was added to achieve a final BSA concentration of 1.42 mg / mL, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized BSA using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a 20°C water bath.

[0204] - Generation of pancreatin-based silica nanoparticles NP-3: SNP (10 mg / mL, 55 nm) and PS80 (8 mg / L) were added to phosphate buffer (20 mM, pH 8), to which APTES (3.9 mM) was added. The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.9 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Sodium taurocholate (2 mM) and pancreatin (20 g / L) were added successively, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized pancreatin using APTES (7.7 mM), TEOS (40.4 mM), and benzyltriethoxysilane (35 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged (1000 rcf for 5 minutes), washed three times in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L) (centrifugation at 1000 rcf for 5 minutes), and resuspended in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 minutes.

[0205] The particles were centrifuged (at 1000 rcf for 5 minutes), washed three times in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L), and resuspended in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L) to obtain NP-3. NP-3 was cured overnight in a 20°C water bath.

[0206] - Generation of inert silica nanoparticles NP-4: SNP (10 mg / mL, 55 nm) and PS80 (8 mg / L) were added to phosphate buffer (20 mM, pH 8), to which APTES (3.9 mM) was added. The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.9 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.9 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. BSA (11.1 g / L) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized BSA using APTES (7.7 mM), TEOS (40.4 mM), and benzyltriethoxysilane (35 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were centrifuged (at 1000 rcf for 5 minutes), washed three times in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L) (by centrifugation at 1000 rcf for 5 minutes), and resuspended in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to achieve a final chitosan concentration of 121 μg / mL. The reaction mixture was incubated at 20°C and 400 rpm for 30 minutes.

[0207] The particles were centrifuged (at 1000 rcf for 5 minutes), washed three times in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L), and resuspended in phosphate buffer (0.120 M, pH 6) and PS80 (8 mg / L) to obtain NP-4. NP-4 was cured overnight in a 20°C water bath.

[0208] - Generation of pancreatin-based silica nanoparticles NP-5: APTES (3.7 mM) was added to SNP (10 mg / mL, 58 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 minutes. Then, glutaraldehyde (3.7 mM) was added and the reaction mixture was stirred at 20 °C and 400 rpm for 10 minutes. Priming was carried out by adding APTES (3.7 mM) and stirring the reaction mixture at 20 °C and 400 rpm for 10 minutes. Pancreatin (23.5 mg / mL) containing lipase and / or its fragment, protease and / or its fragment, and amylase and / or its fragment was added and the reaction mixture was reacted at 20 °C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized lactase using APTES (7.2 mM) and TEOS (75.6 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 2 hours. The particles were washed 3 times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 minutes) and resuspended in H2O / PS80 (8 mg / L). A solution of chitosan in acetic acid (0.1 M) was added to the particle suspension to a final chitosan concentration of 103 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 minutes. The particles were centrifuged at 20000 rcf for 5 minutes and washed 3 times in H2O / PS80 (8 mg / L). NP-5 was cured overnight in a water bath at 20 °C.

[0209] Generation of pancreatin-based silica nanoparticle NP-3 variant: In the following experiments, the effects of covalently bonding an enzyme to a protective layer on enzyme stability and enzyme activity were investigated, respectively.

[0210] In the first comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888 to generate pancreatin-based silica nanoparticles (NP-3(2)) in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 69 nm) and PS80 (8 mg / L) were added to phosphate buffer (25 mM, pH 7.5), to which APTES (3.1 mM) was added. The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Then, glutaraldehyde (3.1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Sodium taurocholate (2 mM) and pancreatin (15 g / L) were added successively, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of immobilized pancreatin using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-3(1) was cured overnight in a water bath at 20°C.

[0211] In the second experiment, pancreatin-based silica nanoparticles (NP-3) were generated in phosphate buffer (20 mM, pH 8), PS80 (8 mg / L) according to the section entitled "Generation of NP-3" in "Example 3" above. To keep an excess amount of glutaraldehyde that did not bind to pancreatin in the reaction mixture, the nanoparticles were not washed between each chemical step. Thus, glutaraldehyde was still present during layer growth and caused a covalent bond between the protective layer and pancreatin. The covalent bond between the protective layer and pancreatin can be observed by the appearance of a yellow / orange color with a maximum absorbance at 460 nm. This color is due to the formation of an imine bond by the reaction of the aldehyde group of the glutaraldehyde linker with the primary amine of the amino acids of pancreatin and the organic silica layer. When the absorbance of pancreatin-based silica nanoparticles NP-3(1) and NP-3 at a wavelength of 460 nm was measured after organic silica layer formation and final particle washing (see Figure 24), it was shown that the absorbance of NP-3 at 460 nm was much higher than that of NP-3(1). NP-3(1) still shows a certain degree of absorbance at this wavelength because an imine bond is also formed during enzyme immobilization. However, the absorbance of NP-3 is significantly higher than others, indicating that the further formation of imine bonds was caused by the covalent bond between the protective layer and pancreatin.

[0212] Indium 111( 111 In) radiolabeling: For nanoparticle labeling, an additional step was added to the nanoparticle process described in the sections entitled "Enzyme Shield and Protein Shield, and Surface Functionalization", "Generation of Inactive DOTA-Labeled Silica Nanoparticles Non-Functionalized NP-2", and "Generation of Chitosan-Functionalized Inactive DOTA-Labeled Silica Nanoparticles NP-1".

[0213] The nanoparticles were labeled at a specific activity of 500 - 400 MBq / g nanoparticles according to the following protocol.

[0214] All buffers used were preprocessed with Chelex®.

[0215] Nanoparticles, 111 The nanoparticles were incubated with Lu (0.02 M HCl) and ammonium acetate (1 M, pH 5.4) under continuous stirring at 45°C for 12 hours. The nanoparticles were centrifuged at 5000 g for 5 minutes and resuspended in sodium acetate (20 mM, pH 5) containing polysorbate 80 (8 mg / L). The nanoparticles were then resuspended in DTPA (1 mM, pH 5) and incubated overnight at room temperature (RT) for quenching. The nanoparticles were then washed and resuspended in 0.9% sodium chloride containing polysorbate 80 (8 mg / L).

[0216] Pancreatic lipase activity assay: The olive oil solution was prepared by mixing olive oil / gum arabic / water (1 / 8.25 / 0.75). The buffer solution was prepared by dissolving Trizma base (0.6 g / L) and sodium chloride (2.34 g / L) in nanopure water. The bile salt solution was prepared by dissolving sodium taurocholate (80 g / L) in water.

[0217] This activity assay was performed by mixing olive oil solution (13.8 mL), buffer solution (11 mL), bile salt solution (2.8 mL), and water (12.4 mL) in a bioreactor at 37°C. The pH was adjusted to 9.2 by adding aqueous sodium hydroxide solution (0.1 M). NP-3 (1.4 mL, 10 mg / mL) was washed twice with water and added to the bioreactor. The kinetics of the lipase were monitored for 10 minutes by measuring the amount of sodium hydroxide added to the reaction mixture to maintain the pH at 9.

[0218] Pancreatic protease activity assay: An enzyme sample (50 μL, 0.2 mg / mL) in phosphate buffer (50 mM, pH 7.4) was mixed with casein solution (250 μL, 0.65% w / v). The reaction mixture was incubated at 37°C and 750 rpm for 30 minutes. The sample was centrifuged at 20000 rcf for 5 minutes, and the supernatant was collected. TCA (166.7 μL) was added to the supernatant (200 μL). The sample was incubated at 37°C and 750 rpm for 30 minutes. The sample was centrifuged at 20000 rcf for 5 minutes, and the supernatant was collected. Na2CO3 was added to the supernatant (200 μL). 3( 500 μL (500 mM) and Folin reagent (100 μL, 0.5 M) were added. The sample was incubated at 37°C and 750 rpm for 30 minutes. The absorbance of 200 μL of the resulting solution was measured at 660 nm.

[0219] Pancreatic amylase activity assay: The activity of pancreatin-based silica nanoparticle NP-3 amylase was evaluated using Sigma's amylase assay kit. In a 96-well plate, the enzyme sample (2 μL, 18.2 mg / mL) was mixed with activity buffer (48 μL). Amylase substrate solution (100 μL) was added to the wells, and the kinetics of the sample were monitored with a spectrophotometer at 37°C for 30 minutes at λ=405 nm.

[0220] animal: All animal experiments were conducted under a license approved by the National Animal Testing Service of the Danish Ministry of Food, Agriculture and Fisheries.

[0221] - Rat: This study was conducted on male Wistar rats (8 weeks old) from Janvier, France. 〇Food and drinking water: The rats had free access to the pellet-type complete feed "Altromin 1324." The animals also had free access to drinking water. 〇 Pancreatic duct ligation (PDL) and duodenal catheterization: The animals were anesthetized with isoflurane (2-4%) in an induction chamber and then transferred to a nose cone containing isoflurane for surgery. The rats were placed supine on a heated table, and the abdomen was incised along the midline. The pancreas was located and gently moved to locate the biliary and pancreatic ducts. The pancreatic tissue surrounding the biliary and pancreatic ducts was bluntly dissected to visualize the ducts. The area near the biliary and pancreatic ducts was ligated to stop the flow of pancreatic enzymes. After ligating the pancreatic ducts, a catheter (C30PU-RDD1444, Instech Laboratories) was placed in the duodenum anticortex near the opening of the biliary and pancreatic ducts. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's neck. The abdominal and neck incisions were then closed with sutures. Evaluation of the effectiveness of pancreatin-based silica nanoparticles NP-3 PDL rats were administered inactive nanoparticles NP-4 (7 mg) or pancreatin-based silica nanoparticles NP-3 (7 mg) via oral enteral nutrition through the duodenum. Approximately 150 μL of blood was collected by EDTA at 0.25, 0.5, 1, 1.5, 2, 4, and 6 hours after Triolein administration. Blood samples were centrifuged (10 minutes, 4°C, 2000×g), and at least 50 μL of plasma was transferred to an Eppendorf tube and stored at -80°C until triglyceride content analysis. Measurement of plasma triglycerides in rats: Quantification of the target analyte was performed using an LC system: Thermo Vanquish Horizon Binary Pump and mass spectrometer: Thermo Q Exactive. Plasma samples were prepared according to the BUME method. 10 microliters of plasma sample were mixed with 300 μL of 1-butanol / methanol (3:1, v / v). The sample was incubated with agitation (900 rpm) at 20°C for 1 hour. After centrifugation (16,000 g, 10 min, 20°C), 50 μL was transferred to a glass vial and used for LC-MS.

[0222] The injection volume used was 2.5 uL, and the runtime at a flow rate of 1 mL / min was 7.5 minutes. Mobile phase A was 60% acetonitrile, 40% H2O, 5 mM NH4 acetate, and mobile phase B was 90% isopropanol, 10% acetonitrile, 5 mM NH4 acetate. Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) (gradient: 15% B to 99% B).

[0223] MS was performed by using a Thermo Q Exactive mass spectrometer in the acquisition mode DDA top5. The MS parameters were as follows: MS1 resolution: 70,000 and MS2 resolution: 17,500. HCD fragmentation was performed at normalized stepped collision energies of 10, 20, and 30. The extraction amounts targeted for EIC were TG 54:3 for triolein and the most abundant triglyceride. Data analysis was performed using Thermo quan Browser software.

[0224] - Göttingen minipigs: This study was performed on female Göttingen minipigs from the stock of Ellegaard Goettingen Minipigs A / S in Denmark. 〇Diet and drinking water: The minipigs were regularly fed with the pelleted complete feed "Altromin 9033" at an allotment of approximately 250 g per day. To evaluate the efficacy of NP-3 (chronic administration), the diet was changed to a high-fat diet, and the daily allotment of Altromin 9033 was supplemented with olive oil (olive oil: Altromin = 1:10) and 100 g of apple sauce. The high-fat diet was started 15 days before the start of administration and maintained during the chronic administration of the treatment.

[0225] The animals had free access to good-quality drinking water for household use.

[0226] 〇SPECT / CT imaging SPECT / CT scans (Clinical D670 SPECT / CT, GE) were initiated 15 minutes, 3, 8, 24, 48, and 72 hours (±1 / 2 hours) after administration of In-111-labeled nanoparticles. SPECT / CT scans were not initiated in control animals, and were initiated 15 minutes, 3, 8, and 24 hours after administration of free In-111. SPECT acquisition times were determined based on the counting rate on the scan day. SPECT imaging included two fields of view (FOV) covering the area from the stomach to the rectum. Both FOVs were scanned, with the FOV containing the stomach and duodenum being the first acquisition. Prior to the CT scan procedure, intravenous infusion of iodine-containing contrast agent (Ultravist®, 370 mg / mL, 1 mL / kg, flow rate 2 mL / s) was performed to improve organ visibility for image analysis. The FOV of the CT imaging included the entire animal. The animals were photographed on the spot and then transported from the enclosure to the scanner under anesthesia. To quantify the uptake of In-111-labeled compounds, regions of interest were defined in the relevant areas of the gastrointestinal organs (three compartments: small intestine, colon, and rectum) identified from CT image data. Uptake is expressed as %ID (percentage injected) and SUV (standardized uptake value). All data analysis was performed by a single viewer to avoid intra-observational deviation.

[0227] 〇 Pancreatic duct ligation (PDL) and duodenal catheterization: In the animal lab, the animals were premedicated with a Zoletil mixture (6.25 mL of Rompun Vet (xylazine, 20 mg / mL) + 1.25 mL of Ketaminol Vet (ketamine, 100 mg / mL) + 2.5 mL of Torbugesic Vet (butorphanol, 10 mg / mL)) from one vial of Zoletil Vet50 (125 mg of tyletamine + 125 mg of zolazepam) to be administered via IM / IV injection. After sedation, the pigs were intubated and anesthesia was maintained with inhalation of 2-4% isoflurane. An ear vein catheter was placed on one side. The animals were placed in a left lateral decubitus position, and the right flank was prepared for surgery according to standard procedure. An incision was made in the right flank, and the location of the duodenum and pancreas was gently confirmed. Two ligatures were placed around the pancreatic duct at intervals of 2-3 mm, and the pancreatic duct was incised between the ligatures to stop the flow of pancreatic enzymes. After ligating the pancreatic duct, a catheter (Dog duodenal catheter 7F, SAI Infusion Technologies) was placed in the duodenum on the antiintestinal side, near the opening of the biliary and pancreatic ducts. The catheter was ligated to the intestinal wall and exposed by creating a subcutaneous tunnel in the animal's back. The flank incision was then sutured. A protective bandage was applied to the abdomen.

[0228] Pre- and post-operative painkillers and antibiotics Animals received prophylactic analgesia in the form of intramuscular NSAIDs prior to surgery. Postoperative analgesia consisted of oral NSAIDs once daily for four days, with the addition of low-dose transdermal opioid patches for 72 hours as needed. The antibiotic (amoxicillin) was administered intramuscularly before the surgery, and the treatment was completed with once-daily oral administration for four days after the surgery.

[0229] Evaluation of the effectiveness of pancreatin-based silica nanoparticles NP-3 ■Single dose: PDL miniature pigs were administered either inactive nanoparticles NP-4 (1 g) or pancreatin-based silica nanoparticles NP-3 (1 g) via an intraduodenal catheter, followed by administration of olive oil (14 g) through the same catheter. Following the administration of olive oil, 5 mL of blood was collected by EDTA at 0, 0.0833, 0.25, 0.5, 1, 2, 3, 4, and 6 hours. Blood samples were centrifuged (10 minutes, 4°C, 2000×g), and at least 500uL of plasma was transferred to an Eppendorf tube and stored at -80°C until triglyceride content analysis. ■Chronic administration: PDL miniature pigs were administered either inactive nanoparticles NP-4 (1g) or pancreatin-based silica nanoparticles NP-3 (1g) twice daily via a duodenal catheter, followed by a diet for 10 days. Fecal samples from individual animals were collected over a 72-hour period before treatment (days -3, -2, and -1) and at the end of treatment (days 8, 9, and 10). Fecal samples were stored at -20°C until analysis.

[0230] Measurement of plasma triglycerides in miniature pigs: The quantification of triglycerides in miniature pig plasma was evaluated using the Konelab system according to the supplier's specifications.

[0231] Measurement of fat content in the feces of miniature pigs Fecal fat content was quantified by near-infrared spectroscopy (NIR) using an Impana FT 9700 (Perkin Elmer). For NIR (700-2500 nm) analysis, stool samples were weighed, homogenized, and placed in open glass petri dishes. Measurements were taken at three different spots for each sample. The results were expressed as the average fat concentration (grams of fat per 100 g of wet stool) from the three spot samples.

[0232] Cell culture: In all experiments, cells were cultured at 37°C and 5% CO2.

[0233] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) cells were cultured in DMEM supplemented with 10% thermo-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids, and 100 U / mL penicillin / streptomycin.

[0234] For the development of an intestinal barrier model, a Transwell PET insert (pore size 1 μm) was used to collect 2.6 × 10⁶ of material. 5 cells / cm 2 Cells were seeded at the following density. All cell models were used in the experiment on day 21. Caco-2 cells and HT-29-MTX-E12 cells were used in co-culture at a ratio of 75% to 25%.

[0235] Transepithelial electrical resistance Cell barrier integrity was evaluated by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). After refreshing the cell medium and treating it with nanoparticles, TEER was automatically measured every 15 minutes for up to 24 hours in the range of 1 Hz to 100,000 Hz.

[0236] Confocal microscope Cells on the insert were washed with phosphate-buffered saline and fixed with 4% buffered formalin. The fixed cells were permeabilized in PBS solution containing 1% Triton-X 100 (reference). Specific binding was blocked using PBS solution containing 3% bovine serum albumin (BSA). The cells were incubated with primary antibody against zonula occludens 1 (ZO-1; 1:100) at RT for 2 hours. After washing, the cells were incubated with the corresponding fluorescently labeled secondary antibody (goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1000)) at RT in the dark for 2 hours. For imaging, the membrane was cut from the insert and placed between two coverslips loaded with mounting medium containing DAPI, and imaged using a confocal laser scanning microscope (FluoView, FV3000, Olympus, Tokyo, Japan).

[0237] result: Example 1: In vivo distribution of chitosan-functionalized nanoparticles in miniature pigs To evaluate the advantage of chitosan-functionalized nanoparticles in specifically retaining in the digestive tract of miniature pigs via mucous binding, inactive DOTA-labeled silica nanoparticles (NP-2) and chitosan-functionalized inactive DOTA-labeled silica nanoparticles (NP-1) were administered endoscopically into the duodenum of miniature pigs. SPECT / CT images were acquired at different time points (15 minutes to 24 hours), and the in vivo distribution of nanoparticles in the digestive tract was analyzed using a three-compartment analysis including the small intestine, colon, and rectal portion of the colon. Quantification of NP-2 and NP-1 in the small intestine is shown in Figure 17A. Comparing the in vivo distribution data obtained from SPECT / CT of animals administered NP-1 and NP-2, it was shown that NP-1 remained in the small intestine 2.7 times longer than the unfunctionalized nanoparticles (AUC: 170.7 vs. 460.9; NP-2 vs. NP-1, respectively) (Figure 17B). These results demonstrate that NP-1 is specifically retained in the digestive tract in vivo. In summary, these in vivo data suggest that surface functionalization of chitosan-shielded nanoparticles may enable specific targeting of intestinal mucus, potentially allowing the nanoparticles to temporarily implant in the intestinal wall.

[0238] Example 2: Biocatalytic activity of pancreatin-based silica nanoparticles NP-3 and NP-5 Exocrine pancreatic insufficiency is a condition in which the exocrine function of the pancreas is impaired, resulting in a deficiency in its ability to effectively deliver digestive enzymes to the duodenum. The standard medical treatment for clinical symptoms and malabsorption is oral pancreatic enzyme replacement therapy (PERT). Currently approved treatments consist of porcine-derived pancreatic enzyme products (lipase, amylase, and protease).

[0239] The pancreatin-based silica nanoparticles NP-3 and NP-5 were evaluated for their enzymatic activity. The lipase and amylase enzymatic activity of NP-3 is shown in Figures 18A and 18C. The protease enzymatic activity of NP-5 is shown in Figure 18B.

[0240] These data demonstrate the ability to immobilize multiple enzymes onto nanoparticles while maintaining the complete activity of each enzyme. Verification of these biocatalytic activities in nanoparticles demonstrates the potential for applying strategies for enzyme immobilization and protection to restore digestive function, which may be useful for therapeutic purposes.

[0241] Example 3: In vivo activity of pancreatin-based silica nanoparticles NP-3 in PDL rats Considering the importance of lipid digestion for patients with exocrine pancreatic insufficiency (EPI), we evaluated the validity of pancreatin-based silica nanoparticles NP-3 by focusing on their in vivo lipid digestive capacity, using PDL-rats as the first animal model. Before administering triolein enterally to PDL-rats, either pancreatin-based silica nanoparticles NP-3 or inactive nanoparticles NP-4 were administered intraduodenally (hereinafter referred to as the administration sequence). After the single-dose administration sequence, PDL-rats administered with NP-3 (active nanoparticles) showed an increase in plasma TG compared to PDL-rats administered with inactive nanoparticles (NP-4) (Figure 19). This result demonstrates the ability of pancreatin-based silica nanoparticles NP-3 to restore lipase digestive function in an EPI animal model.

[0242] Example 4: In vivo activity of pancreatin-based silica nanoparticles NP-3 in PDL-miniature pigs From an anatomical and physiological standpoint, it is widely recognized that pig models share a high degree of similarity with the human gastrointestinal tract. Therefore, to evaluate the efficacy of pancreatin-based silica nanoparticles NP-3, PDL-miniature pigs were generated. As previously described in the PDL-rat model, the initial validation of NP-3 involved administering a single dose of nanoparticles to PDL-miniature pigs, followed by administration of olive oil. Compared to healthy miniature pigs, the increase in plasma TG levels in PDL-miniature pigs administered pancreatin-based silica nanoparticles NP-3 showed similar dynamics, reaching a peak level 3 hours after feeding (Figure 20A). Calculations of the area under the curve (AUC) demonstrate an extremely remarkable digestibility of 62% for pancreatin-based silica nanoparticles NP-3 compared to healthy miniature pigs (Figure 20B). These results confirm the ability of pancreatin-based silica nanoparticles NP-3 to restore lipase digestive function in the EPI animal model.

[0243] Example 5: In vivo therapeutic efficacy of pancreatin-based silica nanoparticles NP-3 in PDL-miniature pigs To further demonstrate the ability of pancreatin-based silica nanoparticles NP-3 to restore lipase digestive function, PDL-miniature pigs fed a fat-rich diet were administered pancreatin-based silica nanoparticles NP-3 daily for 10 days. Since the primary endpoint of current treatments for EPI patients is the quantification of fat absorption, the benefits of pancreatin-based silica nanoparticles NP-3 were evaluated by measuring unabsorbed fat excreted in the feces. A comparison of fecal fat content between healthy and untreated PDL-miniature pigs showed that the EPI animal model exhibits a buildup of fat in the feces that reflects a lack of lipase activity (3.42g vs. 5.55g per 100g of feces for healthy and untreated PDL-miniature pigs, respectively) (Figure 21A). Importantly, PDL-miniature pigs treated with NP-3 had reduced fecal fat content compared to untreated PDL-miniature pigs (Figure 21A). In fact, PDL-miniature pigs administered pancreatin-based silica nanoparticles NP-3 daily were able to digest 53.4% ​​of the fat they ingested (Figure 21B). These results clearly demonstrate the therapeutic efficacy of pancreatin-based silica nanoparticles NP-3 for pancreatic enzyme replacement therapy.

[0244] Enzyme replacement therapy is available to treat clinical symptoms and malabsorption. A minimum dose of 40,000–50,000 units of PERT-lipase is recommended with each main meal, and half that amount is recommended for snacks. Such doses can be a burden for patients due to the heavy tablets. However, this treatment is not efficient and has several limitations (persistence of symptoms, short duration of intraluminal activity of lipase, potential intolerance due to large enzyme loads, and gastrointestinal damage due to large protease loads).

[0245] The efficacy of pancreatin-based silica nanoparticles NP-3 was compared with that of free pancreatin. The amount of lipase activity administered with NP-3 was calculated based on fat intake (1300U per dose per 14g of olive oil), while the amount of lipase activity administered with pancreatin followed standard treatment (40000U per dose). Surprisingly, administration of pancreatin-based silica nanoparticles NP-3 at a volume of 1300U twice daily significantly improved digestive efficiency by 25% compared to standard treatment with pancreatin administered at a much higher dose of 40000U twice daily. In fact, 80000U of pancreatin per day promoted 43.6% fat digestion, while 2600U of NP-3 per day achieved 53.4% ​​fat digestion (Figure 21B). Pancreatin-based silica nanoparticles NP-3 consist of nanoparticles functionalized with mucosal adhesive components that enable interaction between the nanoparticles and intestinal mucus (PCT / EP2023 / 051194). As a result, NP-3 temporarily engrafts, and lipase activity in the intestinal wall is sustained. In addition, by immobilizing and protecting lipase on the nanoparticles, the enzyme is protected from external stress (International Publication No. 2022 / 223699) and its activity is stabilized. In short, this series of data demonstrates the added value of pancreatin-based silica nanoparticles NP-3 for lipase digestion and highlights the therapeutic potential of NP-3 for EPI patients.

[0246] Example 6: In vitro biocompatibility of pancreatin-based silica nanoparticles NP-5 Currently, patients with exocrine pancreatic insufficiency are taking enormous amounts of pancreatin (10-20 tablets per day), resulting in a daily intake of large amounts of proteases. Despite their digestive role, gastrointestinal proteases contribute to gut homeostasis. An imbalance in protease levels can lead to gastrointestinal complications (Vergnolle N et al., Gut 2016;65:1215-1224).

[0247] To evaluate the safety of pancreatin-based silica nanoparticles NP-5, the inventors focused on the maintenance of intestinal barrier integrity in the presence of the nanoparticles and compared it with pancreatin (Figure 22A). Transepithelial electrical resistance (TEER) measurements across a monolayer of Caco2-HT29-MTX-E12 cells showed that intestinal epithelial barrier integrity was maintained even after 24 hours of contact with NP-5, while treatment with pancreatin resulted in a dose-dependent loss of barrier integrity. To correlate TEER measurements with the morphological characteristics of the cell monolayer, the tight junction protein Zonula-Occludens-1 (ZO-1) was evaluated by confocal microscopy (Figure 22B). When cultured in the presence of NP-5, ZO-1 staining showed a continuous ring-like appearance at the cell boundaries, while in the presence of pancreatin, ZO-1 staining showed a diffused, punctate protein distribution around intestinal cells. These morphological data confirmed the safety of pancreatin-based silica nanoparticles NP-5 for application to the gastrointestinal tract. Furthermore, these data highlight the added value of our technology compared to pancreatic enzyme replacement therapy.

[0248] Example 7: Improvement of pancreatin stability by covalent bonding to a protective layer In the first experiment, pancreatin-based silica nanoparticles NP-3(1) were produced under buffered conditions, followed by washing after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, pancreatin-based silica nanoparticles NP-3 were produced under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0249] To determine the pancreatin immobilization yield on NP-3(1) and its surface, protein quantification was performed on the reaction supernatant. Surprisingly, the results showed that enzyme immobilization yield doubled under conditions where glutaraldehyde was maintained (NP-3) (Figure 23A), and consequently, the enzyme load per dry weight of SNP doubled compared to buffer conditions where glutaraldehyde was removed by the washing step (NP-3(1)) (Figure 23B).

[0250] The lipase biocatalytic activity of NP-3(1) and pancreatin immobilized and protected on NP-3 was evaluated. Even more surprising than the increase in enzyme immobilization load while maintaining the presence of glutaraldehyde, the specific activity of the nanoparticles increased 11-fold compared to buffered conditions in which glutaraldehyde was removed by a washing step (Figure 23C). This astonishing 11-fold increase in nanoparticle specific activity is accompanied by an extremely astonishing 7-fold increase in the specific activity of the enzyme protected in the presence of glutaraldehyde (units per gram of pancreatin) compared to buffered conditions in which glutaraldehyde was removed by a washing step (Figure 23D). In summary, covalent bonding of the protective layer to the pancreatin surface unexpectedly improves its load, stability, and enzyme specific activity compared to pancreatin protected by an organosilica layer via electrostatic interactions alone.

[0251] Lipase Materials and methods reagent: - 99% tetraethyl orthosilicate (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (grade I, 25% in water), polysorbate 80, recombinant human pancreatic lipase (HRL, certified reference substance), porcine pancreatic lipase (4x USP standard), 1,2-di-O-lauryl-rac-glycero-3-(6-methylresorphin glutarate), Tris base, collipase, and sodium taurocholate hydrate were purchased from Sigma-Aldrich. HRL, porcine pancreatic lipase, and collipase were dissolved in water to reconstitute the stock buffer. - Benzyltriethoxysilane (B, 96%) was purchased from abcr GmbH. - The trifluoroacetate salt of peptide Glu-Leu-Gly-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Glu-Gly-Gly-Gly-Glu-Arg-Gly-Gly-Gly-Gly-Asn-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6), which has an E-azide-Nle-OH at its carboxyl terminus, was purchased from Bachem.

[0252] Synthesis of silica nanoparticles: Silica nanoparticles (50 nm) were synthesized according to the original Stoeber process as described in International Publication No. 2015 / 014888. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 hour. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm at 20°C for 22 hours. The solution was then centrifuged at 20000 g for 20 minutes and subsequently washed with ethanol and water. Particle size was measured using SEM micrographs acquired at a magnification of 150,000x with the image analysis software Olympus Stream Motion.

[0253] Free recombinant human pancreatic lipase activity assay: Recombinant human pancreatic lipase (3.43 μL, 1 mg / mL) in Tris-buffer (0.1 M, pH 8.4, 56.6 μL) was mixed with 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester) (60 μL, 100 μM). The kinetics of lipase activity were measured by steady-state fluorescence (λ) in a dark 96-well plate at 37°C for 30 minutes. ex / λ em Monitoring was performed using (=529 / 600nm).

[0254] Assay on free recombinant human pancreatic lipase activity in the presence of colipase: Recombinant human pancreatic lipase (3.43 μL, 1 mg / mL) and colipase (0.72 μL, 3 μg / mL) were dissolved in Tris-buffer (0.1 M, pH 8.4, 55.8 μL), to which 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester) (60 μL, 100 μM) was added. The kinetics of lipase activity were measured by steady-state fluorescence (λ) in a dark 96-well plate at 37°C for 30 minutes. ex / λ em Monitoring was performed using (=529 / 600nm).

[0255] Immobilized and protected recombinant human pancreatic lipase activity assay: To a solution of immobilized and protected recombinant human pancreatic lipase (20 μL, 10 mg / mL) in Tris-buffer (0.1 M, pH 8.4, 40 μL), 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester) (60 μL, 100 μM) was added. The kinetics of lipase activity were measured by steady-state fluorescence (λ) in a dark 96-well plate at 37°C for 30 minutes. ex / λ em Monitoring was performed using (=529 / 600nm).

[0256] Immobilized and protected porcine pancreatic lipase activity assay: To a solution of immobilized and protected porcine pancreatic lipase (15 μg / mL enzyme) in Tris-buffer (0.1 M, pH 8.4), 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester) (50 μM) was added. The kinetics of lipase activity were measured by steady-state fluorescence (λ) in a dark 96-well plate at 37°C for 30 minutes. ex / λ em Monitoring was performed using (=529 / 600nm).

[0257] Example 1 A) Immobilization and shielding of recombinant human pancreatic lipase (HRL) SNP (10 mg / mL) in H2O / PS80 (8 mg / L) was mixed with APTES (3.8 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Recombinant human pancreatic lipase solution (525 μg / mL, 11 μM) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized lipase using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-HRL-ATB was cured overnight in a 20°C water bath.

[0258] B) Co-immobilization and shielding of recombinant human pancreatic lipase (HRL) and colipase (CLPS): SNPs (10 mg / mL) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. A solution containing recombinant human pancreatic lipase (525 μg / mL, 11 μM) and collipase (110 μg / mL) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized protein using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-HRL-CLPS-ATB was cured overnight in a 20°C water bath.

[0259] Activation of recombinant human pancreatic lipase (HRL) by colipase The activation of recombinant human pancreatic lipase by a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorphin ester), was investigated (Figure 27a). The faster rate of hydrolysis of the lipase substrate by lipase in the presence of a lipase compared to lipase alone indicates appropriate activation of the lipase enzyme by the lipase.

[0260] To generate activated lipase nanoparticles, recombinant human pancreatic lipase was co-immobilized with a colipase and protected with a hydrophobic organic silica shield on the surface of the silica nanoparticles, as described in B) above. The activation of lipase by the colipase was evaluated using a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(6-methylresorphin glutarate) (Figure 27b). The fact that the hydrolysis rate of the lipase substrate by the lipase co-immobilized with the colipase, as described in B), was faster than that of lipase immobilized without the colipase, as described in A), indicates that the activation of the lipase enzyme by the colipase in the shielded nanoparticles was surprisingly more than three times faster than that of lipase without the colipase. These results confirm the strategy of co-immobilizing lipase and colipase on the surface of silica nanoparticles to generate nanoparticles that retain immobilized and protected lipase maintained within its active conformation.

[0261] Example 2 A) Immobilization and shielding of porcine pancreatic lipase (PL) SNP (10 mg / mL) in H2O / PS80 (8 mg / L) was mixed with APTES (3.8 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Porcine pancreatic lipase solution (1.9 mg / mL, 39 μM) was added, and the reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of the immobilized lipase using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was incubated at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20,000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-PL-ATB was cured overnight in a 20°C water bath.

[0262] B) Co-immobilization and shielding of porcine pancreatic lipase (PL) and colipase (CLPS) according to the present invention: SNPs (10 mg / mL) in H2O / PS80 (8 mg / L) were mixed with APTES (3.8 mM). The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.8 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. A solution containing porcine pancreatic lipase (1.9 mg / mL, 39 μM) and colipase (150 μg / mL) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized protein using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-PL-CLPS-ATB was cured overnight in a 20°C water bath.

[0263] Activation of porcine pancreatic lipase (PL) by colipase To generate activated lipase nanoparticles, porcine pancreatic lipase was co-immobilized with a colipase and protected with a hydrophobic organic silica shield on the surface of silica nanoparticles, as described in B) above. The activation of lipase by the colipase was evaluated using a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorphin ester) (Figure 28). As described in B) above, the rate of hydrolysis of the lipase substrate by the lipase co-immobilized with the colipase was faster than that of lipase immobilized without the colipase, as described in A) above, indicating that the activation of the lipase enzyme by the colipase in the shielded nanoparticles was, surprisingly, three times greater than that of the lipase enzyme without the colipase. These results confirm the strategy of co-immobilizing lipase and colipase on the surface of silica nanoparticles to generate nanoparticles that hold immobilized and protected lipase maintained within its active conformation. This also demonstrates the versatility of this method in terms of the origin of the enzyme used.

[0264] Example 3 Immobilization and shielding of porcine pancreatic lipase (PL) in the presence of sodium taurocholate (NaTc): SNP (10 mg / mL) was added to a mixture of NaTc (2 mM), H2O, and H2O / PS80 (8 mg / L), to which APTES (3.8 mM) was added. The reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.8 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Porcine pancreatic lipase (1.9 mg / mL, 39 μM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized lipase using APTES (4.2 mM), TEOS (21.8 mM), and benzyltriethoxysilane (18.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were centrifuged at 20000 rcf for 5 minutes and washed three times in H2O / PS80 (8 mg / L). SNP-PL-NaTc-ATB was cured overnight in a 20°C water bath.

[0265] Activation of recombinant human pancreatic lipase (HRL) by sodium taurocholate (NaTc) The activation of recombinant human pancreatic lipase by sodium taurocholate was investigated using the fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(glutaric acid 6-methylresorfin ester) (Figure 29). The higher rate of hydrolysis of the lipase substrate by lipase in the presence of gradually increasing concentrations of sodium taurocholate compared to lipase alone indicates appropriate activation of the lipase enzyme by bile salts.

[0266] Activation of porcine pancreatic lipase (PL) by sodium taurocholate (NaTc) To generate activated lipase nanoparticles, porcine pancreatic lipase was immobilized in the presence of sodium taurocholate and protected with a hydrophobic organic silica shield on the surface of silica nanoparticles. The activation of lipase by sodium taurocholate was evaluated using a fluorescent lipase substrate, 1,2-di-O-lauryl-rac-glycero-3-(6-methylresorphin glutarate) (Figure 30). The rate of hydrolysis of the lipase substrate by lipase immobilized in the presence of sodium taurocholate was faster than that of lipase immobilized without bile salts, indicating that the activation of the lipase enzyme by sodium taurocholate in shielded nanoparticles was, surprisingly, nearly three times faster than that of lipase without sodium taurocholate. These results confirm the strategy of immobilizing lipase on the surface of silica nanoparticles in the presence of sodium taurocholate to generate nanoparticles that retain immobilized and protected lipase maintained within its active conformation.

[0267] Example 4: Activation of pancreatic lipase (PL) by a colipase-mimicking peptide Analysis of the lipase-colipase complex made it possible to identify the amino acid residues responsible for the lipase-colipase interaction. Based on these findings, the inventors designed a peptide represented by Sequence ID No. 6 (Glu-Leu-Gly-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Glu-Gly-Gly-Gly-Glu-Arg-Gly-Gly-Gly-Gly-Asn-Gly-Gly-Gly-Gly-Gly) that mimics the interaction between the collipase and the lipase structure and induces conformational changes around the lid and lipase lid opening (Figure 31). This peptide was chemically modified at its carboxyl terminus by adding an E-azide-Nle-OH group, enabling crosslinking of the lipase enzyme on the surface of silica nanoparticles by click chemistry.

[0268] Example 5: Generation of HRL-based silica nanoparticle NP-1 variants: The following experiments investigated the effects of covalently bonding enzymes to a protective layer on enzyme stability and enzyme activity.

[0269] In the initial experiment, HRL-based silica nanoparticles (NP-1(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L) was mixed with APTES (3.1 mM). The reaction mixture was incubated at 20°C and 400 rpm for 10 minutes. The particles were then mixed with H2O / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Then, glutaraldehyde (3.1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. / The particles were washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Sodium taurocholate (2 mM) and human recombinant lipase (0.732 g / L, 15.2 μM) were added sequentially, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized HRL using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then removed from H2O / The sample was washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). NP-1(1) was cured overnight in a 20°C water bath.

[0270] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to International Publication No. 2015 / 014888, and HRL-based silica nanoparticles (NP-1(2)) were generated in buffer. The nanoparticles were washed after each chemical step, resulting in the removal of glutaraldehyde. SNP (10 mg / mL, 69 nm) and PS80 (8 mg / L) were added to phosphate buffer (25 mM, pH 7.5), to which APTES (3.1 mM) was added. The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Then, glutaraldehyde (3.1 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). Sodium taurocholate (2 mM) and human recombinant lipase (0.732 g / L, 15.2 μM) were added successively, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organic silica layer was grown on the surface of immobilized HRL using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were washed three times in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L), and then resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-1(2) was cured overnight in a water bath at 20°C.

[0271] In the third experiment, HRL-based silica nanoparticles (NP-1) were generated in H2O / PS80 (8 mg / L). The nanoparticles were not washed between each chemical step to retain excess glutaraldehyde in the reaction mixture, which did not bind the solid support to the manipulated HRL. Therefore, glutaraldehyde remained present during layer growth, inducing covalent bonding between the protective layer and the human recombinant lipase. SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L) was mixed with APTES (3.1 mM). The reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. Glutaraldehyde (3.1 mM) was then added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 minutes. Priming was performed by adding APTES (3.1 mM) and stirring the reaction mixture at 20°C and 400 rpm for 10 minutes. Sodium taurocholate (2 mM) and human recombinant lipase (0.732 g / L, 15.2 μM) were added sequentially, and the reaction mixture was reacted at 20°C and 400 rpm for 10 minutes. An organosilica layer was grown on the surface of the immobilized HRL using APTES (5.7 mM), TEOS (29.9 mM), and benzyltriethoxysilane (25.9 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 hours. The particles were then subjected to H2O / The NP-1 was washed three times in PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). NP-1 was cured overnight in a 20°C water bath.

[0272] The covalent bond between the protective layer and HRL can be observed by the appearance of a yellow / orange color with a maximum absorbance at 460 nm. This color is due to the formation of imine bonds through the reaction of the aldehyde group of the glutaraldehyde linker with the primary amine amino acids in the HRL and organic silica layers. The absorbance of HRL-based silica nanoparticles NP-1(1), NP-1(2), and NP-1 at a wavelength of 460 nm was measured after the formation of the organic silica layer and the final particle washing (see Figure 33A). The results showed that NP-1 absorbed much more light than NP-1(2) at this wavelength. Interestingly, NP-1 appeared darker to the naked eye than NP-1(1), but their absorbances were similar. This suggests that the instrument could not distinguish the formation of imine bonds due to strong interference from the particles themselves (due to the small amount of enzyme used). To overcome this limitation, the inventors used an inverted microscope to image the NPs (Figure 33B). At the same concentration, NP-1 appeared significantly darker under a microscope than NP-1(1). This visual confirmation indicates that NP-1 has more imine bonds, which is likely due to covalent bonding of a protective layer against human recombinant lipase.

[0273] Improvement of the stability and specific activity of human recombinant lipase by covalent binding to the protective layer. In the first experiment, HRL-based silica nanoparticles NP-1(1) were produced under unbuffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the second experiment, HRL-based silica nanoparticles NP-1(2) were produced under buffered conditions, and washing was performed after each chemical step (i.e., glutaraldehyde removal before layer growth). In the third experiment, HRL-based silica nanoparticles NP-1 were produced under unbuffered conditions without an intermediate washing step (i.e., unreacted glutaraldehyde was still present in the reaction mixture during layer growth).

[0274] To determine the HRL immobilization yield on the HRL-based silica nanoparticles NP-1(1), NP-1(2), and the surface of NP-1, protein quantification was performed on the reaction supernatant. Surprisingly, the results showed that enzyme immobilization yield increased fourfold under conditions where the presence of glutaraldehyde was maintained (NP-1) (Figure 32A), and consequently, the enzyme load per dry weight of SNPs increased fourfold compared to buffer conditions where glutaraldehyde was removed by the washing step (NP-1(2)) (Figure 32B).

[0275] The lipase biocatalytic activity of NP-1(1), NP-1(2), and HRL immobilized and protected on NP-1 was evaluated. Even more surprising than the increased enzyme immobilization load when glutaraldehyde was maintained was the 37-fold increase in the specific activity of the nanoparticles compared to buffered conditions in which glutaraldehyde was removed by the washing step, and the 2-fold increase compared to unbuffered conditions in which glutaraldehyde was removed by the washing step (Figure 32C). These astonishing 37-fold and 2-fold increases in the specific activity of the nanoparticles correspond to the HR specific activity (U per 1g of HRL) of the enzyme protected in the presence of glutaraldehyde compared to buffered and unbuffered conditions in which glutaraldehyde was removed by the washing step, respectively. μM / min This is accompanied by an extremely surprising 10-fold and 2-fold increase (Figure 32D). This result is completely unexpected, as the enzyme is expected to exhibit significantly higher activity in the presence of the buffer.

[0276] In summary, covalent bonding of a protective layer to the HRL surface unexpectedly improves the load, stability, and specific enzyme activity of enzymes protected by an organic silica layer via electrostatic interactions alone.

Claims

1. A method for producing a composition, the composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and optionally 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 containing at least one amino group and / or at least one thiol group, and the method is: (a) A step of providing a solid carrier, wherein the solid carrier is provided in a suspension; (b) A step of immobilizing a protein or fragment thereof on a solid carrier, comprising: i) adding a linker to a suspension of the solid carrier; ii) adding a solution of the protein or fragment thereof to the suspension containing the solid carrier and the linker, wherein the linker connects the solid carrier and the protein or fragment thereof; (c) A step of forming a protective layer on the surface of a solid carrier in order to protect a protein or fragment thereof immobilized on the solid carrier, wherein a linker or part thereof that did not connect the solid carrier and the protein or fragment thereof in step (b) covalently bonds the protective layer to the protein or fragment thereof; and optionally (d) A step of immobilizing a functional component on the surface of a protective layer, wherein the functional component immobilized on the surface of the protective layer is a polymer containing repeating units, and each repeating unit contains at least one amino group and / or at least one thiol group. A method that includes this.

2. The method according to claim 1, wherein there is no washing step between adding a linker to the suspension of the solid carrier in step (b)(i) and adding a protein or a fragment thereof to the suspension containing the solid carrier and the linker in step (b)ii).

3. The method according to claim 1 or 2, wherein there is no washing step between any of steps (a) to (c).

4. The method according to any one of claims 1 to 3, wherein the solid carrier is provided suspended in water, a buffer, a nonionic surfactant, or a mixture thereof.

5. The method according to any one of claims 1 to 3, wherein the solid carrier is provided suspended in a mixture of water and a nonionic surfactant.

6. The method according to any one of claims 1 to 5, wherein the linker is added to the suspension of the solid carrier in step (b)(i) in an amount that is in molar excess with respect to the protein or fragment thereof added to the suspension containing the solid carrier and the linker in step (b)ii).

7. The method according to any one of claims 1 to 5, wherein the linker is added to the suspension of the solid carrier in step (b) i) in an amount that is 1 to 1000 times more molar than the protein or fragment thereof added to the suspension containing the solid carrier and the linker in step (b) ii).

8. The method according to any one of claims 1 to 5, wherein the linker is added to the suspension of the solid carrier in step (b)(i) in an amount that is 4 to 250 times molar excess relative to the protein or fragment thereof added to the suspension containing the solid carrier and the linker in step (b)ii).

9. The method according to any one of claims 1 to 8, wherein a linker that did not connect the solid carrier to the protein or a fragment thereof in step (b) is present during the formation of a protective layer on the surface of the solid carrier in step (c).

10. The method according to any one of claims 1 to 9, wherein, after the addition of the protein or fragment thereof in ii), the amount of linker that did not link the solid carrier and the protein or fragment thereof in step (b) is 30% to 70% of the amount of linker added to the suspension of the solid carrier in i) in step (b).

11. The method according to any one of claims 1 to 10, wherein a protein or a fragment thereof is immobilized on a solid support by covalent bonding via a linker in step (b).

12. The method according to any one of claims 1 to 11, wherein the surface of the solid carrier is at least partially modified before the protein or fragment thereof is immobilized on the solid carrier in step (b).

13. The linker is glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl]sverate, ethylene glycol bis(sulfosuccinimidyl succinate), dimethyl adipimidate, dimethyl pimelidate, sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimodoxycarbonyloxy)ethyl]sulfone), DSP (dithiobis[succinimidyl]propionate), DTSSP (3,3'-dithiobis[sulfosuccinimidyl]propionate), DTBP (dimethyl 3,3'-dithiobispropionimidate-2) The method according to any one of claims 1 to 12, selected from the group consisting of HCl, DST (disuccinimidyl tartrate), and BMDB (1,4-bismaleimidyl-2,3-dihydroxybutane).

14. The method according to any one of claims 1 to 12, wherein the linker is glutaraldehyde.

15. A composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer protecting the protein or fragment thereof by embedding it, and optionally 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 comprising at least one amino group and / or at least one thiol group, and the composition can be obtained by the method according to any one of claims 1 to 14.

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