Preparation method and application of a compartmentalized multi-chamber ferritin delivery system
By adjusting the concentration and pH of the ferritin solution, adding genipin solution dropwise and heating the reaction, a stable compartmentalized multi-chamber ferritin delivery system was prepared. This solved the problems of poor stability and easy leakage of active molecules in the ferritin delivery system, realizing compartmentalized loading and targeted release of active substances, which is suitable for the food and pharmaceutical fields.
Patent Information
- Application Number
- CN202310738189.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-21
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2043-06-21
AI Technical Summary
Existing ferritin delivery systems suffer from poor stability and are prone to leakage of active molecules, making it difficult to achieve compartmentalized multi-chamber loading and targeted release of active substances.
By adjusting the concentration and pH of the ferritin solution, adding genipin solution dropwise and raising the temperature to control the reaction conditions, a segmented multi-compartment ferritin delivery system was prepared. The system utilizes the cross-linking of genipin with the ferritin solution to form a stable multi-compartment structure, thereby achieving segmented loading and targeted release of active substances.
The prepared compartmentalized multi-chamber ferritin delivery system maintains structural stability over a wide pH range and under high ionic strength, preventing leakage of active substances and enabling ordered loading and targeted release of active substances, making it suitable for the food and pharmaceutical fields.
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Figure CN116999404B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of ferritin delivery, and more particularly to a method for preparing and applying a compartmentalized multi-chamber ferritin delivery system. Background Technology
[0002] Ferritin is a universal, naturally occurring iron storage protein widely found in various animals, plants, and microorganisms. It is a large protein molecule with a molecular weight of approximately 450 kDa, composed of 24 subunits that self-assemble into a hollow cage-like structure. As a novel protein nanomaterial, ferritin has been widely used in inorganic nanoparticle synthesis, biosensors, bioimaging, and drug delivery. Based on its unique nanocage-like structure, ferritin's internal nanoscale spatial structure can accommodate various functional active substances found in food.
[0003] Ferritin itself possesses pH-regulated reversible assembly properties, meaning that ferritin molecular cages can dissociate into subunits under extreme conditions of strong acid or strong alkalinity. When the pH of the ferritin molecular solution is readjusted to neutral, recombination can occur, thus enabling its successful encapsulation of macromolecules. However, current ferritin delivery systems still have certain problems. For example, the stability and self-assembly properties of ferritin cages may change after modification. In vivo, the active substances encapsulated in the ferritin system may suffer structural damage due to the acidity and enzymatic action of the gastrointestinal tract, leading to leakage of contents. Furthermore, the cage structure is difficult to maintain under acidic or alkaline conditions and different ionic strengths; the delivery system is prone to dissociation in strongly acidic or alkaline environments. In addition, current ferritin encapsulation of multiple active substances is basically a mixed encapsulation, resulting in poor practical performance and difficulty in achieving targeted release. Therefore, to solve the above problems, this invention discloses a novel method for preparing a segmented multi-compartment ferritin delivery system. Summary of the Invention
[0004] In view of this, the purpose of this invention is to provide a structurally stable segmented multi-chamber ferritin delivery system to solve the problems of poor stability, easy leakage of active molecules, difficulty in achieving segmented multi-chamber loading of active substances, and difficulty in targeted release of current ferritin delivery systems.
[0005] A method for preparing a compartmentalized multi-chamber ferritin delivery system includes the following preparation steps:
[0006] After adjusting the concentration and pH of the ferritin solution, genipin solution was added dropwise, and the reaction was carried out by heating. After the reaction was completed, dialysis was performed to obtain a compartmentalized multi-chamber ferritin delivery system.
[0007] Of course, after adjusting the pH of the ferritin solution, you can stir it for about 2 hours before homogenizing to disperse the ferritin molecules more evenly.
[0008] Furthermore, the concentration of the ferritin solution is 0.5-2 μM, the concentration of the genipin solution is 100-1000 mM, and the volume ratio of the ferritin solution to the genipin solution is 1:0.05.
[0009] Furthermore, during the reaction, the pH of the ferritin solution was adjusted to 4-6, and the temperature of the reaction system was raised to 30-60℃. The reaction conditions were strictly controlled to inhibit the intramolecular cross-linking reaction of the ferritin nanocages.
[0010] Furthermore, the reaction time is 4-6 hours. Of course, stirring, ultrasound, or other methods can be used to promote the reaction.
[0011] Furthermore, the ferritin solution is one of an empty-shell ferritin solution or a ferritin composite particle solution in which functional active molecules are embedded in the cavity.
[0012] When genipin and empty-shell ferritin solution are used as raw materials, the prepared segmented multi-chamber ferritin delivery system has a cage-like structure without loading active substances. However, when genipin, empty-shell ferritin solution, and active substances are used as raw materials and the reaction conditions are strictly controlled, the prepared segmented multi-chamber ferritin delivery system can achieve segmented loading in different cross-linked ferritin cavities. This means that two or more functional active molecules can be ordered loaded into different cavities without interference and can be released in a targeted manner.
[0013] Furthermore, when the ferritin solution is a ferritin complex particle solution, the preparation method of the ferritin complex particle solution is as follows:
[0014] Adjust the pH of the empty shell ferritin solution to 2, add the active solution dropwise, stir the reaction, and then adjust the pH of the reaction system to 7 to obtain a mixed solution. Dialyze the mixed solution to obtain a ferritin complex particle solution.
[0015] Furthermore, the active ingredients in the active solution include one or more of the following: bioactive substances, polypeptides, amino acids, fatty acids, vitamins, minerals, and drugs.
[0016] Ferritin delivery systems can be loaded with different active substances. For example, in the pharmaceutical field, they can be loaded with different targeted drugs, and in the food field, they can be loaded with fatty acids, vitamins, etc. The ferritin delivery system prepared by this invention can be freely loaded according to the substances to be delivered.
[0017] The application of the compartmentalized multi-chamber ferritin delivery system prepared in this invention in the food or medical fields.
[0018] Furthermore, the prepared segmented multi-chamber ferritin delivery system is applicable in a wide pH range of 2-10 and in systems with an ionic strength ≤600mM.
[0019] Beneficial effects:
[0020] 1. This invention utilizes genipin and ferritin solution to prepare a segmented multi-compartment ferritin delivery system. The prepared delivery system has a diameter of <1 micrometer and a stable structure. It has digestive stability in the gastrointestinal tract and is not easily dissociated due to changes in the external environment. It can effectively prevent the leakage of active substances loaded in the ferritin cavities, thereby improving loading and delivery efficiency.
[0021] 2. The compartmentalized multi-chamber ferritin delivery system prepared by this invention is a multi-chambered molecular particle that can achieve compartmentalized encapsulation between molecules. It can rationally load and deliver food or pharmaceutical functional active substances in different compartmentalized cavities according to different application requirements. Therefore, this delivery system has broad application prospects in food, pharmaceuticals, health products, and medical fields.
[0022] 3. The prepared delivery system can maintain the stability of the particles under strong acid, strong base and strong ion concentration, that is, it can maintain the complete cage structure of the delivery system particles without dissociation or aggregation.
[0023] 4. The proportion of active substances in the delivery system prepared by this invention can be freely adjusted, which broadens the flexibility of the prepared delivery system and lays the foundation for applications in other fields. Attached Figure Description
[0024] Figure 1 SDS-PAGE electrophoresis image of the compartmentalized multi-chamber ferritin delivery system prepared in Example 1;
[0025] Figure 2 Laser scanning confocal microscopy image of the compartmentalized multi-chamber ferritin delivery system containing active substances prepared in Example 2, showing the compartmentalized loading.
[0026] Figure 3 Transmission electron microscope image of the compartmentalized multi-chamber ferritin delivery system prepared in Example 2;
[0027] Figure 4 The particle size distribution diagram is shown for the segmented multi-compartment ferritin delivery system prepared in Example 2.
[0028] Figure 5 The particle size distribution of the compartmentalized multi-chamber ferritin delivery system prepared in Example 2 under different pH conditions is shown in the diagram.
[0029] Figure 6 The particle size distribution of the compartmentalized multi-chamber ferritin delivery system prepared in Example 2 under different ionic intensities is shown in the diagram.
[0030] Figure 7The particle size distribution diagram shows the segmented multi-compartment ferritin delivery system containing chlorogenic acid and iron prepared in Example 3. Detailed Implementation
[0031] The present invention will now be described in detail with reference to the embodiments and accompanying drawings:
[0032] Example 1:
[0033] (1) Adjust the concentration of the purified empty shell ferritin solution to 0.5 μM and adjust the pH of the empty shell ferritin solution to 5. After stirring for 2 h, homogenize. After homogenization, take 5 mL of the above empty shell ferritin solution and place it in a 45℃ water bath and stir at a constant rate for 2 h.
[0034] (2) Accurately weigh the genipin reagent and dissolve it in anhydrous ethanol solution to prepare a genipin solution with a concentration of 800 mM;
[0035] (3) Take 250 μL of the prepared genipin solution and slowly add it dropwise to the empty shell ferritin solution. Place the mixed solution in an ultrasonic cleaner with an ultrasonic power of 120 W for reaction. Control the reaction temperature to 45 °C and react for 4 h. After the reaction, dialyze the resulting mixed solution into Tris-HCl (pH 8.0) solution to remove free genipin molecules, and a structurally stable compartmentalized ferritin delivery system can be obtained.
[0036] In addition, a control experiment was also conducted in this embodiment:
[0037] Experiment 1: Instead of adding genipin solution, the empty shell ferritin solution was subjected to self-crosslinking, and the reaction conditions remained unchanged;
[0038] Experiment 2: The reaction times were set to 1 h, 2 h, 4 h, and 6 h.
[0039] Based on the above experiment, the results are as follows: Figure 1 As shown, the cross-linking reaction between ferritin solution and genipin was analyzed by SDS-PAGE, and the results indicate that:
[0040] pass Figure 1 The SDS-PAGE electrophoresis results show that genipin promotes covalent cross-linking between subunits of ferritin molecules, and a stable and uniform delivery system can only be generated after 4-6 hours of reaction.
[0041] Example 2:
[0042] The active substance in the delivery system prepared in this embodiment is a fluorescent substance, so care must be taken to avoid light during operation.
[0043] (1) Accurately weigh Nile Red fluorescent dye and FITC reagent and dissolve them in DMSO solution to prepare Nile Red fluorescent solution and FITC fluorescent solution respectively. The concentration of the prepared fluorescent solution is 10mM.
[0044] (2) Adjust the concentration of the purified empty shell ferritin solution to 2 μM and adjust the pH of the empty shell ferritin solution to 2.0. Add the prepared Nile Red fluorescent solution dropwise into the empty shell ferritin solution and stir at a constant speed for 20 min under magnetic stirring. Then adjust the pH of the above Nile Red fluorescent solution to 7 and dialyze the mixed solution into Tris-HCl (pH 8.0) solution to remove free fluorescent molecules, thereby obtaining a ferritin complex particle solution embedded with Nile Red fluorescent molecules. The steps for preparing a ferritin complex particle solution embedded with FITC fluorescent molecules are the same.
[0045] (3) Adjust the concentration of the two purified ferritin complex particle solutions to 0.5 μM, mix them in a molar ratio of 1:1 and adjust the pH of the solution to 5 to obtain a mixed ferritin complex particle solution. Take 5 mL of the mixed ferritin complex particle solution and place it in a water bath at 45°C and stir at a constant rate for 2 h or homogenize it for later use.
[0046] (4) Accurately weigh the genipin reagent and dissolve it in anhydrous ethanol solution to prepare a genipin solution concentration of 800 mM. Take 250 μL of the prepared genipin solution and slowly add it dropwise to the mixed ferritin complex particle solution. Place it in an ultrasonic cleaner for reaction, control the reaction temperature to 45℃, and react for 5 h under ultrasonic power of 120 W. After the reaction, dialyze the obtained solution into Tris-HCl (pH 8.0) solution to remove free genipin molecules, and a structurally stable compartmented multi-chamber ferritin delivery system containing fluorescent active substances can be obtained.
[0047] The prepared compartmentalized multi-chamber ferritin delivery system containing fluorescent active substances was subjected to laser scanning, and the results were obtained using laser scanning confocal microscopy. Figure 2 The results show that fluorescent substances exist separately in different chambers, indicating that the prepared multi-chamber delivery system can achieve compartmentalized loading.
[0048] Experiment 3:
[0049] The prepared compartmentalized multi-chamber ferritin delivery system was analyzed by transmission electron microscopy using dynamic light scattering, and the results are as follows: Figure 3-4 As shown:
[0050] The specific covalent cross-linking mechanism between ferritin molecules was analyzed using transmission electron microscopy, and the aggregation state of the compartmentalized multi-chamber ferritin delivery system in solution was analyzed using dynamic light scattering results. Genipin-mediated covalent cross-linking between ferritin molecules is mainly intermolecular cross-linking. This method of cross-linking does not destroy the independent structure of the ferritin molecular cage, and the prepared compartmentalized multi-chamber ferritin delivery system is stable and uniform.
[0051] Experiment 4:
[0052] The prepared compartmentalized ferritin delivery system was immersed in environments with pH values of 2, 4, 6, 8, and 10, respectively, and the results are as follows. Figure 5 As shown in the analysis results, the following can be concluded:
[0053] Within the pH range of 2-10, dynamic light scattering results showed that the particle size in the compartmentalized multi-chamber ferritin delivery system was still <1 micrometer. The results indicate that ferritin molecules maintain their original cage-like structure and do not undergo structural dissociation under extreme acidic and alkaline conditions.
[0054] Experiment 5:
[0055] The prepared compartmentalized ferritin delivery system was immersed in environments with salt concentrations of 100, 200, 400, and 600 mM, respectively, and the results are as follows. Figure 6 As shown in the analysis results, the following can be concluded:
[0056] In environments with salt concentrations less than or equal to 600 mM, dynamic light scattering results showed that the particle structure in the compartmentalized multi-chamber ferritin delivery system remained stable, without dissociation or aggregation under different ionic intensities, maintaining the original cage-like structure.
[0057] Example 3:
[0058] The active substances in the delivery system prepared in this embodiment are chlorogenic acid molecules and iron.
[0059] (1) Adjust the concentration of the purified empty shell ferritin solution to 2 μM and adjust the pH of the empty shell ferritin solution to 2.0. Add the prepared chlorogenic acid (ChA) molecule active solution to the empty shell ferritin solution and stir at a constant speed for 20 min under magnetic stirring. Then adjust the pH of the above mixed solution to 7 and dialyze the mixed solution into Tris-HCl (pH 8.0) solution to remove free chlorogenic acid molecules, so as to obtain the ferritin complex particle solution embedded with chlorogenic acid molecules (ChA). Holoferritin is prepared by the existing ferritin iron oxidation precipitation method, that is, 1 mole of ferritin internal cavity can be loaded with 3000 moles of iron ions, thereby preparing the ferritin complex particle solution embedded with iron element.
[0060] (3) Adjust the concentration of the above ferritin complex particle solution to 0.5 μM, mix them according to a certain molar ratio, adjust the pH of the solution to 5, and obtain a mixed ferritin complex particle solution. Take 5 mL of the above mixed ferritin complex particle solution and place it in a water bath at 45°C and stir at a constant rate for 2 h for later use.
[0061] (4) Accurately weigh the genipin reagent and dissolve it in anhydrous ethanol solution to make the final concentration of the prepared genipin solution 20mM. Take 250uL of genipin solution and slowly add it dropwise to the mixed ferritin complex particle solution. Place it in an ultrasonic cleaner for reaction and control the temperature of the reaction to be 45℃. React for 5h under ultrasonic power of 120W. After the reaction is completed, dialyze the obtained solution into Tris-HCl (pH 8.0) solution to remove free genipin molecules, and a structurally stable compartmented multi-chamber ferritin delivery system containing chlorogenic acid and iron can be obtained.
[0062] In this embodiment, when preparing the mixed ferritin composite particle solution, the molar ratio of the ferritin composite particle solution encapsulated with chlorogenic acid molecules (ChA) and the ferritin composite particle solution encapsulated with iron was set to: 5:0, 3:2, 1:1, 2:3, and 0:5. The results obtained are as follows. Figure 7 As shown in the analysis results, the following can be concluded:
[0063] The proportion of active substances in the compartmentalized multi-chamber ferritin delivery system containing active substances prepared by this invention can be freely adjusted, and the prepared delivery system has a uniform particle size.
[0064] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.
Claims
1. A method for preparing a compartmentalized multi-chamber ferritin delivery system, characterized in that, The preparation steps include the following: After adjusting the concentration and pH of the ferritin solution, genipin solution was added dropwise, and the reaction was carried out by heating. After the reaction was completed, dialysis was performed to obtain a compartmentalized multi-chamber ferritin delivery system. Adjust the concentration of the ferritin solution to 0.5-2 µM and the concentration of the genipin solution to 100-1000 mM; Adjust the pH of the ferritin solution to 4-6, raise the temperature to 30-60℃, and allow the reaction time to be 4-6 hours. The volume ratio of the ferritin solution to the genipin solution is 1:0.
05.
2. The method for preparing a compartmentalized multi-compartment ferritin delivery system according to claim 1, characterized in that, The ferritin solution is an empty-shell ferritin solution.
3. The method for preparing a compartmentalized multi-compartment ferritin delivery system according to claim 1, characterized in that, The ferritin solution is a solution of ferritin composite particles in which functional active molecules are embedded in the cavity of empty shell ferritin. The functional active molecules include one or more of fluorescent molecules, chlorogenic acid, and iron.
4. The method for preparing a compartmentalized multi-chamber ferritin delivery system according to claim 3, characterized in that, When the ferritin solution is a ferritin complex particle solution, the preparation method of the ferritin complex particle solution is as follows: The pH of the empty shell ferritin solution was adjusted to 2, and the functional active molecule solution was added dropwise. After stirring the reaction, the pH of the reaction system was adjusted to 7 to obtain a mixed solution. The mixed solution was then dialyzed to obtain a ferritin complex particle solution.
5. The method for preparing a compartmentalized multi-chamber ferritin delivery system according to claim 4, characterized in that, The prepared compartmentalized multi-chamber ferritin delivery system is suitable for use in systems with pH values of 2-10.
6. The method for preparing a compartmentalized multi-compartment ferritin delivery system according to claim 5, characterized in that, The prepared compartmentalized multi-chamber ferritin delivery system is suitable for systems with an ionic strength ≤ 600 mM.
7. The application of the compartmentalized multi-chamber ferritin delivery system prepared according to claim 2 in food delivery systems.
8. The application of the compartmentalized multi-chamber ferritin delivery system prepared according to any one of claims 1-3 in the preparation of a delivery system for targeted medical drugs.