Method for preparing NMN hydrogel microcapsule based on ionic gelation technology

Abstract

The invention relates to a method for preparing an NMN hydrogel microcapsule based on an ionic gelation technology, which comprises the following steps: taking an NMN solution as an active molecule solution, respectively carrying out ionized cross-linking reaction on a cationic hydrogel polymer and an anionic hydrogel polymer under the triggering action of a cationic cross-linking agent, and embedding NMN in a hydrogel film layer to form the NMN hydrogel microcapsule. The NMN hydrogel microcapsule prepared by the method has excellent slow-release performance, the particle size of the prepared microcapsule is uniform, the whole preparation process is green and environment-friendly, an organic solvent and a heating reaction are not needed, and natural and nontoxic cationic hydrogel polymer and anionic hydrogel polymer are selected as materials for preparing a hydrogel film layer, so that the hydrogel film layer is more stable in performance. And the hydrogel has good biocompatibility and biodegradability, is harmless to a human body and is high in safety.

Description

Technical Field

[0001] The present invention relates to a method for preparing microcapsules, and in particular to a method for preparing NMN hydrogel microcapsules based on ion gelation technology. Background Art

[0002] β-Nicotinamide adenine mononucleotide (NMN), as a precursor for the synthesis of NAD+ (nicotinamide adenine dinucleotide), has the effects of anti-aging, enhancing immunity, and treating cardiovascular and cerebrovascular diseases.

[0003] At present, the most commonly used method is to make NMN into microcapsules by spray drying granulation technology. However, this method has certain disadvantages, which may cause NMN to remain on the surface of the microcapsules, which is easy to oxidize and cause the components to be destroyed. In addition, the spray drying granulation technology also needs to be carried out at a high temperature of 150-170°C, and the NMN raw material is not heat-resistant and is easily destroyed by light and oxygen. It is less stable in a light, high temperature, and oxidative environment. Granulation under such high temperature conditions will cause great damage to the activity of NMN and affect the efficacy of the finished product.

[0004] Furthermore, the NMN products currently on the market do not have a sustained-release effect and generally need to be taken multiple times a day. Multiple doses a day can easily cause large fluctuations in blood drug concentrations, making it difficult to obtain a stable and effective blood drug concentration. It also often results in insufficient or missed doses, thus failing to achieve its therapeutic and health-care effects.

[0005] It can be seen that the primary issue to be solved by the present invention is to develop an NMN microcapsule that can release NMN for a long time, improve user compliance, and has an environmentally friendly preparation method. Summary of the invention

[0006] One of the main purposes of the present invention is to provide a method for preparing NMN hydrogel microcapsules with sustained release effect based on ion gelation technology. To achieve the above-mentioned purpose, the present invention provides a method for preparing NMN hydrogel microcapsules based on ion gelation technology, and the preparation steps are as follows:

[0007] Step 1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution, wherein the mass fraction of NMN in the NMN solution is 1% to 5%;

[0008] Step 2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in an acidic solution with a mass fraction of 0.5% to 2% to obtain a cationic hydrogel polymer solution, wherein the mass fraction of the cationic hydrogel polymer in the cationic hydrogel polymer solution is 0.1% to 1%;

[0009] Step 3, preparing an anionic hydrogel polymer solution, dissolving an anionic hydrogel polymer in an alkaline solution with a pH value of 8 to 10, wherein the mass fraction of the anionic hydrogel polymer in the anionic hydrogel solution is 0.05% to 0.5%;

[0010] Step 4, preparing an NMN gel precursor solution, mixing the NMN solution with the anionic hydrogel polymer solution at a weight ratio of 1:1 to 5:1, and stirring to obtain an NMN gel precursor solution;

[0011] Step 5: Prepare NMN hydrogel microcapsules by cross-linking reaction. The NMN gelation precursor solution and the cationic hydrogel polymer solution are mixed and stirred evenly in a volume ratio of 1:5 to 1:20, and then the cationic cross-linking agent is slowly dropped while stirring at a low speed to form NMN hydrogel microcapsules.

[0012] The present invention uses a cationic crosslinking agent to catalyze the ionic crosslinking reaction between the cationic hydrogel polymer and the anionic hydrogel polymer to form a hydrogel film layer to coat NMN to obtain NMN hydrogel microcapsules, and the porous grid structure of the hydrogel film layer is used to reduce the release rate of NMN, thereby achieving a long-term sustained release effect. During preparation, a corresponding mass fraction concentration of NMN solution is prepared according to the expected content of NMN in the NMN hydrogel microcapsules finally obtained. In order to ensure that the subsequent ionic crosslinking reaction is complete, the cationic hydrogel polymer is dissolved in the acidic solution, and the amino groups in the cationic hydrogel polymer are protonated to form a positively charged ionic state, thereby increasing its reaction activity and facilitating the subsequent ionic crosslinking reaction with the anionic hydrogel polymer. Similarly, since anionic hydrogel polymers are usually polymerized from monomers with anions, in an alkaline solution, the anions they possess can interact with the cations in the alkaline solution to promote their dissolution. Next, the NMN solution and the anionic hydrogel polymer solution are mixed in proportion to prepare an NMN gelation precursor solution. Since the viscosity of the anionic hydrogel polymer solution is higher, a higher proportion of NMN solution is required to achieve the desired NMN concentration. Therefore, the NMN solution is mixed with the anionic hydrogel polymer solution at a weight ratio of 1:1 to 5:1 to ensure that the NMN, cationic hydrogel polymer and anionic hydrogel polymer in the solution are fully mixed and uniform to facilitate the ionic crosslinking reaction. The present invention can adjust the concentration of the ionic hydrogel polymer solution according to actual conditions to obtain the ideal hydrogel microcapsule size and encapsulation rate. During preparation, slowly dripping the cationic crosslinking agent while stirring at a low speed is conducive to the formation of smaller and regular microcapsules and avoiding the formation of microcapsules with large particle sizes.

[0013] Preferably, the cationic crosslinker is prepared by dissolving any one of glutathione, ethylenediamine and guanidine phosphate in water. It is further explained that glutathione, ethylenediamine and guanidine phosphate are small molecule crosslinkers, which are non-toxic and have good biocompatibility. In the solution state, it can diffuse rapidly to trigger the ionic hydrogel polymer to undergo ionic crosslinking reaction to quickly form a hydrogel film layer coated with NMN, and can also further adjust the crosslinking degree and pore size of the hydrogel film, thereby affecting the permeability characteristics of the NMN hydrogel microcapsules.

[0014] Preferably, the cationic cross-linking agent is prepared by dissolving glutathione in water at a mass fraction of 0.1% to 0.2%.

[0015] It is further explained that if the mass fraction of glutathione in the solution is greater than 2%, the viscosity will increase, which is not conducive to diffusion to ensure sufficient reaction, while the mass fraction is less than 0.1%, resulting in insufficient cross-linking strength and poor stability of the hydrogel microcapsules generated by the reaction.

[0016] Preferably, the cationic crosslinking agent is prepared by dissolving 1% to 5% by mass of ethylenediamine or guanidine phosphate in water.

[0017] It is further explained that if the mass fraction of ethylenediamine or guanidine phosphate in the solution is greater than 5%, it is easy to cause the cross-linking reaction to be too fast and the particle size of the formed hydrogel microcapsules cannot be controlled. If the mass fraction is less than 1%, the cross-linking reaction efficiency is reduced, the reaction time is prolonged, and the generation efficiency is reduced.

[0018] Preferably, the dropping rate of the cationic crosslinking agent in step 5 is 1 g / min to 3 g / min, and the rotation speed of the low-speed stirring is 30 r / min to 60 r / min.

[0019] Further explanation: When the drop rate of NMN hydrogel precursor is greater than 3g / min, the reaction will generate large-size microcapsules, and the particle size of the microcapsules is uneven; while when the drop rate is less than 1g / min, the reaction time will be prolonged and the efficiency will be reduced. Similarly, when the speed of low-speed stirring is higher than 60r / min, irregular microcapsules will be generated, while when the speed is lower than 30r / min, large-size microcapsules will be easily generated, resulting in large batch differences.

[0020] Preferably, in step 5, when the viscosity of the monitored solution reaches 102-103 mPa·s, the addition of the cationic crosslinking agent is stopped.

[0021] It is further explained that the viscosity change of the reaction solution is monitored in real time by a viscometer. As the microcapsules are formed, the viscosity increases accordingly. When the viscosity value is reached, it means that the anionic hydrogel polymer and the cationic hydrogel polymer in the solution have basically reacted completely, and the addition of the cationic crosslinking agent can be stopped.

[0022] Another main purpose of the present invention is to achieve efficient encapsulation of NMN active ingredients in ion gel microcapsules through a microfluidic chip device. To achieve the aforementioned purpose, the present invention provides a method for preparing NMN hydrogel microcapsules based on ion gelation technology, and the preparation steps are as follows:

[0023] S1, preparing an NMN aqueous solution, dissolving NMN in water to prepare an NMN aqueous solution, wherein the mass fraction of NMN in the NMN aqueous solution is 1% to 5%;

[0024] S2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in an acidic solution having a mass fraction of 0.5% to 2% to obtain a cationic hydrogel polymer solution, wherein the mass fraction of the cationic hydrogel polymer in the cationic hydrogel polymer solution is 0.1% to 1%;

[0025] S3, preparing an anionic hydrogel polymer solution, which is prepared by dissolving an anionic hydrogel polymer in an alkaline solution with a pH value of 8 to 10, wherein the mass fraction of the anionic hydrogel polymer in the anionic hydrogel solution is 0.05% to 0.5%;

[0026] S4, preparing an NMN gel precursor solution, mixing the NMN aqueous solution, anionic hydrogel polymer and cationic hydrogel polymer solution in a mass ratio of 1:1:2 to 1:2:4, and stirring to obtain an NMN gel precursor solution;

[0027] S5, preparing NMN hydrogel microcapsules by cross-linking reaction, injecting the above-mentioned NMN gelation precursor solution as the core flow into the microfluidic chip device, and injecting the cationic cross-linking agent as the isolation fluid at the same time, when the two fluids meet, an ionic gelation reaction occurs to form NMN hydrogel microcapsules.

[0028] The present invention uses microfluidic technology to realize the ion gel film forming process through a microfluidic chip device. The microfluidic chip device has two or more small flow channels, which can be used to accurately control the flow and mixing of different liquid fluids. The pre-formulated NMN gel precursor solution contains both anionic hydrogel polymers and cationic hydrogel polymers, which are injected into a flow channel of the microfluidic chip as a core flow, and the cationic cross-linking agent is injected as an isolation fluid. Inside the nanoscale flow channel of the microfluidic chip device, the core flow (i.e., the NMN gel precursor solution) and the isolation fluid (cationic cross-linking agent) meet under the precise control of the set flow rate and flow rate ratio to form a fluid / fluid interface. The core flow velocity is 1-10 mm / s. Too fast a flow velocity will destroy the fluid interface, and too much diffusion will lead to diffusion control; the isolation flow velocity is 10-100 mm / s, which is higher than the core flow velocity to keep the fluid cross section stable; and the flow rate ratio is the ratio of the core flow velocity to the isolation fluid flow velocity. The flow rate ratio ranges from 1 / 10 to 1 / 2. A moderate flow ratio is conducive to the formation of a regular fluid / fluid interface. Since the isolation fluid (cationic crosslinker) is generally a small molecule compound such as ethylenediamine, guanidine phosphate and glutathione, it can quickly diffuse and penetrate into the core flow (NMN gelation precursor solution) in a solution state, so that the anions in the core flow and the cations in the isolation fluid begin to undergo an ionic crosslinking reaction driven by electrostatic forces at this fluid / fluid interface, and form a crosslinked grid structure membrane.

[0029] The present invention uses a microfluidic chip device to accurately control the flow ratio, flow rate and other kinetic parameters of the two fluids during the cross-linking reaction, so that the cross-linking reaction continues to diffuse inward in the core flow fluid, and finally completely encapsulates the NMN active molecules in the core flow to form hydrogel microcapsules with spherical or other morphological structures. The prepared NMN hydrogel microcapsules have uniform and controllable particle size and narrow size distribution, and the ionic cross-linking reaction can give the hydrogel microcapsules good mechanical stability, controlled release performance and biocompatibility.

[0030] Preferably, in step S5, the cationic crosslinking agent is prepared by dissolving any one of glutathione, ethylenediamine and guanidine phosphate in water to prepare a corresponding aqueous solution, the glutathione solution is prepared by dissolving glutathione at a mass fraction of 0.1% to 0.2% in water, the ethylenediamine solution is prepared by dissolving ethylenediamine at a mass fraction of 1% to 5% in water, and the guanidine phosphate solution is prepared by dissolving guanidine phosphate at a mass fraction of 1% to 5% in water.

[0031] Preferably, in step S5, the glutathione solution is first injected as the isolation fluid, and meets the NMN gelation precursor solution as the core flow to undergo an ion gelation reaction to form an NMN single-layer membrane microcapsule having an inner biocompatible membrane. The NMN single-layer membrane microcapsule is then used as a secondary gelation precursor and injected into the microfluidic chip device as the core flow. At the same time, the ethylenediamine solution or the guanidine phosphate solution is injected as the isolation fluid, so that the two fluids meet and undergo a secondary ion cross-linking reaction, and a dense controlled release membrane is formed on the inner biocompatible membrane, thereby obtaining an NMN hydrogel microcapsule having a double-layer hydrogel membrane.

[0032] It is further explained that in the microfluidic process of ion gelation film preparation, two cationic crosslinker solutions of different properties are used as isolation fluids. After one of the cationic crosslinkers is used as the isolation fluid to form the inner biocompatible membrane, it is then treated with another crosslinker solution to form a controlled release membrane on the outer layer of the hydrogel inner membrane layer, thereby obtaining a double-layer structure of NMN hydrogel microcapsules. Two different cationic crosslinkers are used to construct two layers of hydrogel membranes with different compositions and properties on the microcapsules, and then the inner biocompatible membrane controls the initial rapid release behavior and the release rate in the initial stage, while the outer controlled release membrane can control the subsequent sustained release process, so that a more sophisticated dual controlled release is achieved with this double-layer structure, and the cross-regulation of each layer of the membrane can optimize and meet more complex sustained release curve requirements. From the overall release curve of the double-layer hydrogel membrane microcapsule, it can be seen that it presents a two-stage release, that is, a slow release in the initial stage, the curve is relatively gentle, and the accelerated release gradually enters the later stage, and the curve rises, which can achieve an ideal double peak or "zigzag" release curve.

[0033] Preferably, when preparing the inner biocompatible membrane, the flow rate of the core flow is 1-5 mL / min, the flow rate of the isolation fluid is 5-25 mL / min, and the flow ratio is the flow rate ratio of the core flow to the isolation flow, and the flow ratio is 1 / 10-1 / 3.

[0034] Preferably, when preparing the outer controlled release membrane, the flow rate of the core flow is 5-20 mL / min, the flow rate of the isolation fluid is 10-50 mL / min, and the flow ratio is the flow rate ratio of the core flow to the isolation flow, and the flow ratio is 1 / 20-1 / 2.

[0035] Further explanation, the present invention realizes the orderly construction of the inner and outer two-layer membrane structure by controlling the flow ratio in two steps. Compared with the NMN single-layer hydrogel membrane microcapsules, the NMN double-layer hydrogel membrane microcapsules have the following advantages:

[0036] (1) More precise control of release behavior: The inner biocompatible membrane controls the rapid release in the initial stage, while the outer controlled release membrane adjusts the subsequent sustained release process, so that the prepared NMN double-layer hydrogel membrane microcapsules can achieve a more ideal bimodal or zigzag release curve.

[0037] (2) Stronger structural adjustability: During the preparation process of NMN double-layer hydrogel membrane microcapsules, the flow rate and flow ratio of the core flow and isolation fluid are adjusted respectively to adjust the composition, cross-linking degree and other properties of the inner and outer membranes respectively, so that the biocompatibility and controlled release performance of the NMN hydrogel microcapsules can be independently optimized.

[0038] (3) Improve mechanical and chemical stability; the outer controlled release membrane has a dense cross-linked structure that can enhance the mechanical strength of the capsule, while the double-layer hydrogel membrane structure improves the overall resistance of the microcapsule to enzyme degradation and chemical degradation.

[0039] (4) The preparation operation is relatively flexible; a single-layer hydrogel membrane is formed at one time, while the preparation process of the double-layer hydrogel membrane structure controls the kinetic parameters of the core flow and the isolation fluid on the basis of the single-layer hydrogel membrane formation to react so as to cross-link the original single-layer hydrogel membrane layer to form another hydrogel membrane layer, which has high operational flexibility.

[0040] (5) Meet more complex drug release requirements; use a double-layer hydrogel membrane to achieve a sophisticated dual-controlled release mode, and the performance of the obtained NMN double-layer hydrogel membrane microcapsules is controllable and excellent, with a wide range of applications.

[0041] Preferably, the mass concentration ratio of the glutathione solution to the ethylenediamine solution or the guanidine phosphate solution is 1:5 to 1:20.

[0042] It is further explained that ethylenediamine and guanidine phosphate are small molecule crosslinking agents, and the crosslinking reaction with the polymer is relatively fast. Glutathione is a large molecule crosslinking agent, and the crosslinking reaction with the ion gel polymer is slow. Therefore, when the amount of small molecule crosslinking agent is greater than the amount of large molecule crosslinking agent, a reasonable double crosslinking network structure can be obtained, which enhances the mechanical strength and chemical stability of the microcapsule.

[0043] Preferably, the acidic solution is prepared by dissolving any one of acetic acid, citric acid and phosphoric acid in water.

[0044] Further explanation: the acidic solutions prepared by acetic acid, citric acid and phosphoric acid in the present invention all have good biocompatibility and can dissolve NMN. In addition, their acidic strength is moderate, which can effectively dissolve cationic hydrogel polymers, enhance ionic state and activation reaction, and facilitate subsequent ionic gelation cross-linking reaction.

[0045] Preferably, the cationic hydrogel polymer is any one or more of chitosan, sodium alginate, chitin, acetylated cell wall enzyme, and polyethyleneimine.

[0046] Further explanation: chitosan comes from the shell of crustaceans and is a natural cationic polysaccharide with good biocompatibility and biodegradability. It is often used to prepare biomedical hydrogel materials. Sodium alginate is extracted from seaweed and is a linear cationic polysaccharide with good ionic cross-linking ability. It is widely used in drug sustained release systems. In addition, natural cationic derivatives such as chitin, acetylated cell wall enzymes, polyethyleneimine, or starch can also be used. These cationic polymers are positively charged in aqueous solution and can react with anionic hydrogel polymers to form hydrogel films.

[0047] Preferably, the anionic hydrogel polymer is any one or more of gelatin, carrageenan, sodium polyacrylate and polyacrylamide, and the alkaline solution is prepared by dissolving any one of triethylamine, sodium hydroxide and potassium hydroxide in water.

[0048] It is further explained that anionic hydrogel polymers are usually polymerized from monomers with anions, which can interact with cations in alkaline solutions to promote the dissolution of anionic hydrogel polymers. It is particularly explained that the anions of sodium polyacrylate are carboxyl ions, while the anions of polyacrylamide are amide ions, and the hydrophilicity of carboxyl ions is stronger than that of amide ions, so the solubility of sodium polyacrylate is better than that of polyacrylamide.

[0049] Preferably, the prepared NMN hydrogel microcapsules are in liquid state and freeze-dried or vacuum-dried to prepare dry NMN hydrogel microcapsule powder.

[0050] Further explanation: freeze drying or vacuum drying can avoid high temperature affecting the stability of the formed hydrogel film layer. The beneficial effects of the present invention are:

[0051] First, it is natural and harmless, and has high safety. The present invention selects natural and non-toxic cationic hydrogel polymers and anionic hydrogel polymers as materials for preparing the hydrogel film layer, which have good biocompatibility and biodegradability, are harmless to the human body, and are highly safe.

[0052] Second, it is green and environmentally friendly. The entire preparation process of the present invention does not require the use of organic solvents, nor does it require violent mechanical action to catalyze the reaction. It is green and environmentally friendly.

[0053] Third, the reaction is carried out at room temperature to ensure the efficacy. The present invention does not require heating reaction to avoid high temperature destroying the activity of NMN and ensure the efficacy of the finished product.

[0054] Fourth, it has excellent sustained-release performance; the hydrogel membrane formed by the ion gelation reaction of the present invention acts as a semipermeable membrane barrier, allowing NMN to gradually diffuse from the microcapsule to prevent a sudden burst of NMN.

[0055] Ensure sustained release over a longer period of time. The release rate of NMN can also be adjusted by controlling the properties of the hydrogel film, such as the type of ionic hydrogel polymer, cross-linking density, and film thickness, thereby optimizing the release curve to meet the desired therapeutic or nutritional needs. The hydrogel film can also protect NMN from degradation by enzymes or harsh environmental conditions, ensuring the stability and efficacy of encapsulated NMN.

[0056] Fifth, NMN hydrogel microcapsules with uniform particle size distribution are prepared by combining microfluidic technology; the present invention also utilizes a microfluidic chip device to cut the hydrogel precursor solution into tiny fields through fluid dynamics, and contacts it with a cationic crosslinker solution to undergo an ionic gelation reaction to form a hydrogel membrane wall, thereby obtaining NMN hydrogel microcapsules with uniform particle size distribution.

[0057] Description of the drawings

[0058] Figure 1 This is an electron microscope image of the NMN single-layer hydrogel membrane microcapsule of the present invention;

[0059] Figure 2 Schematic diagram of the release curve of the NMN single-layer hydrogel film microcapsule of the present invention;

[0060] Figure 3 Schematic diagram of the double-layer membrane structure of the NMN double-layer hydrogel membrane microcapsule of the present invention;

[0061] Figure 4 Schematic diagram of the structure of the microfluidic chip device used in the present invention.

[0062] Inner biocompatible film 1; outer controlled release film 2; microfluidic chip device; DETAILED DESCRIPTION

[0063] The preferred embodiments of the present invention are described in detail in conjunction with the embodiments. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0064] Example 1

[0065] This example is used to illustrate the first method for preparing NMN hydrogel microcapsules based on ionic gelation technology.

[0066] 1. Components:

[0067] Active ingredient: NMN (Nicotinamide Mononucleotide, Chinese for β-nicotinamide mononucleotide); cationic hydrogel polymer: chitosan; anionic hydrogel polymer: sodium polyacrylate; cationic crosslinker: prepared by dissolving glutathione in water at a mass fraction of 0.1%.

[0068] 2. Preparation method

[0069] Step 1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution with a mass fraction of 1%;

[0070] Step 2, preparing a cationic hydrogel polymer solution, dissolving the cationic hydrogel polymer in an acetic acid solution with a mass fraction of 1%, to obtain a cationic hydrogel polymer solution with a mass fraction of 0.5%;

[0071] Step 3, preparing an anionic hydrogel polymer solution, dissolving the anionic hydrogel polymer in a triethylamine solution with a pH value of 8 to prepare an anionic hydrogel polymer solution with a mass fraction of 0.1%;

[0072] Step 4, preparing an NMN gel precursor solution, mixing the NMN solution of step 1 and the anionic hydrogel polymer solution of step 3 in a weight ratio of 1:1, and stirring to obtain an NMN gel precursor solution;

[0073] Step 5: Prepare NMN hydrogel microcapsules by crosslinking reaction. The NMN capsule precursor solution of step 4 and the cationic hydrogel polymer solution of step 2 are mixed at a volume ratio of 1:5 and stirred evenly. Then, the cationic crosslinker is added at a drop rate of 1 g / min, and the mixture is continuously stirred at a low speed of 30 r / min. When the viscosity of the solution reaches 102 mPa·s by continuous monitoring with a viscometer, the addition of the cationic crosslinker is stopped to generate NMN hydrogel microcapsules with a single-layer hydrogel membrane layer.

[0074] Step six, freeze-dry the NMN microcapsule solution prepared in step five to prepare dry NMN hydrogel microcapsule powder.

[0075] Example 2

[0076] 1. Components:

[0077] Active ingredient: NMN (Nicotinamide Mononucleotide, β-nicotinamide mononucleotide in Chinese); cationic hydrogel polymer: sodium alginate; anionic hydrogel polymer: polyacrylamide; cationic crosslinker: prepared by dissolving 5% by mass of ethylenediamine in water.

[0078] 2. Preparation method

[0079] Step 1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution with a mass fraction of 3%;

[0080] Step 2, preparing a cationic hydrogel polymer solution, dissolving the cationic hydrogel polymer in a citric acid solution with a mass fraction of 2%, to obtain a cationic hydrogel polymer solution with a mass fraction of 1%;

[0081] Step 3, preparing an anionic hydrogel polymer solution, dissolving the anionic hydrogel polymer in a sodium hydroxide solution with a pH value of 9 to prepare an anionic hydrogel polymer solution with a mass fraction of 0.5%;

[0082] Step 4, preparing an NMN gel precursor solution, mixing the NMN solution of step 1 and the anionic hydrogel polymer solution of step 3 at a weight ratio of 2:1, and stirring to obtain an NMN gel precursor solution;

[0083] Step 5: Prepare NMN hydrogel microcapsules by crosslinking reaction. The NMN capsule precursor solution of step 4 and the cationic hydrogel polymer solution of step 2 are mixed at a volume ratio of 1:10 and stirred evenly. Then, the cationic crosslinker is added at a drop rate of 3 g / min, and the mixture is continuously stirred at a low speed of 60 r / min. When the viscosity of the solution reaches 103 mPa·s by continuous monitoring with a viscometer, the addition of the cationic crosslinker is stopped to generate NMN hydrogel microcapsules with a single-layer hydrogel membrane layer.

[0084] Step six, vacuum drying the NMN microcapsule solution prepared in step five to prepare dry NMN hydrogel microcapsule powder.

[0085] Example 3

[0086] This example is used to illustrate the second method for preparing NMN hydrogel microcapsules based on ion gelation technology.

[0087] 1. Components:

[0088] Active ingredient: NMN (Nicotinamide Mononucleotide, Chinese for β-nicotinamide mononucleotide); cationic hydrogel polymer: chitosan; anionic hydrogel polymer: carrageenan; cationic crosslinking agent: prepared by dissolving guanidine phosphate in water at a mass fraction of 2%.

[0089] 2. Preparation method

[0090] S1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution with a mass fraction of 4%;

[0091] S2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in a 0.8% by mass phosphoric acid solution to prepare a cationic hydrogel polymer solution, and preparing a 0.8% by mass cationic hydrogel polymer solution;

[0092] S3, preparing an anionic hydrogel polymer solution, dissolving an anionic hydrogel polymer in a potassium hydroxide solution with a pH value of 10, to prepare an anionic hydrogel polymer solution with a mass fraction of 0.3%;

[0093] S4, preparing an NMN gel precursor solution, mixing the NMN solution, anionic hydrogel polymer solution and cationic hydrogel polymer solution prepared in the above steps S1 to S3 at a mass ratio of 1:1:2, and stirring to obtain an NMN gel precursor solution;

[0094] S5, preparing NMN microcapsules by cross-linking reaction, injecting the NMN gelation precursor solution of step S4 into the microfluidic chip device as the core flow, and injecting the cationic cross-linking agent as the isolation flow, wherein the flow rate of the core flow is 1mL / min, the flow rate of the isolation fluid is 5mL / min, and the flow rate ratio of the core flow to the isolation fluid is 1 / 5. When the two fluids meet, an ion gelation reaction occurs to generate NMN hydrogel microcapsules with a single hydrogel membrane layer;

[0095] S6, freeze-drying the NMN microcapsule solution prepared in step S5 to prepare dry NMN hydrogel microcapsule powder.

[0096] Example 4

[0097] 1. Components:

[0098] Active ingredient: NMN (Nicotinamide Mononucleotide, β-nicotinamide mononucleotide in Chinese); cationic hydrogel polymer: sodium alginate; anionic hydrogel polymer: gelatin; cationic cross-linking agent: prepared by dissolving glutathione in water at a mass fraction of 0.15%.

[0099] 2. Preparation method

[0100] S1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution with a mass fraction of 3%;

[0101] S2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in an acetic acid solution having a mass fraction of 2% to prepare a cationic hydrogel polymer solution, and preparing a cationic hydrogel polymer solution having a mass fraction of 1%;

[0102] S3, preparing an anionic hydrogel polymer solution, dissolving an anionic hydrogel polymer in a triethylamine solution with a pH value of 8 to prepare an anionic hydrogel polymer solution with a mass fraction of 0.4%;

[0103] S4, preparing an NMN gel precursor solution, mixing the NMN solution prepared in the above steps S1 to S3, the anionic hydrogel polymer solution and the cationic hydrogel polymer solution in a mass ratio of 1:2:4, and stirring to obtain an NMN gel precursor solution;

[0104] S5, preparing NMN microcapsules by cross-linking reaction, injecting the NMN gelation precursor solution of step S4 into the microfluidic chip device as the core flow, and injecting the cationic cross-linking agent as the isolation flow, wherein the flow rate of the core flow is 5mL / min, the flow rate of the isolation fluid is 15mL / min, and the flow rate ratio of the core flow to the isolation fluid is 1 / 3. When the two fluids meet, an ion gelation reaction occurs to generate NMN hydrogel microcapsules with a single hydrogel membrane layer;

[0105] S6, vacuum drying the NMN microcapsule solution prepared in step S5 to prepare dry NMN hydrogel microcapsule powder.

[0106] Example 5

[0107] This example is used to illustrate the third method for preparing NMN hydrogel microcapsules based on ion gelation technology.

[0108] 1. Components:

[0109] Active ingredient: NMN (Nicotinamide Mononucleotide, Chinese for β-nicotinamide mononucleotide); cationic hydrogel polymer: sodium alginate; anionic hydrogel polymer: polyacrylamide; first cationic crosslinker: prepared by dissolving 0.15% of glutathione in water. Second cationic crosslinker: prepared by dissolving 3% of ethylenediamine in water.

[0110] 2. Preparation method

[0111] S1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution with a mass fraction of 4%;

[0112] S2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in a citric acid solution with a mass fraction of 1% to prepare a cationic hydrogel polymer solution, and preparing a cationic hydrogel polymer solution with a mass fraction of 1%;

[0113] S3, preparing an anionic hydrogel polymer solution, dissolving an anionic hydrogel polymer in a triethylamine solution with a pH value of 10, to prepare an anionic hydrogel polymer solution with a mass fraction of 0.2%;

[0114] S4, preparing an NMN gel precursor solution, mixing the NMN solution, anionic hydrogel polymer solution and cationic hydrogel polymer solution prepared in the above steps S1 to S3 at a mass ratio of 1:1:2, and stirring to obtain an NMN gel precursor solution;

[0115] S5, prepare NMN hydrogel microcapsules by crosslinking reaction, inject the NMN gelation precursor solution of step S4 into the microfluidic chip device as the core flow, and inject the first cationic crosslinker as the isolation flow, wherein the flow rate of the core flow is 2mL / min, the flow rate of the isolation fluid is 20mL / min, and the flow rate ratio of the core flow to the isolation fluid is 1 / 10. The first cationic crosslinker meets the NMN gelation precursor solution to undergo an ionization crosslinking reaction, forming an NMN single-layer membrane microcapsule with an inner biocompatible membrane, and then the NMN single-layer membrane microcapsule is used as a secondary gelation precursor solution and injected into the microfluidic chip device as the core flow, and the second cationic crosslinker is injected as the isolation fluid, wherein the flow rate of the core flow is 20mL / min, the flow rate of the isolation fluid is 40mL / min, and the flow rate ratio of the core flow to the isolation fluid is 1 / 2, so that the two fluids meet and undergo a secondary ion crosslinking reaction, and a dense controlled release membrane is formed on the inner biocompatible membrane, thereby preparing an NMN hydrogel microcapsule with a double hydrogel membrane layer;

[0116] S6, freeze-drying the NMN microcapsule solution prepared in step S5 to prepare dry NMN hydrogel microcapsule powder.

[0117] Example 6

[0118] 1. Components:

[0119] Active ingredient: NMN (Nicotinamide Mononucleotide, Chinese for β-nicotinamide mononucleotide); cationic hydrogel polymer: chitosan; anionic hydrogel polymer: sodium polyacrylate; the first cationic crosslinker: prepared by dissolving 0.2% of glutathione in water. The second cationic crosslinker: prepared by dissolving 4% of guanidine phosphate in water.

[0120] 2. Preparation method

[0121] S1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution with a mass fraction of 5%;

[0122] S2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in an acetic acid solution with a mass fraction of 1.5% to prepare a cationic hydrogel polymer solution, and preparing a cationic hydrogel polymer solution with a mass fraction of 0.5%;

[0123] S3, preparing an anionic hydrogel polymer solution, dissolving an anionic hydrogel polymer in a triethylamine solution with a pH value of 8 to prepare an anionic hydrogel polymer solution with a mass fraction of 0.5%;

[0124] S4, preparing an NMN gel precursor solution, mixing the NMN solution prepared in the above steps S1 to S3, the anionic hydrogel polymer solution and the cationic hydrogel polymer solution in a mass ratio of 1:2:4, and stirring to obtain an NMN gel precursor solution;

[0125] S5, prepare NMN hydrogel microcapsules by crosslinking reaction, inject the NMN gelation precursor solution of step S4 into the microfluidic chip device as the core flow, and inject the first cationic crosslinker as the isolation flow, wherein the flow rate of the core flow is 4mL / min, the flow rate of the isolation fluid is 12mL / min, and the flow rate ratio of the core flow to the isolation fluid is 1 / 3. The first cationic crosslinker meets the NMN gelation precursor solution to undergo an ionization crosslinking reaction, forming an NMN single-layer membrane microcapsule with an inner biocompatible membrane, and then the NMN single-layer membrane microcapsule is used as a secondary gelation precursor solution and injected into the microfluidic chip device as the core flow, and the second cationic crosslinker is injected as the isolation fluid, wherein the flow rate of the core flow is 15mL / min, the flow rate of the isolation fluid is 45mL / min, and the flow rate ratio of the core flow to the isolation fluid is 1 / 3, so that the two fluids meet and undergo a secondary ion crosslinking reaction, and a dense controlled release membrane is formed on the inner biocompatible membrane, thereby preparing an NMN hydrogel microcapsule with a double hydrogel membrane layer;

[0126] S6, vacuum drying the NMN microcapsule solution prepared in step S5 to prepare dry NMN hydrogel microcapsule powder.

[0127] Please refer to Figure 1 for an electron microscope image of the NMN single-layer hydrogel film microcapsule prepared by the present invention. It can be seen from the figure that the formed hydrogel film layer is dense and porous. Figure 2 is a release curve of the NMN single-layer hydrogel film microcapsule prepared by the present invention. It can be clearly seen that there will be a burst release stage at the beginning, and the NMN active molecules will diffuse and release rapidly, and then enter the slow release stage. The release rate gradually decreases, and the overall release curve presents a steep initial stage, and then changes and tends to be flat. Figure 3 is a schematic diagram of the double-layer membrane structure of the NMN double-layer hydrogel film microcapsule prepared by the present invention. It is obvious that the double-layer hydrogel film has an inner biocompatible film and an outer controlled release film. Due to the presence of the inner biocompatible film, the initial burst release behavior will be appropriately delayed, wherein the inner biocompatible film controls the release rate in the initial stage, and the outer controlled release film determines the subsequent sustained release process, and the overall release curve presents a two-stage release to meet the needs of complex drug release.

[0128] The method of NMN hydrogel microcapsule based on ion gelation technology disclosed in the present invention is to perform ion crosslinking reaction between NMN and two oppositely charged hydrogel polymers to generate hydrogel microcapsules coated with NNM, and the good biocompatibility and biodegradability of the hydrogel are used to improve the utilization rate and safety of the active ingredients. The formed hydrogel membrane acts as a semipermeable membrane, allowing NMN to gradually diffuse from the microcapsule to prevent NMN from dissolving and releasing rapidly, ensuring continuous release over a long period of time to achieve a sustained release effect. At the same time, two cationic crosslinkers of different properties can be used to form an inner biocompatible membrane and an outer dense controlled release membrane, and then the initial rapid release behavior is controlled by the inner membrane layer, and the subsequent sustained release process is controlled by the outer membrane layer, thereby achieving a dual release mode, which can prolong the circulation time of the active molecule NMN in the body, so as to achieve a better complex release curve to meet the required treatment or nutritional needs. The formed hydrogel membrane layer can also protect NMN from degradation caused by enzymes or harsh environmental conditions, ensuring the stability and efficacy of the encapsulated NMN.

[0129] The present invention also utilizes the microfluidic chip device shown in FIG. 4 to prepare NMN hydrogel microcapsules. The microfluidic technology can accurately control the kinetics of the ion gelation reaction, thereby preparing regular spherical or other morphological microcapsules, which have the advantages of good stability and controllable release performance. At the same time, the process route is simple, green and environmentally friendly, and has significant innovative advantages.

[0130] Although the present invention has been described in detail with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

[0131] In summary, it should be clear to those familiar with the art that the present invention can indeed achieve the aforementioned objectives and is in compliance with the provisions of the Patent Law, and therefore an application is filed in accordance with the law.

Claims

1. A method for preparing NMN hydrogel microcapsules based on ion gelation technology, characterized in that: The preparation steps are as follows: Step 1, preparing an NMN solution, dissolving NMN (Nicotinamide Mononucleotide, Chinese for β-nicotinamide mononucleotide) in water to prepare an NMN solution, wherein the mass fraction of NMN in the NMN solution is 1% to 5%; Step 2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in an acidic solution with a mass fraction of 0.5% to 2% to obtain a cationic hydrogel polymer solution, wherein the mass fraction of the cationic hydrogel polymer in the cationic hydrogel polymer solution is 0.1% to 1%; Step 3, preparing an anionic hydrogel polymer solution, dissolving an anionic hydrogel polymer in an alkaline solution with a pH value of 8 to 10, wherein the mass fraction of the anionic hydrogel polymer in the anionic hydrogel solution is 0.05% to 0.5%; Step 4, preparing an NMN gel precursor solution, mixing the NMN solution with the anionic hydrogel polymer solution at a weight ratio of 1:1 to 5:1, and stirring to obtain an NMN gel precursor solution; Step 5: Prepare NMN hydrogel microcapsules by cross-linking reaction. The NMN gelation precursor solution and the cationic hydrogel polymer solution are mixed and stirred evenly in a volume ratio of 1:5 to 1:20, and then the cationic cross-linking agent is slowly dropped while stirring at a low speed to form NMN hydrogel microcapsules.

2. The method for preparing NMN hydrogel microcapsules based on ion gelation technology as claimed in claim 1, characterized in that: The cationic cross-linking agent is prepared by dissolving any one of glutathione, ethylenediamine and guanidine phosphate in water.

3. The method for preparing NMN hydrogel microcapsules based on ion gelation technology as claimed in claim 2, characterized in that: The cationic cross-linking agent is prepared by dissolving glutathione in water at a mass fraction of 0.1% to 0.2%.

4. The method for preparing NMN hydrogel microcapsules based on ion gelation technology as claimed in claim 2, characterized in that: The cationic crosslinking agent is prepared by dissolving ethylenediamine or guanidine phosphate in water at a mass fraction of 1% to 5%.

5. The method for preparing NMN hydrogel microcapsules based on ion gelation technology as claimed in claim 1, characterized in that: In step 5, the dropping rate of the cationic crosslinking agent is 1 g / min to 3 g / min, and the rotation speed of the low-speed stirring is 30 r / min to 60 r / min.

6. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 1, characterized in that: When the viscosity of the monitored solution reaches 102-103 mPa·s in step 5, the addition of the cationic crosslinking agent is stopped.

7. A method for preparing NMN hydrogel microcapsules based on ion gelation technology, characterized in that: The preparation steps are as follows: S1, preparing an NMN solution, dissolving NMN in water to prepare an NMN solution, wherein the mass fraction of NMN in the NMN solution is 1% to 5%; S2, preparing a cationic hydrogel polymer solution, dissolving a cationic hydrogel polymer in an acidic solution having a mass fraction of 0.5% to 2% to obtain a cationic hydrogel polymer solution, wherein the mass fraction of the cationic hydrogel polymer in the cationic hydrogel polymer solution is 0.1% to 1%; S3, preparing an anionic hydrogel polymer solution, dissolving an anionic hydrogel polymer in an alkaline solution with a pH value of 8 to 10, wherein the mass fraction of the anionic hydrogel polymer in the anionic hydrogel solution is 0.05% to 0.5%; S4, preparing an NMN gel precursor solution, mixing the NMN solution, anionic hydrogel polymer and cationic hydrogel polymer solution in a mass ratio of 1:1:2 to 1:2:4, and stirring to obtain an NMN gel precursor solution; S5, preparing NMN hydrogel microcapsules by cross-linking reaction, injecting the above-mentioned NMN gelation precursor solution as the core flow into the microfluidic chip device, and injecting the cationic cross-linking agent as the isolation fluid at the same time, when the two fluids meet, an ionic gelation reaction occurs to form NMN hydrogel microcapsules.

8. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 7, characterized in that: In step S5, the cationic crosslinking agent is prepared by dissolving any one of glutathione, ethylenediamine and guanidine phosphate in water to prepare a corresponding aqueous solution, the glutathione solution is prepared by dissolving glutathione at a mass fraction of 0.1% to 0.2% in water, the ethylenediamine solution is prepared by dissolving ethylenediamine at a mass fraction of 1% to 5% in water, and the guanidine phosphate solution is prepared by dissolving guanidine phosphate at a mass fraction of 1% to 5% in water.

9. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 8, characterized in that: In step S5, the glutathione solution is first injected as the isolation fluid, and meets the NMN gelation precursor solution as the core flow to undergo an ion gelation reaction, thereby forming an NMN single-layer membrane microcapsule having an inner biocompatible membrane. The NMN single-layer membrane microcapsule is then used as a secondary gelation precursor and injected into the microfluidic chip device as the core flow. At the same time, the ethylenediamine solution or the guanidine phosphate solution is injected as the isolation fluid, so that the two fluids meet and undergo a secondary ion cross-linking reaction, thereby forming a dense controlled release membrane on the inner biocompatible membrane, thereby obtaining an NMN hydrogel microcapsule having a double-layer hydrogel membrane.

10. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 9, characterized in that: When preparing the inner biocompatible membrane, the flow rate of the core flow is 1-5 mL / min, the flow rate of the isolation fluid is 5-25 mL / min, and the flow ratio is the flow rate ratio of the core flow to the isolation flow, and the flow ratio is 1 / 10-1 / 3.

11. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 9, characterized in that: When preparing the outer controlled release membrane, the flow rate of the core flow is 5-20 mL / min, the flow rate of the isolation fluid is 10-50 mL / min, and the flow ratio is the flow rate ratio of the core flow to the isolation flow, and the flow ratio is 1 / 20-1 / 2.

12. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 9, characterized in that: The mass concentration ratio of the glutathione solution to the ethylenediamine solution or the guanidine phosphate solution is 1:5 to 1:

20.

13. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 1 or 7, characterized in that: The acid solution is prepared by dissolving any one of acetic acid, citric acid and phosphoric acid in water.

14. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 1 or 7, characterized in that: The cationic hydrogel polymer is any one or more of chitosan, sodium alginate, chitin, acetylated cell wall enzyme, and polyethyleneimine.

15. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 1 or 7, characterized in that: The anionic hydrogel polymer is any one or more of gelatin, carrageenan, sodium polyacrylate and polyacrylamide, and the alkaline solution is prepared by dissolving any one of triethylamine, sodium hydroxide and potassium hydroxide in water.

16. The method for preparing NMN hydrogel microcapsules based on ion gelation technology according to claim 1 or 7, characterized in that: The prepared NMN hydrogel microcapsules are in liquid state, and dry NMN hydrogel microcapsule powder is prepared by freeze drying or vacuum drying.

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