An enzymatic metabolic delivery system for reducing enrofloxacin residues in fish skin tissue and a preparation method thereof

Abstract

The application discloses an enzymatic metabolic delivery system for reducing enrofloxacin residues in fish skin tissue and a preparation method thereof, and belongs to the technical field of aquatic breeding drug residue control and biological preparation preparation. The system is composed of an enzyme system nanolipid core formed by arachidonic acid and cytochrome P450 enzyme, a targeting intermediate layer formed by collagen binding peptide (TKKTLRT) and hyaluronic acid, and an outer protective shell formed by low molecular weight chitosan. After oral feeding, the system can be targeted to fish skin tissue, and the residues of enrofloxacin can be significantly reduced through enzyme synergistic metabolism. The degradation rate reaches 85% in the embodiment, and the system has good gastrointestinal stability and biological safety. The application can be widely applied to the field of aquatic breeding drug residue control.

Description

Technical Field

[0001] This invention belongs to the field of drug residue control and biological agent preparation technology in aquaculture, specifically relating to an enzymatic metabolic delivery system that can be orally administered to reduce enrofloxacin residues in fish skin tissue and its preparation method. Background Technology

[0002] Enrofloxacin belongs to the quinolone class of drugs and is widely used in aquaculture disease prevention and control due to its broad-spectrum antibacterial properties. According to the "National Food Safety Standard for Maximum Residue Limits of Veterinary Drugs in Food" (GB 31650-2019), the limit for enrofloxacin in fish is ≤100 μg / kg. Due to its stable chemical properties and long metabolic half-life, it readily accumulates in the skin, muscle, and liver of fish. Multiple studies have shown that the highest concentration of enrofloxacin in fish skin tissue is found in fish, while lower concentrations are found in muscle. Furthermore, the proportion of ciprofloxacin, a metabolite of enrofloxacin, is also high in the skin. Research indicates that the skin is the tissue with the most severe enrofloxacin accumulation and the slowest elimination; therefore, the skin is recommended as a target tissue for residue monitoring. Similarly, while the residue in fish muscle may be below the limit, enrofloxacin in the skin may still exceed the limit by 2-10 times. Therefore, the slow metabolism of enrofloxacin in skin tissue is the most significant factor affecting the quality and safety of farmed aquatic products.

[0003] Enrofloxacin's quinolone ring skeleton is hydrophobic, and the dermis of fish skin contains numerous adipocytes and lipid layers. Skin tissue provides abundant binding sites for enrofloxacin, leading to slow metabolic elimination. Furthermore, tissues with fewer blood vessels, slower blood flow, and lower metabolic enzyme activity often become the "final destination" for drug accumulation during fish metabolism. Conventional withdrawal or hepatic metabolism is insufficient to rapidly clear deeply bound enrofloxacin residues. The elimination half-life of enrofloxacin in fish skin is significantly longer than in muscle and liver. Therefore, developing efficient removal technologies for bound enrofloxacin in the fish skin matrix has significant practical value.

[0004] Current technologies for eliminating drug residues in aquatic organisms mainly include withdrawal period management, probiotic degradation, and physical adsorption. However, existing technologies have the following shortcomings: poor targeting: traditional methods mostly promote systemic metabolism, making it difficult to specifically target fish skin tissue with high residue concentrations and poor metabolism. Low degradation efficiency: single enzyme preparations are easily inactivated in the acidic environment of the gastrointestinal tract and under the action of digestive enzymes, making it difficult to maintain activity and reach the target site. Strong residue binding: enrofloxacin binds non-specifically to collagen and lipid layers in fish skin tissue, making it difficult to achieve efficient removal by relying solely on natural metabolism. Therefore, developing a delivery system that utilizes enzymatic synergy to degrade enrofloxacin residues in fish skin tissue has significant market application value. Summary of the Invention

[0005] This invention aims to solve the technical problem of slow metabolism of enrofloxacin in fish skin tissue, and provides a dual-enzyme metabolic delivery system for targeted replacement of enrofloxacin residues in fish skin tissue via oral administration, as well as its preparation method.

[0006] Enrofloxacin's main metabolic pathway in fish relies on the cytochrome P450 enzyme system. The key to solving the above problem is how to accurately deliver cytochrome P450 enzymes into fish skin tissue.

[0007] To solve the above-mentioned technical problems, the present invention first provides a collagen-binding peptide, the amino acid sequence of which is shown in SEQ ID No.1, specifically the sequence TKKTLRT.

[0008] The present invention provides an enzymatic metabolic delivery system for reducing drug residues in aquatic products, comprising an enzyme system nanolipid core and the collagen-binding peptide.

[0009] This invention is based on the principle of hierarchical self-assembly and constructs an enzyme-catalyzed metabolic delivery system through a three-step synergistic mechanism: The construction principle of the enzyme metabolic delivery system provided by this invention includes the following three levels: 1. First-stage self-assembly: Formation of a functional enzyme system nanolipid core, utilizing hydrophobic interactions and metal ion coordination as the driving force, arachidonic acid (a long-chain unsaturated fatty acid with an exposed hydrophobic tail) and cytochrome P450 enzyme in a Zn-containing... 2+ Mix in the buffer solution. Zn 2+ It can act as a bridge, coordinating / electrostatically with the enzyme surface and fatty acid carboxyl groups. At the same time, the hydrophobic chains of fatty acids spontaneously aggregate, encapsulating or embedding the P450 enzyme within the hydrophobic core. This forms a nanoscale lipid-enzyme complex core ("nanolipid core"), protecting enzyme activity and providing a catalytic microenvironment.

[0010] 2. Second-stage self-assembly: Constructing a targeted intermediate layer (from nanocore to functionalized core-shell structure). Utilizing electrostatic adsorption and covalent cross-linking of mixed amide bonds as the driving force, negatively charged nanolipid cores are dispersed in a solution containing collagen-binding peptides (positively charged peptides) and hyaluronic acid (a polysaccharide with carboxyl groups). First, a primary coating layer is formed through positive and negative electrostatic adsorption; then, using a cross-linking agent (EDC / NHS), stable amide bonds are formed between the peptides and hyaluronic acid and core surface groups, forming a "targeted intermediate layer" that endows the system with the ability to recognize collagen-exposed tissues.

[0011] 3. Third-level self-assembly: Constructing the outer protective shell (functionalized core → final microspheres), driven by layer-by-layer self-assembly (LbL), primarily through the alternating adsorption of oppositely charged polyelectrolytes. Particles with an intermediate layer (potentially negatively charged due to hyaluronic acid) are dispersed in a low-molecular-weight chitosan (positively charged polysaccharide) solution. The outer protective shell is formed through electrostatic layer-by-layer adsorption, improving system stability, controlling enzyme metabolite release, and reducing protein degradation.

[0012] Specifically, the preparation method of the enzyme-catalyzed metabolic delivery system provided by the present invention includes the following steps: (1) Arachidonic acid and cytochrome P450 enzyme were mixed at a mass ratio of 1:0.5-1:1 and dissolved in a solution containing Zn. 2+ The enzyme system nanolipid core was prepared in a buffer solution; (2) The enzyme system nanolipid core is dispersed in a solution containing collagen-binding peptides and hyaluronic acid, and a targeted intermediate layer is constructed by electrostatic adsorption and amide bond cross-linking; (3) The product obtained in step (2) is dispersed in a low molecular weight chitosan solution, and an outer protective shell is constructed by layer-by-layer self-assembly technology to obtain enzyme metabolism delivery microspheres; (4) The enzyme metabolism delivery microspheres obtained in step (3) are freeze-dried to obtain an enzyme metabolism delivery system with an average particle size of 600nm-1000nm.

[0013] Furthermore, the preparation method of the enzyme system nanolipid core in step (1) is as follows: Arachidonic acid was dissolved in ultrapure water at 30–50°C under constant stirring in a Zn-containing solution. 2+ The buffer solution was then subjected to continuous constant temperature and stirring conditions, and cytochrome P450 enzyme was quantitatively added through a precision feeding system. The mixture was continuously and gently stirred for 40 minutes within the above temperature range, so that cytochrome P450 enzyme and arachidonic acid could form a stable enzyme system nanolipid core through hydrophobic interaction.

[0014] Furthermore, the cytochrome P450 enzyme described in this invention is the CYP1A2 subtype, with a specific activity ≥60 U / mg.

[0015] Furthermore, the low molecular weight chitosan described in this invention has a molecular weight of 5-10 kDa.

[0016] Furthermore, the buffer solution described in this invention is a phosphate buffer solution with a pH of 7.4 and containing 0.2 mM zinc sulfate.

[0017] Furthermore, in the construction of the targeted intermediate layer described in this invention, the reaction system pH is 5.5, and EDC and NHS are added as crosslinking agents.

[0018] Furthermore, the freeze-drying process parameters of the present invention are: pre-freezing at -50℃ for 3 hours, sublimation drying at -25℃ for 18 hours, desorption drying at 25℃ for 6 hours, and vacuum degree ≤15Pa.

[0019] This invention provides an application of an enzyme-catalyzed metabolic delivery system, specifically for reducing drug residues in aquatic products. The aquatic products include farmed fish, shrimp, and shellfish.

[0020] Furthermore, the drug mentioned is enrofloxacin.

[0021] The beneficial effects of this invention are: High targeting: By introducing collagen-binding peptides and CYP enzyme-responsive linkers, the delivery system can specifically anchor on residual and accumulated collagen fibers after reaching fish skin tissue via blood circulation, which significantly improves the elimination efficiency of bound enrofloxacin and reduces potential toxicity.

[0022] Enzyme-assisted metabolism: Enrofloxacin's main metabolic pathway in fish relies on the cytochrome P450 enzyme system. The addition of arachidonic acid forms oil droplets that encapsulate the enzymes, ensuring their stability and simultaneously providing raw materials for cytochrome P450 synthesis. This dual system ensures the stability of enzyme-assisted metabolism.

[0023] Layered protection and controllable release: By utilizing chitosan delivered through the blood, the enzyme is protected and released in response to the pH of the fish skin, greatly improving the effective utilization rate of the enzyme. Attached Figure Description

[0024] Figure 1 Scanning electron microscope image of the delivery system.

[0025] Figure 2 Pharmacokinetic curves of enrofloxacin in the skin of largemouth bass. The curves in the figure, from top to bottom, represent the blank control group, the single enzyme group, and the delivery system group prepared in Example 1 of this invention.

[0026] Figure 3 Results of CYP enzyme activity release experiment. Detailed Implementation

[0027] The technical solution of the present invention will be further explained below through embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0028] Example 1: Preparation of an enrofloxacin metabolic enzyme delivery system based on fish skin-targeting peptides 1. Preparation of AA / CYP@Lipid-Zn nanolipid core 15 mg of arachidonic acid (AA, purity ≥99%), 5 mg of soybean lecithin (PC-98T, as a film-forming framework material), and 2 mg of cholesterol (stabilizer) were dissolved together in 3 mL of anhydrous ethanol to form the organic phase. Separately, 10 mg of cytochrome P450 enzyme (CYP1A2 isotype, specific activity ≥60 U / mg) was dissolved in 7 mL of phosphate-buffered saline (PBS, pH 7.4, containing 0.2 mM zinc sulfate) to form the aqueous phase.

[0029] Under magnetic stirring (600 rpm), the organic phase was slowly and uniformly injected into the aqueous phase through a syringe needle (27G), and stirring was continued for 30 minutes to allow spontaneous formation of a promulgation. Subsequently, the promulgation was placed in a probe-type ultrasonic cell disruptor and sonicated for 5 minutes at 20% power (2s intervals on / 3s off) under ice bath conditions to obtain a pale blue, opalescent nanolipid core suspension. Residual ethanol was removed by rotary evaporation (water bath temperature 35℃, vacuum degree -0.08MPa) under reduced pressure, finally yielding AA / CYP@Lipid-Zn nanolipid cores with an average hydrated particle size of approximately 150 nm.

[0030] 2. Construct a targeted intermediate layer The AA / CYP@Lipid-Zn suspension prepared in step 1 was concentrated to 10 mL using an ultrafiltration centrifuge tube (molecular weight cutoff 100 kDa). 0.6 mg / mL collagen-binding peptide (amino acid sequence: TKKTLRT, purity >95%) and 1.0 mg / mL hyaluronic acid (HA, molecular weight 35 kDa) were added sequentially. The pH of the system was adjusted to 5.5 (using 0.1 M MES buffer), followed by the addition of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC, final concentration 3 mM) and N-hydroxysuccinimide (NHS, final concentration 1.5 mM). The reaction was carried out at room temperature in the dark with gentle stirring for 2 hours, allowing the carboxyl groups on HA to crosslink with the amino groups of the TKKTLRT peptide and trace amounts of amino groups on the lipid core surface via amide bonds. Simultaneously, electrostatic adsorption anchored the peptide fragments to the core surface. After the reaction was completed, the microspheres were dialyzed overnight in 1L of deionized water at 4°C using a dialysis bag (molecular weight cutoff 300kDa) (with two solution changes) to remove unreacted crosslinking agents and free molecules, thus obtaining AA / CYP@Lipid-Zn-THA microspheres modified with a targeting intermediate layer.

[0031] 3. Constructing a low molecular weight chitosan that penetrates the outer layer Transfer the dialysis product from step 2 to a beaker, and add 5 mL of low molecular weight chitosan solution (1.0% w / v, molecular weight 6 kDa, degree of deacetylation ≥92%, dissolved in 0.1 M acetate buffer, pH 5.0) dropwise under gentle magnetic stirring. After the addition is complete, continue stirring and incubation for 25 minutes using layer-by-layer self-assembly technology to allow the positively charged low molecular weight chitosan to tightly encapsulate the negatively charged hyaluronic acid outer layer. After the reaction is complete, centrifuge at 10,000 rpm for 15 minutes (4°C), carefully discard the supernatant, and resuspend the precipitate in a small amount of lyophilization protectant solution containing 2% trehalose (w / v).

[0032] 4. Freeze-drying and finished product characterization The resuspended solution obtained in step 3 was dispensed into glass vials and placed in a freeze dryer. The freeze-drying program parameters were: pre-freezing at -50℃ for 3 hours, sublimation drying at -25℃ for 18 hours, and desorption drying at 25℃ for 6 hours (vacuum degree ≤15Pa). After freeze-drying, a loose, white powdery metabolic enzyme delivery system was obtained.

[0033] Scanning electron microscopy after reconstitution revealed that the delivery system exhibits a typical spherical core-shell structure with a low-density chitosan halo visible on the outer layer (see details). Figure 1 Dynamic light scattering (DLS) measured the average hydrated particle size to be 680 nm ± 80 nm, the polydispersity index (PDI) to be 0.12, and the zeta potential to be reversed from negative (-18.7 mV, after modification) to +15.3 mV (outer layer of chitosan), confirming the successful assembly of chitosan.

[0034] Example 2: Effect Verification Healthy largemouth bass weighing approximately 200g were selected and fed a diet containing enrofloxacin (20 mg / kg body weight) for 5 consecutive days to establish an enrofloxacin residue model in fish skin tissue (the residue level was detected to be approximately 0.80 mg / kg). The experimental fish were then divided into three groups: a blank control group (fed a basic diet without the drug), a single enzyme group (fed uncoated free enzyme), and the delivery system group prepared in Example 1 of this invention.

[0035] Feeding method: Mix the feed with 100 mg / kg (calculated as enzyme protein) and feed continuously for 5 days.

[0036] Detection method: Fish skin tissue was collected on the 1st, 3rd and 5th day after drug withdrawal, and the residual amount of enrofloxacin was detected by high performance liquid chromatography-tandem mass spectrometry.

[0037] Experimental Results: After 5 days of feeding, the enrofloxacin residue in the fish skin of the fish group treated with the delivery system of this invention decreased to below 0.10 mg / kg, with a degradation rate as high as 85%. Residue in the fish muscle also decreased simultaneously, and there was no significant impact on fish growth. In contrast, the degradation rate of the single enzyme group was only 20%, and some enrofloxacin metabolites, ciprofloxacin, were detected. The degradation rate of the blank control group was only 5%.

[0038] For details, please see [link / details]. Figure 2 The enzyme metabolic delivery system prepared by this invention can effectively target fish skin tissue, significantly reduce enrofloxacin residue, and has high safety with no tissue damage.

[0039] Example 3 Stability Test The delivery system prepared in Example 1 was incubated in simulated gastric fluid (SGF, pH 1.2, containing pepsin) for 2 hours, and the enzyme activity retention rate was measured. The results are shown below. Figure 3 After treatment with simulated gastric juice, the enzyme metabolic system embedded in this system retained more than 80% of its activity; while the uncoated free enzyme retained less than 15% of its activity under the same conditions. This indicates that the hierarchical protection structure of this invention significantly improves the gastrointestinal stability of enzymes.

Claims

1. A collagen-binding peptide, characterized in that, The amino acid sequence of the collagen-binding peptide is TKKTLRT.

2. An enzymatic metabolic delivery system for reducing drug residues in aquatic products, characterized in that, It includes an enzyme system nanolipid core and the collagen-binding peptide as described in claim 1.

3. A method for preparing the enzyme-catalyzed metabolic delivery system according to claim 2, characterized in that, Includes the following steps: (1) Arachidonic acid and cytochrome P450 enzyme were mixed at a mass ratio of 1:0.5-1:1 and dissolved in a solution containing Zn. 2+ The enzyme system nanolipid core was prepared in a buffer solution; (2) The enzyme system nanolipid core is dispersed in a solution containing collagen-binding peptides and hyaluronic acid, and a targeted intermediate layer is constructed by electrostatic adsorption and amide bond cross-linking; (3) The product obtained in step (2) is dispersed in a low molecular weight chitosan solution, and an outer protective shell is constructed by layer-by-layer self-assembly technology to obtain enzyme metabolism delivery microspheres; (4) The enzyme metabolism delivery microspheres obtained in step (3) are freeze-dried to obtain an enzyme metabolism delivery system with an average particle size of 600nm-1000nm.

4. The preparation method according to claim 3, characterized in that, The preparation steps of the enzyme system nanolipid core are as follows: Arachidonic acid was dissolved in ultrapure water at 30–50°C under constant stirring in a Zn-containing solution. 2+ The buffer solution was then subjected to continuous constant temperature and stirring conditions, and cytochrome P450 enzyme was quantitatively added through a precision feeding system. The mixture was continuously and gently stirred for 40 minutes within the above temperature range, so that cytochrome P450 enzyme and arachidonic acid could form a stable enzyme system nanolipid core through hydrophobic interaction.

5. The preparation method according to claim 3, characterized in that, The cytochrome P450 enzyme is the CYP1A2 subtype, with a specific activity ≥60 U / mg.

6. The preparation method according to claim 3, characterized in that, The low molecular weight chitosan has a molecular weight of 5-10 kDa.

7. The preparation method according to claim 3, characterized in that, The buffer solution is a phosphate buffer solution with a pH of 7.4 and contains 0.2 mM zinc sulfate.

8. The preparation method according to claim 3, characterized in that, In the construction of the targeted intermediate layer, the reaction system pH was 5.5, and EDC and NHS were added as crosslinking agents.

9. The preparation method according to claim 3, characterized in that, The freeze-drying process parameters are as follows: pre-freezing at -50℃ for 3 hours, sublimation drying at -25℃ for 18 hours, desorption drying at 25℃ for 6 hours, and vacuum degree ≤15Pa.

10. The enzyme-catalyzed metabolic delivery system prepared by the preparation method of claim 3.

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