ATP-loaded polyelectrolyte microcapsule and preparation method thereof

Abstract

The invention discloses an ATP (adenosine triphosphate) loaded polyelectrolyte microcapsule and a preparation method thereof. Belongs to the technical field of polyelectrolyte microcapsule preparation. The polyelectrolyte microcapsule is composed of a capsule wall and a core material, wherein the capsule wall is composed of a cationic polyelectrolyte layer and an anionic polyelectrolyte layer which are alternately adsorbed; the core material is ATP molecules and can be loaded on the hollow part of the multi-layer polyelectrolyte capsule and / or the capsule wall of the multi-layer polyelectrolyte capsule. The ATP capsule provided by the invention can effectively protect the stability of ATP molecules, so that the ATP molecules are prevented from being quickly degraded in organisms, the preparation method and process of the ATP capsule are simple, the size of the capsule, the loading rate and the release rate of ATP can be regulated and controlled by changing the size of a template, the variety of the template, the adding concentration of an ATP solution, the number of layers of the capsule and the external environment, and the ATP capsule has a good application prospect. The application range of ATP in disease treatment is greatly expanded, and the use efficiency and safety of ATP are improved.

Description

Technical Field

[0001] The invention relates to the technical field of polyelectrolyte microcapsule preparation, and more particularly to ATP-loaded polyelectrolyte microcapsule and a preparation method thereof. Background Art

[0002] Adenosine triphosphate (ATP) plays a vital role in biological processes such as energy transduction, cellular respiration, enzyme catalysis and signal transduction. The change of ATP concentration is closely related to the cellular energy supply. Many studies have found that in the development of diseases such as sarcopenia, heart failure, and neurodegenerative diseases, patients will have insufficient energy supply, and the synthesis and release of ATP will be significantly downregulated. Exogenous addition of ATP can promote the proliferation and differentiation of various cells to alleviate or treat diseases including but not limited to the above diseases, but direct intake of ATP for medicinal purposes still faces many difficulties: 1. ATP is highly sensitive to enzymatic hydrolysis, unstable in the body, easily hydrolyzed, and rapidly degraded into adenosine after injection into the body, which reduces the efficacy; 2. Direct intake of ATP can easily lead to a surge in ATP levels, causing an imbalance in the cellular energy state and inducing diseases; 3. Organisms are very sensitive to ATP levels. Too high or too low ATP levels are not conducive to the normal survival of organisms. Only by supplementing exogenous ATP equivalent to the lack of ATP can the therapeutic effect be truly achieved. Therefore, how to protect ATP from rapid degradation, long-term sustained release, and how to control the dosage of ATP need to be solved urgently.

[0003] Polyelectrolyte microcapsules (PEMs) can change the size of ATP capsules and the amount of ATP loaded due to the controllability of their template types and synthesis conditions. At the same time, by adjusting the pH, temperature, number of capsule wall layers and other conditions, the permeability of the microcapsule wall can be changed, thereby achieving controlled release of ATP molecules. Polyelectrolyte microcapsules are an ideal sustained-release drug carrier that has been widely studied. Only by performing layer-by-layer self-assembly (LBL) on template particles can core-shell structured microparticles with polyelectrolyte layered ultra-thin films as shells be obtained. It can not only effectively protect drug molecules including ATP from environmental influences and improve their bioavailability, but also has good biocompatibility. Therefore, it has great application prospects in drug delivery systems and is one of the preferred structures for loading and delivering biomolecules.

[0004] Therefore, how to provide a polyelectrolyte microcapsule with controllable size, controllable drug loading, and controllable release rate that can effectively protect and release ATP in a long-term sustained manner and a preparation method thereof is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides a polyelectrolyte microcapsule loaded with ATP and a preparation method thereof. The polyelectrolyte microcapsule has controllable size, drug loading amount, and release rate, can effectively protect and sustainably release ATP for a long time, expands the scope of ATP used in disease treatment, and improves its use efficiency and safety.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A polyelectrolyte microcapsule loaded with ATP, comprising a capsule wall and a core material; the capsule wall is composed of alternately adsorbed cationic polyelectrolyte layers and anionic polyelectrolyte layers; the core material is ATP molecules, and the ATP molecules can be loaded in the hollow of the multilayer polyelectrolyte capsule and / or the capsule wall of the multilayer polyelectrolyte capsule.

[0008] Another object of the present invention is to provide: a preparation method of a polyelectrolyte microcapsule loaded with ATP. When the ATP molecules are loaded in the hollow of the multilayer polyelectrolyte capsule, the specific preparation method is as follows:

[0009] (1) Using an inorganic substance loaded with ATP molecules as template particles;

[0010] (2) Immersing the template particles in step (1) in a cationic polyelectrolyte solution, shaking, centrifuging, and washing the precipitate to obtain polyelectrolyte particles with a layer of cationic polyelectrolyte adsorbed as an anchoring layer;

[0011] (3) Immersing the polyelectrolyte particles obtained in step (2) in an anionic polyelectrolyte solution, shaking, centrifuging, and washing the precipitate to obtain microcapsules with a layer of anionic polyelectrolyte adsorbed. Placing the obtained microcapsules in a cationic polyelectrolyte solution, shaking, centrifuging, and washing the precipitate to obtain microcapsules containing one polyelectrolyte bilayer assembly except for the anchoring layer;

[0012] (4) Repeating step (3) to obtain polyelectrolyte multilayer assembled microcapsules containing ATP molecules;

[0013] (5) Removing the template to obtain a polyelectrolyte microcapsule loaded with ATP.

[0014] Another object of the present invention is to provide: a preparation method of a polyelectrolyte microcapsule loaded with ATP. When the ATP molecules are loaded in the capsule wall of the multilayer polyelectrolyte capsule, the specific preparation method is as follows:

[0015] (1) Using an inorganic substance as template particles;

[0016] (2) Immersing the template particles in step (1) in a cationic polyelectrolyte solution, shaking, centrifuging, and washing the precipitate to obtain polyelectrolyte particles with a layer of cationic polyelectrolyte adsorbed as an anchoring layer;

[0017] (3) Immerse the polyelectrolyte particles obtained in step (2) into the ATP solution. After shaking and centrifuging, immerse the obtained polyelectrolyte particles into the anionic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain microcapsules adsorbed with a layer of ATP and a layer of anionic polyelectrolyte. Place the obtained microcapsules into the cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain microcapsules with a three-layer assembly of one ATP and polyelectrolytes except for the anchoring layer;

[0018] (4) Repeat step (3) to obtain microcapsules with a multilayer assembly of polyelectrolytes containing ATP molecules;

[0019] (5) Remove the template to obtain polyelectrolyte microcapsules loaded with ATP.

[0020] Another object of the present invention is to provide: A method for preparing polyelectrolyte microcapsules loaded with ATP. When ATP molecules are loaded into the hollow and capsule wall of the multilayer polyelectrolyte capsule, the specific preparation method is as follows:

[0021] (1) Use inorganic substances loaded with ATP molecules as template particles;

[0022] (2) Immerse the template particles described in step (1) into the cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain polyelectrolyte particles with a layer of cationic polyelectrolyte adsorbed as the anchoring layer;

[0023] (3) Immerse the polyelectrolyte particles obtained in step (2) into the ATP solution. After shaking and centrifuging, immerse the obtained polyelectrolyte particles into the anionic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain microcapsules adsorbed with a layer of ATP and a layer of anionic polyelectrolyte. Place the obtained microcapsules into the cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain microcapsules with a three-layer assembly of one ATP and polyelectrolytes except for the anchoring layer;

[0024] (4) Repeat step (3) to obtain microcapsules with a multilayer assembly of polyelectrolytes containing ATP molecules;

[0025] (5) Remove the template to obtain polyelectrolyte microcapsules loaded with ATP.

[0026] Preferably, when ATP molecules are loaded into the hollow of the multilayer polyelectrolyte capsule, the template particles described in step (1) are CaCO3 / ATP or MnCO3 / ATP template particles; the particle size of the template is 1-4 μm.

[0027] Further preferably, the preparation method of the CaCO3 / ATP template particles is as follows: Add a starch solution to a CaCl2 solution while stirring, then add an ATP solution and stir evenly, and then add a Na2CO3 solution, continuously stir for 5 - 10 min, and centrifuge and wash to obtain the CaCO3 / ATP template particles;

[0028] Further preferably, the preparation method of the MnCO3 / ATP template particles is as follows: Add absolute ethanol to a MnSO4 solution while stirring, then add an ATP solution and stir evenly, and then add an NH4HCO3 solution and continuously stir for 5 - 10 min, and centrifuge and wash to obtain the MnCO3 / ATP template particles.

[0029] Preferably, when ATP molecules are loaded on the wall of the multilayer polyelectrolyte capsule, the template particles in step (1) are CaCO3 or MnCO3 template particles; the particle size of the template is 1 - 4 μm.

[0030] Preferably, the concentration of the CaCl2 solution is 0.02 - 0.05 M; the concentration of the sodium carbonate solution is 0.02 - 0.05 M; the concentration of the starch solution is 0.02 - 0.06 g / mL; the concentration of the MnSO4 solution is 0.01 - 0.02 M; the concentration of the NH4HCO3 solution is 0.1 - 0.2 M; the concentration of the ATP solution is 0.5 - 4 mg / mL.

[0031] Preferably, the cationic polyelectrolyte in steps (2) - (3) is polyethyleneimine PEI or polyacrylamine hydrochloride PAH; the anionic polyelectrolyte is sodium polystyrene sulfonate PSS; the concentrations of the anionic / cationic polyelectrolytes are both 0.5 - 2 mg / mL, and are both prepared with a 0.5 M NaCl solution as the solvent; the concentration of the ATP solution is 0.5 - 4 mg / mL; the number of repetitions in step (4) is 4 - 12 times;

[0032] The oscillation time in steps (2) - (3) is 6 h, and the centrifugation speed is 5000 - 10000 rpm; wash with a NaCl solution;

[0033] The removal process of the template in step (5) is as follows: Place the polyelectrolyte multilayer - assembled microcapsules containing ATP molecules prepared in step (4) in a dissolution solution, oscillate to dissolve the template until no gas is generated;

[0034] The ATP loading rate of the ATP - loaded polyelectrolyte microcapsules in step (5) is 28.25 - 97.95%.

[0035] As can be seen from the above technical solutions, compared with the prior art, the present invention has the following beneficial effects: Compared with the existing technology, the ATP capsules provided by the present invention can effectively protect the stability of ATP molecules, enabling them to avoid rapid degradation in organisms. The preparation method and process of the ATP capsules are simple. The size of the capsules and the loading rate of ATP can be controlled by changing the size of the template, the type of template, and the addition concentration of the ATP solution to meet the treatment requirements for different diseases. In addition, by changing the number of layers of the ATP capsules or the external environment during their release, such as temperature, pH, ionic strength, etc., the release rate of ATP from the ATP capsules can be regulated to achieve the effect of long-acting and sustained release of ATP. This continuous and slow release will not cause a significant increase in the ATP concentration compared with direct ATP injection, nor will it cause the hydrolysis and inactivation of unutilized ATP. Therefore, the polyelectrolyte microcapsules loaded with ATP provided by the present invention are expected to provide continuous treatment or relief for diseases related to ATP deficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0037] Figure 1 It is: a schematic diagram of the preparation of hollow ATP-loaded polyelectrolyte microcapsules;

[0038] Figure 2 It is: a schematic diagram of the preparation of polyelectrolyte microcapsules with ATP loaded on the capsule wall;

[0039] Figure 3 It is: the TEM images of the ATP capsules in Examples 1 and 3;

[0040] Figure 4 It is: the scanning electron microscope images and particle size distribution diagrams of the template particles in Example 1, Comparative Examples 1 - 5, and when the ATP addition concentration is 0;

[0041] Figure 5 It is: the scanning electron microscope images and particle size distribution diagrams of the template particles in Example 2, Comparative Examples 6 - 10, and when the ATP addition concentration is 0;

[0042] Figure 6 It is: the scanning electron microscope images and particle size distribution diagrams of Example 4(c), Comparative Example 13(a), and Comparative Example 14(b);

[0043] Figure 7For: ATP loading rate graphs (a) of ATP capsules in Example 1 and Comparative Examples 1-5; ATP loading rate graphs (b) of ATP capsules in Example 2 and Comparative Examples 6-10; ATP loading rate graphs (c) of ATP capsules in Example 3 and Comparative Examples 11, 12; ATP loading rate graphs (d) of ATP capsules in Example 4 (small) and Comparative Examples 13 (large), 14 (medium);

[0044] Figure 8 For: Effects of temperature, pH, and number of adsorption layers on the in vitro release rate of polyelectrolyte microcapsules loaded with ATP;

[0045] Figure 9 For: Release results of polyelectrolyte microcapsules loaded with ATP in a cellular environment;

[0046] Figure 10 For: Comparison of release curves between ATP solution and ATP capsules. Detailed implementation manners

[0047] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0048] Example 1

[0049] In this example, the preparation process of the polyelectrolyte microcapsules with empty load of ATP is as Figure 1 shown and includes the following steps:

[0050] (1) Synthesize template particles loaded with ATP molecules:

[0051] Stir 50 mL of a 0.05 M CaCl2 solution at a rotation speed of 800 rpm, add 30 mL of a 0.04 g / mL starch solution thereto, and stir for 30 s; then add 12 mL of a 2 mg / mL ATP solution and continue stirring for 30 s; add 50 mL of a 0.05 M Na2CO3 solution and continue stirring for 5 - 10 min, centrifuge at 5000 rpm and wash with a 0.5 M NaCl solution at least three times to obtain CaCO3 / ATP template particles for standby;

[0052] (2) Using the CaCO3 / ATP particles in step (1) as a template, soak the template with a polyethyleneimine (PEI) solution, place it on an oscillator, centrifuge after shaking for 6 h, discard the supernatant, and wash three times with 0.5 M NaCl solution to obtain polyelectrolyte particles with a layer of polyethyleneimine (PEI) adsorbed as an anchoring layer;

[0053] The concentration of polyethyleneimine (PEI) is 2 mg / mL for all, and it is prepared with a 0.5 M NaCl solution as the solvent;

[0054] (3) Soak the particles with the adsorbed anchoring layer polyelectrolyte in step (2) above with a sodium polystyrene sulfonate (PSS) solution, place it on an oscillator and shake for 6 h, centrifuge to discard the supernatant, and wash three times with 0.5 M NaCl solution to obtain microcapsules adsorbed with a layer of sodium polystyrene sulfonate (PSS); then soak the particles with the adsorbed two layers of polyelectrolytes above with a polyallylamine hydrochloride (PAH) solution, place it on an oscillator and shake for 6 h, centrifuge, discard the supernatant, and wash three times with 0.5 M NaCl solution to obtain a layer-by-layer assembled capsule that completes a polyelectrolyte bilayer except for the anchoring layer;

[0055] The concentrations of the sodium polystyrene sulfonate (PSS) solution and the polyallylamine hydrochloride (PAH) solution are both 2 mg / mL, and they are both prepared with a 0.5 M NaCl solution as the solvent;

[0056] (4) Repeat step (3) 6 times to obtain 6 bilayer polyelectrolyte microcapsules with CaCO3 / ATP particles as the template;

[0057] (5) Place the 6 bilayer polyelectrolyte microcapsules obtained in step (4) in a 0.1 M disodium EDTA solution, shake to dissolve the template, change the solution multiple times to dissolve until no gas is generated, indicating that the de-nucleation is complete, centrifuge at 7000 rpm, discard the supernatant, and wash multiple times to obtain hollow polyelectrolyte microcapsules loaded with ATP. The TEM image is shown in Figure 3 .

[0058] Example 2

[0059] The preparation process of the hollow polyelectrolyte microcapsules loaded with ATP in this example includes the following steps:

[0060] (1) Synthesize template particles loaded with ATP molecules:

[0061] Stir 100 mL of a 0.016 M MnSO4 solution at 800 rpm, add 10 mL of absolute ethanol thereto, and stir for 30 s; then add 23 mL of a 2 mg / mL ATP solution and continue stirring for 30 s; add 100 mL of a 0.16 M NH4HCO3 solution and continue stirring for 5 - 10 min, centrifuge at 8000 rpm, and wash with a 0.5 M NaCl solution at least three times to obtain MnCO3 / ATP template particles for standby;

[0062] (2) Using the MnCO3 / ATP particles in step (1) as a template, soak the template with a polyethyleneimine (PEI) solution, place it on an oscillator, centrifuge after oscillating for 6 h, discard the supernatant, and wash three times with a 0.5 M NaCl solution to obtain polyelectrolyte particles with a layer of adsorbed polyethyleneimine (PEI) as an anchoring layer;

[0063] The concentration of polyethyleneimine (PEI) is 2 mg / mL, and it is prepared with a 0.5 M NaCl solution as a solvent;

[0064] (3) Soak the particles with the adsorbed anchoring layer polyelectrolyte in step (2) above with a sodium polystyrene sulfonate (PSS) solution, place it on an oscillator and oscillate for 6 h, centrifuge to discard the supernatant, and wash three times with a 0.5 M NaCl solution to obtain microcapsules with a layer of adsorbed sodium polystyrene sulfonate (PSS); then soak the particles with the adsorbed two - layer polyelectrolyte above with a polyallylamine hydrochloride (PAH) solution, place it on an oscillator and oscillate for 6 h, centrifuge to discard the supernatant, and wash three times with a 0.5 M NaCl solution to obtain microcapsules with a polyelectrolyte bilayer assembly except for the anchoring layer;

[0065] The concentrations of the sodium polystyrene sulfonate (PSS) solution and the polyallylamine hydrochloride (PAH) solution are both 2 mg / mL, and they are both prepared with a 0.5 M NaCl solution as a solvent;

[0066] (4) Repeat step (3) 6 times to obtain 6 bilayer polyelectrolyte microcapsules with MnCO3 / ATP particles as a template;

[0067] (5) Place the 6 bilayer polyelectrolyte microcapsules obtained in step (4) in a 0.1 M disodium EDTA solution, oscillate to dissolve the template, change the solution multiple times until no gas is generated, indicating that the nucleus removal is complete, centrifuge at 7000 rpm to discard the supernatant, and wash multiple times to obtain hollow polyelectrolyte microcapsules loaded with ATP.

[0068] Example 3

[0069] The preparation process of the polyelectrolyte microcapsules with ATP loaded on the capsule wall in this example is as Figure 2 shown, including the following steps:

[0070] (1) Synthesis of inorganic template particles:

[0071] Place 500 mL of MnSO4 solution in an ultrasonic cleaner, add 50 mL of absolute ethanol to it, set the ultrasonic power to 120 W, and ultrasonicate for 5 min; then add 500 mL of NH4HCO3 solution and continue ultrasonication for 50 min, centrifuge and wash with NaCl solution at least three times to obtain MnCO3 template particles for standby;

[0072] (2) Using the MnCO3 particles in step (1) as a template, soak the template with a polyethyleneimine (PEI) solution, place it on an oscillator, shake for 6 h, then centrifuge, discard the supernatant, and wash three times with 0.5 M NaCl solution to obtain polyelectrolyte particles with a layer of polyethyleneimine (PEI) adsorbed as an anchoring layer;

[0073] The concentration of polyethyleneimine (PEI) is 2 mg / mL, and it is prepared with a 0.5 M NaCl solution as the solvent;

[0074] (3) Soak the particles with an adsorbed anchoring layer of polyelectrolyte in step (2) with a 1 mg / mL ATP solution, place it on an oscillator and shake for 6 h, centrifuge to collect the supernatant, without washing, directly add a sodium polystyrene sulfonate (PSS) solution to it, soak the particles, place it on an oscillator and shake for 6 h, centrifuge to collect the supernatant, and wash three times with 0.5 M NaCl solution to obtain microcapsules adsorbed with a layer of ATP and a layer of sodium polystyrene sulfonate (PSS); then soak the particles with the two layers of adsorbed polyelectrolytes above with a polyacrylamide hydrochloride (PAH) solution, place it on an oscillator and shake for 6 h, centrifuge to discard the supernatant, and wash three times with 0.5 M NaCl solution to obtain microcapsules containing a bilayer assembly of one ATP layer plus one polyelectrolyte layer except for the anchoring layer;

[0075] The concentrations of the sodium polystyrene sulfonate (PSS) solution and the polyacrylamide hydrochloride (PAH) solution are both 2 mg / mL, and they are both prepared with a 0.5 M NaCl solution as the solvent;

[0076] (4) Repeat step (3) six times to obtain multi-layer polyelectrolyte microcapsules with a wall containing ATP molecules using MnCO3 particles as a template;

[0077] (5) Place the multi-layer polyelectrolyte microcapsules with a wall containing ATP molecules obtained in step (4) in a 0.1 M disodium EDTA solution, shake to dissolve the template, change the solution multiple times to dissolve until no gas is produced, indicating that the de-nucleation is complete, centrifuge at 7000 rpm to discard the supernatant, and wash multiple times to obtain polyelectrolyte microcapsules with ATP loaded on the wall. The TEM image is shown in Figure 3 .

[0078] Example 4

[0079] The preparation of the polyelectrolyte microcapsules with ATP loaded on the capsule wall in this example includes the following steps:

[0080] (1) Synthesize inorganic template particles:

[0081] Place 500 mL of MnSO4 solution in an ultrasonic cleaner, add 50 mL of absolute ethanol to it, set the ultrasonic power to 60 W, and ultrasonicate for 5 min; then add 500 mL of NH4HCO3 solution and continue ultrasonication for 50 min, centrifuge and wash with NaCl solution at least three times to obtain MnCO3 template particles for standby;

[0082] (2) Using the MnCO3 in step (1) as a template, soak the template with a polyethyleneimine (PEI) solution, place it on an oscillator, centrifuge after oscillating for 6 h, discard the supernatant, and wash three times with 0.5 M NaCl solution to obtain polyelectrolyte particles with a layer of adsorbed polyethyleneimine (PEI) as an anchoring layer;

[0083] The concentration of polyethyleneimine (PEI) is 2 mg / mL, and it is prepared with 0.5 M NaCl solution as the solvent;

[0084] (3) Soak the polyelectrolyte particles with the adsorbed anchoring layer in step (2) with a 1 mg / mL ATP solution, place it on an oscillator and oscillate for 6 h, centrifuge to collect the supernatant, directly add a sodium polystyrene sulfonate (PSS) solution to soak the particles without washing, place it on an oscillator and oscillate for 6 h, centrifuge to collect the supernatant, and wash three times with 0.5 M NaCl solution to obtain microcapsules adsorbed with a layer of ATP and a layer of sodium polystyrene sulfonate (PSS); then soak the particles with the adsorbed two layers of polyelectrolytes with a polyallylamine hydrochloride (PAH) solution, place it on an oscillator and oscillate for 6 h, centrifuge to discard the supernatant, and wash three times with 0.5 M NaCl solution to obtain microcapsules containing one ATP layer plus one polyelectrolyte bilayer assembly except for the anchoring layer;

[0085] The concentrations of the sodium polystyrene sulfonate (PSS) solution and the polyallylamine hydrochloride (PAH) solution are both 2 mg / mL, and they are both prepared with 0.5 M NaCl solution as the solvent;

[0086] (4) Repeat step (3) 4 times to obtain multi-layered polyelectrolyte microcapsules with ATP molecules in the capsule wall using MnCO3 particles as a template;

[0087] (5) The multilayer polyelectrolyte microcapsules with ATP molecules in the capsule wall obtained in (4) were placed in a 0.1 M disodium EDTA solution, and the template was dissolved by shaking. The solution was changed several times until no gas was generated, indicating that the core removal was complete. The supernatant was removed by centrifugation at 7000 rpm, and after washing several times, the polyelectrolyte microcapsules with ATP loaded in the capsule wall were obtained.

[0088] Comparative Example 1

[0089] It is basically the same as Example 1, except that:

[0090] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected was 0.5 mg / mL.

[0091] Comparative Example 2

[0092] It is basically the same as Example 1, except that:

[0093] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected was 1.0 mg / mL.

[0094] Comparative Example 3

[0095] It is basically the same as Example 1, except that:

[0096] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected was 1.5 mg / mL.

[0097] Comparative Example 4

[0098] It is basically the same as Example 1, except that:

[0099] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected was 3 mg / mL.

[0100] Comparative Example 5

[0101] It is basically the same as Example 1, except that:

[0102] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected was 4 mg / mL.

[0103] Comparative Example 6

[0104] It is basically the same as Example 2, except that:

[0105] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected was 0.5 mg / mL.

[0106] Comparative Example 7

[0107] It is basically the same as Example 2, except that:

[0108] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected is 1.0 mg / mL.

[0109] Comparative Example 8

[0110] Basically the same as Example 2, the difference is only that:

[0111] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected is 1.5 mg / mL.

[0112] Comparative Example 9

[0113] Basically the same as Example 2, the difference is only that:

[0114] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected is 3 mg / mL.

[0115] Comparative Example 10

[0116] Basically the same as Example 2, the difference is only that:

[0117] When synthesizing the template particles loaded with ATP molecules, the concentration of the ATP solution selected is 4 mg / mL.

[0118] Comparative Example 11

[0119] Basically the same as Example 3, the difference is only that:

[0120] In this comparative example, step (4) is repeated 4 times, and the capsule wall with MnCO3 particles as the template contains 4 multi-layered polyelectrolyte microcapsules with ATP molecules.

[0121] Comparative Example 12

[0122] Basically the same as Example 3, the difference is only that:

[0123] In this comparative example, step (4) is repeated 8 times, and the capsule wall with MnCO3 particles as the template contains 8 multi-layered polyelectrolyte microcapsules with ATP molecules.

[0124] Comparative Example 13

[0125] Basically the same as Example 4, the difference is only that:

[0126] When synthesizing the MnCO3 inorganic template particles, the set ultrasonic power is 150 W.

[0127] Comparative Example 14

[0128] Basically the same as Example 4, the difference is only that:

[0129] When synthesizing the MnCO3 inorganic template particles, the ultrasonic power was set to 120W.

[0130] Comparative Example 15

[0131] It is basically the same as Example 1, except that:

[0132] In this comparative example, the process was repeated four times to obtain four double-layer polyelectrolyte microcapsules using the ATP molecule-containing particles as templates.

[0133] Comparative Example 16

[0134] It is basically the same as Example 1, except that:

[0135] In this comparative example, the process was repeated 8 times to obtain 8 double-layer polyelectrolyte microcapsules using the ATP molecule-containing particles as templates.

[0136] Comparative Example 17

[0137] It is basically the same as Example 1, except that:

[0138] In this comparative example, the process was repeated 12 times to obtain 12 double-layer polyelectrolyte microcapsules using the ATP molecule-containing particles as templates.

[0139] The template particles in Example 1 and Comparative Examples 1 to 5 were scanned by electron microscope and the particle size was counted. The experimental results are as follows: Figure 4 shown.

[0140] Results: Scanning electron microscopy and particle size distribution showed that within the ATP concentration range of 0-4 mg / mL, as the ATP concentration increased, the size of the CaCO3 / ATP template particles first increased, then decreased, and finally broke. The size reached the maximum value at a concentration of 1.5 mg / mL, then began to decrease, and began to break at 3 mg / mL. With the increase of ATP concentration, the uniformity of the size of the prepared CaCO3 / ATP template particles increased, and the size distribution of the particles narrowed. The particle uniformity was best at a concentration of 2.0. The SEM image showed that the particles showed an irregular structure at low ATP concentrations, and gradually tended to form a more regular spherical shape as the concentration increased. When the concentration reached 2.5 mg / mL, it began to show an irregular shape again, and when it reached 3 mg / mL, the particles had already shown a tendency to break. Therefore, in general, when the ATP concentration was 2 mg / mL, the prepared CaCO3 / ATP template particles had more application advantages.

[0141] The template particles in Example 2 and Comparative Examples 6 to 10 were subjected to electron microscope scanning and particle size statistics. The experimental results are as follows: Figure 5 As shown;

[0142] Result analysis: It can be obtained from the scanning electron microscope and particle size distribution diagram that within the ATP concentration range of 0 - 4 mg / mL, as the ATP concentration increases, the particle size of the MnCO3 / ATP template particles continuously decreases and finally ruptures. Rupture has occurred at 3 mg / mL. As the ATP concentration increases, the uniformity of the particle size of the prepared MnCO3 / ATP template particles first increases and then decreases, and the particle size distribution first becomes narrower and then wider. The particle size distribution uniformity is the best at a concentration of 2.0. The SEM image shows that the particles present an irregular structure at low ATP concentrations and gradually tend to form a more regular spherical shape as the concentration increases. When the concentration reaches 2.5 mg / mL, it begins to show an obvious irregular shape again, and the particles have a tendency to rupture at 3 mg / mL. Generally speaking, when the ATP concentration is 2 mg / mL, the prepared MnCO3 / ATP template particles have more application advantages.

[0143] Because the balls in Comparative Examples 4, 5, 9, and 10 have broken and are no longer spherical, the particle size cannot be statistically analyzed, and only the scanning electron microscope can be provided.

[0144] Perform electron microscope scanning and particle size statistics on Example 4 and Comparative Examples 13 and 14. The experimental results are as Figure 6 shown.

[0145] Result analysis: It can be obtained from the scanning electron microscope and particle size distribution diagram that the size and particle size distribution of the MnCO3 template particles (without ATP) prepared by the ultrasonic method are related to the ultrasonic power. The smaller the ultrasonic power, the smaller the template size and the larger the particle size distribution. The particle size is the most uniform at a power of 150 W, but its size is too large and its biocompatibility is not as good as that of the template particles prepared at low power. There is little difference in the particle size distribution at powers of 120 W and 60 W, but the particle size is smaller at 60 W, which has more application advantages.

[0146] Statistical analysis was carried out on the ATP loading rates of the capsules in Examples 1 - 4 and Comparative Examples 1 - 14. The experimental results are as Figure 7 shown.

[0147] Result analysis: a: The loading rate of the hollow ATP-loaded polyelectrolyte microcapsules with CaCO3 / ATP as the template shows a continuous increase as the added ATP concentration increases. When the added ATP concentration is lower than 2 mg / mL, as the ATP concentration increases, the loading rate of the prepared microcapsules increases at a relatively fast rate. When the added ATP concentration is higher than 2 mg / mL, as the ATP concentration increases, the loading rate of the prepared microcapsules increases at a relatively slow rate, and the increase is not obvious. The loading rate results are the same as Figure 4In response to the results of the preparation of the middle template, when the added ATP concentration is lower than 2 mg / mL, the CaCO3 / ATP template tends to be uniform and stable with the increase of the ATP concentration, and the corresponding loading rate increases rapidly; when the added ATP concentration is higher than 2 mg / mL, the CaCO3 template begins to become unstable with the increase of the ATP concentration, and even voids appear in the template. At this time, the loading rate should decrease compared with that at 2 mg / mL, but the actual situation does not decrease but increases. This may be because starch is added during the preparation of the template. The starch solution is a high-viscosity colloid, and it is very likely that ATP is adsorbed in the ruptured template with starch present, showing a phenomenon that all ATP molecules can be loaded. In fact, the template is already incomplete and non-uniform, and the quality of the prepared capsules is also poor.

[0148] b: The loading rate of the hollow ATP-loaded polyelectrolyte microcapsules with MnCO3 / ATP as the template shows a phenomenon of first increasing and then decreasing with the increase of the added ATP concentration. When the added ATP concentration is lower than 2 mg / mL, the loading rate of the microcapsules prepared with the increase of the ATP concentration increases at a relatively fast rate. When the added ATP concentration is higher than 2 mg / mL, the loading rate of the microcapsules prepared with the increase of the ATP concentration begins to decrease. When the added ATP concentration is lower than 2 mg / mL, the MnCO3 / ATP template tends to be uniform, stable, and the size decreases with the increase of the ATP concentration, meaning that more template numbers are generated, and the corresponding loading rate gradually increases; when the ATP concentration is greater than 2 mg / mL, the MnCO3 / ATP template begins to gradually show cracks and even collapses and cannot form a sphere, so the loading rate of the capsules decreases accordingly. Different from the CaCO3 / ATP template, the MnCO3 / ATP template does not add a solution with a high viscosity such as a starch solution. Therefore, the unloaded ATP molecules due to template damage can only exist in the solution and cannot be adsorbed by other substances.

[0149] c: It can be seen from the figure that the loading rate of the polyelectrolyte microcapsules with ATP loaded on the capsule wall is related to the number of adsorption layers. With the increase of the number of adsorption layers, its loading rate shows a trend of first increasing and then decreasing. This is related to the structural stability of microcapsules with different numbers of layers. From the Zeta potential results, within the range of seven bilayers adsorbed on the polyelectrolyte microcapsules with ATP loaded on the capsule wall, the absolute value of the surface potential of the capsules increases with the increase of the number of adsorption layers, indicating that the capsule system tends to be stable; when the number of adsorption layers is greater than seven layers, the absolute value of the potential decreases significantly with the increase of the number of layers, and the system begins to become unstable. This may be due to the incomplete coverage of the new layer of polyelectrolyte or the detachment of the adsorbed layer of polyelectrolyte. Therefore, when the number of adsorption layers is four to six layers, the loading rate increases, and when adsorbed to eight layers, the loading rate decreases.

[0150] d: As can be seen from the figure, the loading rate of the polyelectrolyte microcapsules with ATP-loaded capsule walls is related to the template size. The smaller the template size, the higher the ATP loading rate. This is because in the way of loading the capsule wall, ATP is adsorbed layer by layer on the surface based on the manganese carbonate template. Then its loading rate is actually related to the specific surface area of the template. When preparing the template, after the reaction of exactly the same initial solution, the smaller the template size, that is, the more the number of templates, the larger the specific surface area, which is more conducive to the loading of ATP molecules. Therefore, the result shows a trend that the smaller the template size, the higher the ATP loading rate.

[0151] Effect of Temperature, pH and Number of Adsorption Layers on the in Vitro Release Rate of Polyelectrolyte Microcapsules Loaded with ATP

[0152] The polyelectrolyte microcapsules loaded with ATP prepared in Examples 1 and 3 were respectively placed under the conditions of 4 °C, room temperature, and 37 °C for release, and the in vitro release curves related to temperature can be obtained ( Figure 8 ).

[0153] The polyelectrolyte microcapsules loaded with ATP prepared in Examples 1 and 3 were respectively placed in phosphate buffer solutions with pH values of 5.8, 6.5, and 7.4 and the same ionic strength for release, and the in vitro release curves related to pH can be obtained ( Figure 8 ).

[0154] The polyelectrolyte microcapsules loaded with ATP prepared in Comparative Examples 15, 16, 17, Example 3, Comparative Examples 11 and 12 were respectively placed in the same aqueous solution for release, and the in vitro release curves related to the number of capsule layers can be obtained ( Figure 8 );

[0155] Result Analysis:

[0156] Temperature: From Figure 8 it can be seen that the ATP capsules obtained by the hollow and capsule wall loading methods have a long-acting and sustained release effect in vitro under the three selected temperature conditions, and the in vitro release rate increases with the increase of temperature. In fact, the increase of temperature will cause the capsule wall to thicken, the voids to disappear, and the capsule wall to become more dense, which is not conducive to its release; but at the same time, the increase of temperature will increase the thermal motion of ATP molecules and the diffusion kinetics. Since the effect of the thermal motion of ATP molecules is stronger than the influence of temperature on the capsule, the comprehensive performance is that the release rate gradually increases with the increase of temperature.

[0157] pH: From Figure 8It can be seen that the ATP capsules obtained by the hollow and capsule wall loading methods have a long-acting and sustained release effect in vitro under the three selected pH conditions. The in vitro release rate of the hollow-loaded ATP polyelectrolyte microcapsules decreases with the increase of pH. This is because within the range of pH 5.8 - 7.4, as the pH decreases, the degree of protonation of PAH increases, the positive charge density increases, and electrostatic repulsion occurs, resulting in the expansion of the capsule structure and easy penetration. On the contrary, as the pH increases, the charge densities of PAH and PEI decrease, the electrostatic repulsion decreases, and the molecules tend to curl conformation, which promotes the thickening of the multilayer and may cause it to contract due to the fixed composition of the capsule. On the other hand, many studies have observed that (PSS / PAH) microcapsules or polyelectrolyte multilayers containing weak electrolytes such as PAH will generate nano-pores in acidic solutions but not in alkalis. Then, during the process of pH decrease, there may be a process from a continuous film to the appearance of pores, which is beneficial to the release of the encapsulated substances. The mechanism of this process may be related to the above-mentioned charge imbalance. The release rate of the capsule wall-loaded ATP polyelectrolyte microcapsules increases with the increase of pH value. This is because as the pH value increases, the degree of protonation of PAH decreases, that is, the positive charge decreases, and the electrostatic interaction with ATP molecules weakens, thus promoting the release.

[0158] Number of adsorbed layers: The release rate of the hollow-loaded ATP polyelectrolyte microcapsules increases with the increase of the number of layers. This may be because the more layers there are, the longer the de-nucleation time, which allows ATP molecules to gradually diffuse into the polyelectrolyte multilayer. The release rate of ATP molecules in the polyelectrolyte multilayer is faster than that of the ATP molecules in the hollow of the polyelectrolyte capsule. Therefore, the more molecules in the polyelectrolyte multilayer, the faster the release rate.

[0159] The release rate of the capsule wall-loaded ATP polyelectrolyte microcapsules is the highest at four layers, decreases significantly at six layers, and then increases at eight layers. This may be related to the structural stability of the polyelectrolyte multilayer. From the Zeta potential results, it can be seen that within the range of adsorbing seven bilayers for the capsule wall-loaded ATP polyelectrolyte microcapsules, the absolute value of the capsule surface potential increases with the increase of the number of adsorbed layers, indicating that the capsule system tends to be stable; while when the number of adsorbed layers is greater than seven, the absolute value of the potential decreases significantly with the increase of the number of layers, and the system begins to become unstable. This may be due to the incomplete coverage of the new layer of polyelectrolyte or the shedding of the adsorbed layer of polyelectrolyte. Therefore, when the number of layers changes from four to six, the release rate decreases significantly because the capsule structure is more stable due to the thickening of the layers. When the number of adsorbed layers rises to eight bilayers, the structure is less stable than when the number of layers is small, and ATP molecules, especially those in the outermost layer, are more likely to escape, resulting in an increase in the release rate.

[0160] Release of ATP-loaded polyelectrolyte microcapsules in the cell environment

[0161] Using 293T cells as samples, they were cultured on 6-well plates. A control group (cultured in complete medium) and an experimental group (complete medium + ATP hollow-loaded capsules) were set up respectively, and they were incubated in a cell culture incubator. The ATP concentration in the cell environment was measured at 24 h and 48 h respectively. The results are shown in Figure 9 ;

[0162] Result analysis: From Figure 9 it can be obtained that the polyelectrolyte microcapsules loaded with ATP in the hollow have obvious ATP release in the cell environment, and this release process can be sustained.

[0163] Comparison of cell release of polyelectrolyte microcapsules loaded with ATP and ATP solution

[0164] Using 293T cells as samples, they were cultured on 6-well plates. A blank group (cultured in complete medium), a control group (complete medium + ATP solution), and an experimental group (complete medium + ATP hollow-loaded capsules) were set up respectively, and they were incubated in a cell culture incubator. The ATP concentration in the cell environment was measured at 45 min, 3 h, 24 h, and 48 h respectively. The results are shown in Figure 10 ;

[0165] Result analysis: From Figure 10 it can be obtained that the exogenous addition of ATP aqueous solution will cause a rapid and significant increase in the ATP concentration in the cell environment, but it is difficult to maintain. The ATP level has decreased significantly at 3 h, and it has approached the initial concentration (ATP) at 24 h; while the exogenous addition of polyelectrolyte microcapsules loaded with ATP in the hollow will not cause a sharp increase in the ATP level in the cell environment and cause excitotoxicity. A slow and continuous increase in the ATP concentration can be observed within 48 h, which means that the ATP microcapsules can achieve long-acting and sustained release of ATP (cap). Compared with the direct addition of ATP solution, the application of the polyelectrolyte microcapsules loaded with ATP in the present invention will be more extensive, safer, and longer-lasting in related diseases.

[0166] In addition, during the research and development of polyelectrolyte microcapsules loaded with ATP in the process of this invention, it was found that:

[0167] (1) A control experiment for the preparation of polyelectrolyte microcapsules with and without a PEI anchoring layer was set. The experimental results showed that the polyelectrolyte microcapsules without a PEI anchoring layer were not successfully prepared within the time of depositing the polyelectrolyte layer in this experiment, and there was no milky white capsule precipitate after removing the nucleus. While the polyelectrolyte microcapsules with a PEI anchoring layer could be successfully prepared.

[0168] (2) During the research and development of polyelectrolyte microcapsules with ATP loaded in the capsule wall, it was found that the preparation of polyelectrolyte microcapsules using only the ATP layer as the negatively charged layer was not successful. There was no milky white capsule precipitate after removing the nucleus, but there were flocculent precipitates.

[0169] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. For the same or similar parts among the various embodiments, reference can be made to each other.

[0170] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A polyelectrolyte microcapsule loaded with ATP, characterized in that, It includes a capsule wall and a core material; the capsule wall is composed of alternately adsorbed cationic polyelectrolyte layers and anionic polyelectrolyte layers; the core material is ATP molecules, and the ATP molecules can be loaded in the hollow of the multilayer polyelectrolyte capsule and / or the capsule wall of the multilayer polyelectrolyte capsule.

2. A method for preparing an ATP-loaded polyelectrolyte microcapsule, characterized in that, When the ATP molecules are loaded in the hollow of the multilayer polyelectrolyte capsule, the specific preparation method is as follows: (1) Use an inorganic substance loaded with ATP molecules as a template particle; (2) Immerse the template particle in step (1) in a cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a polyelectrolyte particle with a layer of adsorbed cationic polyelectrolyte as an anchoring layer; (3) Immerse the polyelectrolyte particle obtained in step (2) in an anionic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a microcapsule with a layer of adsorbed anionic polyelectrolyte. Place the obtained microcapsule in a cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a microcapsule containing a polyelectrolyte bilayer assembly except for the anchoring layer; (4) Repeat step (3) to obtain a microcapsule with a multilayer polyelectrolyte assembly containing ATP molecules; (5) Remove the template to obtain a polyelectrolyte microcapsule loaded with ATP.

3. A method for preparing an ATP-loaded polyelectrolyte microcapsule, characterized in that, When the ATP molecules are loaded in the capsule wall of the multilayer polyelectrolyte capsule, the specific preparation method is as follows: (1) Use an inorganic substance as a template particle; (2) Immerse the template particle in step (1) in a cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a polyelectrolyte particle with a layer of adsorbed cationic polyelectrolyte as an anchoring layer; (3) Immerse the polyelectrolyte particle obtained in step (2) in an ATP solution, shake and centrifuge, then immerse the obtained polyelectrolyte particle in an anionic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a microcapsule with a layer of adsorbed ATP and a layer of anionic polyelectrolyte. Place the obtained microcapsule in a cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a microcapsule containing a three-layer assembly of ATP and polyelectrolyte except for the anchoring layer; (4) Repeat step (3) to obtain a microcapsule with a multilayer polyelectrolyte assembly containing ATP molecules; (5) Remove the template to obtain a polyelectrolyte microcapsule loaded with ATP.

4. A method for preparing an ATP-loaded polyelectrolyte microcapsule, characterized in that, When the ATP molecules are loaded in the hollow and capsule wall of the multilayer polyelectrolyte capsule, the specific preparation method is as follows: (1) Use an inorganic substance loaded with ATP molecules as a template particle; (2) Immerse the template particle in step (1) in a cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a polyelectrolyte particle with a layer of adsorbed cationic polyelectrolyte as an anchoring layer; (3) Immerse the polyelectrolyte particle obtained in step (2) in an ATP solution, shake and centrifuge, then immerse the obtained polyelectrolyte particle in an anionic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a microcapsule with a layer of adsorbed ATP and a layer of anionic polyelectrolyte. Place the obtained microcapsule in a cationic polyelectrolyte solution, shake, centrifuge, and wash the precipitate to obtain a microcapsule containing a three-layer assembly of ATP and polyelectrolyte except for the anchoring layer; (4) Repeat step (3) to obtain a microcapsule with a multilayer polyelectrolyte assembly containing ATP molecules; (5) Remove the template to obtain the polyelectrolyte microcapsules loaded with ATP.

5. The preparation method of the polyelectrolyte microcapsule loaded with ATP according to any one of claims 2 or 4, characterized in that, The template particles in step (1) are CaCO3 / ATP or MnCO3 / ATP template particles; the particle size of the template is 1-4 μm.

6. The preparation method of the ATP-loaded polyelectrolyte microcapsule according to claim 5, wherein The preparation method of the CaCO3 / ATP template particles is as follows: Add a starch solution to a CaCl2 solution while stirring, then add an ATP solution and stir evenly, and then add a Na2CO3 solution, and continuously stir for 5-10 min, and centrifuge and wash to obtain the CaCO3 / ATP template particles; The preparation method of the MnCO3 / ATP template particles is as follows: Add absolute ethanol to a MnSO4 solution while stirring, then add an ATP solution and stir evenly, and then add an NH4HCO3 solution and continuously stir for 5-10 min, and centrifuge and wash to obtain the MnCO3 / ATP template particles.

7. The preparation method of the ATP-loaded polyelectrolyte microcapsule according to claim 3, characterized in that The template particles in step (1) are CaCO3 or MnCO3 template particles; the particle size of the template is 1-4 μm.

8. The preparation method of the ATP-loaded polyelectrolyte microcapsule according to claim 6, wherein The concentration of the CaCl2 solution is 0.02-0.05 M; the concentration of the Na2CO3 solution is 0.02-0.05 M; the concentration of the starch solution is 0.02-0.06 g / mL; the concentration of the MnSO4 solution is 0.01-0.02 M; the concentration of the NH4HCO3 solution is 0.1-0.2 M; the concentration of the ATP solution is 0.5-4 mg / mL.

9. The preparation method of the polyelectrolyte microcapsule loaded with ATP according to any one of claims 2-4, characterized in that, The cationic polyelectrolyte in steps (2)-(3) is polyethyleneimine PEI or polyacrylamine hydrochloride PAH; the anionic polyelectrolyte is sodium polystyrene sulfonate PSS; the concentrations of the anionic / cationic polyelectrolytes are both 0.5-2 mg / mL, and are both prepared with a NaCl solution with a concentration of 0.5 M as the solvent; the concentration of the ATP solution is 0.5-4 mg / mL; the number of repetitions in step (4) is 4-12 times; The oscillation time in steps (2)-(3) is 6 h, and the centrifugation speed is 5000-10000 rpm; washing is carried out with a NaCl solution; The process of removing the template in step (5) is as follows: Place the microcapsules of the polyelectrolyte multilayer assembly containing ATP molecules prepared in step (4) in a dissolution solution, and oscillate to dissolve the template until no gas is generated; The ATP loading rate of the polyelectrolyte microcapsules loaded with ATP in step (5) is 28.25-97.95%.

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