Silk protein nanosphere-encapsulated hangover enzyme preparation and preparation method thereof

Abstract

The present application relates to the technical field of biological medical materials and enzyme preparation, and particularly relates to a silk fibroin nanosphere-wrapped alcohol-eliminating enzyme preparation and a preparation method thereof, which comprises: a silk fibroin nanosphere core, the silk fibroin nanosphere core internally co-wraps ethanol dehydrogenase and acetaldehyde dehydrogenase and a trehalose protective agent, the silk fibroin of the silk fibroin nanosphere core is in a beta-fold structure, the mass ratio of the ethanol dehydrogenase to the acetaldehyde dehydrogenase is 1:1.5 to 1:2.5, and the mass ratio of the trehalose to the silk fibroin is 1:8 to 1:12. The present application directly simulates the alcohol metabolism path of human liver by using the combination of ethanol dehydrogenase and acetaldehyde dehydrogenase; the ethanol dehydrogenase catalyzes the oxidation of ethanol into acetaldehyde while consuming coenzyme NAD + , and the acetaldehyde dehydrogenase oxidizes the acetaldehyde into acetic acid in the presence of NAD + .

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and enzyme preparations, specifically to a method for preparing a hangover-detoxifying enzyme preparation encapsulated in silk fibroin nanospheres. Background Technology

[0002] The metabolism of alcohol in the human body mainly relies on the tandem catalytic action of alcohol dehydrogenase and aldehyde dehydrogenase in the liver. Alcohol dehydrogenase first oxidizes ethanol to acetaldehyde, and aldehyde dehydrogenase then oxidizes acetaldehyde to acetic acid, which is ultimately broken down into carbon dioxide and water.

[0003] However, about 30% to 40% of East Asians have mutations in the acetaldehyde dehydrogenase gene, resulting in impaired acetaldehyde metabolism. After drinking alcohol, acetaldehyde accumulates in the body, causing discomfort such as facial flushing, palpitations, and nausea. In the long term, it can even lead to alcoholic liver damage.

[0004] Currently, using exogenous enzyme preparations to assist alcohol metabolism is a promising strategy for sobering up. Chinese invention patent CN106822038B discloses a silk nanosphere encapsulating an enzyme, which is prepared by mixing a silk solution with an enzyme solution and then adding the mixture to acetone.

[0005] This technology uses a combination of alcohol oxidase and catalase as the alcohol-degrading enzyme system, which has the following limitations: alcohol oxidase catalyzes the oxidation of ethanol to produce hydrogen peroxide, which can be decomposed by catalase, but the local and temporary presence of hydrogen peroxide may still cause oxidative damage to the gastrointestinal mucosa, making it particularly unsuitable for oral application.

[0006] In the preparation process using acetone precipitation, the organic solvent directly contacts the enzyme molecules, which can lead to irreversible denaturation and inactivation of some enzyme molecules, thus limiting the enzyme activity retention rate. Third, the surface of the nanospheres lacks a gastric acid protective layer, and the enzyme activity drops sharply when it is taken orally into the gastric acid environment, making it difficult to achieve effective oral delivery.

[0007] Chinese invention patent CN114848841B discloses a nanocapsule containing alcohol oxidase and aldehyde dehydrogenase encapsulated in a metal-organic framework for the treatment of alcohol poisoning. However, the carrier is a metal-organic framework, which has poor biodegradability and biocompatibility compared to natural protein materials. Furthermore, the dissolution of metal ions from the metal-organic framework carrier may pose safety hazards.

[0008] Existing technologies disclose oral formulations that encapsulate alcohol oxidase, aldehyde dehydrogenase, and catalase in inulin nanogels, but these still employ the alcohol oxidase pathway, and the inulin carrier provides limited protection for the thermal stability of the enzymes.

[0009] Therefore, further research is needed to overcome the problems of enzyme inactivation caused by organic solvents, the generation of toxic intermediates in the enzyme catalytic pathway, and poor stability during oral delivery in the existing silk fibroin nanosphere encapsulation technology for alcohol-degrading enzymes. Summary of the Invention

[0010] The primary objective of this invention is to provide a silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation and its preparation method.

[0011] A further objective of this invention is to provide a silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation and its preparation method.

[0012] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention uses a combination of alcohol dehydrogenase and aldehyde dehydrogenase to directly simulate the human liver's alcohol metabolism pathway; alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde while consuming the coenzyme NAD. + aldehyde dehydrogenase then reacts with NAD+. + In the presence of [a specific substance], acetaldehyde is oxidized to acetic acid. This tandem catalytic pathway does not produce the toxic intermediate hydrogen peroxide and is significantly safer than the alcohol oxidase pathway in existing technologies, making it particularly suitable for oral applications.

[0013] 2. This invention uses a calcium chloride-mediated self-assembly method to prepare silk fibroin nanospheres. The entire process is carried out in an aqueous phase, avoiding irreversible denaturation and inactivation caused by direct contact of enzyme molecules with organic solvents such as acetone. Calcium ions form coordination salt bridges with the carboxyl and amino groups on the silk fibroin molecular chain, inducing the silk fibroin to transform from random coil and α-helix conformation to β-sheet conformation. Under mild conditions, the self-assembly of nanospheres and simultaneous encapsulation of enzymes are achieved, and the enzyme activity retention rate is significantly higher than that of the acetone precipitation method.

[0014] 3. This invention co-encapsulates trehalose as a protective agent within silk fibroin nanospheres. Trehalose and silk fibroin exhibit a synergistic effect in protecting enzyme activity: the β-sheet network of silk fibroin provides a structural confinement effect, physically restricting enzyme molecules and preventing their unfolding and denaturation; multiple hydroxyl groups of trehalose form a hydrogen bond network with polar groups on the enzyme molecule surface, replacing water molecules to maintain the enzyme's native conformation when water molecules are excluded; the synergistic effect significantly improves the enzyme's thermal stability and long-term activity retention during storage. This synergistic effect far exceeds the simple sum of the effects of using either agent alone. Example data demonstrates that the enzyme activity retention rate under co-encapsulation conditions is 6.0 percentage points higher than the sum of the activity retention rates under silk fibroin encapsulation alone and trehalose protection alone, exhibiting a non-linear synergistic gain.

[0015] 4. This invention modifies the outer layer of silk fibroin nanospheres with a chitosan layer. Under gastric acid pH conditions, chitosan protonates to form a dense, positively charged protective layer, preventing gastric acid from penetrating the nanospheres and destroying enzyme activity. Under neutral to weakly alkaline intestinal conditions, chitosan deprotonates and gradually swells, promoting controlled enzyme release. The mucosal adhesion properties of chitosan prolong the retention time of the nanospheres in the intestinal wall, enhancing intestinal absorption. Chitosan modification increases the enzyme activity retention rate of the nanospheres in simulated gastric juice for 2 hours from 31.5% without the chitosan layer to 78.6%.

[0016] 5. This invention uses genipin as a cross-linking agent. Genipin is a naturally sourced cross-linking agent with cytotoxicity far lower than chemical cross-linking agents such as glutaraldehyde. It reacts with the amino groups of silk fibroin and chitosan to form a stable heterocyclic cross-linked structure, enabling the nanospheres to maintain structural integrity during sustained release in vivo. The drug loading remains above 60% of the initial drug loading within 14 days, achieving a long-lasting hangover relief effect. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Calcium chloride-mediated silk fibroin template crystallization and physical encapsulation mechanism: In calcium chloride aqueous solution, calcium ions form coordination salt bridges with the carboxyl groups of aspartic acid and glutamic acid residues on the regenerated silk fibroin molecular chain, as well as the amino group at the N-terminus of the silk fibroin. This coordination triggers a conformational transition of the silk fibroin molecular chain from random coils and α-helices to β-sheets, leading to ordered arrangement and self-assembly of the silk fibroin molecules along the calcium ion bridging sites. At a low temperature of 4°C, the self-assembly rate of silk fibroin is moderate, allowing alcohol dehydrogenase and acetaldehyde dehydrogenase molecules pre-mixed in the silk fibroin solution to be physically encapsulated within the microregions of the nanospheres by the gradually growing β-sheet network.

[0019] During self-assembly, enzyme molecules act as nanoscale templates in the crystallization of silk fibroin. Their surface polar groups form non-covalent interactions with the silk fibroin molecular chains, anchoring the enzyme molecules within the amorphous regions between β-sheet crystalline domains. This templated crystallization encapsulation tightly confines the enzyme molecules within a nanoscale space, physically restricting their conformational unfolding under thermal stress, thereby significantly improving the enzyme's thermal stability.

[0020] This mechanism differs from the organic solvent desolvation mechanism of the existing acetone precipitation method: acetone directly strips the hydration layer on the surface of silk fibroin, leading to rapid precipitation. The precipitation process is violent and uneven, and some enzyme molecules undergo irreversible denaturation under the action of acetone. In contrast, the calcium ion-mediated self-assembly process is mild and controllable. Calcium ions coordinate only with specific functional groups of silk fibroin and do not directly destroy the native conformation of the enzyme.

[0021] Synergistic protective mechanism of trehalose water substitution and vitrification: Trehalose and silk fibroin exhibit a deep synergistic effect in protecting enzyme activity, with the following pathway: The nanoscale confinement space formed by the β-sheet network of silk fibroin provides the first layer of protection for enzyme molecules, namely the spatial constraint effect, which limits the conformational unfolding range of enzyme molecules during thermal motion. Within this confinement space, trehalose molecules form a dense hydrogen bond network with polar groups on the enzyme molecule surface, replacing water molecules that detach from the enzyme surface during dehydration or thermal stress, thus maintaining the natural hydration shell of the enzyme molecule—this is the water substitution effect. When both are present simultaneously, the spatial confinement of silk fibroin maintains a high concentration of trehalose within local micro-regions, enhancing the probability and stability of hydrogen bond formation between trehalose and enzyme molecules. Simultaneously, the presence of trehalose reduces the rigidity and contraction of the silk fibroin β-sheet network, maintaining appropriate flexibility within the confinement space and preventing excessive rigidity that could lead to mechanical stress and deformation of the enzyme molecule.

[0022] In addition, trehalose forms an amorphous glassy matrix during freeze-drying, which fixes enzyme molecules in a rigid environment, greatly reducing molecular mobility and inhibiting degradation reactions.

[0023] The synergistic protective effect was confirmed by a specific verification experiment: after heat treatment at 60℃ for 2 hours, the activity retention rate of alcohol dehydrogenase was 41.3% when only silk fibroin was coated, and 35.6% when only trehalose solution was soaked. The sum of the activity retention rates of the two was 76.9%, while the activity retention rate under the condition of co-coating silk fibroin and trehalose reached 82.9%, which exceeded the simple summation value by 6.0 percentage points, proving that the synergistic effect has a non-linear gain characteristic.

[0024] Chitosan pH-responsive protection and mucosal adhesion targeting mechanism: Chitosan molecules contain a large number of amino groups, exhibiting distinctly different protonation states under varying pH conditions, thus endowing the nanospheres with pH-responsive protective functions. In the acidic environment of the stomach (pH 1.2 to 2.0), the chitosan amino groups are fully protonated and positively charged. Electrostatic repulsion between adjacent molecular chains causes the chitosan layer to form a dense, swollen gel layer on the nanosphere surface. This gel layer effectively blocks the penetration of hydrogen ions from gastric acid through steric hindrance, protecting the enzyme molecules inside the nanospheres from acid denaturation. Furthermore, the positive charge of the chitosan layer tightly binds to the negative charge on the surface of the silk fibroin nanospheres through electrostatic attraction, further enhancing the density and stability of the coating.

[0025] Under intestinal conditions of pH 6.0 to 7.4, chitosan amino groups are deprotonated, hydrogen bonds between molecular chains are restored, and the chitosan layer changes from a swollen state to a contracted state and gradually dissolves, exposing the internal silk fibroin nanospheres. The silk fibroin is gradually degraded and released by pancreatic enzymes, achieving targeted and controlled release in the intestine.

[0026] Meanwhile, chitosan retains some positive charge under intestinal pH conditions, electrostatically adhering to the negatively charged sialic acid residues of mucin on the intestinal mucosa surface. This prolongs the retention time of the nanospheres on the intestinal wall by 3 to 5 times, enhancing the contact time and absorption probability of enzyme molecules with the intestinal wall. This pH-responsive protective mechanism increased the enzyme activity retention rate of the nanospheres from 31.5% without the chitosan layer to 78.6% after 2 hours in simulated gastric fluid, while the cumulative release rate in simulated intestinal fluid was 68.3% after 6 hours, exhibiting intelligent behavior of protecting the enzyme in the stomach while releasing it in the intestine.

[0027] Nanoscale spatial cascade catalytic mechanism of alcohol dehydrogenase and acetaldehyde dehydrogenase: By co-encapsulating alcohol dehydrogenase and aldehyde dehydrogenase within the same silk fibroin nanosphere, the two enzyme molecules are placed in close proximity within a nanoscale space, establishing a highly efficient cascade catalytic system. Alcohol dehydrogenase consumes NAD+ during the oxidation of ethanol to acetaldehyde. + The production of NADH is necessary when acetaldehyde dehydrogenase catalyzes the oxidation of acetaldehyde to acetic acid. + .

[0028] Within the confined space inside the nanospheres, NAD + The enzyme rapidly diffuses and cycles between the two enzyme molecules, resulting in a coenzyme reuse efficiency far exceeding that of the two enzymes when they are freely dispersed in solution. Simultaneously, acetaldehyde produced by alcohol dehydrogenase is captured and catalytically oxidized by adjacent acetaldehyde dehydrogenase before diffusing out of the nanospheres, preventing localized accumulation of acetaldehyde.

[0029] The spatial cascade effect makes the ethanol degradation efficiency of the co-encapsulated system much higher than that of a simple physical mixture of two enzymes. In in vitro experiments, the ethanol degradation rate reached 81.5% after 4 hours.

[0030] In systems where only alcohol dehydrogenase is encapsulated, acetaldehyde accumulates excessively and cannot be further metabolized. In systems where only aldehyde dehydrogenase is encapsulated, catalysis cannot be initiated due to the lack of the substrate acetaldehyde. The essential difference between this cascade catalytic pathway and the alcohol oxidase pathway in existing technologies lies in the following: alcohol oxidase catalyzes the oxidation of ethanol to produce hydrogen peroxide intermediates, which require catalase for removal. However, hydrogen peroxide can cause oxidative damage to the enzyme itself and surrounding biomolecules even in its brief local presence. Although acetaldehyde, the intermediate product of the alcohol dehydrogenase and aldehyde dehydrogenase pathways, has some toxicity, it does not produce free radical oxidative damage and is immediately converted by aldehyde dehydrogenase within the nanospheres, making it significantly safer than the alcohol oxidase pathway.

[0031] Raw material source: Silkworm cocoons: White cocoons of domestic silkworms (Bombyxmori), purchased from Guangxi Sericultural Research Institute.

[0032] Lithium bromide: analytical grade, molecular weight 86.85 g / mol, purchased from Sigma-Aldrich Trading Co., Ltd., product number L9651.

[0033] Ethanol dehydrogenase: derived from Saccharomyces cerevisiae, specific activity 120 U / mg, solid powder, purchased from Sigma-Aldrich Trading Co., Ltd., product number A7011.

[0034] Acetaldehyde dehydrogenase: derived from Saccharomyces cerevisiae, specific activity 65 U / mg, solid powder, purchased from Sigma-Aldrich Trading Co., Ltd., product number A9115.

[0035] Trehalose: dihydrate, purity greater than 99%, purchased from Hayashibara Biochemical Co., Ltd., Japan.

[0036] Calcium chloride: analytical grade, purity greater than 96%, purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 10005818; Genipin: purity greater than 98%, purchased from Sichuan Weikeqi Biotechnology Co., Ltd., catalog number WKQ-10022306.

[0037] Chitosan: Deacetylation degree 90%, molecular weight 100kDa, purchased from Zhejiang Jinke Pharmaceutical Co., Ltd.; Sodium sulfate: Analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0038] Sodium carbonate: analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0039] Alcohol oxidase: derived from Pichia pastoris, specific activity 30 U / mg, purchased from Sigma-Aldrich Trading Co., Ltd., product number A2404.

[0040] Catalase: derived from bovine liver, specific activity 2000 U / mg, purchased from Sigma-Aldrich Trading Co., Ltd., product number C40.

[0041] Acetone: analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0042] Performance testing methods 1. Particle size and Zeta potential: Dynamic light scattering method was used with a Malvern Zetasizer NanoZS90 nanoparticle size and Zeta potential analyzer at a test temperature of 25℃. Each group of samples was tested 3 times and the average value was taken.

[0043] 2. Enzyme activity retention rate: The nanosphere formulation was dispersed in phosphate buffer (pH 7.4), and the NADH generation rate was determined by ultraviolet spectrophotometry at a wavelength of 340 nm. The activity retention rate was calculated with the activity of the same amount of free enzyme as 100%.

[0044] Ethanol dehydrogenase activity assay: The reaction system contained 50 mmol / L ethanol and 2 mmol / L NAD. + The assay was performed in phosphate buffer solution at pH 7.5 at 25°C.

[0045] Acetaldehyde dehydrogenase activity assay: The reaction system contained 5 mmol / L acetaldehyde and 2 mmol / L NAD. + The assay was performed in phosphate buffer solution at pH 7.5 at 25°C.

[0046] Alcohol oxidase activity assay: The reaction system contained 50 mmol / L ethanol in phosphate buffer at pH 7.5 and the assay was performed at 25°C. The oxygen consumption rate was determined by oxygen electrode method.

[0047] Catalase activity assay: The reaction system contained 10 mmol / L hydrogen peroxide in phosphate buffer pH 7.0 and was measured at 25℃. The decomposition rate of hydrogen peroxide was determined by ultraviolet spectrophotometry at a wavelength of 240 nm.

[0048] 3. Drug loading: The nanosphere formulation was dissolved in a 9.3 mol / L lithium bromide aqueous solution to destroy the nanosphere structure and release the encapsulated enzyme. The protein content was determined by the BCA protein quantification method. The drug loading was equal to the mass of the encapsulated enzyme divided by the total mass of the nanospheres multiplied by 100%.

[0049] 4. Stability of simulated gastric juice: Disperse the preparation in simulated gastric juice at pH 1.2, incubate in a water bath at 37°C with shaking for 2 hours, centrifuge and collect the precipitate to determine residual enzyme activity.

[0050] 5. Simulated intestinal fluid release: Disperse the formulation in simulated intestinal fluid at pH 6.8, shake in a 37°C water bath, and take samples at 1, 2, 4, 6, 8, 12, and 24 hours for centrifugation. Measure the protein content in the supernatant and calculate the cumulative release rate.

[0051] 6. In vitro alcohol degradation experiment: The preparation was dispersed in phosphate buffer solution (pH 7.4) containing 5% ethanol (v / v) and reacted with shaking at 37°C. The ethanol concentration was determined by gas chromatography at 0, 0.5, 1, 2 and 4 h.

[0052] 7. Verification experiment on the synergistic protection of trehalose and silk fibroin: A single-factor control group was designed to test the activity retention rate of the enzyme after heat treatment at 60℃ for 2 hours under four conditions: silk fibroin only without trehalose, trehalose solution soaking only without silk fibroin, silk fibroin and trehalose co-encapsulation, and no protection of the free enzyme.

[0053] 8. Determination of β-sheet crystallinity: Fourier transform infrared spectroscopy was used. Freeze-dried nanosphere powder was pressed into a KBr pellet and subjected to β-sheet crystallinity determination at 4000 to 4000 cm⁻¹. -1 Range scan, resolution 4cm -1 32 scans were performed. The amide I band (1590 to 1710 cm⁻¹) was observed. -1 Deconvolution and peak fitting were performed, with peak values ​​ranging from 1615 to 1630 cm⁻¹. -1 The percentage of the area of ​​the characteristic β-sheet peak relative to the total peak area of ​​the amide I band is used as the β-sheet crystallinity.

[0054] 9. Determination of chitosan deposition amount: The chitosan-modified nanosphere suspension was centrifuged, the supernatant was collected, and the residual chitosan concentration in the supernatant was determined by ultraviolet spectrophotometry at a wavelength of 280 nm. The chitosan deposition amount was calculated based on the difference between the amount of feed and the amount of residue, and then divided by the amount of silk fibroin to obtain the mass ratio of chitosan deposition amount to silk fibroin.

[0055] Example 1:

[0056] Raw material formula: 100 mL of 3% regenerated silk fibroin solution (w / v), 4 parts by weight of ethanol dehydrogenase (120 U / mg, derived from Saccharomyces cerevisiae), 8 parts by weight of acetaldehyde dehydrogenase (65 U / mg, derived from Saccharomyces cerevisiae), 10 parts by weight of trehalose dihydrate (greater than 99% purity), appropriate amount of calcium chloride (analytical grade, greater than 96% purity), 0.08 parts by weight of genipin (greater than 98% purity), 5 parts by weight of chitosan (90% degree of deacetylation, molecular weight 100 kDa), appropriate amount of sodium sulfate (analytical grade), and 500 mL of 1% acetic acid aqueous solution (v / v).

[0057] Preparation process: Step 1: Cut 5g of silkworm cocoons into approximately 1cm square pieces, place them in a 0.5% sodium carbonate aqueous solution, and boil for 30 minutes. Repeat twice to thoroughly remove the sericin. After each boiling, wash three times with 60℃ deionized water. Dry the degummed silk in a 60℃ oven for 12 hours. Weigh 1g of the dried degummed silk and add 10mL of a 9.3mol / L lithium bromide aqueous solution. Stir and dissolve in a 60℃ water bath for 4 hours. Transfer the solution to a dialysis bag with a molecular weight cutoff of 3500Da and dialyze with deionized water for 48 hours, changing the water every 4 hours. After dialysis, concentrate the silk fibroin solution to 3% (w / v).

[0058] Step 2: Take 100 mL of 3% regenerated silk fibroin solution, add 4 parts by weight of alcohol dehydrogenase, 8 parts by weight of acetaldehyde dehydrogenase and 10 parts by weight of trehalose, and mix magnetically at 4℃ for 30 min to ensure complete dissolution and uniformity, thus obtaining a mixed solution.

[0059] Step 3: Prepare a 0.1 g / mL calcium chloride aqueous solution. Under magnetic stirring at 300 rpm, add the calcium chloride aqueous solution dropwise to the mixture prepared in Step 2 at a rate of 0.5 mL / min. The mass ratio of calcium chloride to silk fibroin is 1:10. After the addition is complete, allow it to stand at 4℃ for 3 hours to self-assemble. Dynamic light scattering monitoring confirms that the particle size is stable at approximately 130 nm and the polydispersity index is less than 0.2, forming a silk fibroin nanosphere core suspension encapsulating alcohol-degrading enzymes.

[0060] Step 4: Add 0.08 parts by weight of genipin to the suspension obtained in Step 3, and perform a cross-linking reaction with magnetic stirring at 25°C for 8 hours. The free amino content is reduced to 25% of the initial value by ninhydrin colorimetric method, and a cross-linked nanosphere suspension is obtained.

[0061] Step 5: Dissolve 5 parts by mass of chitosan in 500 mL of 1% (v / v) acetic acid aqueous solution and stir until completely dissolved to obtain a chitosan solution (concentration 10 mg / mL). Disperse the cross-linked nanosphere suspension obtained in Step 4 in the chitosan solution, and add 10 mL of 2% (v / v) sodium sulfate aqueous solution dropwise under magnetic stirring. The sulfate ions provided by sodium sulfate will undergo ionic cross-linking with the amino groups of chitosan, promoting the uniform deposition of chitosan on the surface of the nanospheres. React at 4℃ for 3 h, and confirm that the potential is stable at a positive value (approximately +23 mV) by Zeta potential monitoring, thus forming a chitosan-modified nanosphere suspension.

[0062] Step 6: Centrifuge the nanosphere suspension obtained in Step 5 at 15000 r / min for 20 min, discard the supernatant, collect the precipitate, wash it 3 times with deionized water, and freeze-dry it at -50℃ for 48 h to obtain the silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation.

[0063] The formulation prepared in this embodiment has an average particle size of 132 nm, a Zeta potential of 23 mV, an alcohol dehydrogenase activity retention rate of 86.3%, an acetaldehyde dehydrogenase activity retention rate of 82.7%, and a drug loading of 9.8%. The mass ratio of chitosan deposition to silk fibroin is 0.52:1.

[0064] β-sheet crystallinity 28.6%.

[0065] Example 2:

[0066] Raw material formula: 100 mL of 4% regenerated silk fibroin solution (w / v), 3 parts by weight of ethanol dehydrogenase (120 U / mg specific activity from Saccharomyces cerevisiae), 5 parts by weight of acetaldehyde dehydrogenase (65 U / mg specific activity from Saccharomyces cerevisiae), 12 parts by weight of trehalose dihydrate (purity >99%), appropriate amount of calcium chloride (analytical grade >96%), 0.06 parts by weight of genipin (purity >98%), 4 parts by weight of chitosan (degree of deacetylation 90%, molecular weight 100 kDa), appropriate amount of sodium sulfate (analytical grade), and 400 mL of 1% acetic acid aqueous solution (volume percentage).

[0067] Preparation process: Same as in Example 1, except that: Step 1: the silk fibroin solution is concentrated to 4% by mass volume; Step 3: the mass ratio of calcium chloride to silk fibroin is 0.5:10, and the self-assembly time is 4 hours; Step 4: 0.06 parts by mass of genipin is cross-linked for 10 hours; Step 5: 4 parts by mass of chitosan is dissolved in 400 mL of acetic acid aqueous solution (concentration 10 mg / mL) and 8 mL of sodium sulfate aqueous solution, and the reaction is carried out for 4 hours.

[0068] The formulation prepared in this embodiment has an average particle size of 168 nm, a Zeta potential of 18 mV, an alcohol dehydrogenase activity retention rate of 83.5%, an acetaldehyde dehydrogenase activity retention rate of 80.1%, and a drug loading of 7.6%. The mass ratio of chitosan deposition to silk fibroin is 0.41:1. The β-sheet crystallinity is 24.3%.

[0069] Example 3:

[0070] Raw material formula: 100 mL of 3% regenerated silk fibroin solution (w / v), 5 parts by weight of ethanol dehydrogenase (120 U / mg specific activity from Saccharomyces cerevisiae), 10 parts by weight of acetaldehyde dehydrogenase (65 U / mg specific activity from Saccharomyces cerevisiae), 8 parts by weight of trehalose dihydrate (purity >99%), appropriate amount of calcium chloride (analytical grade >96%), 0.12 parts by weight of genipin (purity >98%), 8 parts by weight of chitosan (degree of deacetylation 90%, molecular weight 100 kDa), appropriate amount of sodium sulfate (analytical grade), and 600 mL of 1% acetic acid aqueous solution (volume percentage).

[0071] Preparation process: Same as in Example 1, except that: in step 3, the mass ratio of calcium chloride to silk fibroin is 1.5:10, and the self-assembly time is 2h; in step 4, 0.12 parts by mass of genipin is cross-linked for 6h; in step 5, 8 parts by mass of chitosan is dissolved in 600mL of acetic acid aqueous solution (concentration 13.3mg / mL) and 15mL of sodium sulfate aqueous solution, and the reaction is carried out for 2h.

[0072] The formulation prepared in this embodiment has an average particle size of 97 nm, a Zeta potential of 31 mV, an alcohol dehydrogenase activity retention rate of 84.1%, an acetaldehyde dehydrogenase activity retention rate of 81.9%, and a drug loading of 12.3%. The mass ratio of chitosan deposition to silk fibroin is 0.68:1. The β-sheet crystallinity is 32.1%.

[0073] Comparative Example 1: The raw material formula is the same as in Example 1, except that trehalose is not added.

[0074] The preparation process is the same as in Example 1, except that trehalose is not added in step two.

[0075] This comparative example, without the addition of trehalose as a protective agent, retained 58.2% of alcohol dehydrogenase activity and 53.6% of acetaldehyde dehydrogenase activity.

[0076] Compared with Example 1, the retention rate of alcohol dehydrogenase activity decreased by 28.1 percentage points and the retention rate of acetaldehyde dehydrogenase activity decreased by 29.1 percentage points, indicating that trehalose has a significant protective effect on enzyme activity.

[0077] Comparative Example 2: The raw material formulation is the same as in Example 1, except that the chitosan modification in step five is not performed.

[0078] The cross-linked nanosphere suspension obtained in step four was centrifuged, washed, and freeze-dried to directly obtain a silk fibroin nanosphere encapsulated with an alcohol-degrading enzyme preparation without a chitosan-modified layer.

[0079] This comparative example has no chitosan-modified layer and a Zeta potential of -18mV.

[0080] After incubation for 2 hours in simulated gastric fluid at pH 1.2, the alcohol dehydrogenase activity retention rate was only 31.5%, while the cumulative release rate in simulated intestinal fluid at pH 6.8 was 92.3% after 6 hours. Compared with the enzyme activity retention rate of 78.6% in simulated gastric fluid in Example 1 after 2 hours, the gastric acid protection ability of nanospheres without chitosan layers is significantly insufficient, and there is a burst release problem in intestinal fluid.

[0081] Comparative Example 3: The raw material formula is the same as in Example 1.

[0082] The difference in the preparation process is that step three uses acetone precipitation instead of calcium chloride-mediated self-assembly: the mixture obtained in step two is slowly added to acetone at a rate of 25 μL per drop, with a volume ratio of 1.2:5 between the mixture and acetone. The resulting solution is centrifuged at 18000 r / min for 30 min, the supernatant is discarded, deionized water is added and ultrasonically washed and dispersed, and the centrifugation and washing are repeated 3 times to obtain a suspension of silk fibroin nanospheres encapsulating alcohol degrading enzymes.

[0083] Steps four through six are the same as in Example 1.

[0084] In this comparative example, the acetone precipitation method was used instead of the calcium chloride-mediated self-assembly method. The activity retention rate of ethanol dehydrogenase was 61.8%, and the activity retention rate of acetaldehyde dehydrogenase was 57.3%.

[0085] Compared with Example 1, the retention rate of ethanol dehydrogenase activity decreased by 24.5 percentage points, and the retention rate of acetaldehyde dehydrogenase activity decreased by 25.4 percentage points, indicating that acetone organic solvent causes irreversible denaturation and inactivation of enzyme molecules. The calcium chloride-mediated aqueous phase self-assembly method is significantly better than the acetone precipitation method in protecting enzyme activity.

[0086] Comparative Example 4: Raw material formula: 100 mL of 3% regenerated silk fibroin solution (w / v), 4 parts by weight of alcohol oxidase with a specific activity of 30 U / mg derived from Pichia pastoris, 8 parts by weight of catalase with a specific activity of 2000 U / mg derived from bovine liver, 10 parts by weight of trehalose dihydrate with a purity greater than 99%, appropriate amount of calcium chloride with an analytical purity greater than 96%, 0.08 parts by weight of genipin with a purity greater than 98%, 5 parts by weight of chitosan with a degree of deacetylation of 90% and a molecular weight of 100 kDa, appropriate amount of sodium sulfate with an analytical purity, and 500 mL of 1% acetic acid aqueous solution (v / v).

[0087] The preparation process is the same as in Example 1, except that in step two, alcohol oxidase and catalase are used instead of alcohol dehydrogenase and acetaldehyde dehydrogenase.

[0088] This comparative example uses a combination of alcohol oxidase and catalase as the alcohol-degrading enzyme system.

[0089] The retention rate of alcohol oxidase activity was 79.6%, and the retention rate of catalase activity was 76.8%.

[0090] Alcohol oxidase catalyzes the oxidation of ethanol and produces hydrogen peroxide intermediates. After reacting in simulated intestinal fluid in vitro for 30 minutes, the concentration of hydrogen peroxide in the supernatant was detected to be 0.15 mmol / L.

[0091] In Example 1, the tandem catalytic pathway of alcohol dehydrogenase and acetaldehyde dehydrogenase did not produce hydrogen peroxide, and no hydrogen peroxide was detected in the supernatant.

[0092] The results indicate that the combination of alcohol dehydrogenase and acetaldehyde dehydrogenase used in this invention is significantly safer than the alcohol oxidase pathway.

[0093] Comparative Example 5: The raw material formula is the same as in Example 1.

[0094] The preparation process is the same as in Example 1, except that in step three, the calcium chloride aqueous solution is replaced by the direct addition of solid calcium chloride powder and rapid stirring. The mass ratio of calcium chloride to silk fibroin is still 1:10, but the addition method is a one-time addition rather than a slow dripping.

[0095] In this comparative example, the calcium chloride was added in a single, continuous process instead of slowly dripping. The resulting nanospheres had an average particle size of 287 nm and an uneven particle size distribution, with a polydispersity index of 0.43. The retention rates of alcohol dehydrogenase activity were 71.2% and acetaldehyde dehydrogenase activity were 67.8%.

[0096] Compared with Example 1, the activity retention rate decreased by 15.1 and 14.9 percentage points, respectively, indicating that the dripping rate of calcium chloride has a significant impact on the uniformity of nanosphere particle size and enzyme activity retention rate. Rapid addition leads to excessively high local calcium ion concentration, causing rapid aggregation of silk fibroin, and the enzyme molecules are not fully encapsulated and are partially exposed to the outside.

[0097] Comparative Example 6: Raw material formula: 100 mL of 3% regenerated silk fibroin solution (w / v), 4 parts by weight of alcohol oxidase with a specific activity of 30 U / mg derived from Pichia pastoris, 8 parts by weight of catalase with a specific activity of 2000 U / mg derived from bovine liver, 500 mL of analytical grade acetone, 0.08 parts by weight of genipin with a purity greater than 98%, 5 parts by weight of chitosan with a degree of deacetylation of 90% and a molecular weight of 100 kDa, appropriate amount of analytical grade sodium sulfate, and 500 mL of 1% acetic acid aqueous solution (v / v).

[0098] Preparation process: The steps are the same as in Example 1.

[0099] Step 2: Take 100 mL of 3% regenerated silk fibroin solution, add 4 parts by weight of alcohol oxidase and 8 parts by weight of catalase, mix evenly at 4℃, without adding trehalose.

[0100] Step 3 uses the acetone precipitation method. The mixture obtained in step 2 is slowly added to acetone at a rate of 25 μL per drop. The volume ratio of the mixture to acetone is 1.2:5. The mixture is centrifuged at 18000 r / min for 30 min, and then ultrasonically washed and dispersed with deionized water. The centrifugation and washing are repeated 3 times.

[0101] Steps four through six are the same as in Example 1.

[0102] This comparative example combines the existing acetone precipitation method and the combination of alcohol oxidase and catalase with chitosan surface modification.

[0103] The retention rates of alcohol oxidase activity were 49.3%, catalase activity was 44.8%, and enzyme activity retention rates were 72.1% after 2 hours in simulated gastric juice.

[0104] Compared with Example 1, the enzyme activity retention rate decreased significantly, indicating that simply adding chitosan modification to the acetone precipitation method and alcohol oxidase system cannot achieve the technical effect of the present invention.

[0105] The problems of enzyme damage caused by acetone precipitation and hydrogen peroxide generated by the alcohol oxidase pathway are not solved in this solution. Although chitosan modification provides gastric acid protection, the enzyme it protects is itself largely inactivated by acetone treatment. Furthermore, the hydrogen peroxide generated by alcohol oxidase causes oxidative damage to other enzyme molecules inside the nanospheres, resulting in an overall effect far lower than that of this invention.

[0106] Comparative Example 7: The raw material formula is the same as in Example 1, except that trehalose is not added in step two and the chitosan modification in step five is not performed.

[0107] The cross-linked nanosphere suspension obtained in step four was directly washed by centrifugation and freeze-dried.

[0108] This comparative example lacked both trehalose as a protective agent and a chitosan modification layer, and was used to verify the synergistic effect of the two protective measures of trehalose and chitosan. The retention rates of alcohol dehydrogenase activity were 42.7%, acetaldehyde dehydrogenase activity was 38.9%, and enzyme activity retention rates after 2 hours in simulated gastric fluid were 22.6%.

[0109] Comparative analysis: Removing trehalose alone (Comparative Example 1) reduced the ADH activity retention rate by 28.1 percentage points, while removing chitosan alone (Comparative Example 2) reduced it by only 1.6 percentage points. The simple sum of the two was expected to reduce the ADH activity retention rate by 29.7 percentage points, but removing both at the same time actually reduced it by 43.6 percentage points, exceeding the expected sum by 13.9 percentage points. This indicates that trehalose and chitosan have a synergistic effect in protecting enzyme activity: the lack of a chitosan layer makes the nanospheres more susceptible to environmental factors during storage after preparation. If the molecular-level protection of trehalose is also lacking at this time, enzyme inactivation is accelerated. The combined effect of the two missing is much greater than the sum of the effects of their individual missing effects.

[0110] Table 1. Results of enzyme activity retention rate and drug loading test:

[0111] Note: ADH = alcohol dehydrogenase, ALDH = acetaldehyde dehydrogenase, AO = alcohol oxidase, CAT = catalase.

[0112] Comparative Examples 4 and 6 used a combination of alcohol oxidase and catalase, and the activity retention rates of alcohol oxidase and catalase are reported in the table.

[0113] Table 2. Results of simulated gastrointestinal stability and in vitro alcohol degradation tests:

[0114] Table 3. Results of the experimental verification of the synergistic protection of trehalose and silk fibroin:

[0115] Results analysis showed that the alcohol dehydrogenase activity retention rates in Examples 1 to 3 were 83.5% to 86.3%, and the acetaldehyde dehydrogenase activity retention rates were 80.1% to 82.7%, with drug loading rates ranging from 7.6% to 12.3%. In Comparative Example 1, without the addition of trehalose, the alcohol dehydrogenase activity retention rate was 58.2%, and the acetaldehyde dehydrogenase activity retention rate was 53.6%, representing decreases of 28.1 and 29.1 percentage points, respectively, compared to Example 1. In Comparative Example 3, using acetone precipitation instead of calcium chloride-mediated self-assembly, the alcohol dehydrogenase activity retention rate was 61.8%, and the acetaldehyde dehydrogenase activity retention rate was 57.3%, representing decreases of 24.5 and 25.4 percentage points, respectively, compared to Example 1. In Comparative Example 4, using a combination of alcohol oxidase and catalase instead of ADH and ALDH, the alcohol oxidase activity retention rate was 79.6%, and the catalase activity retention rate was 76.8%. Although the enzyme activity retention rates were acceptable, the catalytic pathway produced hydrogen peroxide, posing a safety risk. Comparative Example 6, which combined acetone precipitation with a combination of alcohol oxidase and catalase, exhibited only 49.3% alcohol oxidase activity retention and 44.8% catalase activity retention, despite the addition of chitosan modification. These figures were significantly lower than in Example 1, indicating that the enzyme damage caused by acetone precipitation could not be compensated for by chitosan modification. Furthermore, the hydrogen peroxide produced by the alcohol oxidase pathway also caused oxidative damage to the enzyme itself. Comparative Example 7, lacking both trehalose and chitosan, showed activity retention rates of only 42.7% and 38.9%, respectively. After 2 hours in simulated gastric fluid, the activity retention rate was only 22.6%, demonstrating that the absence of these two protective measures resulted in a severe decrease in both enzyme activity and oral stability.

[0116] Regarding simulated gastric fluid stability, the enzyme activity retention rates of Examples 1 to 3 were 75.2% to 80.1% after 2 hours, while that of Comparative Example 2 without the chitosan modification layer was only 31.5%, indicating that the chitosan modification layer is crucial for gastric acid protection. Regarding simulated intestinal fluid release, the cumulative release rates of Examples 1 to 3 after 6 hours were 63.7% to 72.5%, exhibiting good sustained-release characteristics, while the cumulative release rate of Comparative Example 2 without the chitosan layer was as high as 92.3% after 6 hours, indicating a burst release problem.

[0117] Regarding in vitro alcohol degradation, the 4-hour ethanol degradation rates in Examples 1 to 3 ranged from 76.8% to 84.3%. Comparative Example 4, using a combination of alcohol oxidase and catalase, achieved a 4-hour ethanol degradation rate of 73.6%. Although this degradation rate was close to that of the examples, its reaction system produced 0.15 mmol / L hydrogen peroxide, posing a safety risk. Comparative Example 7, lacking both trehalose and chitosan, achieved a 4-hour ethanol degradation rate of only 37.2%, significantly lower than the 81.5% of Example 1.

[0118] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation, characterized in that, The formulation comprises: a silk fibroin nanosphere core, wherein alcohol dehydrogenase, aldehyde dehydrogenase, and trehalose are co-encapsulated within the silk fibroin nanosphere core; the silk fibroin in the silk fibroin nanosphere core has a β-sheet structure; the mass ratio of alcohol dehydrogenase to aldehyde dehydrogenase is 1:1.5 to 1:2.5; and the mass ratio of trehalose to silk fibroin is 1:8 to 1:12; a genipin cross-linking layer, which coats the outer surface of the silk fibroin nanosphere core; and a chitosan modification layer, which coats the outer surface of the genipin cross-linking layer; the average particle size of the formulation is 80 to 200 nm, and the zeta potential is ±15 mV to ±35 mV.

2. The silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation as described in claim 1, characterized in that, The alcohol dehydrogenase has a specific activity greater than 100 U / mg and is derived from Saccharomyces cerevisiae; the acetaldehyde dehydrogenase has a specific activity greater than 50 U / mg and is derived from Saccharomyces cerevisiae.

3. The silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation as described in claim 1, characterized in that, The silk fibroin in the core of the silk fibroin nanospheres has a molecular weight of 10 kDa to 200 kDa, and the β-sheet crystallinity of the core is greater than 20%. This β-sheet crystallinity was determined by Fourier transform infrared spectroscopy, with values ​​in the amide I band from 1615 to 1630 cm⁻¹. -1 The percentage of the area of ​​the β-sheet characteristic peak relative to the total peak area of ​​the amide I band.

4. The silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation as described in claim 1, characterized in that, The mass ratio of genipin to silk fibroin in the genipin crosslinking layer is 0.05:1 to 0.15:1; the mass ratio of chitosan deposition to silk fibroin in the chitosan modified layer is 0.3:1 to 0.8:1, the degree of deacetylation of chitosan is greater than 85%, and the molecular weight is 50kDa to 150kDa.

5. A method for preparing a wine-degrading enzyme preparation encapsulated in silk fibroin nanospheres as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: After degumming the silkworm cocoons, dissolve them in a lithium bromide aqueous solution, and obtain a regenerated silk fibroin solution through dialysis and concentration; Step 2: Mix the regenerated silk fibroin solution obtained in Step 1 with alcohol dehydrogenase, acetaldehyde dehydrogenase and trehalose at 4°C to obtain a mixed solution. Step 3: Under stirring conditions, calcium chloride aqueous solution is slowly added dropwise to the mixture prepared in Step 2 at a rate of 0.3 to 0.8 mL / min. The mass ratio of calcium chloride to silk fibroin is 0.5:10 to 1.5:

10. The mixture is allowed to stand at 4℃ for 2 to 4 hours for self-assembly. The self-assembly is considered complete when the particle size no longer decreases significantly and the distribution is stable, as monitored by dynamic light scattering. This forms a silk fibroin nanosphere core suspension encapsulating alcohol-degrading enzymes. Step 4: Add genipin to the suspension obtained in Step 3, and perform a cross-linking reaction at 25°C for 6 to 10 hours. When the free amino content drops to less than 30% of the initial value by ninhydrin colorimetric method, the cross-linking is considered complete, and a cross-linked nanosphere suspension is obtained. Step 5: Dissolve chitosan in a 1% (v / v) aqueous acetic acid solution to prepare a chitosan solution. Disperse the cross-linked nanosphere suspension obtained in Step 4 in the chitosan solution. Add sodium sulfate aqueous solution dropwise under stirring to promote the ionic cross-linking deposition of chitosan on the surface of the nanospheres. React at 4°C for 2 to 4 hours. When the zeta potential is monitored and stabilized at a positive value, the chitosan modification is considered complete, and a chitosan-modified nanosphere suspension is formed. Step 6: Centrifuge the nanosphere suspension obtained in Step 5, collect the precipitate, wash with deionized water, and freeze-dry to obtain the silk fibroin nanosphere-encapsulated alcohol-degrading enzyme preparation.

6. The preparation method according to claim 5, characterized in that, The degumming process in step one involves cutting the silkworm cocoons into small pieces, boiling them in a 0.5% sodium carbonate aqueous solution for 30 minutes, repeating this process twice, washing them with deionized water, and then drying them at 60°C. The lithium bromide aqueous solution has a concentration of 9.3 mol / L, a dissolution temperature of 60°C, and a dissolution time of 4 hours. The dialysis time is 48 hours, and the concentration of the concentrated silk fibroin solution is 3% to 4% by mass / volume.

7. The preparation method according to claim 5, characterized in that, The mixing ratio of the regenerated silk fibroin solution with alcohol dehydrogenase, aldehyde dehydrogenase and trehalose in step two is as follows: based on 100 parts by weight of silk fibroin, alcohol dehydrogenase is 3 to 5 parts by weight, aldehyde dehydrogenase is 5 to 10 parts by weight, and the mass ratio of alcohol dehydrogenase to aldehyde dehydrogenase is in the range of 1:1.5 to 1:2.5, and trehalose is 8 to 12 parts by weight.

8. The preparation method according to claim 5, characterized in that, In step five, the concentration of the chitosan solution is 5 mg / mL to 15 mg / mL, the mass percentage concentration of the sodium sulfate aqueous solution is 2%, and the volume ratio of the chitosan solution to the cross-linked nanosphere suspension is 5:1 to 10:

1.

9. The preparation method according to claim 5, characterized in that, In step six, the centrifugation speed is 15000 r / min, the centrifugation time is 20 min, the washing is performed 3 times, the freeze-drying temperature is -50℃, and the time is 48 h.

10. The use of the silk fibroin nanospheres encapsulating an alcohol-degrading enzyme preparation as described in any one of claims 1 to 4 in the preparation of an oral remedy for relieving acute alcohol poisoning.

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