Protein immobilization method

Abstract

The present invention relates to a method of producing a composition comprising a solid support, a protein or fragment thereof immobilized on the surface of the solid support, a protective layer protecting the protein or fragment thereof by embedding the protein or fragment thereof, and optionally a functional ingredient immobilized on the surface of the protective layer, wherein the functional component fixed on the surface of the protective layer is a polymer comprising repeating units, and each repeating unit comprises at least one amino group and / or at least one mercapto group. The invention also relates to a composition obtainable by this process.

Description

Technical Field

[0001] This 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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. The invention also relates to compositions obtainable by this method. Background Technology

[0002] Proteins, such as enzymes, are frequently required in applications such as industrial, diagnostic, or therapeutic applications. To stabilize proteins and / or provide resistance to various types of stress, it has been suggested in the prior art to immobilize proteins on a carrier surface and protect them with a protective layer. This method has been described, for example, in WO2015 / 014888 A1, which discloses a biocatalytic composition comprising a solid carrier, a functional component such as an enzyme, and a protective layer, as well as a method for producing such a biocatalytic composition, wherein the protective layer protects the functional component by at least partially embedding it. However, the method described in WO2015 / 014888A1 is difficult to use on a large scale because of the low protein loading per unit dry weight particle. Therefore, there is a need for improved immobilization methods to obtain high protein loading per unit dry weight particle, resulting in particles with high enzyme activity. Summary of the Invention

[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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension; (b) Immobilizing a protein or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect the protein or fragment thereof fixed on the solid support, wherein a linker or portion thereof that did not connect the solid support to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof; and optionally (d) Fixing a functional component onto the surface of a protective layer, wherein the functional component fixed onto the surface of the protective layer is a polymer comprising repeating units, wherein each repeating unit comprises 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 for protecting the protein or fragment thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition can be obtained by the methods described herein.

[0005] The inventors of this application have unexpectedly discovered that the methods provided and described in this invention can be used for large-scale protein immobilization because these methods enable a high loading of immobilized protein per unit dry weight of particle, thereby significantly improving particle activity. Attached Figure Description

[0006] Figure 1 The diagram shows a method for producing the composition of the present invention: a) immobilizing an engineered PAL or a fragment thereof on a solid carrier; b) and c) growing a protective layer around the immobilized engineered PAL or a fragment thereof, thereby encapsulating the immobilized engineered PAL or a fragment thereof; and d) immobilizing the functional component on the surface of the protective layer.

[0007] Figure 2 (A) PAL quantification of reaction supernatants of PAL-based silica nanoparticles NP-1(1), NP-1(2), and NP-1. (B) PAL loading per unit dry weight of SNP. (C) PAL activity of SNP expressed as U / g SNP. (D) PAL-specific activity expressed as U / g PAL.

[0008] Figure 3 The activity of phenylalanine ammonia-lyase (PAL) in nanoparticles was demonstrated. The biocatalytic activity (in U / g) of engineered PAL immobilized and protected on nanoparticles was quantified after exposure to phenylalanine.

[0009] Figure 4The resistance of PAL to external stress was demonstrated. PAL-based silica nanoparticles NP-1 and engineered PAL were exposed to (AB) acidic conditions (pH 4) or (C) proteases, and their stability was assessed by measuring PAL enzyme activity at different time points.

[0010] Figure 5 shows the in vitro biocompatibility and efficacy of PAL-based silica nanoparticles NP-1 in an intestinal barrier model. (A) In vitro assessment of intestinal barrier integrity by measuring transepithelial resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to PAL-based silica nanoparticles NP-1 (9.7 mU) or to trypsin alone (30 mU) for 6 h, with or without trypsin (30 mU). The figure shows the time-course curve evolution of mean normalized TEER data over 6 h. The dashed line represents the untreated condition. (B) In vitro metabolism of phenylalanine (Phe) in the intestinal barrier model. Differentiated Caco-2 / HT29-MTX-E12 cocultures cultured in cell culture medium containing 0.4 mM Phe were exposed to the top side of the barrier to NP-1 (9.7 mU) or engineered PAL (9.7 mU) (with or without trypsin (30 mU)) for 6 h. Phe metabolism was evaluated by quantifying trans-cinnamic acid (TCA) at the base of the barrier. The figure shows the evolution of the time-series curve of TCA accumulation over 6 hours.

[0011] Figure 6 The figures show the quantification of trans-cinnamic acid (TCA) in rat urine. Wistar rats were administered PAL-based silica nanoparticles NP-1 (n=5) or inactive nanoparticles NP-2 (n=5) via duodenal administration, while simultaneously receiving d5-Phe via tube feeding. Urine was collected within 24 h post-administration and analyzed by LC-MS. The figures show the concentration of D5-hippuric acid in urine. p < 0.01, passing the t-test.

[0012] Figure 7 shows BTBR- Pah enu2 Plasma concentration of Phe in J mice. BTBR- mice with free access to drinking water containing L-Phe were compared twice daily over 12 days. Pah enu2 / J Mice were administered PAL-based silica nanoparticles NP-1 (0.581U; 7mg), inactive nanoparticles NP-2 (7mg), or engineered PAL (0.581U) via intraduodenal administration. Blood samples were collected on days 0, 4, 6, 8, 10, and 12 for plasma extraction and LC-MS analysis. (A) Figure shows BTBR- Pah enu2The plasma concentration of Phe in J mice. (B) Figure shows the BTBR- Pah enu2 Normalized plasma concentration of Phe in J mice.

[0013] Figure 8 The absorbance of PAL-based silica nanoparticles NP-1, NP-1(1) and NP-1(2) at 460 nm is shown.

[0014] Figure 9 The diagram shows a method for producing the composition of the present invention: a) immobilizing a disaccharidase or a fragment thereof (referred to as "protein") on a solid support; b) and c) growing a protective layer around the immobilized disaccharidase or a fragment thereof, thereby embedding the immobilized disaccharidase or a fragment thereof; and d) immobilizing the functional component on the surface of the protective layer.

[0015] Figure 10 (A) Quantification of lactase in the reaction supernatant of lactase-based silica nanoparticles NP-2(1), NP-2(2) and NP-2. (B) Lactase loading per unit dry weight of SNP.

[0016] Figure 11 The following figures illustrate the disaccharidase activities of the nanoparticles: (A) Lactase activity (in U / g) of lactase-based silica nanoparticle NP-2 after exposure to lactose. (B) Invertase activity (in U / mg) of invertase-based silica nanoparticle NP-3 after exposure to sucrose. (C) Isomaltase activity (in U / g) of isomaltase-based silica nanoparticle NP-4 after exposure to isomaltose. (D) Isomaltase and invertase activities (in U / g) of invertase / isomaltase-based silica nanoparticle NP-5 after exposure to isomaltose and sucrose, respectively.

[0017] Figure 12(A) In vitro biocompatibility of a representative model of nanoparticles on the intestinal barrier. (B) In vitro assessment of intestinal barrier integrity by measuring transepithelial resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to inactive nanoparticles NP-1 (0.5 mg / mL and 1 mg / mL) for 16 h. TEER data were normalized relative to a control point, which was the equilibrium value before the addition of inactive nanoparticles NP-1 (defined as control), and set to 100%. The figure shows the time-course curve evolution of the mean normalized TEER data over 16 h. The dashed line represents the untreated condition. (C) In vitro evaluation of the effect of inflammation on the intestinal epithelial barrier. Differentiated Caco2-HT29-MTX-E12 and M0-differentiated THP-1 were cocultured and exposed to inactive nanoparticles NP-1 (1 mg / mL) at 37 °C for 16 h. Lipopolysaccharide (LPS) (50 and 100 μg / mL) were used as positive controls to induce an inflammatory response. TEER data were normalized to 100% with the equilibrium value before the addition of inactive nanoparticles NP-1 or LPS. The figure shows the evolution of the time-series curve of the average normalized TEER data over 16 hours in the presence of inactive nanoparticles NP-1.

[0018] Figure 13 (A) Photograph of the rat gastrointestinal tract. The cecum is shown in the following figures. Wistar rats were administered lactase-based silica nanoparticles N-2, inactive silica nanoparticles NP-1, or a solvent into the duodenum daily over a 15-day period, immediately followed by lactose feeding via a lactose tube. Cecum size was evaluated at the end of the period. (B) MRI image of the rat gastrointestinal tract. The cecum is indicated by a circle. (C) Bar chart showing the size of the cecum as assessed by MRI imaging in cm. 3 The cecum size is represented. A one-way ANOVA test was performed. p<0.05, p<0.01.

[0019] Figure 14 This figure illustrates the in vitro digestion of sucrose in an intestinal barrier model. Differentiated Caco-2 / HT29-MTX-E12 co-cultures were exposed to different amounts of invertase-based silica nanoparticles NP-3 (0.5 mU or 1 mU) for 4 h on the top of the barrier in the presence of sucrose. Sucrose hydrolysis was evaluated by quantifying glucose levels on the bottom of the barrier. The figure shows the evolution of glucose accumulation over 4 h.

[0020] Figure 15 The absorbance of the lactase-based silica nanoparticles NP-2, NP-2(1) and NP-2(2) at 460 nm is shown.

[0021] Figure 16 The diagram shows a method for producing the composition of the present invention: a) adding lipase, protease, amylase and an agent (shown as a ring) having a closed cap to a solid carrier and immobilizing lipase, protease and amylase having an open cap on the solid carrier; b) and c) growing a protective layer around the immobilized lipase, protease and amylase having an open cap, thereby encapsulating all three enzymes; and d) immobilizing the functional component on the surface of the protective layer.

[0022] Figure 17 This shows silica nanoparticles labeled with inactive DOTA and functionalized with chitosan. 111 In-NP-1 (n=5) and unfunctionalized, inactive DOTA-labeled silica nanoparticles 111 Biodistribution of In-NP-2 (n=4) (%ID) in miniature pigs. (A) Intraduodenal administration 111 In-NP-1 and 111 SPECT / CT images were acquired at 0.25, 3, 8, and 24 hours after In-NP-2. The graphs represent the relative quantification (in %) of nanoparticles at imaging time points in the small intestinal compartment. (B) Histograms show the nanoparticles in the small intestine. 111 In-NP-1 and 111 Area under the residence time curve (AUC) of In-NP-2. (C) Intraduodenal administration. 111 In-NP-1 or free 111 Blood samples were collected at 0.25, 3, 8, and 24 hours post-infection. The bar chart represents the relative quantification of radioactivity in the blood compartment.

[0023] Figure 18 (A) Lipase activity (in U / g) of trypsin-based silica nanoparticles NP-3 after exposure to olive oil. (B) Protease activity (in U / mg) of trypsin-based silica nanoparticles NP-5 after exposure to casein. (C) Amylase activity (in U / g) of trypsin-based silica nanoparticles NP-3 after exposure to amylase substrate solution.

[0024] Figure 19The relative quantification of plasma triglycerides (TG) in pancreatic duct ligation (PDL) rats is shown. PDL rats were administered pancreatic enzyme-based silica nanoparticles NP-3 (n=1) or inactive nanoparticles NP-4 (n=1) via intraduodenal administration, followed by trioleic triglyceride via gavage 5 minutes later. Blood samples were collected before treatment and at 0.25, 0.5, 1, 1.5, 2, 4, and 6 h after treatment for plasma extraction and LC-MS analysis. The graph shows the peak area of ​​trioleic triglyceride (TG(54:3)).

[0025] Figure 20 This paper compares plasma triglyceride (TG) concentrations between healthy miniature pigs and pancreatic duct ligation (PDL) miniature pigs treated with NP-3 based pancreatic enzyme silica nanoparticles. Healthy miniature pigs (n=1) and PDL miniature pigs (n=1) received olive oil, followed by administration of NP-3 based pancreatic enzyme silica nanoparticles. Blood samples were collected before administration and at 0.083, 0.25, 0.5, 1, 2, 3, 4, and 6 h after administration for TG analysis. (A) The graph shows plasma TG concentrations in miniature pigs as determined by a Konelab analyzer. (B) The histogram shows the area under the curve (AUC) for plasma TG concentrations in miniature pigs.

[0026] Figure 21) shows the fecal fat content of miniature pigs on a high-fat diet. Healthy and PDL miniature pigs were fed a high-fat diet for 25 days. PDL miniature pigs were administered NP-3, a trypsin-based silica nanoparticle, twice daily for 10 days. Feces were collected on days 8, 9, and 10, and fecal homogenates were analyzed by near-infrared spectroscopy for fecal fat quantification. (A) The histogram shows the absolute fecal fat measurements. (B) The graph shows the normalized fecal fat homogenate fat content relative to healthy and untreated PDL miniature pigs.

[0027] Figure 22 (A) The in vitro biocompatibility of trypsin-based silica nanoparticles NP-5 on the intestinal barrier was demonstrated. (B) In vitro, intestinal barrier integrity was assessed by measuring transepithelial electrical resistance (TEER). Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to increasing amounts of trypsin-based silica nanoparticles NP-5 or trypsin (32.9 to 263.6 U / m³). 2(A) TEER data were normalized to 100% equilibrium value before the addition of trypsin-based silica nanoparticles NP-5 or trypsin. The figure shows the time-course evolution of the mean normalized TEER data over 20 hours. The dashed line represents the untreated condition. (B) Confocal image of tight junctions on the intestinal barrier model. Differentiated Caco-2 / HT29-MTX-E12 cocultures were exposed to trypsin-based silica nanoparticles NP-5 or trypsin (263.6 U / m³). 2 Cells were stained with tight junction band 1 (ZO-1) for up to 20 hours and evaluated by confocal microscopy (white signal on the image).

[0028] Figure 23 (A) Protein quantification of reaction supernatants from trypsin-based silica nanoparticles NP-3 and NP-3(1). (B) Trypsin loading per unit dry weight of SNP. (C) Lipase activity of SNP expressed in μmol / min. (D) Specific activity of trypsin expressed in U / g trypsin.

[0029] Figure 24 The absorbance of trypsin-based silica nanoparticles NP-3 and NP-3(1) at 460 nm is shown.

[0030] Figure 25 The diagram shows a method for producing the composition of the present invention: a) providing a lipase or a fragment thereof having a closed cap and an agent (shown as a circle) interacting with the cap domain of the lipase or fragment thereof to a solid support, and immobilizing a lipase or a fragment thereof having an open cap on the solid support; b) and c) growing a protective layer around the immobilized lipase or fragment thereof having an open cap, the protective layer embedding the immobilized lipase or fragment thereof.

[0031] Figure 26 The image shows the 3D structure of pancreatic lipase in its a) inactive conformation (with a closed cap) and b) active conformation (with an open cap). The active site of pancreatic lipase is covered by the cap, which prevents substrate from reaching the enzyme's active site. Figure 26 a). Lipase cap opening is induced by interaction with an agent that interacts with the cap domain of the lipase, such as bile salts and / or protein cofactors called colipases, thereby allowing for the stabilization of the active conformation of pancreatic lipase. Figure 26 b).

[0032] Figure 27The kinetics of hydrolysis of lipase substrates by recombinant human pancreatic lipase (HRL) with or without colipase (CLPS) were shown: a) in the free form of colipase; b) the colipase was immobilized on the surface of silica nanoparticles (SNP) and protected in an organosilicon layer made of APTES, TEOS and benzyltriethoxysilane (ATB).

[0033] Figure 28 The study demonstrates the kinetics of porcine pancreatic lipase (PL) hydrolysis of lipase substrates with or without co-lipase (CLPS), the porcine pancreatic lipase being immobilized on the surface of silica nanoparticles (SNP) and protected in an organosilicon layer made of APTES, TEOS and benzyltriethoxysilane (ATB).

[0034] Figure 29 This study demonstrates the kinetics of lipase substrate hydrolysis via free human recombinant lipase (HRL) using increasing concentrations of sodium taurocholate (NaTc).

[0035] Figure 30 The study demonstrated the kinetics of porcine pancreatic lipase (PL) hydrolysis of its substrate with or without sodium taurocholate (NaTc), the porcine pancreatic lipase being immobilized on the surface of silica nanoparticles (SNP) and protected in an organosilicon layer made of APTES, TEOS and benzyltriethoxysilane (ATB).

[0036] Figure 31 The image shows the 3D structure of pancreatic lipase activated by a colipase mimic peptide.

[0037] Figure 32 (A) Protein quantification of the reaction supernatant of HRL-based silica nanoparticles NP-1, NP-1(1), and NP-1(2). (B) HRL loading per unit dry weight of SNP. (C) Lipase activity of nanoparticles expressed in U. µM / min / g SNP is used to express (D)HRL specific activity in U µM / min / g HRL indicates.

[0038] Figure 33 The absorbance of HRL-based silica nanoparticles NP-1, NP-1(1), and NP-1(2) at 460 nm is shown. Detailed Implementation

[0039] This 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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension; (b) Immobilizing a protein or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect the protein or fragment thereof fixed on the solid support, wherein a linker or portion thereof that did not connect the solid support to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof; and optionally (d) Fixing a functional component onto the surface of a protective layer, wherein the functional component fixed onto the surface of the protective layer is a polymer comprising repeating units, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0040] 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 for protecting the protein or fragment thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition can be obtained by the methods described herein.

[0041] For the purpose of interpreting this specification, the following definitions will be used, and where appropriate, terms used in the singular will also include the plural, and vice versa. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] Features, integrals, properties, and compounds described in connection with specific aspects, embodiments, or examples of the invention should be understood to be applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith. All features disclosed in this specification (including any appended claims, abstract, and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except for combinations in which at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments.

[0043] The term “comprise” and its variations, such as “comprises” and “comprising”, are generally used in the sense of inclusion, that is, “including but not limited to”, meaning that one or more features or components are allowed to be present.

[0044] Unless the context clearly indicates otherwise, the singular forms “a” and “the” include plural references.

[0045] The term “about” refers to a range of ±10% of a specified value. For example, the phrase “about 200” includes ±10% of 200, or 180 to 220.

[0046] As used herein, the term "solid support" generally refers to particles. Preferably, the solid support is monodisperse or polydisperse particles, more preferably monodisperse particles. Solid supports typically include organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, titanium particles, and are preferably silica particles, more preferably silica nanoparticles (SNPs). The particle size of the solid support is typically from 1 nm to 1000 µm, preferably from 10 nm to 100 µm, and particularly about 50 nm.

[0047] As used herein, the terms "linker" or "cross-linker" refer to any linking agent containing a group capable of binding a specific functional group (e.g., primary amine, thiol, etc.). In the context of this invention, a linker typically links the surface of a solid support to a protein or fragment thereof, i.e., to an enzyme such as a lipase or fragment thereof, a protease or fragment thereof, and an amylase or fragment thereof. For example, the linker can be immobilized on the surface of a solid support, such as a silica surface as the support material, and then the protein or fragment thereof, i.e., the enzyme such as a lipase or fragment thereof, a protease or fragment thereof, and an amylase or fragment thereof, can be bound to an unoccupied binding site of the linker. Alternatively, the linker may first bind to the protein or fragment thereof, i.e., the enzyme such as a lipase or fragment thereof, a protease or fragment thereof, and an amylase or fragment thereof, and then the linker bound to the enzyme such as a lipase or fragment thereof, a protease or fragment thereof, and an amylase or fragment thereof can bind to the solid support at its unoccupied binding site. Various types of linkers are known in the art, including but not limited to linear or branched carbon linkers, heterocyclic carbon linkers, peptide linkers, polyether linkers, and linkers known in the art as labeled linkers.

[0048] As used herein, the term "protective layer" refers to a layer used to protect the functional properties of proteins or fragments thereof (e.g., lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof) immobilized on the surface of a solid support. The protective layer of the present invention is generally composed of building units, at least a portion of which are monomers generally capable of interacting with each other by covalent bonding, and monomers generally interacting with the immobilized proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, by non-covalent bonding. A protective layer is formed on the surface of a solid support to protect proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, immobilized on the solid support. The protective layer is generally 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 lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are embedded.

[0049] The term "protein or a fragment thereof" includes naturally occurring proteins or fragments thereof, and also includes artificially engineered proteins or fragments thereof. Artificially engineered proteins or fragments thereof are, for example, variants or functionally active fragments of proteins. Therefore, the terms "fragment of a protein," "fragment thereof" in relation to a protein, and "functionally active fragment of a protein" are used synonymously herein. A "variant or functionally active fragment thereof" in relation to the protein of the present invention means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the protein. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Consideration is given to the addition, deletion, substitution, and derivation of one or more amino acids, 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 protein, 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. Protein fragments as defined herein generally have the same functional properties as the protein, i.e., the full-length protein from which it is derived. 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 in this invention are enzymes or fragments thereof, more preferably enzymes or fragments thereof selected from hydrolases and ammonia-lyases or fragments thereof. Even more preferred proteins or fragments thereof in this invention are selected from lipases or fragments thereof, proteases or fragments thereof, amylases or fragments thereof, pancreatic enzymes or proteins or fragments thereof composed of pancreatic enzymes, engineered phenylalanine ammonia-lyase (PAL) or fragments thereof, and disaccharidases or fragments thereof.

[0050] The term "lipase or fragment thereof" includes naturally occurring lipases or fragment thereof, and also includes artificially modified lipases or fragment thereof. Artificially modified lipases or fragments thereof are, for example, variants or functionally active fragments of lipases. Therefore, the terms "fraction of lipase," "fraction thereof" associated with lipases, and "functionally active fragment of lipase" are used synonymously herein. A "variant or functionally active fragment thereof" associated with the lipase of the present invention means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the lipase. Such variants include naturally occurring allelic variants and non-naturally occurring variants. The addition, deletion, substitution, and derivation of one or more amino acids are considered, 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. Lipase fragments, as defined herein, typically possess the same functional properties as the lipase (i.e., the full-length enzyme from which the fragment originates) and include at least a cap domain and a substrate-binding region. Lipase fragments typically contain 100 to 450 amino acids, preferably 150 to 400 amino acids, more preferably 200 to 350 amino acids. Preferred lipases or fragments thereof are lipases or fragments extracted from the pancreas, more preferably from porcine pancreas, and even more preferably lipases or fragments thereof comprised of pancreatic enzymes. In a preferred embodiment, the lipase or fragment thereof is recombinant human pancreatic lipase (HRL) or a fragment thereof, or porcine pancreatic lipase or a fragment thereof, preferably recombinant human pancreatic lipase (HRL) or a fragment thereof, more preferably full-length recombinant human pancreatic lipase (HRL).

[0051] The term "protease or fragment thereof" includes naturally occurring proteases or fragments thereof, and also includes artificially modified proteases or fragments thereof. Artificially modified proteases or fragments thereof are, for example, variants or functionally active fragments of proteases. Therefore, the terms "fraction of a protease," "fraction thereof" associated with a protease, and "functionally active fragment of a protease" are used synonymously herein. A "variant or functionally active fragment thereof" associated with the protease of the present invention 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 non-naturally occurring variants. The addition, deletion, substitution, and derivatization of one or more amino acids are considered, 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 generally have the same functional properties as the protease from which they are derived. Fragments of proteases 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 extracted from the pancreas of pigs, and even more preferably proteases or fragments thereof comprised of pancreatic enzymes.

[0052] The term "amylase or a fragment thereof" includes naturally occurring amylases or fragments thereof, and also includes artificially modified amylases or fragments thereof. Artificially modified amylases or fragments thereof are, for example, variants or functionally active fragments of amylases. Therefore, the terms "fraction of amylase," "fraction thereof" in relation to amylases, and "functionally active fragment of amylase" are used synonymously herein. A "variant or functionally active fragment thereof" in relation to the amylase of the present invention means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the amylase. Such variants include naturally occurring allelic variants and non-naturally occurring variants. The addition, deletion, substitution, and derivation of one or more amino acids are considered, 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% sequence identity, even more preferably at least about 95% sequence identity, and most preferably at least about 98% sequence identity. The amylase fragments defined herein generally have the same functional properties as the amylase from which they are derived. Fragments of amylase 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 extracted from the pancreas of pigs, and even more preferably amylases or fragments thereof contained in pancreatic enzymes.

[0053] As used herein, the term "pancreatin" is also referred to as and is used interchangeably with "pancreatic enzyme" to mean a pancreatic enzyme derived from the porcine pancreas and containing lipase or fragments thereof, protease or fragments thereof, and amylase or fragments thereof. As used herein, the term "pancreatin" also includes formulated pancreatic enzymes, such as capsules containing pancreatic enzymes, for example, Zenpep®.

[0054] 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 ammonia-lyase family (EC 4.3.1.23, EC 4.3.1.24, and EC 4.3.1.25), which also includes histidine and tyrosine ammonia-lyases. PAL is sometimes also referred to as phenylalanine / tyrosine ammonia-lyase because some PALs can use both tyrosine and phenylalanine as substrates. 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 contain the cofactor 3,5-dihydro-5-methylene-4H-imidazol-4-one (MIO). This cofactor may be required for catalytic activity and is formed through the cyclization and dehydration of the conserved active site Alal67-Serl68-Glyl69 tripeptide fragment.

[0055] When used to refer to phenylalanine ammonia-lyase or fragments thereof as used herein, the terms "engineered" and "non-natural" refer to natural or naturally occurring forms of phenylalanine ammonia-lyase or fragments thereof that are modified in a manner not found in nature. The term "engineered phenylalanine ammonia-lyase or fragments thereof" does not include or encompass "wild-type" and "naturally occurring" phenylalanine ammonia-lyase or fragments thereof. As used herein, "wild-type" and "naturally occurring" refer to forms of phenylalanine ammonia-lyase or fragments thereof found in nature. For example, wild-type phenylalanine ammonia-lyase or fragments thereof are polypeptides present in organisms that can be isolated from natural sources and have not been intentionally modified by human intervention. Engineered PAL or fragments thereof are, for example, variants or functionally active fragments of engineered phenylalanine ammonia-lyase. Therefore, the terms "fraction of engineered phenylalanine ammonia-lyase," "fraction thereof" in relation to engineered phenylalanine ammonia-lyase, and "functionally active fragment of engineered phenylalanine ammonia-lyase" are used synonymously herein. The term "variant or functionally active fragment thereof" in relation to the engineered phenylalanine ammonia-lyase of the present invention refers to a fragment or variant (e.g., an analogue, derivative, or mutant not found in nature) that performs the same or improved physiological functions as wild-type phenylalanine ammonia-lyase. The addition, deletion, substitution, and derivatization of one or more amino acids are considered, provided that the modification does not result in a loss of functional activity of the fragment or variant. The PAL fragment comprises a homotetramerase, wherein at least one monomer of the homotetramerase, preferably all four monomers, typically contains 100 to 550 amino acids, preferably 200 to 500 amino acids, and more preferably 300 to 450 amino acids. "Improved physiological function" or "improved enzymatic properties" means that the engineered PAL exhibits any improved enzymatic properties compared to a reference PAL polypeptide such as a wild-type PAL polypeptide. Improved properties include, but are not limited to, the following: increased protein surface area; increased thermal activity; increased thermal stability; increased pH activity; increased stability; increased enzyme activity; increased substrate specificity and / or affinity; increased specific activity; increased resistance to substrate and / or end-product inhibition; increased chemical stability; improved chemoselectivity; improved solvent stability; increased tolerance to acidic pH; increased tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis); reduced aggregation; increased solubility; reduced immunogenicity; and altered temperature profile. The preferred engineered phenylalanine ammonia-lyase or fragment thereof of the present invention is the engineered phenylalanine ammonia-lyase described in WO2018 / 148633 A1. In a preferred embodiment, the engineered phenylalanine ammonia-lyase or fragment thereof comprises or is composed of an amino acid sequence having at least 90%, at least 95%, at least 96%, or at least 97% sequence identity with the sequence of SEQ ID NO: 1.In one embodiment, the engineered phenylalanine ammonia-lyase or a fragment thereof is SEQ ID NO: 2, 3, 4, or 5. In a particularly preferred embodiment, the engineered phenylalanine ammonia-lyase or a fragment thereof comprises a polypeptide as shown in SEQ ID NO: 5.

[0056] 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 intended engineered phenylalanine ammonia-lyase is covered by the protective layer, although typically at least 70% is covered, thus 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 intended engineered phenylalanine ammonia-lyase 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 intended engineered phenylalanine ammonia-lyase is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the engineered phenylalanine ammonia-lyase is covered by a protective layer. In a more particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the engineered phenylalanine ammonia-lyase is covered by a protective layer in which the active site is not covered.

[0057] As used herein, the term "fully encapsulated engineered phenylalanine ammonia-lyase" means that the engineered phenylalanine ammonia-lyase according to the present invention is completely, i.e., 100% covered by the protective layer, meaning that the active site is also covered. Preferably, the engineered phenylalanine ammonia-lyase or a fragment thereof according to the present invention is completely, i.e., 100% covered by the protective layer, meaning that the active site is also covered.

[0058] As used herein, the term "engineered phenylalanine ammonia-lyase that is at least partially embedded" means that the engineered phenylalanine ammonia-lyase is at least partially embedded and can be completely embedded by a protective layer. Therefore, "engineered phenylalanine ammonia-lyase that is at least partially embedded" means that the protective layer covers about 30% to 100% of the engineered phenylalanine ammonia-lyase or fragments thereof, 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%, wherein the active site is preferably covered.

[0059] The term "disaccharidase or fragment thereof" includes naturally occurring disaccharidases or fragment thereof, and also includes artificially modified disaccharidases or fragment thereof. Disaccharidases are glycoside hydrolases, enzymes that break down certain types of sugars called disaccharides into simpler sugars called monosaccharides. In humans, disaccharidases are primarily produced in a region of the small intestinal wall called the brush border. Disaccharidases include, for example, lactase, maltase, isomaltase, trehalase, and sucrase (also known as invertase). Artificially modified disaccharidases or fragments thereof are, for example, variants or functionally active fragments of disaccharidases. Therefore, the terms "fraction of disaccharidase," "fraction thereof" associated with a disaccharidase, and "functionally active fragment of a disaccharidase" are used synonymously herein. "Variant or functionally active fragment thereof" associated with the disaccharidase of the present invention means that the fragment or variant (such as an analog, derivative, or mutant) is capable of performing the same physiological function as the disaccharidase. Such variants include naturally occurring allelic variants and non-naturally occurring variants. The addition, deletion, substitution, and derivation of one or more amino acids are permissible, 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 moiety of the disaccharidase, 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 disaccharidase fragments defined herein generally have the same functional properties as the disaccharidase from which they are derived. Disaccharidase fragments typically contain 100 to 1000 amino acids, preferably 300 to 800 amino acids, more preferably 500 to 700 amino acids.

[0060] As used herein, the term "partially embedded disaccharidase" means that the disaccharidase is covered by a protective layer, and therefore, the disaccharidase is not completely embedded in the protective layer. In one embodiment, less than 50% of the target disaccharidase is covered by the protective layer, although typically at least 70% is covered, thus 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 target disaccharidase 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 target disaccharidase is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the target disaccharidase is covered by the protective layer. In a more particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the target disaccharidase is covered by a protective layer in which the active site is not covered.

[0061] As used herein, the term "fully encapsulated disaccharidase" means that the disaccharidase according to the present invention is completely, i.e., 100% covered by the protective layer, i.e., the active site is also covered.

[0062] As used herein, the term "at least partially embedded disaccharidase" means that the disaccharidase is at least partially embedded and can be completely embedded by the protective layer. Thus, "at least partially embedded disaccharidase" means that the protective layer covers about 30% to 100% of the disaccharidase or fragments thereof, 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%, wherein the active site is preferably covered.

[0063] As used herein, the term "partially embedded protein" refers to a protein, such as a lipase or a fragment thereof, a protease or a fragment thereof, and an amylase or a fragment thereof, that is not completely covered by the protective layer, and therefore is not completely embedded in the protective layer. In one embodiment, less than 50% of the protein, such as a lipase or a fragment thereof, a protease or a fragment thereof, and an amylase or a fragment thereof, is covered by the protective layer, although typically at least 70% is covered, thus improving protein protection. 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 a lipase or a fragment thereof, a protease or a fragment thereof, and an amylase or a fragment thereof, is covered by the protective layer. In another preferred embodiment, about 70% to about 95%, more preferably about 80% to about 95%, even more preferably about 90% to about 95%, and most preferably about 90% to about 95%, 96%, 97%, 98%, or 99% of the protein, such as a lipase or a fragment thereof, a protease or a fragment thereof, and an amylase or a fragment thereof, is covered by the protective layer. In a particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the proteins, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are covered by a protective layer. In a more particularly preferred embodiment, about 70%, particularly about 80%, more particularly about 90%, and most particularly about 95% of the proteins, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are covered by a protective layer, wherein the active sites are not covered.

[0064] As used herein, the term "fully encapsulated protein" means that the proteins of the present invention, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are completely, i.e., 100% covered by the protective layer, meaning that the active site is also covered. Preferably, the lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof according to the present invention are completely, i.e., 100% covered by the protective layer, meaning that the active site is also covered.

[0065] As used herein, the term "at least partially embedded protein" means a protein, such as a lipase or a fragment thereof, a protease or a fragment thereof, and an amylase or a fragment thereof, that is at least partially embedded and can be completely embedded by a protective layer. Therefore, "at least partially embedded protein" means that the protective layer covers about 30% to 100% of the protein or a fragment thereof, such as a lipase or a fragment thereof, a protease or a fragment thereof, and an amylase or a fragment thereof, 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%, wherein the active site is preferably covered.

[0066] As used herein, the term "agent which interacts with the lid domain of the lipase or a fragment thereof" refers to an agent that typically binds to the lid domain of the lipase or a fragment thereof and / or binds to the region surrounding the lid domain, thereby causing the lid domain to convert the lipase or fragment thereof to an open conformation and / or maintain the open conformation of the lipase or fragment thereof. The lid domains of lipases are typically amphiphilic; in the closed conformation, their hydrophilic sides face the solvent, while their hydrophobic sides point towards the catalytic pocket (Brocca S., Secundo F., Ossola M., Alberghina L., Carrea G., Lotti M. (2003). The lid sequence affects *Candida rhombifolia* (…). Candida rugosa (Activity and specificity of lipase isoenzymes. Protein Sci. 12, 2312-2319.10.1110 / ps.0304003). When a lipase transitions to its open conformation, the hydrophobic surface becomes exposed and acts on the substrate-binding region. Preferably, agents that interact with the cap domain of a lipase or fragment thereof cause the lipase or fragment to be locked in its active conformation. When locked in its active conformation, the lipase is usually fully activated. Agents that interact with the cap domain of a lipase or fragment thereof to cause the lipase or fragment thereof to be in an open conformation include colipases or fragments thereof, colipase mimic peptides, and amphiphilic molecules.

[0067] As used herein, the term "amphiphilic molecule" refers to a molecule, such as a compound, that contains both polar (water-soluble) and nonpolar (water-insoluble) moieties in its structure. It can also refer to molecules, such as compounds, that possess both hydrophobic and hydrophilic regions. Amphiphilic molecules include bile salts, phospholipids, and nonionic detergents.

[0068] The terms "open conformation" or "open conformation of a lipase or a fragment thereof" are used interchangeably herein and refer to a conformation of a lipase or a fragment thereof in which the substrate can enter the active site of the lipase and be converted. In a closed conformation, the substrate enters the active site of the lipase or a fragment thereof, and its conversion is limited or impossible. The conformation of a lipase or a fragment thereof can be determined by X-ray crystallography, enzyme activity studies, site-directed spin labeling (SDSL) methods, and electron paramagnetic resonance (EPR). That is, whether the lipase is in an open or closed conformation.

[0069] As used herein, the term "colipase or fragment thereof" includes natural colipases or fragments thereof, as well as artificially modified colipases or fragments thereof. Artificially modified colipases or fragments thereof are, for example, variants or functionally active fragments of lipases. "Variants or functionally active fragments thereof" in relation to the colipase of the present invention 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 non-naturally occurring variants. The addition, deletion, substitution, and derivatization of one or more amino acids are considered, 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 colipase fragment as defined herein has the same functional properties as the colipase from which it is derived. A preferred colipase is Uniprot number: P02703.

[0070] As used herein, the term "colipase mimic peptide" refers to a peptide consisting of 10 to 40 amino acids that allows specific amino acid residues to be geometrically positioned in the correct order to interact with the amino acids in the pancreatic lipase structure, inducing the extension of the lipase conformation and the opening of the cap, thereby possessing the same functional properties as colipase. The colipase mimic peptide that can be used in this invention is preferably the peptide shown in SEQ ID NO: 6.

[0071] As used herein, the term "bile salt" refers to bile acids conjugated with taurine or glycine, including sodium taurocholate, sodium glycine deoxycholate, sodium taurocholate, sodium glycine chenodeoxycholate, and sodium taurocholate.

[0072] As used herein, the term "nonionic detergent" refers to a surfactant including tetraethylene glycol monooctyl ether, octyl-β-D-glucopyranoside, N,N-dimethyldodecylamine-N-oxide, and β-octylglucomaloside.

[0073] As used herein, the term "phospholipid" refers to a class of lipids whose molecules have a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, linked by alcohol residues (usually glycerol molecules). Phospholipids include lecithin and lysophospholipid.

[0074] As used herein, the term "functional component" refers to a component that retains its characteristic and functional properties after being fixed to the surface of a protective layer. In the context of this invention, a functional component is a polymer comprising repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0075] As used herein, the term "polymer comprising repeating units, wherein each repeating unit contains at least one amino group" refers to a polymer comprising several repeating units (monomers), wherein each repeating unit contains at least one amino group. Preferred polymers comprise several repeating units (monomers), wherein each repeating unit contains one amino group, particularly a primary amino group.

[0076] As used herein, the term "polymer comprising repeating units, wherein each repeating unit contains at least one thiol group" refers to a polymer comprising a plurality of repeating units (monomers), wherein each repeating unit contains at least one thiol group. Preferred polymers comprise a plurality of repeating units (monomers), wherein each repeating unit contains one thiol group.

[0077] As used herein, the term "polycarbophil-cysteine ​​conjugate" refers to a conjugate containing a cysteine ​​residue covalently linked to polycarbophil. Such conjugates can be generated as described in Bernkop-Schnurch and Thaler, 2000, Journal of Pharmaceutical Sciences 89(7):901-9.

[0078] 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 that can 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.

[0079] As used herein, the term "polycysteine" may consist of L-cysteine ​​or D-cysteine, preferably L-cysteine, and preferably contains 2 to 30 cysteine ​​residues, more preferably 2 to 5 cysteine ​​residues.

[0080] As used herein, the term "polyglucosamine" refers to a linear amino-polysaccharide composed of D-glucosamine and N-acetyl-D-glucosamine units linked by (1-4) glycosidic bonds. Polyglucosamines contain free amine (-NH2) groups and can be characterized by the ratio of N-acetyl-D-glucosamine units to D-glucosamine units, expressed as the degree of deacetylation (DDA) of a fully acetylated polymeric chitosan. Preferred polyglucosamines of this invention are selected from chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan, or derivatives thereof. Chitosan or its derivatives are most preferred.

[0081] As used herein, the term "chitosan or a derivative thereof" refers to chitosan or chitosan derivatives thereof, including its salts, preferably having a molecular weight of 2,000 Da or greater, more preferably in the range of 25,000 to 2,000,000 Da, more preferably about 50,000 to 350,000 Da, and most preferably about 50,000 to 190,000 Da or 190,000 to 310,000 Da. The term chitosan derivatives include esters, ethers, or other derivatives formed by the reaction of an acyl or alkyl group with an OH group. Examples are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Suitable derivatives are shown, for example, in GAE Roberts, Chitin Chemistry, MacMillan Press Ltd, London, 1992. Suitable chitosan salts include nitrates, phosphates, sulfates, xanthates, hydrochlorides, glutamates, lactates, and acetates.

[0082] In a first aspect, 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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension; (b) Immobilizing a protein or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect the protein or fragment thereof fixed on the solid support, wherein a linker or portion thereof that did not connect the solid support to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof; and optionally (d) Fixing a functional component onto the surface of a protective layer, wherein the functional component fixed onto the surface of the protective layer is a polymer comprising repeating units, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0083] In one embodiment, a method of generating a composition includes the steps of: wherein the composition comprises 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 the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension; (b) Immobilizing a protein or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid carrier to protect the protein or fragment thereof fixed on the solid carrier, wherein a linker or portion thereof that did not connect the solid carrier to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof.

[0084] In other aspects, 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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension; (b) Immobilizing a protein or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect the protein or fragment thereof fixed on the solid support, wherein a linker or portion thereof that did not connect the solid support to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof; and optionally (d) 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, provided that the protein or a fragment thereof is not a lipase or a fragment thereof, a protease or a fragment thereof, an amylase or a fragment thereof, a pancreatic enzyme or a protein or a fragment thereof composed of pancreatic enzymes, an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof, or a disaccharidase or a fragment thereof, preferably provided that the protein or a fragment thereof is not a lipase or a fragment thereof, a protease or a fragment thereof, an amylase or a fragment thereof, a pancreatic enzyme or a protein or a fragment thereof composed of pancreatic enzymes, an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof, or a disaccharidase or a fragment thereof as described herein.

[0085] In one embodiment, a method of generating a composition includes the steps of: wherein the composition comprises 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 the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension; (b) Immobilizing a protein or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid carrier to protect a protein or fragment thereof fixed on the solid carrier, wherein the protective layer is covalently bound to the protein or fragment thereof by a linker or portion thereof that did not connect the solid carrier to the protein or fragment thereof in step (b), provided that the protein or fragment thereof is not a lipase or a fragment thereof, a protease or a fragment thereof, an amylase or a fragment thereof, a pancreatic enzyme or a protein or fragment thereof composed of pancreatic enzymes, an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof, or a disaccharidase or a fragment thereof, preferably provided that the protein or fragment thereof is not a lipase or a fragment thereof, a protease or a fragment thereof, an amylase or a fragment thereof, a pancreatic enzyme or a protein or fragment thereof composed of pancreatic enzymes, an engineered phenylalanine ammonia-lyase (PAL) or a fragment thereof, or a disaccharidase or a fragment thereof as described herein.

[0086] 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 binding. Non-covalent binding includes pp (aromatic) interactions, van der Waals interactions, H-bonding interactions, and electrostatic interactions, such as 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 the solid support by covalent binding or by covalent binding via a linker.

[0087] Solutions of proteins or fragments thereof are typically contained in a buffer solution. Commonly used buffers include phosphates, chlorides, citrates, MES, MOPS, HEPES, PIPES, ACES, or mixtures thereof. The solution may additionally contain sugar alcohols or nonionic surfactants, as described herein. Solutions of proteins or fragments thereof can be prepared, for example, by dissolving the protein or fragment in water to reconstitute a stock buffer for the protein or fragment.

[0088] In one embodiment, the solid support is selected from organic particles, inorganic particles, organic-inorganic particles, self-assembled organic particles, silica particles, gold particles, titanium particles, and preferably silica particles, more preferably silica nanoparticles (SNPs). Particle size is typically 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. When the solid support is monodisperse particles, the particle size is typically from 1 nm to 1000 nm, preferably from 10 nm to 100 nm, and particularly about 50 nm. When the solid support is polydisperse particles, the particle size is typically from 1 nm to 1000 μm, preferably from 10 nm to 100 μm, and particularly 50 nm to 50 μm. In one embodiment, the composition comprises a solid carrier, wherein the solid carrier comprises at least 4%, preferably at least 10%, more preferably at least 20%, even more preferably 4% to 50%, particularly 10% to 40%, even more particularly 25% to 35%, even more particularly 15% to 25% of immobilized protein or fragments thereof per dry weight of solid carrier.

[0089] Typically, monodisperse or polydisperse particles, preferably monodisperse particles, are used as the solid carrier in this invention. In a preferred embodiment, the monodisperse particles are spherical monodisperse particles. In other preferred embodiments, the polydisperse particles are non-spherical polydisperse particles.

[0090] Solid carriers are typically provided in suspension form. The suspension of the solid carrier can be, for example, in water, a buffer solution, or a nonionic surfactant or a mixture thereof, preferably in a mixture of water and a nonionic surfactant. The nonionic surfactant is typically selected from ethoxylated sorbitol esters such as PEG-40 sorbitol diisostearate, polysorbate 80 (PS80), polysorbate 20 (PS20), polysorbate 40 (PS40), and polysorbate 60 (PS60); and block copolymers such as poloxamer 124, poloxamer 188, poloxamer 331, and poloxamer 40. 7. Fatty acid ethoxylates such as PEG-5 oleate, PEG-8 stearate, polyoxyethylene 40 stearate, polyoxyethylene 15 hydroxy stearate; fatty alcohol ethoxylates such as stearyl alcohol polyether (steareth) 40; fatty acid esters such as palmitic acid ascorbate, beeswax, polyglycerol 3-oleate, propylene glycol monocaprylate, propylene glycol monolaurate; fatty alcohols such as cetearyl alcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol; glycerides; polyethylene glycol-modified triglycerides; sugar esters, preferably polysorbate, more preferably polysorbate 80 (PS80). The buffer solutions that can be used in the method of this invention are 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)propyl-2-yl]amino]ethanesulfonic acid, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, 3-(N,N-bis[2-hydroxyethyl]propanesulfonic acid, etc. [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, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid.

[0091] In one embodiment, an agent that interacts with the capping domain of the lipase or a fragment thereof is added to a suspension of the solid support. Preferably, the agent that interacts with the capping domain of the lipase or a fragment thereof is added to the suspension of the solid support together with the lipase or a fragment thereof, the protease or a fragment thereof, and the amylase or a fragment thereof. Before immobilizing the lipase or a fragment thereof, the protease or a fragment thereof, and the amylase or a fragment thereof onto the solid support, the lipase or a fragment thereof, the protease or a fragment thereof, and the amylase or a fragment thereof may be added to the suspension of the solid support in the form of trypsin.

[0092] Typically, proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or pancreatic enzymes or solutions thereof, are immobilized on a solid carrier by adding a suspension of the solid carrier. In a preferred embodiment, proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or pancreatic enzymes or solutions thereof, are immobilized on the solid carrier by providing a suspension of the solid carrier and providing a solution containing proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or pancreatic enzymes or solutions thereof, wherein the suspension of the solid carrier is incubated with a solution containing proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or pancreatic enzyme preparations or solutions thereof, to allow the proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, to bind to the surface of the solid carrier. In a more preferred embodiment, the protein or fragments thereof, such as lipase, protease or fragments thereof, and amylase or fragments thereof, or pancreatase or solution thereof, are immobilized on the solid support by providing a suspension of the solid support, a solution containing a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, or pancreatase or solution thereof, and a solution containing an agent that interacts with the capping domain of the lipase or fragment thereof, wherein the suspension of the solid support is incubated together with a solution containing a protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, or pancreatase or solution thereof, and a solution containing an agent that interacts with the capping domain of the lipase or fragment thereof, to allow the protein or fragment thereof, such as lipase or fragment thereof, protease or fragment thereof, and amylase or fragment thereof, to bind to the surface of the solid support.

[0093] In other preferred embodiments, the protein or its fragments are immobilized on the solid support by a linker, preferably a bifunctional crosslinker, that binds to the protein or its fragments and the surface of the solid support, or preferably a linker that covalently binds to the protein or its fragments and the surface of the solid support, or preferably a bifunctional crosslinker.

[0094] In one embodiment, the surface of a solid support is modified to introduce molecular or functional chemical groups as anchoring sites, i.e., as anchoring sites for proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, or as anchoring sites for binders that link proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, to the solid support. Preferably, the anchoring sites are amine functional chemical groups or portions. As a non-limiting example, an amino-modified surface of the 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 with a silica surface with an aminosilane, such as APTES. Therefore, in a preferred embodiment, the solid support is a solid support with a silica surface containing an amino-modified surface, more preferably a solid support obtained by reacting a solid support with a silica surface with an aminosilane (e.g., with APTES). Such modified carriers can form amide bonds between proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, and amine groups on the surface of the carrier material, or between the linker and amine groups on the surface of the carrier material. In one embodiment, the introduced molecules or functional chemical groups are uniformly distributed on the surface of the solid carrier as anchoring sites.

[0095] In one embodiment, the agent interacting with the cap domain of a lipase or a fragment thereof is selected from colipase or a fragment thereof, colipase mimic peptides, and amphiphilic molecules. Preferably, the agent interacting with the cap domain of a lipase or a fragment thereof is selected from colipase or a fragment thereof, colipase mimic peptides, and bile salts; more preferably, it is selected from colipase or a fragment thereof, colipase mimic peptides, and sodium taurocholate; even more preferably, it is selected from colipase or a fragment thereof, colipase mimic peptides as shown in SEQ ID NO:6, and sodium taurocholate. Most preferably, the agent interacting with the cap domain of a lipase or a fragment thereof is a bile salt, particularly sodium taurocholate.

[0096] In one embodiment, an agent that interacts with the capping domain of a lipase or a fragment thereof specifically interacts with the capping domain of the lipase or a fragment thereof, causing the lipase or a fragment thereof to switch to an open conformation and / or maintain an open conformation.

[0097] 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 fragments thereof immobilized on the surface of the solid carrier are in an open conformation.

[0098] In some embodiments, the protective layer has a defined thickness of about 1 to about 200 nm, typically 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, particularly about 1 to about 15 nm. The most preferred defined thickness is about 1 to about 10 nm. In some embodiments, the layer has a defined thickness of about 5 to about 100 nm, preferably about 5 to about 50 nm, more preferably about 5 to about 25 nm, even more preferably about 5 to about 20 nm, particularly about 5 to about 15 nm. The most preferred defined thickness is about 5 to about 10 nm. The protective layer is typically porous with a pore size of 1 to 100 nm, preferably 1 to 20 nm.

[0099] In one embodiment, proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are partially embedded in a protective layer. In a preferred embodiment, proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are at least partially embedded in a protective layer. In a more preferred embodiment, proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are completely embedded in a protective layer.

[0100] In one embodiment, the protective layer embeds a solid carrier and embeds proteins or fragments thereof immobilized on the surface of the solid carrier, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof. In one embodiment, functional components immobilized on the surface of the protective layer are not embedded by the protective layer. Preferably, the protective layer completely embeds the solid carrier and completely embeds the proteins or fragments thereof immobilized on the surface of the solid carrier, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof. More preferably, the protective layer completely embeds the solid carrier and completely embeds the proteins or fragments thereof immobilized on the surface of the solid carrier, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, and functional components immobilized on the surface of the protective layer are not embedded by the protective layer. If the protective layer completely embeds the solid carrier and completely embeds the proteins or fragments thereof immobilized on the surface of the solid carrier, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, then the proteins or fragments thereof, such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, are completely, i.e., 100% covered by the protective layer, that is, the active sites are also covered, and the solid carrier is completely, i.e., 100% covered by the protective layer.

[0101] In a preferred embodiment, the lipase or a fragment thereof, the protease or a fragment thereof, and the amylase or a fragment thereof used in the present invention are contained in a pancreatic enzyme. In a more preferred embodiment, the pancreatic enzyme is used to immobilize the lipase or a fragment thereof, the protease or a fragment thereof, and the amylase or a fragment thereof on the surface of a solid carrier.

[0102] The thickness of the protective layer can be measured using microscopes such as scanning electron microscopes (SEM), transmission electron microscopes (TEM), scanning probe microscopes (SPM), light scattering methods, or elliptic polarization techniques.

[0103] The compositions of the present invention are typically produced in a reaction vessel such as a reactor. The formation of the protective layer is generally carried out by the formation of a corresponding protective layer from building units, wherein the building units construct the protective layer during a polycondensation reaction. Polycondensation can be carried out in various solvents, preferably in aqueous solutions. If suitable, polycondensation can be easily controlled and stopped, allowing for the achievement of a defined thickness of the protective layer. The selection of building units that can be used to construct the protective layer may depend on the known structures of proteins such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, in order to adapt the affinity of the protective layer according to optimal and / or desired parameters. As building units for the protective layer, both structural building units and protective building units are typically used to construct the protective layer. A structural building unit that can be used is, for example, tetraethyl orthosilicate (referred herein to as “TEOS” or “T”). The protective building blocks that can be used are, for example, 3-aminopropyltriethoxysilane (referred to herein as “APTES” or “A”), propyltriethoxysilane (referred to herein as “PTES” or “P”), isobutyltriethoxysilane (referred to herein as “IBTES”), hydroxymethyltriethoxysilane (referred to herein as “HTMEOS” or “H”), benzyltriethoxysilane (referred to herein as “BTES”), ureopropyltriethoxysilane (referred to herein as “UPTES”), or carboxyethyltriethoxysilane (referred to herein as “CETES”). The structural building blocks are typically precursors of inorganic silica capable of forming four covalent bonds in the formed layers. The protective building blocks are typically organosilanes carrying an organic moiety conferred with the ability to interact with proteins such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof. Preferred structural building blocks are tetravalent silanes, particularly tetraalkoxysilanes. Preferred protective building blocks are trivalent silanes, particularly trialkoxysilanes. More preferably, the structural building blocks are mixtures of tetravalent and trivalent silanes, particularly mixtures of tetraalkoxysilanes and trialkoxysilanes. Even more preferably, the structural building blocks are selected from tetraethyl orthosilicate, tetra-(2-hydroxyethyl)silane, and tetramethyl orthosilicate.Even more preferred protective building blocks are selected from carboxyethyl silanetriol, benzyl silane, propyl silane, isobutyl silane, n-octyl silane, hydroxysilane, bis(2-hydroxyethyl)-3-aminopropyl silane, aminopropyl silane, ureopropyl silane, (N-acetylglycyl)-3-aminopropyl silane, hydroxy(polyvinyloxy)propyl]triethoxysilane, especially selected from benzyltriethoxysilane (BTES), propyltriethoxysilane, isobutyltriethoxysilane, n-octyltriethoxysilane, hydroxymethyltriethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 3-aminopropyltriethoxysilane, ureopropyltriethoxysilane, and (N-acetylglycyl)-3-aminopropyltriethoxysilane. Or selected from benzyltrimethoxysilane, propyltrimethoxysilane, isobutyltrimethoxysilane, n-octyltrimethoxysilane, hydroxymethyltrimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrimethoxysilane, aminopropyltrimethoxysilane, ureopropyltrimethoxysilane, (N-acetylglycyl)-3-aminopropyltrimethoxysilane, or selected from benzyltrihydroxyethoxysilane, propyltrihydroxyethoxysilane, isobutyltrihydroxyethoxysilane, n-octyltrihydroxyethoxysilane, hydroxymethyltrihydroxyethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltrihydroxyethoxysilane, aminopropyltrihydroxyethoxysilane, ureopropyltrihydroxyethoxysilane, (N-acetylglycyl)-3-aminopropyltrihydroxyethoxysilane.

[0104] Particularly preferred building blocks are TEOS as structural building blocks, and APTES, BTES, and / or HTMEOS, with APTES and / or BTES being preferred as protective building blocks. Specifically, TEOS is used as the structural building block and APTES and / or BTES as the protective building blocks to construct the protective layer.

[0105] If a linker is used, the reaction time between the building blocks and the solid carrier carrying the immobilized enzyme can depend on the length of the linker and the size of the protein, such as lipase or a fragment thereof, protease or a fragment thereof, and amylase or a fragment thereof. The reaction typically proceeds for 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 or about 20°C at about 5 to about 25°C. The formation of the protective layer can be stopped by actively halting the polycondensation reaction, for example by removing unreacted building blocks, such as through a washing step; or by self-stopping the polycondensation reaction caused by a limited amount of building blocks.

[0106] In other preferred embodiments, the protein or its fragments, such as lipase or its fragments, protease or its fragments and amylase or its fragments, are immobilized on the solid support by introducing molecules as described above for proteins or fragments thereof, such as lipase or its fragments, protease or its fragments and amylase or its fragments, as anchoring sites, and by using a linker, preferably a crosslinking linker, that binds to the anchoring sites and proteins such as lipase or its fragments, protease or its fragments and amylase or its fragments, to at least partially modify the surface of the solid support.

[0107] In one embodiment, the introduced molecules and / or binders serving as anchoring points are uniformly distributed on the surface of the solid carrier.

[0108] Step (a) of the method is typically performed by providing a solid support in a suspension of water, a buffer solution, or a suspension of a nonionic surfactant or a mixture thereof, preferably in a suspension of water and / or a nonionic surfactant, more preferably in a suspension of water and / or a nonionic surfactant (wherein no buffer solution is present in the suspension), even more preferably in a suspension of a mixture of water and a nonionic surfactant, particularly in a suspension of a mixture of water and a nonionic surfactant (wherein no buffer solution is present in the suspension). In step (b) of the method of the invention, the immobilization of the protein or its fragments on the solid support is typically performed by adding a solution of the protein or its fragments to the suspension of the solid support. Preferably, a linker connecting the solid support to the protein or its fragments is added to the suspension of the solid support before adding the solution of the protein or its fragments to the suspension of the solid support. In a preferred embodiment, the immobilization of the protein or its fragments on the solid support is performed by providing a suspension of the solid support and adding a solution of the protein or its fragments, wherein the suspension containing the solution of the protein or its fragments is incubated to allow the protein to bind to the surface of the solid support. In a more preferred embodiment, the immobilization of the protein or its fragments on the solid support in step b) is performed by i) adding a linker to a suspension containing the solid support provided in step (a), and ii) adding a solution of the protein or its fragments to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or its fragments, preferably wherein the suspension containing the solution of the protein or its fragments is incubated to allow the protein to bind to the surface of the solid support. In one embodiment, before adding the solution of the protein or its fragments, building blocks of the protective layer, preferably monomers of the building blocks of the protective layer, more preferably organosilanes, even more preferably triethoxysilanes, particularly APTES, are added to the suspension containing the solid support and the linker. In a preferred embodiment, the surface of the solid support is at least partially modified to improve the immobilization of the protein or its fragments on the solid support. In particular, the surface of the solid support is at least partially modified before immobilizing the protein or its fragments. As described above, the surface of the solid support can be at least partially modified by adding molecules that serve as anchoring sites for the protein or its fragments to the surface of the solid support.

[0109] Typically, after each of the above-described addition steps, a suspension containing a solid carrier is incubated to allow reactions, such as between the solid carrier and / or molecules serving as anchoring points, between the solid carrier and the linker, and between the solid carrier containing the linker and the protein or fragment thereof, respectively, such that the protein or fragment thereof is attached to the solid carrier via the linker, preferably by covalent bonding, preferably to the surface of the solid carrier, thereby immobilizing the protein or fragment thereof on the solid carrier.

[0110] In one embodiment, in step (b), the protein or a fragment thereof is immobilized on the solid support by linking the solid support to the protein or the protein or the fragment thereof via a linker, wherein the solid support is linked to the protein or the fragment thereof by covalent bonding between the linker and the solid support and between the linker and the protein or the fragment thereof. Preferably, the linker links the surface of the solid support to the protein or the fragment thereof via covalent bonding. More preferably, the linker is added to the suspension of the solid carrier in step (b) i) in an amount that is a molar excess of the protein or its fragments in the suspension containing the solid carrier and the linker added in step (b) ii). Preferably, the linker is added to the suspension of the solid carrier in step (b) in an amount that is 1 to 1000 times the molar excess of the protein or its fragments in the suspension containing the solid carrier and the linker added in step (b) ii). More preferably, the linker is added to the suspension of the solid carrier in step (b) in an amount that is 2 to 300 times the molar excess of the protein or its fragments in the suspension containing the solid carrier and the linker added in step (b) ii). Even more preferably, the linker is added to the suspension of the solid carrier in step (b) in an amount that is 4 to 250 times the molar excess of the protein or its fragments in the suspension containing the solid carrier and the linker added in step (b) ii).

[0111] In a preferred embodiment, the binder that did not link the solid carrier to the protein or its fragment in step (b) is present during step (c) when a protective layer is formed on the surface of the solid carrier. In other more preferred embodiments, the binder that did not link the solid carrier to the protein or its fragment in step (b) is not removed in or between steps (b) and (c). In a specific embodiment, the binder that did not link the solid carrier to the protein or its fragment in step (b) is not removed in or between steps (b) and (c), and a portion of the binder that did not link the solid carrier to the protein or its fragment in step (b) covalently binds the protective layer to the protein or its fragment in step (c). After the protein in ii) is added, the amount of binder that did not link the solid carrier to the protein or its fragment in step (b) is typically 30% to 70%, preferably 40% to 60%, and more preferably about 50%, of the binder added to the solid carrier in step (b). In one embodiment, there is no washing step between adding the linker to the suspension of the solid carrier provided in step (a) in step (b) (i) and adding the solution of the protein or a fragment thereof to the suspension containing the solid carrier and the linker in step (b) (ii). In one embodiment, there is no washing step between any of steps (a) to (c). In one embodiment, there is no washing step between adding the linker to the suspension of the solid carrier provided in step (a) in step (b) (i) and adding the protein or a fragment thereof to the suspension containing the solid carrier and the linker in step (b) (ii), and there is no washing step between any of steps (a) to (c).

[0112] In a preferred embodiment, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl pimelimidate, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated thiol, thiol-reactive 2-pyridyl dithiol, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[succinimide] propionate), DTBP (3,3'-dithiobispropionylimine dimethyl ester·2 HCl), DST (disuccinimidyl tartrate), sulfonyl-LC-SMPT (4-succinimidyl-6-methyl-a-(2-pyridyldithio)toluamide]hexanoate), SPDP (N-succinimidyl-3-(2-pyridyldithio)-propionate), LC-SPDP (succinimidyl-6-(3-[2-pyridyldithio]-propamido)hexanoate), SMPT (4-succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DDPPB (1,4-di-[3'-2'-pyridyldithio)-propionamide]butane), DTME (dithio-bismaleimide ethane), BMDB (1,4-bismaleimide-2,3-dihydroxybutane). More preferably, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated thiol groups (e.g., thiol-reactive 2-pyridine dithiol), and colipase mimic peptides, wherein the colipase mimic peptides may be functionalized with chemical groups capable of covalently binding to the surface of a solid carrier. In a more preferred embodiment, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[sulfosuccinimide] propionate), DTBP (3,3'-dithiobispropionine dimethyl ester·2HCl), DST (disuccinimide tartrate), and BMDB (1,4-bismaleimide-2,3-dihydroxybutane).More preferably, the crosslinking agent is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide) succinate, dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, and activated thiol groups (e.g., thiol-reactive 2-pyridinedithiol). Glutaraldehyde is most preferred.

[0113] In one embodiment, after the protective layer is formed, a solid carrier containing proteins such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, as well as the protective layer, can be stored. Storage is typically accomplished, for example, by washing the formed composition with a buffer and suspending or dissolving it in the buffer for the required storage time. In a preferred embodiment, the solid carrier containing proteins such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, as well as the protective layer, is stored at a constant temperature of 2 to 25°C. In other preferred embodiments, the solid carrier containing proteins such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, as well as the protective layer, is stored for 5 to 48 hours, preferably 10 to 30 hours. More preferably, the solid carrier containing proteins such as lipases or fragments thereof, proteases or fragments thereof, and amylases or fragments thereof, as well as the protective layer, is stored at a constant temperature of 2 to 25°C, preferably at room temperature for 10 to 30 hours.

[0114] A preferred method of the present invention is a method of 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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension, preferably wherein the solid carrier is provided in the form of a suspension in water and / or a nonionic surfactant, more preferably wherein the solid carrier is provided in the form of a suspension in a mixture of water and a nonionic surfactant; (b) Immobilizing a protein or a fragment thereof onto a solid support, wherein preferably the surface of the solid support is at least partially modified before immobilizing the protein or a fragment thereof onto the solid support, wherein i) a linker is added to a suspension of the solid support, or i) a linker is added to a suspension of the solid support after at least partially modifying the surface of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension comprising the solid support and the linker, wherein the linker links the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect the protein or fragment thereof fixed on the solid support, wherein a linker or portion thereof that did not connect the solid support to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof; and optionally (d) Fixing a functional component onto the surface of a protective layer, wherein the functional component fixed onto the surface of the protective layer is a polymer comprising repeating units, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0115] Another preferred method of the present invention is a method of 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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension, preferably wherein the solid carrier is provided in the form of a suspension in water and / or a nonionic surfactant, more preferably wherein the solid carrier is provided in the form of a suspension in a mixture of water and a nonionic surfactant; (b) Immobilizing a protein or a fragment thereof onto a solid support, wherein preferably the surface of the solid support is at least partially modified before the protein or a fragment thereof is immobilized onto the solid support, wherein i) a linker is added to a suspension of the solid support, or i) a linker is added to a suspension of the solid support after at least partially modifying the surface of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension comprising the solid support and the linker, wherein the linker links the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect the protein or fragment thereof fixed on the solid support, wherein a linker or portion thereof that did not connect the solid support to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof. In one implementation, the optional functional component binds to the mucus.

[0116] In one embodiment, a polymer comprising repeating units in which each repeating unit contains at least one amino group and / or at least one thiol group is a polymer comprising repeating units in which each repeating unit contains at least one amino group.

[0117] In one embodiment, a polymer comprising repeating units wherein each repeating unit contains at least one amino group and / or at least one thiol group is a polymer comprising repeating units wherein each repeating unit contains at least one thiol group.

[0118] In one embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, and amino-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a preferred embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, and polymeric APTES, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0119] In a more preferred embodiment, the polymer comprising repeating units is selected from polyglucosamines such as chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan or derivatives thereof; polymeric silanes-PEG-NH2; and amino-containing polymeric silanes, preferably polymeric APTES, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In an even more preferred embodiment, the polymer comprising repeating units is a polyglucosamine, preferably selected from polyglucosamines such as chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan or derivatives thereof, more preferably chitosan or derivatives thereof, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0120] The preferred polyglucosamine of this invention is selected from chitosan, chitosan, polyglucosaminoglycan, chondroitin, heparin, keratin, and dermatan, or derivatives thereof. Chitosan or its derivatives are most preferred. The preferred silane-PEG-NH2 is selected from silane-PEG4-NH2, silane-PEG2000-NH2, and silane-PEG5000-NH2. Preferred amino-containing polysilanes are selected from APTES, amino-butyl-TES, amino-pentyl-TES, amino-hexyl-TES, amino-heptyl-TES, and amino-octyl-TES, especially APTES.

[0121] In other embodiments, the polymer comprising repeating units is selected from polyglucosamine, polysilane-PEG-NH2, amino-containing polysilanes, thiol-containing polysilanes, polycarbofibril-cysteine ​​conjugates, polysilane-PEG-thiols, and polycysteine, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In other more preferred embodiments, the polymer comprising repeating units is selected from polyglucosamine of chitosan, chitosan, polyglucosamine polysaccharide, chondroitin, heparin, keratin, and dermatan or derivatives thereof; polysilane-PEG-NH2; thiol-containing polysilanes, preferably polyMPTS; polycarbofibril-cysteine ​​conjugates; polysilane-PEG-thiols; and polycysteine, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In even more preferred embodiments, the polymer comprising the repeating unit is polyglucosamine or a thiol-containing polymeric silane, preferably selected from chitosan, chitosan, polyglucosamine polysaccharide, chondroitin, heparin, keratin and dermatan or derivatives thereof, more preferably chitosan or derivatives thereof or thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates and polymeric silane-PEG-thiol, preferably thiol-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0122] In a specific embodiment, the polymer containing repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof, polysilane-PEG-NH2 selected from polysilane-PEG4-NH2, polysilane-PEG2000-NH2, polysilane-PEG5000-NH2, amino-containing polysilanes, preferably polymeric APTES, and thiol-containing polysilanes, preferably polymeric MPTS, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

[0123] In one embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, amino-containing polymeric silanes, and thiol-containing polymeric silanes, wherein each repeating unit comprises at least one amino group and / or at least one thiol group. In a preferred embodiment, the polymer comprising repeating units is selected from polyglucosamine, polymeric silane-PEG-NH2, polymeric APTES, and polymeric MPTS, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0124] In a more preferred embodiment, the polymer containing repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, and dermatan or derivatives thereof; polysaccharide-PEG-NH2; amino-containing polysaccharides, preferably polymeric APTES; and mercapto-containing polysaccharides, preferably polymeric MPTS, wherein each repeating unit contains at least one amino group and / or at least one mercapto group. In a specific embodiment, the polymer containing repeating units is selected from chitosan, chitosan, polyglucosamine, chondroitin, heparin, keratin, dermatan or derivatives thereof, particularly chitosan or derivatives thereof; polysaccharide-PEG-NH2 selected from polysaccharide-PEG4-NH2, polysaccharide-PEG2000-NH2, polysaccharide-PEG5000-NH2; amino-containing polysaccharides, i.e., APTES; and mercapto-containing polysaccharides, i.e., MPTS, wherein each repeating unit contains at least one amino group and / or at least one mercapto group.

[0125] In one embodiment, the polymer comprising the repeating unit is selected from thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates, polymeric silane-PEG-thiol, and polycysteine, and is preferably selected from thiol-containing polymeric silanes, polycarbofil-cysteine ​​conjugates, and polymeric silane-PEG-thiol, and more preferably thiol-containing polymeric silanes, and most preferably polymeric MPTS, wherein each repeating unit comprises at least one thiol group. In one embodiment, the thiol-containing polymeric silane is preferably polymeric MPTS.

[0126] In one embodiment, 5% to 100%, preferably 10% to 100%, more preferably 50% to 100% of the protective layer surface is covered with a polymer comprising repeating units, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0127] In one embodiment, optional functional components are fixed to the surface of the protective layer by bonding, preferably covalent bonding. In a preferred embodiment, optional functional components are fixed to the surface of the protective layer by non-covalent bonding, preferably by electrostatic interaction. In a more preferred embodiment, a polymer comprising repeating units is fixed to the surface of the protective layer by covalent bonding, wherein each repeating unit comprises at least one amino group and / or at least one thiol group.

[0128] In one embodiment, an optional functional component is immobilized on the surface of the protective layer using spacers that bind to both the protective layer surface and the functional component. Therefore, in one embodiment, the invention comprises a composition comprising a solid carrier, a protein or fragment thereof immobilized on the surface of the solid carrier, a protective layer for protecting the protein or fragment thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, and wherein the functional component is immobilized on the surface of the protective layer by spacers. Examples of such spacers include polyethylene, such as PEG4, PEG2000, and PEG5000. The functional component immobilized on the surface of the protective layer by spacers is typically produced by first reacting the spacers with the functional component, such that the spacers bind to the functional component, and then reacting the functional component bound to the spacers with the surface of the protective layer.

[0129] Optional functional components are typically immobilized onto the surface of the protective layer in a reaction vessel, such as a reactor, by suspending a solid carrier carrying proteins, such as enzymes, embedded in the protective layer as described above in, for example, water, a buffer solution, or a nonionic surfactant or a mixture thereof, preferably a mixture of water and nonionic surfactants. Nonionic surfactants are typically selected from ethoxylated sorbitol esters such as PEG-40 sorbitol 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 ethoxylates such as PEG-5 oleate and PEG-8 stearate. Esters, polyoxyethylene 40 stearate, polyoxyethylene 15 hydroxy stearate, fatty alcohol ethoxylates such as stearyl alcohol polyether (steareth) 40; fatty acid esters such as palmitic acid ascorbate, beeswax, polyglycerol 3-oleate, propylene glycol monocaprylate, propylene glycol monolaurate; fatty alcohols such as cetearyl alcohol, cetyl alcohol, myristyl alcohol, stearyl alcohol; glycerides; polyethylene glycol-modified triglycerides; glycol esters, preferably polysorbate, more preferably polysorbate 80 (PS80). The functional component is then added to the suspension and reacts with the surface of the protective layer, usually under stirring, to immobilize the functional component on the surface of the protective layer. The resulting composition is usually washed and resuspended in water, buffer solution, or nonionic surfactant or mixtures thereof. Immobilization is carried out through non-covalent bonding, such as electrostatic bonding, or through covalent bonding of the functional component. Functional components can be immobilized by chemically modifying the protective layer and the surface of the functional component using methods such as "click chemistry" (e.g., copper-catalyzed azide-alkyne cycloaddition, see, for example, Kolb et al., (2001) Angew. Chem. 40(11) 2004-2021) or copper-free click chemistry (Wittig G, A Chem Ber, 1961, 94, 3260). For example, as described above, a solid carrier carrying a protein, such as an enzyme, embedded in the protective layer is first reacted with a reactive compound, such as an acetylenic compound, and the functional component is modified by adding a reactive compound, such as an azide residue. The two components are then reacted to immobilize the functional component on the surface of the protective layer.

[0130] In other respects, the present invention provides compositions as described herein, which are used as pharmaceuticals.

[0131] In other aspects, the present invention provides compositions as described herein for use in methods of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, or for use in methods of preventing, delaying, or treating pancreatic exocrine insufficiency (EPI). In a preferred embodiment, the present invention provides compositions for use in methods of preventing, delaying, or treating pancreatic exocrine insufficiency (EPI). In other preferred embodiments, the present invention provides compositions for use in methods of enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy.

[0132] The use of the compositions described herein in the preparation of a medicament for the prevention, delay of progression, or treatment of pancreatic exocrine insufficiency (EPI) in an individual is also provided. The use of the compositions described herein in the prevention, delay of progression, or treatment of pancreatic exocrine insufficiency (EPI) in an individual is also provided. A method for the prevention, delay of, or treatment of pancreatic exocrine insufficiency (EPI) in an individual is also provided, the method comprising administering a therapeutically effective amount of the compositions described herein to the individual. The use of the compositions described herein in the preparation of a medicament for enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, is also provided. The use of the compositions described herein in enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in an individual is also provided. A method for administering enzyme replacement therapy (ERT), preferably gastrointestinal enzyme replacement therapy, in an individual is also provided, comprising administering a therapeutically effective amount of the compositions described herein to the individual.

[0133] In other respects, the present invention provides a composition as described herein for use in a method of preventing, delaying the progression of, or treating phenylketonuria (PKU). Use of the composition as described herein in the preparation of a medicament for preventing, delaying the progression of, or treating PKU in an individual is also provided. Use of the composition as described herein for preventing, delaying the progression of, or treating PKU in an individual is also provided. A method for preventing, delaying the progression of, or treating PKU in an individual is also provided, the method comprising administering to the individual a therapeutically effective amount of the composition as described herein. Preferably, when administered to an individual according to the method of the present invention, the composition degrades phenylalanine in the individual's intestine.

[0134] In other aspects, the present invention provides compositions as described herein for use in methods of preventing, delaying the progression of, or treating lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance. In one embodiment, the present invention provides compositions for use in methods of preventing, delaying the progression of, or treating 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 preferably includes congenital sucrase-isomaltase deficiency (CSID). The use of the compositions described herein in the preparation of a medicament for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in an individual is also provided. The use of the compositions described herein in the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in an individual is also provided. A method for the prevention, delay of progression, or treatment of lactase deficiency, sucrase-isomaltase deficiency, and / or disaccharide intolerance in an individual is also provided, comprising administering a therapeutically effective amount of the compositions described herein to the individual.

[0135] The compositions according to the invention are preferably pharmaceutical compositions and comprise a therapeutically effective amount of the composition described herein and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical compositions of the invention are suitable for oral administration to an individual. Unless otherwise stated, the pharmaceutical compositions of the invention are prepared in a manner known per se.

[0136] Compositions, such as the pharmaceutical compositions of the present invention, can be administered accordingly for one week or a portion thereof, for two weeks, for three weeks, for four weeks, for five weeks, or for six weeks, followed by a one-week or a portion thereof, for two weeks, for three weeks, for four weeks, for five weeks, or for six weeks. Compositions, such as the pharmaceutical compositions of the present invention, can be conveniently administered in unit dosage forms. Units of enzyme activity (“U”) can be described as the weight or mass of substrate hydrolyzed per unit time.

[0137] As used herein, the term "effective amount" or "therapeutic effective amount" means an amount capable of causing one or more desired effects in an individual receiving a composition of the present invention. The determination of a therapeutically effective amount is entirely within the competence of those skilled in the art, especially based on the detailed disclosure provided herein.

[0138] As used herein, the term “treatment / treating” includes: (1) delaying the onset of clinical symptoms of a state, symptom, or condition in animals, particularly mammals, especially humans, who may have or be susceptible to the state, symptom, or condition but have not yet experienced or exhibited clinical or subclinical symptoms of the state, symptom, or condition; (2) suppressing a state, symptom, or condition (e.g., preventing, reducing, or delaying the development or recurrence of a disease, or at least one clinical or subclinical symptom thereof, in the case of maintenance treatment); and / or (3) alleviating a condition (i.e., causing the resolution of at least one of a state, symptom, or condition, or its clinical or subclinical symptoms). The benefit of treatment to a patient is statistically significant or at least perceptible to the patient or physician. However, it should be understood that when a patient is given medication to treat a disease, the outcome may not always be an effective treatment.

[0139] As used in this article, “delaying progression” means increasing the time to symptom onset or slowing the increase in symptom severity. Furthermore, “delaying progression” as used in this article includes reversing or inhibiting disease progression. “Inhibiting” an individual’s disease progression or complications means preventing or reducing an individual’s disease progression and / or complications.

[0140] Preventive therapy includes preventive treatment. In preventive use, the drug combination of the present invention is administered to an individual suspected of having the aforementioned disease or condition or at risk of developing the aforementioned disease or condition. In therapeutic use, the drug combination is administered to an individual in an amount sufficient to cure or at least partially stop the symptoms of the disease, such as a patient already suffering from the aforementioned disease or condition. The effective amount for this use will depend on the severity and course of the disease, previous treatment, the individual's health condition and response to the drug, and the judgment of the treating physician.

[0141] If the condition of any individual does not improve, The drug combination of the present invention can be administered for a prolonged period of time, including the entire duration of an individual's life, to improve or otherwise control or limit the symptoms of an individual's disease or condition.

[0142] In cases where an individual's condition has indeed improved, the drug combination can be administered continuously; alternatively, the dosage of the administered drugs can be temporarily reduced or temporarily suspended for a period of time (i.e., a "drug holiday"). Once the patient's condition has improved, a maintenance dose of the drug combination of the present invention is administered, if necessary. Subsequently, the dosage or frequency of administration, or both, is preferably reduced to a level that maintains the improvement in the disease, depending on the symptoms.

[0143] In other aspects, 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 the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition can be obtained by the method described above.

[0144] In other aspects, 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 the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition can be obtained by the method described above. The condition is that the protein or fragment thereof is not a lipase or fragment thereof, a protease or fragment thereof, an amylase or fragment thereof, a pancreatic enzyme or a protein or fragment thereof composed of pancreatic enzymes, an engineered phenylalanine ammonia-lyase (PAL) or fragment thereof, or a disaccharidase 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, a pancreatic enzyme or a protein or fragment thereof composed of pancreatic enzymes, an engineered phenylalanine ammonia-lyase (PAL) or fragment thereof, or a disaccharidase or fragment thereof as described herein.

[0145] Solid carriers, proteins or fragments thereof, protective layers protecting proteins or fragments thereof, and optional functional components of the composition are as described above.

[0146] Example

[0147] Phenylalanine ammonia-lyase

[0148] Materials and methods:

[0149] Reagents:

[0150] - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (Grade I, 25% aqueous solution), polysorbate 80, acetic acid, bovine serum albumin (BSA), Tris buffer, L-phenylalanine, trypsin, streptomycin, purchased from Sigma-Aldrich. BSA, trypsin, and streptomycin were dissolved in water to reconstitute the stock buffer.

[0151] Chitosan 95 / 500P, purchased from Heppe Medical Chitosan GmbH

[0152] - The engineered PAL (SEQ ID NO: 5) was provided by Nestlé Health Science at a concentration of 90 mg / mL in 25 mM sodium phosphate, 250 mM sodium chloride, 5% D-mannitol, and 0.2% poloxamer 188 at pH 7.5.

[0153] -Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line), European Collection of Cell Cultures (ECACC).

[0154] -ThinCert™ cell culture inserts (1.0 μm membrane), purchased from Greiner bio-one.

[0155] - 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, white DMEM, purchased from Gibco.

[0156] -Matrigel® Low Growth Factor (GFR) Basement Membrane Matrix, LDEV-free, purchased from Corning.

[0157] - Animal feed Altromin 1324, purchased from Altromin International.

[0158] - Free phenylalanine 5LF2 in animal feed, purchased from LabDiet.

[0159] - Catheter, purchased from Instech Laboratories

[0160] Synthesis of silica nanoparticles (SNPs): Silica nanoparticles (50 nm) were synthesized according to the original Stöber method described in WO2015 / 014888A1. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 h. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm for 22 h at 20 °C. The solution was then centrifuged at 20,000 g for 20 min and washed successively with ethanol and water. Particle size was measured using SEM micrographs obtained at 150,000x magnification using the Olympus flow motion image analysis software.

[0161] Generation of PAL-based silica nanoparticles NP-1: APTES (3.9 mM) was added to SNPs (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Engineered PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized engineered PAL surface using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a 20 °C water bath.

[0162] Generation of inactive nanoparticles NP-2: APTES (3.8 mM) was added to SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. BSA solution was added to bring the final BSA concentration to 1.42 mg / mL, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized BSA surface using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a 20 °C water bath.

[0163] Generation of PAL-based silica nanoparticle NP-1 variant: The following experiments investigated the effects of covalently linking the enzyme to the protective layer on enzyme stability and enzyme activity.

[0164] In the first experiment, PAL-based silica nanoparticles (NP-1(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.9 mM) was added to the SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. 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 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Engineered PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized engineered PAL surface using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). NP-1(1) was cured overnight in a water bath at 20 °C.

[0165] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to WO2015 / 014888 A1 to generate PAL-based silica nanoparticles (NP-1(2)) in buffer. The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.9 mM) was added to SNP (10 mg / mL, 59 nm) in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 min. 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 min. 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). Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. 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). Engineered PAL (11.9 mg / mL, 1 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized engineered PAL surface using APTES (7.7 mM) and TEOS (80.8 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. 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). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20°C and 400 rpm for 30 min. 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). NP-1(2) was cured overnight in a water bath at 20°C.

[0166] 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. To retain excess glutaraldehyde in the reaction mixture that did not bind the solid support to the engineered PAL, the nanoparticles were not washed between each chemical step. Therefore, the glutaraldehyde remained during layer growth, resulting in a covalent bond between the protective layer and the engineered PAL. This covalent bond between the protective layer and the engineered PAL can be observed as a yellow / orange appearance with maximum absorption at 460 nm. This color is due to the formation of imine bonds between the aldehyde functional groups of the glutaraldehyde linker and the primary amines of the amino acids in the engineered PAL and the organosilicon layer. After the formation of the silicone layer and final particle washing, i.e., after the formation of the silicone layer, the absorbance of PAL-based nanoparticles NP-1(1), NP-1(2), and NP-1 at 460 nm was measured. The particles were washed three times in H2O / PS80 and then resuspended in H2O / PS80, as described in the section titled "Generation of NP-1" in "Example 1: Immobilization and Generation of Phenylalanine Ammonia Lyase" above. The results showed that the absorbance of NP-1 at 460 nm was significantly higher than that of NP-1(1) and NP-1(2) (see [link to previous section]). Figure 8 Since imine bonds are also formed during enzyme immobilization, NP-1(1) and NP-1(2) still show a certain degree of absorbance at this wavelength. However, the absorbance of NP-1 is significantly higher, indicating that the covalent bonding between the protective layer and the engineered PAL leads to the formation of additional imine bonds.

[0167] Activity determination 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). PAL kinetics were monitored for 30 min at 37 °C with a spectrophotometer at λ = 290 nm.

[0168] Resistance to external stress: - Resistance to acidic conditions Engineered PAL or PAL-based silica nanoparticles NP-1 were incubated at pH 4 for 24 hours. Enzyme activity was assessed at 0, 1, 3, 6, and 24 h as described in the “NP-1 Activity Assay”.

[0169] - Resistance to proteases

[0170] Engineered PAL or PAL-based silica nanoparticles NP-1 were treated with trypsin (30 mU) or streptomycin (0.8 U) and cultured at 37 °C with shaking at 300 rpm for 4 h. Enzyme activity was assessed at 0, 0.1, 0.25, 0.5, 1, 2, and 4 h as described in the "NP-1 Activity Assay" section.

[0171] Cell culture: For all experiments, cells were cultured at 37°C and 5% CO2.

[0172] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids and 100 U / mL penicillin / streptomycin.

[0173] To develop an intestinal barrier model, cells were stored at a density of 2.6 × 10⁻⁶. 5 cells / cm 2 Density inoculation in Transwell PET inserts (1 (m pore size). All cell models were used for experiments on day 21. For co-culture, Caco-2 and HT-29-MTX-E12 cells were used at a ratio of 75%–25%.

[0174] Transepithelial resistance

[0175] Cell barrier integrity was assessed by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). TEER was automatically measured every 15 minutes for 24 h, in the range of 1 Hz to 100,000 Hz, after cell culture medium renewal and treatment with nanoparticles.

[0176] Phe's in vitro metabolism

[0177] Caco-2 / HT29-MTX-E12 cells were co-cultured in white DMEM (hereinafter referred to as “cell culture medium”) supplemented with 1% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids and 100 U / mL penicillin / streptomycin.

[0178] On the top side of the barrier, the intestinal barrier was exposed to PAL-based silica nanoparticles NP-1 (9.7 mU) or engineered PAL (9.7 mU) for 6 h in the presence of trypsin (30 mU). At each time point, 150 μL aliquots were removed from the outer side of the intestinal barrier and replaced with the same volume of preheated cell culture medium. The barrier was further incubated at 37 °C. The absorbance of the removed aliquots was measured at 290 nm to quantify the level of trans-cinnamic acid (TCA).

[0179] animal:

[0180] All animal experiments were conducted with permission from the National Animal Experiments Inspectorate under the Ministry of Food, Agriculture and Fisheries of Denmark.

[0181] - Wistar rats: The study was conducted on male Wistar rats (8 weeks old) of the original breed from Janvier, France.

[0182] Food and drinking water: Rats were fed a complete pelleted diet, "Altromin 1324," with free access to food and drinking water.

[0183] Duodenal catheter insertion

[0184] The animal was placed in the induction chamber and anesthetized with isoflurane (2-4%), then transferred to a nasal cannula with isoflurane for surgery. A catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the duodenal mesentery, near the opening of the bile and pancreatic ducts. The catheter was ligated and secured to the intestinal wall and then tunneled through a subcutaneous tunnel to the animal's neck, exposing it. The abdominal and cervical incisions were then sutured shut. Throughout the procedure, the animal was kept warm and closely monitored until fully recovered from anesthesia.

[0185] Efficacy evaluation of PAL-based silica nanoparticles NP-1 in rats

[0186] Animals were starved for 4 h before administration of d5-L-Phe. Rats were then administered NP-1 (0.85 U) or NP-2 (8.5 mg) via the duodenum, followed immediately by tube feeding of 3.6 mg of d5-L-Phe. Rats were then housed in metabolic cages for 24 h.

[0187] Urine sampling, metabolic cage

[0188] Urine was collected in a metabolic cage for 24 h. The total urine output was obtained, and urine samples were taken into Eppendorf tubes and stored at -80°C until transport for analysis.

[0189] Measuring D5-hippuric acid in rat urine: Quantification of the target analyte was performed using an LC system: a Thermo Vanquish Horizon binary pump and a Thermo Q Exactive mass spectrometer. Urine samples were prepared as follows: 50 μL of urine sample was mixed with 5 μL of ISTD (100 μM 13C6-HIP, final concentration 2 μM per sample), and 200 μL of 100% methanol was added before vortexing. After incubating on ice for 20 min, the sample was centrifuged at 16000 g for 10 min at 4 °C. The supernatant was transferred to a total recovery MS vial for analysis.

[0190] The injection volume used was 2.5 μL, the run time was 4.8 min, and the flow rate was 1 mL / min. Mobile phase A was H₂O and formic acid (0.1%); 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) with a gradient of 10% B to 100% B.

[0191] MS was performed using a Thermo Q Exactive mass spectrometer in DDA top5 mode. MS parameters were as follows: MS1 resolution: 70,000 Å and MS2 resolution: 17,500 Å. HCD fragmentation was performed using normalized step collision energies of 10, 20, and 30 Å. Data analysis was performed using Thermo Q Exactive Browser software.

[0192] - BTBR-Pah enu2 / J:

[0193] This study involved male and female BTBR-Pah, the original species, at Jackson Laboratory in the United States. enu2 The study was conducted in J mice (8 weeks old).

[0194] Food and drinking water: Mice were fed a phenylalanine-free diet (5LF2, LabDiet) and had free access to food and drinking water.

[0195] Duodenal catheter insertion

[0196] Anesthetize the animal with isoflurane (2-4%). ​​Make an incision along the linea alba and insert a catheter (C19PB-MGI1923, Instech Laboratories) into the duodenum via the contralateral mesentery. Advance the catheter tip close to the opening of the bile and pancreatic ducts. Ligate the catheter to the intestinal wall and tunnel it through a subcutaneous tunnel to the animal's neck, exposing and closing it. Then suture the abdominal wall and neck incisions. Throughout the procedure, keep the animal on a warm bed and monitor closely until fully recovered from anesthesia.

[0197] Efficacy evaluation of PAL-based silica nanoparticles NP-1 in mice

[0198] Prior to the study, mice were kept on a phenylalanine-free diet for at least 3 days, then supplemented with a low concentration (0.03 g / L) of L-Phe in their drinking water for 3 days, after which the concentration of L-Phe in the drinking water was increased to 0.5 g / L. Mice were allowed free access to the L-Phe-supplemented drinking water at night.

[0199] Mice were administered NP-1 (0.581 U, 7 mg), NP-2 (7 mg), or engineered PAL (0.581 U) twice daily via the duodenum over a 12-day period. Blood samples were collected in EDTA on days 0, 4, 6, 8, 10, and 12. The blood samples were then centrifuged (10 min, 4°C, 2000×g), and at least 20 μL of plasma was transferred to Eppendorf tubes and stored at –80°C until Phe content was analyzed.

[0200] Measurement of mouse plasma Phe: Quantification of target analytes using an LC system: Thermo Vanquish Horizon binary pump and Thermo TSQ Quantiva mass spectrometer.

[0201] Plasma samples were prepared as follows: 20 μL of plasma sample was centrifuged at 13.2 krpm for 10 min at 4 °C. 10 μL 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 for 10 min at 4 °C. 50 μL of the supernatant was then dried at 30 °C under a gentle nitrogen atmosphere. 500 μL of 0.1% (v / v) formic acid aqueous solution was added, and the sample was shaken at 900 rpm for 10 min at 15 °C, followed by centrifugation (shaking at 13.2 krpm for 10 min at 4 °C). Finally, 350 μL of the supernatant was transferred to a total recovery vial for analysis.

[0202] The injection volume was 2 μL, the run time was 5 min, and the flow rate was 1 mL / min. Mobile phase A consisted of H₂O and formic acid (0.1%); mobile phase B consisted of methanol and formic acid (0.1%). Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) with a gradient of 100% A to 100% B.

[0203] MS was performed using a Thermo TSQ Quantiva mass spectrometer, in selective reaction monitoring mode.

[0204] MS parameters were as follows: Q§1 resolution: 0.7; Q3 resolution: 0.7; fragmentation: CID fragmentation using argon (1.5 mTorr). Analyte concentrations were calculated from the peak area ratio of Phe to the internal standard d5-Phe. Data analysis was performed using Thermo Quan Browser software.

[0205] result: Example 1: PAL stability is enhanced by covalently linking it to the protective layer. To demonstrate that glutaraldehyde crosslinking, which covalently bonds the protective layer to the PAL surface, further improves enzyme stability compared to immobilized proteins protected by an organosilicon layer (which only interacts electrostatically with the immobilized protein), a series of three experiments were conducted.

[0206] In the first experiment, PAL-based silica nanoparticles NP-1(1) were produced under unbuffered conditions, including washing after each chemical step (i.e., removal of glutaraldehyde before layer growth). In the second experiment, PAL-based silica nanoparticles NP-1(2) were produced under unbuffered conditions, including washing after each chemical step (i.e., removal of glutaraldehyde before layer growth). In the third experiment, PAL-based silica nanoparticles NP-1 were produced under unbuffered conditions without any intermediate washing steps (i.e., unreacted glutaraldehyde remains in the reaction mixture during layer growth).

[0207] Protein quantification was performed on the reaction supernatant to determine the PAL immobilization yield on the surfaces of NP-1(1), NP-1(2), and NP-1. Surprisingly, enzyme immobilization under conditions maintaining glutaraldehyde (NP-1) increased the enzyme immobilization yield by two-fold. Figure 2 A), resulting in a SNP enzyme loading per unit dry weight that is twice that of buffer conditions (NP-1(2)) for removing glutaraldehyde via a washing step. Figure 2 B). Similarly, enzyme immobilization under conditions maintaining glutaraldehyde (NP-1) resulted in an enzyme loading per unit dry weight of SNP ( Figure 2B) is 1.5 times higher than the unbuffered conditions (NP-1(1)) in which glutaraldehyde is removed by a washing step.

[0208] The biocatalytic activity of PAL immobilized and protected on NP-1(1), NP-1(2), and NP-1 was evaluated. Even more surprisingly than the increase in enzyme immobilization load while maintaining glutaraldehyde, the specific activity of the nanoparticles increased threefold compared to buffered conditions where glutaraldehyde was removed via a washing step, and twofold compared to unbuffered conditions where glutaraldehyde was removed via a washing step. Figure 2 C). This astonishing three-fold increase in the specific activity of the nanoparticles is closely correlated with the specific activity (U / g PAL) of the enzyme immobilized under unbuffered conditions maintaining glutaraldehyde, which is comparable to that of the enzyme immobilized under buffered conditions. This result is completely unexpected, as it was presumed that the enzyme would have much higher activity in the presence of buffer ( Figure 2 D). In summary, compared to PAL protected only by an organosilicon layer through electrostatic interactions, the covalent bonding on the surface of the protective layer PAL unexpectedly enhanced its loading capacity and stability.

[0209] Example 2: Phenylalanine ammonia-lyase (PAL) activity of PAL-based silica nanoparticles NP-1

[0210] The biocatalytic activity of immobilized and protected engineered PALs was evaluated. Figure 3 The results shown reported PAL activity on NP-1. This indicates that, despite protection and functionalization, the protected functionalized SNP still possesses the ability to approach and transform L-Phe. Validation of the biocatalytic activity of NP-1 confirms the potential for using nanoparticles for therapeutic purposes.

[0211] Example 3: Resistance to external stress

[0212] NP-1, a silica nanoparticle based on PAL, is a nanoparticle developed for gastrointestinal applications. Due to the physiological characteristics of the gastrointestinal tract, NP-1 will be subjected to various stresses. To ensure the sustained activity of NP-1 in the gastrointestinal tract, the protection of immobilized PAL was evaluated. First, NP-1 or engineered PAL was subjected to acidic conditions (pH 4). Monitoring of PAL activity over 24 hours revealed sustained enzymatic activity of NP-1. Figure 4 B), while the free-form PAL of engineering modifications loses its activity over time. Figure 4 A). These data demonstrate the protection of engineered PALs immobilized and protected on NP-1 in an acidic environment.

[0213] During digestion, pancreatic enzymes are released, particularly proteases that may affect therapeutic enzymes in the gastrointestinal tract. Therefore, NP-1 and engineered PAL were exposed to various proteases at 37°C. First, to simulate physiological digestive conditions, NP-1 or engineered PAL was incubated with pancreatic enzymes (30 mU) (a mixture of pancreatic enzymes extracted from porcine pancreas), and then their PAL activity was evaluated. After 4 h, both NP-1 and engineered PAL exhibited sustained PAL activity (…). Figure 4 C). Then, to further evaluate the benefits of the protective shield under harsh conditions, NP-1 or engineered PAL was exposed to streptomycin (0.88 U), a mixture of purified proteases. Figure 4 The results shown by C indicate that 80% of the PAL retained its activity against NP-1 after 4 h of exposure, while the engineered PAL lost its enzymatic activity after 4 h. In summary, these data demonstrate the added value of engineered PAL for immobilization and protection on nanoparticles and highlight the potential use of NP-1 in gastrointestinal therapeutic applications.

[0214] Example 4: In vitro biocompatibility and efficacy of PAL-based silica nanoparticles NP-1

[0215] Maintaining the integrity of the intestinal barrier is crucial for preventing unwanted lumen contents, such as pathogens or food allergens, from entering the body. To evaluate the biocompatibility of the nanoparticles, Caco2-HT29-MTX-E12 cell monolayers were exposed to NP-1 in the presence or absence of pancreatic enzymes to simulate digestive conditions, and transepithelial electrical resistance (TEER) was measured. Figure 5A The data presented showed that, with or without pancreatic enzymes, the integrity of the intestinal epithelial barrier was maintained after 6 h of exposure to NP-1. These results demonstrate the in vitro biocompatibility of NP-1 in gastrointestinal applications.

[0216] NP-1 has been developed for metabolizing 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 culture medium containing 0.4 mM L-phenylalanine was exposed on its apical side to NP-1 (9.7 mU) or engineered PAL (9.7 mU) for 6 h in the presence or absence of trypsin (30 mU). Figure 5B The figure shows the quantification of Phe metabolites on the basal side of the barrier. The graph also reports the accumulation of TCA on the basal side of the barrier under all conditions. These results demonstrate the in vitro efficacy of NP-1 in the digestive environment and suggest its potential use in therapeutic applications.

[0217] Example 5: In vivo activity of PAL-based silica nanoparticles NP-1 in rats

[0218] To evaluate the transition from in vitro to in vivo application, the efficacy of NP-1 was assessed in rats. Rats were administered NP-1 (nanoparticles containing engineered PAL) or NP-2 (nanoparticles containing bovine serum albumin (BSA), where Phe was not a substrate) via the duodenum, followed by gavage feeding with d5-Phe. In vivo, the Phe metabolite TCA is rapidly metabolized to hippuric acid. The in vivo activity of NP-1 was then evaluated by measuring d5-hippuric acid in rat urine collected 24 h after administration. Figure 6 The results showed that rats receiving NP-1 had significantly increased levels of d5-hippuric acid compared to rats receiving NP-2, indicating that NP-1 can digest Phe in the intestinal lumen and suggesting that NP-1 can be used for therapeutic applications.

[0219] Example 6: In vivo therapeutic efficacy of PAL-based silica nanoparticles NP-1 in mice

[0220] Phenylketonuria (PKU) is characterized by a deficiency of the intracellular liver enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the conversion of the essential amino acid phenylalanine to tyrosine. PAH deficiency leads to abnormally high phenylalanine concentrations, which are toxic to the brain. The basis of PKU treatment is a combination of a low-phenylalanine diet and a phenylalanine-free L-amino acid regimen. Currently, Enzyme replacement therapy using recombinant phenylalanine ammonia-lyase can be administered subcutaneously, but this treatment can cause allergic reactions and immune-mediated acute hypersensitivity reactions.

[0221] NP-1 has been developed to exhibit sustained PAL activity in the gastrointestinal environment (acidic and exposed to proteases). Our approach to controlling Phe levels in patients involves degrading Phe (from dietary intake) in the intestine to prevent its absorption and accumulation in the blood.

[0222] Using a representative animal model of the disease—BTBR- Pah enu2 / J mice—the mice in which the gene encoding PAH was mutated—were used to evaluate this treatment strategy.

[0223] During the 12-day period, Mice given drinking water supplemented with L-phenylalanine were injected twice daily with NP-1, NP-2, or engineered PAL. Impressively, during the study period, plasma Phe levels in mice receiving NP-1 showed a stable decrease, while plasma concentrations in mice receiving engineered PAL were unstable (Figure 7). Figure 7B As shown, the normalization of Phe plasma concentration highlights that at the end of NP-1 treatment, Phe plasma concentration in mice decreased by 30%.

[0224] The interaction between nanoparticles and intestinal mucus leads to the temporary attachment of NP-1 to the intestinal wall, enabling PAL to remain active on the intestinal wall, while the free form of engineered PAL is flushed downstream. These results validate and reinforce the strategy of degrading phenylalanine in the intestine and support the significant therapeutic effect of NP-1 on PKU.

[0225] disaccharidase

[0226] Materials and methods:

[0227] Reagents:

[0228] - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (Grade I, 25% aqueous solution), polysorbate 80, acetic acid, lactase (USP standard), invertase, bovine serum albumin (BSA), invertase activity assay kit, lactose, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), purchased from Sigma-Aldrich. BSA, lactase, and invertase were dissolved in water to reconstitute the stock buffer.

[0229] Chitosan 95 / 500P, purchased from Heppe Medical Chitosan GmbH

[0230] Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were purchased from the European Collection of Cell Cultures (ECACC).

[0231] -THP-1 (human acute monocytic leukemia cell line), purchased from LGC.

[0232] -ThinCert™ cell culture inserts (1.0 μm membrane), purchased from Greiner bio-one.

[0233] 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, EPES, sodium pyruvate, D-glucose, β-mercaptoethanol, purchased from Gibco.

[0234] -Matrigel® Low Growth Factor (GFR) Basement Membrane Matrix, LDEV-free, purchased from Corning.

[0235] - 200g of modified polysaccharides of Altromin 1319 and AIN 93G for animal feed, purchased from Altromin International.

[0236] - Catheter, purchased from Instech Laboratories

[0237] - Blood glucose meter TB100 Holtex reagent kit, purchased from MediSafe

[0238] - Oligo-α-1,6-glucosidase 13A, recombinant (isomaltase), from Bifidobacterium adolescentis, 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.

[0239] Synthesis of silica nanoparticles: Silica nanoparticles (50 nm) were synthesized according to the original Stöber method described in WO2015 / 014888A1. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 h. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm for 22 h at 20 °C. The solution was then centrifuged at 20,000 g for 20 min and washed successively with ethanol and water. Particle size was measured using SEM micrographs obtained at 150,000x magnification using the Olympus flow motion image analysis software.

[0240] Enzyme protection and surface functionalization

[0241] - Generation of NP-2 silica nanoparticles based on lactase: APTES (3.9 mM) was added to the SNP (10 mg / mL, 55 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Lactase (7 mg / mL, 0.1 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized lactase surface using APTES (8.4 mM) and TEOS (125.9 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a 20 °C water bath.

[0242] - Lactase-based silica nanoparticle NP-2 variant: The following experiments investigated the effects of covalently linking the enzyme to the protective layer on enzyme stability and enzyme activity.

[0243] In the first experiment, lactase-based silica nanoparticles (NP-2(1)) were generated in H2O / PS80 (8 mg / L). The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.3 mM) was added to the SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. The particles were washed three times in H2O / 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 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Initiation was performed by adding APTES (3.3 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. The particles were washed three times in H2O / 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 min. An organosilicon layer was grown on the immobilized lactase surface using APTES (6.5 mM) and TEOS (93 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). NP-2(1) was cured overnight in a water bath at 20 °C.

[0244] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to WO2015 / 014888 A1 to generate lactase-based silica nanoparticles (NP-2(2)) in buffer. The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.3 mM) was added to SNP (10 mg / mL, 69 nm) in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. 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 min. 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). Initiation was performed by adding APTES (3.3 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. 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). Lactase (5.2 mg / mL, 0.1 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized lactase surface using APTES (6.5 mM) and TEOS (93 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. 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 water bath at 20°C.

[0245] In the third experiment, following the section titled "Generation of NP-2" above, lactase-based nanoparticles (NP-2) were generated in H2O / PS80 (8 mg / L). To retain excess glutaraldehyde in the reaction mixture that did not bind the solid support to lactase, the nanoparticles were not washed between each chemical step. Therefore, the glutaraldehyde remained during layer growth and resulted in a covalent bond between the protective layer and lactase. This covalent bond between the protective layer and lactase was observable as a yellow / orange appearance with maximum absorbance at 460 nm. This color is due to the formation of imine bonds between the aldehyde functional group of the glutaraldehyde linker and the primary amine of the lactase amino acid and the organosilicon layer. After the formation of the silicone layer and final particle washing, i.e., after the formation of the silicone layer, the absorbance of lactase-based silica nanoparticles NP-2(1), NP-2(2), and NP-2 at 460 nm was measured. The particles were washed three times in H2O / PS80 and then resuspended in H2O / PS80, as described in the section titled "Generation of NP-2" in "Example 2: Immobilization and Generation of Disaccharidase" above. The results showed that the absorbance of NP-2 at 460 nm was significantly higher than that of NP-2(1) and NP-2(2) (see Example 2: Immobilization and Generation of Disaccharidase). Figure 15 Since imine bonds are also formed during enzyme immobilization, NP-2(1) and NP-2(2) still show a certain degree of absorbance at this wavelength. However, the absorbance of NP-2 is significantly higher, indicating that the covalent binding of the protective layer with lactase causes the formation of additional imine bonds.

[0246] - Generation of silica nanoparticles NP-3 based on invertase: APTES (3.8 mM) was added to the SNP (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Invertase (1.726 mg / mL, 0.03 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized invertase surface using APTES (5.4 mM) and TEOS (81.3 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 115 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-3 was cured overnight in a 20 °C water bath.

[0247] - Generation of silica nanoparticles NP-4 based on isomaltase: APTES (3.6 mM) was added to the SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.6 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.6 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Isomaltase (3.55 mg / mL, 0.05 mM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized invertase surface using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 82 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). NP-4 was cured overnight in a 20 °C water bath.

[0248] - Generation of silica nanoparticles NP-5 based on invertase / isomaltase: APTES (3.6 mM) was added to the SNP (10 mg / mL, 59 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.6 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.6 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Isomaltase (1.77 mg / mL, 0.025 mM) and invertase (4.05 mg / mL, 0.07 mM) were added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized enzyme surface using APTES (5.8 mM) and TEOS (88 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 82 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). NP-5 was cured overnight in a 20 °C water bath.

[0249] - Generation of inactive nanoparticles NP-1: APTES (3.8 mM) was added to SNPs (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. BSA solution was added to bring the final BSA concentration to 1.42 mg / mL, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized BSA surface using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20 °C. SNPs-BSA-AT was cured overnight at 20 °C.

[0250] Disaccharidase activity assay: - Lactase activity assay Lactose solution (100 μL, 50 mg / mL) was added to 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). The reaction mixture was incubated in a hot mixer at 37 °C and 750 rpm for 20 min. Samples were collected every 2.5 min, and glucose formation was monitored using a glucometer.

[0251] - Invertase activity assay

[0252] The activity of invertase-based silica nanoparticles NP-3 was evaluated using the Sigma invertase assay kit. NP-3 (94 μL, 147 μg / L) was added to 1X reaction buffer (94 μL). Then, 1X sucrose solution (11.76 μL) was added. The reaction mixture was incubated in a hot mixer at 37 °C, 300 rpm for 20 min. The sample was centrifuged at 20000 rcf for 5 min. The supernatant was collected, and 85 μL was transferred to a 96-well plate. 90 μL of the master reaction mixture (prepared by mixing the enzyme mixture, dye reagent, and assay buffer) was added to each well. The reaction mixture was incubated at room temperature in the dark for 20 min. The absorbance was measured at λ = 570 nm.

[0253] - Isomaltase activity assay

[0254] The activity of isomaltase-based silica nanoparticles NP-4 was assessed using a blood glucose meter. An isomaltose solution (25 μL, 100 mM) was equilibrated at 37 °C and 700 rpm for 5 min. Then, NP-4 (25 μL, 67 μg) in 50 mM pH 6.8 phosphate buffer was added to the isomaltose solution. The reaction mixture was incubated at 37 °C and 700 rpm for 10 min. Samples were collected after 2 min, 5 min, and 10 min, and glucose concentrations were measured using a blood glucose meter.

[0255] - Assay of co-immobilized isomaltase-invertase activity

[0256] The isomaltase activity of NP-5 silica nanoparticles based on invertase / isomaltase was assessed using a blood glucose meter. An isomaltose solution (25 μL, 100 mM) was equilibrated at 37°C and 700 rpm for 5 min. Then, NP-5 (25 μL, 67 μg) in 50 mM pH 6.8 phosphate buffer was added to the isomaltose solution. The reaction mixture was incubated at 37°C and 700 rpm for 10 min. Samples were collected after 2 min, 5 min, and 10 min, and glucose concentrations were measured using a blood glucose meter.

[0257] The activity of NP-5 invertase based on silica nanoparticles containing invertase / isomaltase was assessed using a blood glucose meter. A sucrose solution (25 μL, 100 mM) was equilibrated at 37 °C and 700 rpm for 5 min. Then, NP-5 (25 μL, 67 μg) in 50 mM, pH 6.8 phosphate buffer was added to the sucrose solution. The reaction mixture was incubated at 37 °C and 700 rpm for 10 min. Samples were collected after 2 min, 5 min, and 10 min, and glucose concentrations were measured using a blood glucose meter.

[0258] Cell culture: For all experiments, cells were cultured at 37°C and 5% CO2.

[0259] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids and 100 U / mL penicillin / streptomycin.

[0260] THP-1 (human monocytic leukemia cell line) cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 2 mL M glutamine and 100 U / mL penicillin / streptomycin.

[0261] To differentiate THP-1 cells into macrophages, THP-1 cells were cultured in differentiation medium: RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U / mL penicillin / streptomycin, 10 mM HEPES, 1 mM sodium pyruvate, 2.5 g / L glucose, and 50 pM... Mercaptoethanol. THP-1 cells were differentiated into MO-macrophages by incubation with 150 nM phorbol 12-myristate 13-acetate (PMA) for 24 h, followed by incubation in differentiation medium for 24 h.

[0262] - Intestinal barrier model

[0263] To develop an intestinal barrier model, cells were stored at a density of 2.6 × 10⁻⁶. 5 cells / cm 2 Density inoculation in Transwell PET inserts (1 (m pore size). All cell models were used for experiments on day 21. For co-culture, Caco-2 and HT-29-MTX-E12 cells were used at a ratio of 75%–25%.

[0264] - An immune-active intestinal barrier model

[0265] To develop an immune-active 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 side of a Transwell membrane containing previously differentiated cells co-cultured in 25% Matrigel solution using a drop method.

[0266] Transepithelial resistance

[0267] Cell barrier integrity was assessed by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). TEER was automatically measured every 15 minutes for 24 h, in the range of 1 Hz to 100,000 Hz, after cell culture medium renewal and treatment with nanoparticles.

[0268] In vitro digestion of sucrose

[0269] On the top side of the barrier, 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 h. At each time point, 150 μL aliquots were removed from the outer side of the intestinal barrier and replaced with the same volume of preheated PBS. The barrier was further incubated at 37 °C, and the absorbance of the removed aliquots was measured at 570 nm to quantify glucose levels.

[0270] animal:

[0271] All animal experiments were conducted with permission from the National Animal Experiments Inspectorate under the Ministry of Food, Agriculture and Fisheries of Denmark.

[0272] The study was conducted on male Wistar rats (8 weeks old) of the original breed from Janvier, France.

[0273] - Food and drinking water: To maintain their diet, the rats were fed a complete pelleted diet, "Altromin 1319," with free access to food. They also had free access to drinking water.

[0274] One week before treatment and during the experiment, rats were fed a freely available low-sugar diet (AIN 93G modified 200g polysaccharide).

[0275] - Duodenal catheter insertion

[0276] The animal was placed in the induction chamber and anesthetized with isoflurane (2-4%), then transferred to a nasal cannula with isoflurane for surgery. A catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the duodenal mesentery, near the opening of the bile and pancreatic ducts. The catheter was ligated and secured to the intestinal wall and then tunneled through a subcutaneous tunnel to the animal's neck, exposing it. The abdominal and cervical incisions were then sutured shut. Throughout the procedure, the animal was kept warm and closely monitored until fully recovered from anesthesia.

[0277] - Administration and lactose administration

[0278] Animals were starved for 4 hours before administration of the drug and lactose. Rats were then administered lactase-based silica nanoparticles NP-2 (97 U), inactive nanoparticles NP-1 (54 mg), or a solvent (1.5 mL of NaCl 0.9%-polysorbate 80 mg / mL) via intraduodenal administration, followed immediately by g of lactose via gavage. This administration and gavage were repeated daily for 15 days.

[0279] - Cecal analysis

[0280] After the experiment, all animals were scanned under general anesthesia using a Bruker Pharmascan 7 Tesla equipped with a rat volume coil (scan time approximately 5 minutes) to assess cecal size. Target regions were delineated on the cecum in all obtained sections. Cecal volume was determined using MRI images.

[0281] During the autopsy, photographs of the gastrointestinal tract were taken to observe the extent of cecal dilation.

[0282] result: Example 1: Lactase loading is enhanced by covalently linking to the protective layer. In the first experiment, lactase-based silica nanoparticles NP-2(1) were generated under unbuffered conditions, including washing after each chemical step (i.e., removal of glutaraldehyde before layer growth). In the second experiment, lactase-based silica nanoparticles NP-2(2) were generated under unbuffered conditions, including washing after each chemical step (i.e., removal of glutaraldehyde before layer growth). In the third experiment, lactase-based silica nanoparticles NP-2 were generated under unbuffered conditions without any intermediate washing steps (i.e., unreacted glutaraldehyde remains in the reaction mixture during layer growth).

[0283] Lactase was quantified in the reaction supernatant to determine the lactase immobilization yield on the surfaces of lactase-based silica nanoparticles NP-2(1), NP-2(2), and NP-2. The results showed that, surprisingly, enzyme immobilization on NP-2 (while maintaining glutaraldehyde) increased the enzyme immobilization yield by 26-fold. Figure 10 A), resulting in a 24-fold increase in enzyme loading / SNP dry weight compared to buffered conditions for glutaraldehyde removal via a washing step (NP-2(2)). Figure 10 B). Similarly, enzyme immobilization under conditions maintaining glutaraldehyde (NP-2) resulted in an enzyme loading per unit dry weight of SNP ( Figure 10 B) is 2 times higher than the unbuffered conditions (NP-2(1)) in which glutaraldehyde is removed by a washing step.

[0284] In summary, compared to lactase protected by an organosilicon layer solely through electrostatic interactions, the covalent connection between the protective layer and the lactase surface unexpectedly enhanced its loading capacity.

[0285] Example 2: Disaccharidase 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.

[0286] The biocatalytic activities of four different immobilized and protected disaccharidases were evaluated. Figure 11 The results shown report the enzyme activity of each nanoparticle: lactase activity of lactase-based silica nanoparticles NP-2 ( Figure 11 A) The invertase activity of NP-3 silica nanoparticles based on invertase ( Figure 11 B) Isomaltase activity of silica nanoparticles NP-4 based on isomaltase ( Figure 11 C) and dual enzymatic activity (isomaltase and invertase) of silica nanoparticles NP-5 based on invertase / isomaltase (C) Figure 11 (D) These data indicate that the disaccharide reaches the enzyme's catalytic site and is cleaved with high enzymatic activity. Validation of the biocatalytic activity of NP-2, NP-3, NP-4, and NP-5 confirms that immobilization and protection strategies can be applied to a variety of disaccharidases and may be used for therapeutic purposes.

[0287] Example 3: Biocompatibility of nanoparticles for gastrointestinal applications

[0288] The intestinal mucosa consists of a single layer of epithelial cells, which are tightly attached by intercellular tight junctions adjacent to the subepithelial region containing the lamina propria. It acts as a barrier between the external and internal environments. Its integrity is a key parameter ensuring protection of the body from unwanted contaminants such as microorganisms. Lamina propria ( lamina propria This includes diffuse lymphoid tissue composed of immune cells that maintain homeostasis or respond to disruption of epithelial protection.

[0289] To evaluate the biocompatibility of nanoparticles, we developed representative models of lactase-based silica nanoparticles NP-2 and invertase-based silica nanoparticles NP-3. NP-1 consists of inactive nanoparticles, i.e., nanoparticles with the same functionalized outer surface as NP-2 and NP-3 but without enzymatic activity.

[0290] To evaluate the safety of the nanoparticles, we first focused on maintaining intestinal barrier integrity in the presence of inactive nanoparticles NP-1. Figure 12 A). Transepithelial electrical resistance (TEER) measurements across a Caco2-HT29-MTX-E12 cell monolayer showed that the integrity of the intestinal epithelial barrier remained intact after 24 hours of contact with NP-1. This result demonstrates the in vitro biocompatibility of NP-1.

[0291] To further characterize the impact of nanoparticles on the intestinal barrier, we evaluated the ability of NP-1 to trigger an inflammatory response. For this purpose, we developed an immune-active intestinal barrier model and monitored its integrity. Figure 12 B shows that barrier integrity decreased in a dose-dependent manner when treated with the pro-inflammatory component LPS. This loss of integrity reveals the recruitment of macrophages from the base to the top of the barrier.

[0292] Most importantly, TEER measurements showed that the integrity of the epithelial barrier remained intact upon contact with NP-1. Figure 12 B). NP-1 did not stimulate the recruitment of macrophages on the apical side of the intestinal barrier, leading to the conclusion that NP-1 does not induce inflammation.

[0293] In summary, these results demonstrate the in vitro safety of nanoparticles in gastrointestinal applications.

[0294] Example 4: In vivo efficacy of lactase-based silica nanoparticles NP-2

[0295] Lactose malabsorption is attributed to 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 lactose fermentation leads to the production of short-chain fatty acids and gases, significantly enlarging the cecum.

[0296] Compared to rats fed a normal diet without additional lactose intake, rats that were tube-fed a high dose of lactose daily for 15 consecutive days had more substrate available for fermentation in their large intestine, leading to cecal enlargement (conditions were "solvent group" and "lactose-free group," respectively). Figure 13 Importantly, in rats, administration of lactase-based silica nanoparticles NP-2 significantly reduced cecal size, 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.

[0297] Enzyme replacement therapy with exogenous lactase from microorganisms is feasible, but results regarding its exact efficacy are inconsistent (Montalto et al., World J Gastroenterol 2006, Jan 14;12(2):187-91). A rule of thumb for calculating lactase dosage is 7500 units (U) of lactase per 16 grams of lactose. In addition to this close relationship between the amount of lactose to be hydrolyzed and the required enzyme units, gastric pH and bile salt concentrations influence the efficacy of exogenous lactase and the lack of specific localization of the enzyme in the intestine.

[0298] exist Figure 13 This study demonstrates the in vivo efficacy of NP-2, a lactase-based silica nanoparticle, at a dose of 97 U per 3 g of lactose. This dose surprisingly corresponds to 1 / 14.5 of the dose of currently available lactase preparations. Therefore, these data indicate the high value of NP-2 in lactose digestion and highlight the therapeutic potential of NP-2, a lactase-based silica nanoparticle, for patients with disaccharide digestion disorders, such as lactose malabsorption.

[0299] Example 5: In vitro efficacy of silica nanoparticles NP-3 based on invertase

[0300] Congenital sucrase-isomaltase deficiency (CSID) is characterized by a complete or near-complete lack of sucrose activity and varying degrees of reduced isomaltase activity.

[0301] The efficacy of NP-3 silica nanoparticles based on invertase in digesting sucrose was evaluated in an intestinal barrier model. Differentiated Caco2-HT29-MTX-E12 cell monolayers were exposed to NP-3 on their apical side in the presence of their substrate for 4 h. Figure 14The figure shows the quantification of sucrose hydrolysates on the basolateral side of the intestinal barrier. The figure demonstrates that glucose accumulation on the basolateral side of the barrier is dose-dependent in the presence of increased amounts of invertase-based silica nanoparticles NP-3, while glucose was not detected in the untreated intestinal barrier. These results demonstrate the in vitro efficacy of invertase-based silica nanoparticles NP-3 and highlight the potential applications of NP-3 in therapeutic settings.

[0302] Lipase, amylase and protease

[0303] Materials and methods:

[0304] Reagents:

[0305] - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (Grade I, 25% aqueous solution), Chelex® 100 sodium form, polysorbate 80, trypsin (4xUSP specification), bovine serum albumin (BSA), potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium taurocholate hydrate, acetic acid, olive oil, gum arabic from acacia, sodium chloride, sodium hydroxide, Trizma base, hydrogen chloride, ammonium acetate, sodium acetate, trypsin, bile salts, butanol, methanol, isopropanol, acetonitrile, NH4 acetate, amylase activity assay kit, 4% formalin buffer, Triton X-100, purchased from Sigma-Aldrich. BSA and trypsin were dissolved in water to reconstitute the stock buffer.

[0306] -p-SCN-Bn-DOTA, purchased from Macrocyclics.

[0307] -Benzyltriethoxysilane (B, 96%), purchased from abcr GmbH.

[0308] Chitosan 95 / 500P, purchased from Heppe Medical Chitosan GmbH

[0309] -Indium chloride ( 111 -In), purchased from Curium

[0310] - Animal feed Altromin 1324 and Altromin 9033, purchased from Altromine International

[0311] - Catheter, purchased from Instech Laboratories

[0312] -Caco-2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line), European Collection of Cell Cultures (ECACC).

[0313] -ThinCert™ cell culture inserts (1.0 μm membrane), purchased from Greiner bio-one.

[0314] 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, were purchased from Gibco.

[0315] Anti-tight junction band 1 (ZO-1) antibody, goat anti-rabbit IgG conjugated to Alexa Fluor 488, and DAPI-containing mounting medium were purchased from ThermoFischer Scientific.

[0316] Synthesis of silica nanoparticles (SNPs): Silica nanoparticles (50 nm) were synthesized according to the original Stöber method described in WO2015 / 014888A1. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 h. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm for 22 h at 20 °C. The solution was then centrifuged at 20,000 g for 20 min and washed successively with ethanol and water. Particle size was measured using SEM micrographs obtained at 150,000x magnification using the Olympus flow motion image analysis software.

[0317] Enzyme protection, protein protection, and surface functionalization

[0318] - Generation of NP-1 silica nanoparticles with inactive DOTA labeling and chitosan functionalization: APTES (3.8 mM) was added to the SNP (10 mg / mL, 56 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, DOTA (3.8 mM) was added and the mixture was reacted at 50 °C for 1 h. The DOTA-labeled particles were washed three times in H2O / PS80 (8 mg / L, Chelex) by centrifugation (20000 rcf, 20 min), resuspended in H2O / PS80 (8 mg / L, Chelex), and tip-sonicated. APTES (3.8 mM) was added to the DOTA-labeled particles. The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. BSA solution was added to bring the final BSA concentration to 1.42 mg / mL, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized BSA surface using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 5 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-1 was cured overnight in a water bath at 20 °C. SNPs-BSA-AT was cured overnight at 20 °C.

[0319] - Generation of unfunctionalized NP-2 from inactive DOTA-labeled silica nanoparticles: Enzyme immobilization and protection were performed similarly to those described in WO2015 / 014888:A1. 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 min. Then, DOTA (3.8 mM) was added and the mixture was incubated at 50 °C for 1 h. The DOTA-labeled particles were washed three times by centrifugation (20000 rcf, 20 min) in H2O / PS80 (8 mg / L, Chelex), resuspended in H2O / PS80 (8 mg / L, Chelex), and 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 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. BSA solution was added to bring the final BSA concentration to 1.42 mg / mL, and the reaction mixture was reacted at 20°C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized BSA surface using APTES (7.5 mM) and TEOS (75.4 mM). The resulting suspension was reacted at 20°C and 400 rpm for 5 h. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in NaCl (0.9%) / PS80 (8 mg / L). NP-2 was cured overnight in a 20°C water bath.

[0320] - Generation of NP-3 silica nanoparticles based on trypsin: APTES (3.9 mM) was added to SNP (10 mg / mL, 55 nm) in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Sodium taurocholate (2 mM) and trypsin (20 g / L) were added sequentially, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the surface of the immobilized trypsin 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 h. The particles were centrifuged (1000 rcf, 5 min), washed three times (by centrifuging at 1000 rcf for 5 min) in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L), and resuspended in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was incubated at 20 °C and 400 rpm for 30 min.

[0321] The particles were centrifuged (1000 rcf, 5 min), 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 then cured overnight in a water bath at 20 °C.

[0322] - Generation of inactive silica nanoparticles NP-4: APTES (3.9 mM) was added to SNP (10 mg / mL, 55 nm) in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.9 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.9 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. BSA (11.1 g / L) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon 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 reacted at 20 °C and 400 rpm for 5 h. The particles were centrifuged (1000 rcf, 5 min), washed three times (by centrifuging at 1000 rcf for 5 min) in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L), and resuspended in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 121 μg / mL. The reaction mixture was incubated at 20 °C and 400 rpm for 30 min.

[0323] The particles were centrifuged (1000 rcf, 5 min), 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 then cured overnight in a 20°C water bath.

[0324] - Generation of NP-5 silica nanoparticles based on trypsin: APTES (3.7 mM) was added to the 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 min. Then, glutaraldehyde (3.7 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.7 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Trypsin (23.5 mg / mL) containing lipase and / or its fragments, protease and / or its fragments, and amylase and / or its fragments was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the immobilized lactase surface using APTES (7.2 mM) and TEOS (75.6 mM). The resulting suspension was reacted at 20 °C and 400 rpm for 2 h. The particles were washed three times in H2O / PS80 (8 mg / L) (by centrifugation at 20000 rcf for 5 min) and resuspended in H2O / PS80 (8 mg / L). An acetic acid solution (0.1 M) of chitosan was added to the particle suspension to bring the final chitosan concentration to 103 μg / mL. The reaction mixture was reacted at 20 °C and 400 rpm for 30 min. The particles were centrifuged at 20000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). NP-5 was cured overnight in a 20 °C water bath.

[0325] Generation of NP-3 variants of silica nanoparticles based on trypsin: The following experiments investigated the effects of covalently linking the enzyme to the protective layer on enzyme stability and enzyme activity.

[0326] In the first comparative experiment, enzyme immobilization and protective layer formation were performed according to WO2015 / 014888 A1 to generate trypsin-based silica nanoparticles (NP-3(1)) in buffer. The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.1 mM) was added to SNP (10 mg / mL, 69 nm) in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The reaction mixture was reacted at 20°C and 400 rpm for 10 min. 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 min. 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). Initiation was performed by adding APTES (3.1 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. 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). Sodium taurocholate (2 mM) and trypsin (15 g / L) were added sequentially, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon layer was grown on the surface of the immobilized trypsin 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 resuspended in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). NP-3(1) was solidified overnight in a water bath at 20°C.

[0327] In the second experiment, according to the section titled "Generation of NP-3" in "Example 3: Immobilization and Generation of Lipase, Amylase and Protease" above, trypsin-based nanosilica particles (NP-3) were generated in phosphate buffer (20 mM, pH 8) and PS80 (8 mg / L). To retain excess glutaraldehyde in the reaction mixture that did not bind the solid support to the trypsin, the nanoparticles were not washed between each chemical step. Therefore, glutaraldehyde remained during layer growth and resulted in the covalent binding of the protective layer to the trypsin. The covalent binding of the protective layer to the trypsin could be observed by its yellow / orange appearance with maximum absorbance at 460 nm. This color is due to the formation of imine bonds between the aldehyde functional group of the glutaraldehyde linker and the primary amine of the trypsin amino acid and the organosilicon layer. After organosilicon layer formation and final particle washing, the absorbance of the trypsin-based nanoparticles NP-3(1) and NP-3 at 460 nm was measured (see [link to example 3]). Figure 24 The results show that the absorbance of NP-3 at 460 nm is much higher than that of NP-3(1). Since imine bonds are also formed during enzyme immobilization, NP-3(1) still shows a certain degree of absorbance at this wavelength. However, the absorbance of NP-3 is significantly higher, indicating that the covalent binding of the protective layer with the trypsin causes the formation of additional imine bonds.

[0328] Use 111 ( 111 In) radioactive labeling: For the labeling of nanoparticles, additional steps have been added to the methods for nanoparticles described in the sections “Enzyme Protection and Protein Protection and Surface Functionalization”, “Generation of Unfunctionalized NP-2 of Inactive DOTA-Labeled Silica Nanoparticles”, and “Generation of NP-1 of Inactive DOTA-Labeled Silica Nanoparticles Functionalized with Chitosan”.

[0329] According to the following scheme, nanoparticles with a specific activity of 500-400 MBq / g were labeled.

[0330] All buffer solutions used were pretreated with Chelex®.

[0331] At 45°C, under continuous stirring, nanoparticles were mixed with... 111 The nanoparticles were incubated with Lu (0.02M HCl) and ammonium acetate (1M, pH 5.4) for 12 hours. The nanoparticles were centrifuged at 5000g for 5 min and resuspended in sodium acetate (20mM, pH 5) with polysorbate 80 (8 mg / L). Then, the nanoparticles were 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 with polysorbate 80 (8 mg / L).

[0332] Pancreatic lipase activity assay: An olive oil solution was prepared by mixing olive oil, gum arabic, and water (1 / 8.25 / 0.75). A buffer solution was prepared by dissolving Trizma base (0.6 g / L) and sodium chloride (2.34 g / L) in nano-purified water. A bile salt solution was prepared by dissolving sodium taurocholate (80 g / L) in water.

[0333] Lipase activity was determined by mixing an olive oil solution (13.8 mL), a buffer solution (11 mL), a 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 sodium hydroxide solution (0.1 M). NP-3 (1.4 mL, 10 mg / mL) was washed twice with water and added to the bioreactor. Lipase kinetics were monitored by measuring the amount of sodium hydroxide added to the reaction mixture to maintain the pH at 9 for 10 minutes.

[0334] Trypsin activity assay: Add 250 μL of casein solution (0.65% w / v) to the enzyme sample (50 mM, pH 7.4) in phosphate buffer (50 mM, pH 7.4). Incubate the reaction mixture at 37 °C and 750 rpm for 30 min. Centrifuge the sample at 20,000 rcf for 5 min and collect the supernatant. Add 166.7 μL of TCA to the supernatant (200 μL). Incubate the sample at 37 °C and 750 rpm for 30 min. Centrifuge the sample at 20,000 rcf for 5 min and collect the supernatant. Add 500 µL of Na₂CO₃ (500 mM) and 100 μL of Folin reagent (0.5 M) to the supernatant (200 μL). Incubate the sample at 37 °C and 750 rpm for 30 min. Measure the absorbance of the 200 µL solution at 660 nm.

[0335] Pancreatic amylase activity assay: The activity of NP-3 amylase based on trypsin-containing silica nanoparticles was evaluated using the Sigma amylase assay kit. In 96-well plates, 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 sample kinetics were monitored at 37 °C for 30 min using a spectrophotometer at λ = 405 nm.

[0336] animal:

[0337] All animal experiments were conducted with permission from the National Animal Experiments Inspectorate under the Ministry of Food, Agriculture and Fisheries of Denmark.

[0338] - Rats: The study was conducted on male Wistar rats (8 weeks old) of the original breed from Janvier, France.

[0339] Food and drinking water: Rats were fed a complete pelleted diet, "Altromin 1324," with free access to food and drinking water.

[0340] Pancreatic duct ligation (PDL) and duodenal catheter insertion: The animal was placed in an induction chamber and anesthetized with isoflurane (2-4%), then transferred to a nasal mask with isoflurane for surgery. The rat was placed supine on a heated table, and an incision was made along the midline of the abdomen. The pancreas was located and gently moved to locate the bile-pancreatic duct. The pancreatic tissue surrounding the bile-pancreatic duct was bluntly dissected to visualize the duct. The pancreatic duct was ligated near the bile-pancreatic duct to block the flow of pancreatic enzymes. After ligation of the pancreatic duct, a catheter (C30PU-RDD1444, Instech Laboratories) was placed on the opposite side of the duodenal mesentery, near the opening of the bile-pancreatic duct. The catheter was ligated and secured to the intestinal wall and led through a subcutaneous tunnel to the animal's neck, exposing it. The abdominal and neck incisions were then sutured shut.

[0341] Efficacy evaluation of trypsin-based silica nanoparticles NP-3 in mice

[0342] PDL rats were administered either inactive nanoparticles NP-4 (7 mg) or trypsin-based silica nanoparticles NP-3 (7 mg; 7.5 U) via duodenum, followed by oral administration of triolein (10 mg) via tube feeding 5 minutes later. Approximately 150 μL of blood was sampled in EDTA at 0.25, 0.5, 1, 1.5, 2, 4, and 6 h following triolein administration.

[0343] Centrifuge blood samples (10 min, 4°C, 2000×g), transfer at least 50 μL of plasma to Eppendorf tubes, and store at –80°C until triglyceride content is analyzed.

[0344] Measurement of plasma triglycerides in rats: Quantification of the target analyte was performed using an LC system: a Thermo Vanquish Horizon binary pump and a Thermo Q Exactive mass spectrometer. Plasma samples were prepared according to the BUME method. Ten μL of plasma sample was mixed with 300 μL of 1-butanol / methanol (3:1, v / v). The sample was incubated at 20 °C with stirring (900 rpm) for 1 h. After centrifugation (16000 g, 10 min, 20 °C), 50 μL was transferred to a glass vial for LC-MS.

[0345] The injection volume used was 2.5 μL, the run time was 7.5 min, and the flow rate was 1 mL / min. Mobile phase A consisted of 60% acetonitrile, 40% H₂O, and 5 mM NH₄ acetate, while mobile phase B consisted of 90% isopropanol, 10% acetonitrile, and 5 mM NH₄ acetate. Chromatographic separation was performed using a Waters Premier BEH C18 column (50 mm x 2.1 mm) with a gradient of 15% B to 99% B.

[0346] MS was performed using a Thermo Q Exactive mass spectrometer in DDA top5 mode. MS parameters were as follows: MS1 resolution: 70,000 Å and MS2 resolution: 17,500 Å. HCD fragmentation was performed using normalized stepwise collision energies of 10, 20, and 30 Å. For triolein and the most abundant triglycerides, the target extraction for EIC was TG 54:3. Data analysis was performed using Thermo Q Exactive Browser software.

[0347] - Miniature pigs: The study used female Göttingen minipigs from the Danish company Ellegaard Göttingen Minipigs A / S.

[0348] Food and drinking water: Miniature pigs are regularly fed complete pelleted feed "Altromin 9033" at a daily rate of approximately 250g.

[0349] To evaluate the efficacy of NP-3 (long-term administration), the diet was changed to a high-fat diet, with Altromin 9033 supplemented daily with olive oil (1:10 – olive oil:altromin) and 100g of applesauce. The high-fat diet was started 15 days before administration and maintained throughout the long-term administration period.

[0350] Animals have free access to household-quality drinking water.

[0351] SPECT / CT imaging

[0352] SPECT / CT scans (clinical D670 SPECT / CT, GE) were initiated 15 min, 3, 8, 24, 48, and 72 hours (+ / - ½ h) after administration of In-111-labeled nanoparticles. For control animals, SPECT / CT scans were not initiated but performed 15 min, 3, 8, and 24 hours after administration of free In-111. SPECT acquisition time was determined based on the count rate on the day of scan. SPECT imaging included two fields of view (FOVs) to cover the area from the stomach to the rectum. When scanning both FOVs, the FOV including the stomach and duodenum was used as the first acquisition. Prior to the CT scan procedure, an iodine-containing contrast agent (Ultravist®, 370 mg / mL, 1 mL / kg, flow rate 2 mL / s) was administered intravenously to improve organ visibility and facilitate image analysis. The FOV used for CT imaging included the entire animal.

[0353] The animal was imaged on-site and transported from the waiting room to the scanner under anesthesia.

[0354] To quantify the uptake of In-111-labeled compounds, target regions were plotted on relevant parts of the gastrointestinal tract (three compartments – small intestine, colon, and rectum) identified from CT image data. Uptake was expressed as %ID (percentage of injected dose) and SUV (normalized uptake value). All data analyses were performed by a single observer to avoid internal observational bias.

[0355] Pancreatic duct ligation (PDL) and duodenal catheter insertion: Animals were pre-administered in stable pen via intramuscular / intravenous (IM / IV) Zoltil mixture (6.25 mL Rompun Vet® (toluidine, 20 mg / mL) + 1.25 mL Ketaminol Vet® (ketamine, 100 mg / mL) + 2.5 mL Torbugesic Vet® (ketamine, 10 mg / mL) in a bottle of Zoltil Vet 50® (125 mg Tiletamin + 125 mg zoprazepam). Once sedated, the pigs were intubated and anesthesia was maintained by inhalation of 2–4% isoflurane. A catheter was placed in a unilateral ear vein. The animal was placed in the left lateral decubitus position, and the right flank was prepared according to standard surgical procedures.

[0356] Make an incision on the right side of the abdomen and gently locate the duodenum and pancreas. Place two ligatures 2-3 mm apart around the pancreatic duct and cut the duct between the two ligatures to stop the flow of pancreatic enzymes.

[0357] After ligation of the pancreatic duct, a catheter (canine duodenal catheter 7F, SAI infusion technique) is placed on the opposite side of the duodenal mesentery, near the opening of the bile and pancreatic ducts. The catheter is ligated and secured to the intestinal wall and then led through a subcutaneous tunnel to the animal's back, exposing it. The abdominal incision is then sutured, and a protective bandage is applied around the abdomen.

[0358] Preoperative and postoperative analgesics and antibiotics

[0359] Animals were given preemptive analgesia in the form of intramuscular NSAIDs prior to surgery. Postoperative analgesia consisted of oral NSAIDs once daily for 4 days, and low-dose transdermal opioid patches for 72 hours as needed.

[0360] The antibiotic (amoxicillin) was administered intramuscularly before surgery and continued orally once daily for four days after surgery.

[0361] Efficacy evaluation of trypsin-based silica nanoparticles NP-3 in mice

[0362] Single dose: PDL miniature pigs were administered either inactive nanoparticles NP-4 (1 g) or trypsin-based silica nanoparticles NP-3 (1 g; 1365 U) via intraduodenal catheter, followed by intraduodenal administration of olive oil (14 g). Following olive oil administration, 5 mL of blood was collected in EDTA at 0, 0.0833, 0.25, 0.5, 1, 2, 3, 4, and 6 hours.

[0363] Centrifuge blood samples (10 min, 4°C, 2000×g), transfer at least 500 μL of plasma to Eppendorf tubes, and store at –80°C until triglyceride content is analyzed.

[0364] Long-term administration: PDL miniature pigs were administered twice daily via an intraduodenal catheter either inactive nanoparticles NP-4 (1 g) or trypsin-based silica nanoparticles NP-3 (1 g; 1365 U), each administration before feeding, for 10 days.

[0365] Feces were collected from each animal for 72 hours before treatment (days -3, -2, and -1) and at the end of treatment (days 8, 9, and 10). Fecal samples were kept at -20°C until analysis.

[0366] Measurement of plasma triglycerides in miniature pigs: The quantification of triglycerides in miniature pig plasma was assessed using the Konelab system according to the supplier's instructions.

[0367] Measurement of fat content in miniature pig feces

[0368] Fecal fat content was quantified using near-infrared spectroscopy (NIR) with an Impana FT 9700 (Perkin Elmer). Feces were weighed, homogenized, and placed in open glass petri dishes for NIR analysis (700–2500 nm). Measurements were taken at three distinct points for each sample. Results are expressed as the average fat concentration (grams of fat per 100 grams of wet fecal weight) at the three measurement points.

[0369] Cell culture: For all experiments, cells were cultured at 37°C and 5% CO2.

[0370] Caco2 (human colorectal adenocarcinoma cell line) and HT29-MTX-E12 (human colon cancer cell line) were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1% non-essential amino acids and 100 U / mL penicillin / streptomycin.

[0371] To develop an intestinal barrier model, cells were stored at a density of 2.6 × 10⁻⁶. 5 cells / cm 2 Density inoculation in Transwell PET inserts (1 (m pore size). All cell models were used for experiments on day 21. For co-culture, Caco-2 and HT-29-MTX-E12 cells were used at a ratio of 75%–25%.

[0372] Transepithelial resistance

[0373] Cell barrier integrity was assessed by measuring transepithelial electrical resistance (TEER) using the CellZscope system (NanoAnalytics). TEER was automatically measured every 15 minutes for 24 h, in the range of 1 Hz to 100,000 Hz, after cell culture medium renewal and treatment with nanoparticles.

[0374] Confocal microscopy analysis

[0375] Cells on the insert plate were washed with phosphate-buffered saline and fixed with 4% formalin. The fixed cells were permeabilized in PBS containing 1% Triton-X 100 (reference). Non-specific binding was blocked with PBS containing 3% bovine serum albumin (BSA). Cells were incubated with primary antibody against tight junction band 1 (ZO-1; 1:100) at room temperature for 2 h. After washing, cells were incubated with the corresponding fluorescently labeled secondary antibody: goat anti-rabbit IgG conjugated to Alexa Fluor 488 (1:1000) in the dark at RT for 2 h. To acquire images, a membrane was cut from the insert plate, placed between two coverslips with a drop of DAPI-containing mounting medium, and imaged using a confocal laser scanning microscope (FluoView, FV3000, Olympus, Tokyo, Japan).

[0376] result: Example 1: Biodistribution of chitosan-functionalized nanoparticles in miniature pigs To evaluate the benefits of chitosan-functionalized nanoparticles specifically retained in the gastrointestinal tract of miniature pigs via mucobinding, unfunctionalized inactive DOTA-labeled silica nanoparticles (NP-2) and chitosan-functionalized inactive DOTA-labeled silica nanoparticles (NP-1) were administered to the duodenum of miniature pigs via endoscopy. SPECT / CT images were acquired at different time points (15 min to 24 h), and the biodistribution of nanoparticles in the gastrointestinal tract was analyzed using a 3-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 below. Figure 17 As shown in Figure A. Comparison of biodistribution data of animals given NP-1 and NP-2 using SPECT / CT technology revealed that NP-1 had a 2.7-fold longer retention time in the small intestine than the unfunctionalized nanoparticles (AUC: NP-2). vs NP-1 is 170.7. vs 460.9)( Figure 17 (B) These results demonstrate that NP-1 can be specifically retained in the gastrointestinal tract in vivo. In summary, these in vivo data reveal that surface functionalization of protected nanoparticles with chitosan allows for specific targeting of intestinal mucus, suggesting the potential for temporarily anchoring nanoparticles to the intestinal wall.

[0377] Example 2: Biocatalytic activity of trypsin-based silica nanoparticles NP-3 and NP-5

[0378] Pancreatic exocrine insufficiency refers to a condition in which the pancreas's exocrine function is impaired, resulting in an inability 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 therapies include pancreatic enzyme products derived from pigs (lipase, amylase, protease).

[0379] The enzyme activities of trypsin-based silica nanoparticles NP-3 and NP-5 were evaluated. The lipase and amylase activities of NP-3 are shown in the figure. Figure 18 A and Figure 18 In C, the enzymatic activity of the Np-5 protease is shown in... Figure 18 B.

[0380] These data demonstrate the ability to immobilize multiple enzymes on nanoparticles while perfectly preserving the activity of each enzyme. Validation of the biocatalytic activity on these nanoparticles suggests that enzyme immobilization and protection strategies can be applied to restore digestive function and may be used for therapeutic purposes.

[0381] Example 3: In vivo activity of trypsin-based silica nanoparticles NP-3 in PDL rats

[0382] Given the importance of fat digestion for patients with pancreatic exocrine insufficiency (EPI), PDL rats were used as the first animal model to evaluate the validation of the pancreatic enzyme-based silica nanoparticle NP-3 by focusing on its ability to digest lipids in vivo. PDL rats were administered either the pancreatic enzyme-based silica nanoparticle NP-3 or the inactive nanoparticle NP-4 (hereinafter referred to as the dosing sequence) intraduodenally before being fed with trioleate. Following a single dosing sequence, an increase in plasma TG was observed in PDL rats receiving NP-3 (active nanoparticles) compared to those receiving the inactive nanoparticles (NP-4). Figure 19 This result demonstrates that NP-3 silica nanoparticles based on pancreatic enzymes can restore lipase digestive function in an EPI animal model.

[0383] Example 4: In vivo activity of trypsin-based silica nanoparticles NP-3 in PDL miniature pigs

[0384] From an anatomical and physiological perspective, pig models are widely considered to have a high degree of similarity to the human gastrointestinal tract. Therefore, PDL miniature pigs were generated to evaluate the efficacy of the pancreatic enzyme-based silica nanoparticle NP-3. As described previously in the PDL rat model, the first validation of NP-3 involved a single administration of the nanoparticles to PDL miniature pigs, followed by an application of olive oil. When compared with healthy miniature pigs, the increase in plasma TG levels in PDL miniature pigs administered the pancreatic enzyme-based silica nanoparticle NP-3 showed similar kinetics, reaching its maximum after 3 hours of feeding. Figure 20A). The area under the curve (AUC) calculation shows that the pancreatic enzyme-based silica nanoparticles NP-3 have a remarkably high digestibility of 62% compared to healthy miniature pigs. Figure 20 B). These results confirm that NP-3 silica nanoparticles based on pancreatic enzymes can restore lipase digestive function in EPI animal models.

[0385] Example 5: In vivo therapeutic efficacy of trypsin-based silica nanoparticles NP-3 in PDL miniature pigs

[0386] To further demonstrate that the pancreatic enzyme-based silica nanoparticle NP-3 can restore lipase digestive function, PDL miniature pigs fed a high-fat diet were given the pancreatic enzyme-based silica nanoparticle NP-3 daily for 10 days. Since the primary endpoint for EPI patient treatment is currently the quantification of fat absorption, the benefit of the pancreatic enzyme-based silica nanoparticle NP-3 was evaluated by measuring unabsorbed fat excreted in feces. Comparison of fecal fat content between healthy miniature pigs and untreated PDL miniature pigs showed that the accumulation of fecal fat in the EPI animal model reflected a deficiency in lipase activity (3.42% in healthy miniature pigs and 3.42% in untreated PDL miniature pigs). vs 5.55 g / 100g feces) Figure 21A Importantly, compared with untreated PDL miniature pigs, PDL miniature pigs treated with NP-3 had reduced fecal fat content levels. Figure 21A In fact, PDL miniature pigs that received a 3-day dose of NP-3, a pancreatic enzyme-based silica nanoparticle, were able to digest 53.4% ​​of their fat intake. Figure 21B These results clearly demonstrate the therapeutic efficacy of NP-3 silica nanoparticles based on pancreatic enzymes for pancreatic enzyme replacement therapy.

[0387] Enzyme replacement therapy is currently available to treat clinical symptoms and malabsorption. It is recommended to take at least 40,000 to 50,000 units of PERT-lipase with each main meal, and half the dose with snacks. This dosage places a heavy burden on patients. However, this treatment method is not very effective and has some limitations (persistent symptoms, short survival time of lipase in the intestinal lumen, the possibility of intolerance due to high enzyme loads, and gastrointestinal problems caused by high protease levels).

[0388] The efficacy of NP-3, a silica nanoparticle-based pancreatic enzyme, was compared with that of free pancreatic enzyme. The dosage of NP-3 for lipase activity was calculated based on fat intake (1300 U per dose, 14 g olive oil), while the dosage of pancreatic enzyme for lipase activity followed standard treatment (40,000 U per dose). Surprisingly, administration of NP-3 at a dose of 1300 U twice daily significantly improved digestive efficiency by 25% compared to standard treatment with pancreatic enzyme (administered at a higher dose of 40,000 U twice daily). In fact, 80,000 U of pancreatic enzyme daily promoted 43.6% fat digestion, while 2600 U of NP-3 daily achieved 53.4% ​​fat digestion. Figure 21B The pancreatic enzyme-based silica nanoparticles NP-3 are contained within nanoparticles functionalized with mucosal adhesion components, allowing the nanoparticles to interact with intestinal mucus (PCT / EP2023 / 051194). This results in the temporary attachment of NP-3 to the intestinal wall, where it sustains lipase activity. Furthermore, immobilizing and protecting the lipase on the nanoparticles protects the enzyme from external stress (WO 2022 / 223699 A1) and stabilizes its activity. Therefore, these data demonstrate the added value of the pancreatic enzyme-based silica nanoparticles NP-3 for lipase digestion and highlight the therapeutic potential of NP-3 for patients with EPI.

[0389] Example 6: In vitro biocompatibility of trypsin-based silica nanoparticles NP-5

[0390] Currently, patients with pancreatic exocrine insufficiency are taking large amounts of pancreatic enzymes (10-20 tablets / day), resulting in a high daily intake of proteases. While they play a digestive role, gastrointestinal proteases also contribute to intestinal homeostasis. Any imbalance in protease levels can lead to gastrointestinal disorders (Vergnolle N). people, Gut 2016;65:1215–1224).

[0391] To evaluate the safety of NP-5 silica nanoparticles based on pancreatic enzymes, we focused on maintaining intestinal barrier integrity in the presence of the nanoparticles and compared it with that of pancreatic enzymes. Figure 22 A). Transepithelial electrical resistance (TEER) measurements across the Caco2-HT29-MTX-E12 cell monolayer showed that the integrity of the intestinal epithelial barrier remained intact after 20 h of NP-5 exposure, while treatment with pancreatic enzymes led to a dose-dependent loss of barrier integrity. To correlate TEER measurements with the morphological characteristics of the cell monolayer, tight junction 1 (ZO-1) was evaluated using confocal microscopy. Figure 22(B) When cultured in the presence of NP-5, ZO-1 staining showed a continuous ring pattern at the cell edges; while in the presence of pancreatic enzymes, ZO-1 staining showed a diffuse punctate distribution of proteins around intestinal cells. These morphological data confirm the safety of the pancreatic enzyme-based silica nanoparticle NP-5 for gastrointestinal applications and highlight the added value of our technology in pancreatic enzyme replacement therapy.

[0392] Example 7: Enhance pancreatic enzyme stability by covalently linking to the protective layer.

[0393] In the first experiment, trypsin-based silica nanoparticles NP-3(1) were generated under buffered conditions, including washing after each chemical step (i.e., removal of glutaraldehyde before layer growth). In the second experiment, trypsin-based silica nanoparticles NP-3 were generated under non-buffered conditions without any intermediate washing steps (i.e., unreacted glutaraldehyde remains in the reaction mixture during layer growth).

[0394] Protein quantification was performed on the reaction supernatant to determine the enzyme immobilization yield on NP-3(1) and the NP-3 surface. Surprisingly, enzyme immobilization (NP-3) under the condition of maintaining glutaraldehyde resulted in a two-fold increase in enzyme immobilization yield. Figure 23 A), resulting in a SNP enzyme loading per unit dry weight that is twice that of buffer conditions (NP-3(1)) for removing glutaraldehyde via a washing step. Figure 23 B).

[0395] The lipase biocatalytic activity of pancreatic enzymes immobilized and protected on NP-3(1) and NP-3 was evaluated. Even more surprisingly than the increase in enzyme immobilization load while maintaining glutaraldehyde, the specific activity of the nanoparticles increased by 11-fold compared to buffered conditions where glutaraldehyde was removed via a washing step. Figure 23 C). Compared to buffered conditions where glutaraldehyde is removed via a washing step, these surprising 11-fold increases in the specific activity of the nanoparticles were accompanied by an extremely surprising 7-fold increase in the pancreatic specific activity (enzyme units / g pancreatic enzyme) of the enzyme protected in the presence of glutaraldehyde. Figure 23 D) In ​​summary, compared with pancreatic enzymes protected only by an organosilicon layer through electrostatic interactions, the covalent connection between the protective layer and the surface of the pancreatic enzyme unexpectedly enhanced its loading capacity, stability, and specific enzyme activity.

[0396] Lipase

[0397] Materials and methods:

[0398] Reagents:

[0399] - Tetraethyl orthosilicate 99% (TEOS), (3-aminopropyl)-triethoxysilane (APTES), ammonium hydroxide (ACS grade, 28-30%), ethanol (ACS grade, anhydrous), glutaraldehyde (Grade I, 25% aqueous solution), polysorbate 80, recombinant human pancreatic lipase (HRL, certified reference material), porcine pancreatic lipase (4x USP specification), 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate), Tris base, colipase, sodium taurocholate hydrate, purchased from Sigma-Aldrich. HRL, porcine pancreatic lipase, and colipase were dissolved in water to reconstitute the stock buffer.

[0400] -Benzyltriethoxysilane (B, 96%), purchased from abcr GmbH.

[0401] - A trifluoroacetate compound of the peptide Glu-Leu-Gly-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Arg-Glu-Gly-Gly-Gly-Gly-Gly-Arg-Gly-Gly-Gly-Gly-Asn-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 6) with an E-azido-Nle-OH group at the carboxyl terminus, purchased from Bachem.

[0402] Synthesis of silica nanoparticles: Silica nanoparticles (50 nm) were synthesized according to the original Stöber method described in WO2015 / 014888A1. Briefly, ethanol, distilled water (6 M), and ammonium hydroxide (0.13 M) were mixed and stirred at 400 rpm for 1 h. TEOS (0.28 M) was added, and the solution was stirred at 400 rpm for 22 h at 20 °C. The solution was then centrifuged at 20,000 g for 20 min and washed successively with ethanol and water. Particle size was measured using SEM micrographs obtained at 150,000x magnification using the Olympus flow motion image analysis software.

[0403] Assay of free recombinant human pancreatic lipase activity: Add 60 μL of 1,2-di-O-lauryl-rac-glycerol-3-(6-methyl styrene glutarate) to a Tris buffer solution (0.1 M, pH 8.4, 56.6 μL) containing recombinant human pancreatic lipase (3.43 μL, 1 mg / mL) and 1,2-di-O-lauryl-rac-glycerol-3-(6-methyl styrene glutarate) (100 μM). Measure the steady-state fluorescence (λ) in a dark 96-well plate at 37 °C for 30 min. ex / λ em= 529 / 600 nm) to monitor lipase activity kinetics.

[0404] Assay of free recombinant human pancreatic lipase activity in the presence of colipase: Add 60 μL of 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate) to a Tris buffer (0.1 M, pH 8.4, 55.8 μL) solution of recombinant human pancreatic lipase (3.43 μL, 1 mg / mL) and colipase (0.72 μL, 3 μg / mL) (0.1 M, pH 8.4, 55.8 μL). Steady-state fluorescence (λ) was measured by continuous fluorescence in a dark 96-well plate at 37 °C for 30 min. ex / λ em = 529 / 600 nm) to monitor lipase activity kinetics.

[0405] Assay of immobilized and protected recombinant human pancreatic lipase activity: Add 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate) (60 μL, 100 μM) to a Tris buffer solution (0.1 M, pH 8.4, 40 μL) containing immobilized and protected recombinant human pancreatic lipase (20 μL, 10 mg / mL). Measure steady-state fluorescence (λ) in a dark 96-well plate at 37 °C for 30 min. ex / λ em = 529 / 600 nm) to monitor lipase activity kinetics.

[0406] Assay of immobilized and protected porcine pancreatic lipase activity: 1,2-Di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate) (50 μM) was added to a Tris buffer (0.1 M, pH 8.4) solution of immobilized and protected porcine pancreatic lipase (15 μg / mL enzyme). Steady-state fluorescence (λ) was measured by continuous fluorescence in a dark 96-well plate at 37 °C for 30 min. ex / λ em = 529 / 600 nm) to monitor lipase activity kinetics.

[0407] Example 1

[0408] A) Immobilization and shielding of recombinant human pancreatic lipase (HRL)

[0409] APTES (3.8 mM) was added to SNP (10 mg / mL) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Recombinant human pancreatic lipase solution (525 μg / mL, 11 μM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon 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 h. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). SNPs-HRL-ATB was then cured overnight in a water bath at 20 °C.

[0410] B) Co-immobilization and protection of recombinant human pancreatic lipase (HRL) and co-lipase (CLPS):

[0411] APTES (3.8 mM) was added to SNP (10 mg / mL) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. A solution containing recombinant human pancreatic lipase (525 μg / mL, 11 μM) and colipase (110 μg / mL) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon 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 h. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). SNPs-HRL-CLPS-ATB were then cured overnight in a water bath at 20 °C.

[0412] Activation of recombinant human pancreatic lipase (HRL) with colipase

[0413] The activation of recombinant human pancreatic lipase with colipase was studied using the fluorescent lipase substrate 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate). Figure 27 a) The kinetics of lipase substrate hydrolysis induced by lipase in the presence of colipase are faster than those of lipase alone, indicating that colipase can appropriately activate lipase.

[0414] To generate activated lipase nanoparticles, recombinant human pancreatic lipase was co-immobilized with a colipase and protected within a hydrophobic organosilica shield on the surface of silica nanoparticles, as described in section B) above. The activation effect of the colipase on the lipase was evaluated using the fluorescent lipase substrate 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate). Figure 27 (b) The faster kinetics of lipase substrate hydrolysis by the co-immobilized co-lipase described in (b) above, compared to the lipase without co-lipase immobilization described in (a) above, indicate that the activation of the lipase by the co-lipase in the protected nanoparticles is surprisingly more than three times higher than that of the lipase without co-lipase. These results validate a strategy of co-immobilizing lipase and co-lipase on the surface of silica nanoparticles to produce nanoparticles with immobilized and protected lipase that maintains its active conformation.

[0415] Example 2

[0416] A) Immobilization and shielding of porcine pancreatic lipase (PL)

[0417] APTES (3.8 mM) was added to SNP (10 mg / mL) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Porcine pancreatic lipase solution (1.9 mg / mL, 39 μM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon 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 h. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). SNP-PL-ATB was then cured overnight in a water bath at 20 °C.

[0418] B) According to the present invention, porcine pancreatic lipase (PL) and co-lipase (CLPS) are co-immobilized and screened for protection:

[0419] APTES (3.8 mM) was added to SNP (10 mg / mL) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. A solution containing porcine pancreatic lipase (1.9 mg / mL, 39 μM) and colipase (150 μg / mL) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon 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 h. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). SNPs-PL-CLPS-ATB were then cured overnight in a water bath at 20 °C.

[0420] Activation of porcine pancreatic lipase (PL) using colipase

[0421] To generate activated lipase nanoparticles, porcine pancreatic lipase was co-immobilized with a colipase and protected on the surface of silica nanoparticles in a hydrophobic organosilicon protectant, as described in section B) above. The activation effect of the colipase on the lipase was evaluated using the fluorescent lipase substrate 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate). Figure 28 The faster kinetics of lipase substrate hydrolysis by the co-immobilized co-lipase described in B) above, compared to the lipase without co-lipase immobilization described in A) above, indicate that the co-lipase activates the lipase in the protected nanoparticles at a surprisingly high rate, up to three times, compared to the lipase without co-lipase. These results validate a strategy of co-immobilizing lipase and co-lipase on the surface of silica nanoparticles to produce nanoparticles with immobilized and protected lipases that maintain their active conformation. This also demonstrates the versatility of this method in terms of the source of the enzymes used.

[0422] Example 3

[0423] Immobilization and protection of porcine pancreatic lipase (PL) in the presence of sodium taurocholate (NaTc):

[0424] APTES (3.8 mM) was added to SNP (10 mg / mL) in a mixture of NaTc (2 mM), H2O, and PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.8 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.8 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Porcine pancreatic lipase (1.9 mg / mL, 39 μM) was added, and the reaction mixture was reacted at 20 °C and 400 rpm for 10 min. An organosilicon 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 h. The particles were centrifuged at 20,000 rcf for 5 min and washed three times in H2O / PS80 (8 mg / L). SNPs-PL-NaTc-ATB were then cured overnight in a water bath at 20 °C.

[0425] Activation of recombinant human pancreatic lipase (HRL) with sodium taurocholate (NaTc)

[0426] The activation of recombinant human pancreatic lipase by sodium taurocholate was studied using the fluorescent lipase substrate 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate). Figure 29 With increased sodium taurocholate concentration, the kinetics of lipase substrate hydrolysis induced by lipase are faster than those of lipase alone, indicating that bile salts appropriately activate lipase.

[0427] Activation of porcine pancreatic lipase (PL) with sodium taurocholate (NaTc)

[0428] To generate activated lipase nanoparticles, porcine pancreatic lipase was immobilized in the presence of sodium taurocholate and protected within a hydrophobic organosilicon protectant on the surface of silica nanoparticles. The activation of the lipase by sodium taurocholate was evaluated using the fluorescent lipase substrate 1,2-di-O-lauryl-rac-glycerol-3-(6-methylresorcinol glutarate). Figure 30 The kinetics of substrate hydrolysis of lipase immobilized in the presence of sodium taurocholate were faster than those immobilized in the absence of bile salts, indicating that the activation of lipase by sodium taurocholate in shielded nanoparticles was surprisingly almost three times that of lipase without sodium taurocholate. These results validate a strategy of immobilizing lipase on the surface of silica nanoparticles in the presence of sodium taurocholate to produce nanoparticles with immobilized and protected lipase that maintains its active conformation.

[0429] Example 4: Activation of pancreatic lipase (PL) using colipase mimic peptides.

[0430] Analysis of the lipase-colipase complex allowed for the identification of amino acid residues responsible for the lipase-colipase interaction. Based on these findings, we designed a polypeptide (Glu-Leu-Gly-Gly-Arg-Gly-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) as shown in SEQ ID NO: 6 to mimic the interaction between the colipase and the lipase structure, inducing a conformational change around the cap and the opening of the lipase cap. Figure 31 The peptide is chemically modified at its carboxyl terminus by adding an -E-azido-Nle-OH group to enable the crosslinking of lipases on the surface of silica nanoparticles via click chemistry.

[0431] Example 5: based on HRL Generation of the NP-1 variant of silica nanoparticles: The following experiments investigated the effects of covalently linking the enzyme to the protective layer on enzyme stability and enzyme activity.

[0432] In the first experiment, a product based on H2O / PS80 (8 mg / L) was generated. HRLSilica nanoparticles (NP-1(1)). The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.1 mM) was added to SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. The particles were washed three times in H2O / 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 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Initiation was performed by adding APTES (3.1 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). Sodium taurocholate (2 mM) and recombinant human 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 min. An organosilicon 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 h. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). NP-1(1) was cured overnight in a water bath at 20 °C.

[0433] In the second comparative experiment, enzyme immobilization and protective layer formation were performed according to WO2015 / 014888 A1 to generate HRL-based silica nanoparticles (NP-1(2)) in buffer. The nanoparticles were washed after each chemical step to remove glutaraldehyde. APTES (3.1 mM) was added to SNP (10 mg / mL, 69 nm) in phosphate buffer (25 mM, pH 7.5) and PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. 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 min. 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). Initiation was performed by adding APTES (3.1 mM), and the reaction mixture was stirred at 20°C and 400 rpm for 10 min. 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). Sodium taurocholate (2 mM) and recombinant human 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 min. An organosilicon 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 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). NP-1(2) was cured overnight in a water bath at 20 °C.

[0434] In the third experiment, HRL-based silica nanoparticles (NP-1) were generated in H2O / PS80 (8 mg / L). To maintain an excess of glutaraldehyde in the reaction mixture without the solid support linked to the engineered HRL, the nanoparticles were not washed between each chemical step. Therefore, the glutaraldehyde remained during layer growth and resulted in the covalent binding of the protective layer to the human recombinant lipase. APTES (3.1 mM) was added to the SNP (10 mg / mL, 69 nm) in H2O / PS80 (8 mg / L). The reaction mixture was reacted at 20 °C and 400 rpm for 10 min. Then, glutaraldehyde (3.1 mM) was added, and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Initiation was performed by adding APTES (3.1 mM), and the reaction mixture was stirred at 20 °C and 400 rpm for 10 min. Sodium taurocholate (2 mM) and recombinant human 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 min. An organosilicon 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 h. The particles were washed three times in H2O / PS80 (8 mg / L) and resuspended in H2O / PS80 (8 mg / L). NP-1 was cured overnight in a 20 °C water bath.

[0435] The covalent bonding of the protective layer to HRL can be observed through its yellow / orange appearance with maximum absorbance at 460 nm. This color is due to the formation of imine bonds between the aldehyde functional groups of the glutaraldehyde linker and the primary amine of the amino acid in HRL and the organosilicon layer. After the formation of the organosilicon layer and final particle washing, the absorbance of HRL-based silica nanoparticles NP-1(1), NP-1(2), and NP-1 at 460 nm was measured based on AAV (see AAV). Figure 33 A). The results showed that the absorbance of NP-1 at this wavelength was much higher than that of NP-1(2). Interestingly, although NP-1 appeared to be a darker color visually, the absorbance values ​​of NP-1 and NP-1(1) were similar. This indicates that the instrument could not distinguish the formation of imine bonds due to strong interference from the particles themselves (because of the low amount of enzyme used). To overcome this limitation, we used an inverted microscope to capture images of NP ( Figure 33 B). At the same concentration, NP-1 was significantly darker than NP-1 (1) under a microscope. This visual confirmation suggests that NP-1 contains more imine bonds, which may be due to the covalent connection between the protective layer and human recombinant lipase.

[0436] Enhancing the stability and specific activity of human recombinant lipase by covalently linking it to the protective layer.

[0437] In the first experiment, HRL-based silica nanoparticles NP-1(1) were generated under unbuffered conditions, including washing after each chemical step (i.e., removal of glutaraldehyde before layer growth). In the second experiment, HRL-based silica nanoparticles NP-1(2) were generated under unbuffered conditions, including washing after each chemical step (i.e., removal of glutaraldehyde before layer growth). In the third experiment, HRL-based silica nanoparticles NP-1 were generated under unbuffered conditions without any intermediate washing steps (i.e., unreacted glutaraldehyde remains in the reaction mixture during layer growth).

[0438] Protein quantification was performed on the reaction supernatant to determine the HRL immobilization yield of HRL-based silica nanoparticles NP-1(1), NP-1(2), and the surface of NP-1. The results showed that, surprisingly, enzyme immobilization (NP-1) under the condition of maintaining glutaraldehyde resulted in a 4-fold increase in enzyme immobilization yield. Figure 32 A), resulting in an enzyme loading / SNP dry weight ratio that is four times higher than that under buffered conditions for removing glutaraldehyde via a washing step (NP-1(2)). Figure 32 B).

[0439] The lipase biocatalytic activity of HRL immobilized and protected on NP-1(1), NP-1(2), and NP-1 was evaluated. Even more surprising than the increase in enzyme immobilization load while maintaining glutaraldehyde, the specific activity of the nanoparticles increased by 37-fold compared to buffered conditions where glutaraldehyde was removed via a washing step, and by 2-fold compared to unbuffered conditions where glutaraldehyde was removed via a washing step. Figure 32 C). These surprising 37-fold and 2-fold increases in the specific activity of the nanoparticles compared to buffered and non-buffered conditions where glutaraldehyde was removed via a washing step were accompanied by HRL specific activity (UL) of the enzyme protected in the presence of glutaraldehyde. µM / min The extremely surprising 10-fold and 2-fold increase in / HRL ( Figure 32 D). This result was completely unexpected, as the enzyme was expected to have much higher activity in the presence of buffer.

[0440] In summary, compared with enzymes protected by an organosilicon layer solely through electrostatic interactions, the covalent connection between the protective layer and the HRL surface unexpectedly enhanced their loading capacity, stability, and specific enzyme activity.

Claims

1. A method of 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 thereof by embedding the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, the method comprising the following steps: (a) Providing a solid carrier, wherein the solid carrier is provided in the form of a suspension; (b) Immobilizing a protein or a fragment thereof on the solid support, wherein i) a linker is added to a suspension of the solid support, and ii) a solution of the protein or a fragment thereof is added to a suspension containing the solid support and the linker, wherein the linker connects the solid support to the protein or a fragment thereof; (c) A protective layer is formed on the surface of a solid support to protect a protein or fragment thereof fixed on the solid support, wherein a linker or portion thereof that did not connect the solid support to the protein or fragment thereof in step (b) covalently binds the protective layer to the protein or fragment thereof; and optionally (d) A functional component is fixed to the surface of the protective layer, wherein the functional component fixed to the surface of the protective layer is a polymer containing repeating units, wherein each repeating unit contains at least one amino group and / or at least one thiol group.

2. The method according to claim 1, wherein there is no washing step between adding the linker to the suspension of the solid carrier in step (b) (i) and adding the protein or 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 1-3, wherein the solid carrier is provided in the form of a suspension in water, a buffer solution, or a nonionic surfactant or a mixture thereof.

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

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

7. The method according to any one of claims 1-5, 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 molar excess of the protein or fragment thereof added to the suspension comprising the solid carrier and the linker in step (b) ii).

8. The method according to any one of claims 1-5, 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 of the protein or fragment thereof added to the suspension comprising the solid carrier and the linker in step (b) ii).

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

10. The method according to any one of claims 1-9, wherein after adding the protein or a fragment thereof in step ii), the amount of the linker that does not link the solid carrier to 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 step (b) in step i).

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

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

13. The method according to any one of claims 1-12, wherein the binder is selected from glutaraldehyde, disuccinimide tartrate, bis[sulfosuccinimide] octanoate, ethylene glycol bis(sulfosuccinimide succinate), dimethyl adipate, dimethyl heptamethimide, sulfosuccinimide (4-iodoacetyl)aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, BSOCOES (bis[2-(succinimideoxycarbonyloxy)ethyl] sulfone), DSP (dithiobis[succinimide] propionate), DTSSP (3,3'-dithiobis[sulfosuccinimide] propionate), DTBP (3,3'-dithiobispropionine dimethyl ester·2HCl), DST (disuccinimide tartrate), BMDB (1,4-bismaleimide-2,3-dihydroxybutane).

14. The method according to any one of claims 1-12, wherein the binder 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 the protein or fragment thereof, 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, wherein each repeating unit comprises at least one amino group and / or at least one thiol group, wherein the composition is obtained by the method of any one of claims 1-14.

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