A method for preparing polyelectrolyte capsules using a self-sacrificing soft template

Abstract

The application discloses a method for preparing polyelectrolyte capsules by using a self-sacrificing soft template, fixing the concentration of a polycation solution, titrating the polycation solution with a small-molecule anion solution, drawing a curve of optical density changing with the concentration of the titrating molecules, and taking the concentration range of the small-molecule anion corresponding to the optical density greater than 0.1 as the optimal concentration range; fixing the concentration of the polycation solution, titrating the polycation solution with a polyanion solution, drawing a curve of optical density changing with the concentration of the titrating molecules, and taking the concentration range of the polyanion corresponding to the optical density greater than 0.1 as the optimal concentration range; mixing the polycation solution with the small-molecule anion solution, so that the concentration of the small-molecule anion is in the optimal concentration range, and forming phase separation droplets; adding the polyanion solution to the phase separation coacervate droplets, so that the concentration of the polyanion is in the optimal concentration range, mixing uniformly by using a vortex machine, and then carrying out centrifugal precipitation and dispersion again, so that the polyelectrolyte capsules are obtained.

Description

Technical Field

[0001] This invention belongs to the field of polyelectrolyte capsules and relates to a method for preparing polyelectrolyte capsules using a self-sacrificing soft template. Background Technology

[0002] Polyelectrolyte capsules are a class of micro / nano hollow structures formed by the electrostatic interaction of polyelectrolytes with opposite charges. Their unique hollow chambers and stable outer shell structure make them valuable for molecular delivery and drug sustained release, making them a research hotspot in materials science, biomedicine, and engineering. Currently, common polyelectrolyte capsules are typically prepared using layer-by-layer self-assembly and complex condensation methods. Layer-by-layer self-assembly often requires using silica, calcium carbonate, or similar materials as templates, coating polyelectrolyte layers one by one through electrostatic interaction, followed by etching to remove the internal template to obtain the polyelectrolyte capsule. Layer-by-layer self-assembly allows for precise internal cavity fabrication and high structural controllability, but its reliance on multiple template-based depositions results in relatively low preparation efficiency. Complex condensation primarily utilizes the phase separation of two polyelectrolytes with opposite charges under specific conditions (such as pH or temperature changes) to form a polyelectrolyte-rich "condensed phase" that encapsulates the target substance, which is then cross-linked and solidified to form microcapsules. The core of this method is a phase separation-driven encapsulation process, which requires no template, resulting in lower cost and suitability for large-scale production. However, its structural controllability is poor, and internal cavity ratio and wall thickness cannot be controlled. Inspired by the Kendall effect in the preparation of hollow metal particles, we utilized the differences in binding and diffusion capabilities between polycation / small molecule anionic complexes and polycation / polyanionic complexes to achieve the preparation of polyelectrolyte capsules with a phase-separated droplet soft template through self-sacrifice. This method combines the characteristics of layer-by-layer self-assembly and complex condensation, using phase-separated droplets as soft templates. By introducing highly competitive polyanions to induce competitive cavitation at the interface, the removal of the soft template and encapsulation are achieved in one step. This method offers strong structural controllability and a simple preparation process, showing potential applications in molecular delivery and drug encapsulation. Summary of the Invention

[0003] In view of this, the present invention provides a method for preparing polyelectrolyte capsules using a self-sacrificing soft template. Specifically, the present invention provides the following technical solution:

[0004] A method for preparing polyelectrolyte capsules using a self-sacrificing soft template, comprising the following steps:

[0005] 1) Fix the concentration of the polycationic solution, titrate with a small-molecule anionic solution, and plot the curve of optical density as a function of titration molecular concentration. The small-molecule anion concentration range corresponding to an optical density greater than 0.1 is taken as the optimal concentration range. The small-molecule anion is one or more of adenosine triphosphate disodium salt, pyromellitic acid, or sodium 1,2,3,4,5,6-cyclohexanehexacarboxylate. The polycation is one or more of protamine sulfate, polydiallyldimethylammonium chloride, polylysine, and diethylaminoethyl dextran.

[0006] 2) Fix the concentration of the polycationic solution, titrate with the polyanionic solution, and plot the curve of optical density as a function of the concentration of titrated molecules. The concentration range of the polyanion corresponding to the optical density of the curve greater than 0.1 is taken as the optimal concentration range; the polyanion is heparin sodium, sodium polystyrene sulfonate, or dextran sulfate.

[0007] 3) Mix the polycationic solution with the small molecule anion solution to bring the concentration of the small molecule anion to the optimal concentration range determined in step 1), forming phase-separated droplets;

[0008] 4) Add the polyanion solution to the phase separation and coagulation droplets in step 3) to bring the concentration of polyanion to the optimal concentration range determined in step 2). After mixing evenly with a vortex mixer, centrifuge and precipitate and then disperse again to obtain polyelectrolyte capsules.

[0009] Further, the method for determining the optimal concentration range of the small molecule anion solution in step 1) is as follows: Prepare a polycationic solution, titrate it using small molecule anions, measure the absorbance of the titrated sample at 600 nm using a UV-Vis spectrophotometer as the optical density, and plot the correlation curve between optical density and small molecule anion concentration. Take the portion of the curve located in the region where the optical density is greater than 0.1, and take the small molecule anion concentration range of the curve in this region as the optimal concentration range.

[0010] Further, the method for determining the optimal concentration range of the polyanionic solution in step 2) is as follows: Prepare a polycationic solution, titrate with the polycationic solution, measure the absorbance of the titrated sample at 600 nm using a UV-Vis spectrophotometer as the optical density, and plot the correlation curve between optical density and polycationic concentration. Take the portion of the curve located in the region where the optical density is greater than 0.1, and take the polyanionic concentration range of the curve in this region as the optimal concentration range.

[0011] Furthermore, the preparation method of the phase-separated droplets in step 3) is as follows: prepare a polycationic solution and a small molecule anion solution, with the pH of the solution between the pKa of the small molecule anion and the polycation, and control the formation time of the phase-separated droplets to 0-10 min.

[0012] Further, in step 4), the amplitude of the vortex generator is 4.5 mm, the speed is 200-3000 rpm, and the vortex time is 20 s.

[0013] Furthermore, the polycation is protamine sulfate, and when the concentration is 1 mg / mL, the optimal concentration is 4-7 mM when the small molecule anion is disodium adenosine triphosphate, the optimal concentration is 0.7-1.2 mM when the small molecule anion is pyromellitic acid, the optimal concentration is 0.75-1.2 mg / mL when the polyanion is sodium heparin; the optimal concentration is 0.8-1.8 mg / mL when the polyanion is sodium polystyrene sulfonate, and the optimal concentration is 1.6-2.0 mg / mL when the polyanion is dextran sulfate.

[0014] Furthermore, the polycation is polydiallyldimethylammonium chloride, and when the concentration is 1 mg / mL, the optimal concentration is 2.0-3 mM when the small molecule anion is adenosine triphosphate disodium salt, 1.6-3 mM when the small molecule anion is pyromellitic acid, 1.2-2.4 mg / mL when the polyanion is heparin sodium, 0.6-1.4 mg / mL when the polyanion is sodium polystyrene sulfonate, and 0.7-2.3 mg / mL when the polyanion is dextran sulfate.

[0015] Furthermore, the polycation is polylysine, and when the concentration is 1 mg / mL, the optimal concentration is 3-6 mM when the small molecule anion is disodium adenosine triphosphate, 1.6-3.0 mM when the small molecule anion is pyromellitic acid, 0.8-1.4 mg / mL when the polyanion is sodium heparin, 0.7-1.7 mg / mL when the polyanion is sodium polystyrene sulfonate, and 0.5-2.0 mg / mL when the polyanion is dextran sulfate.

[0016] Furthermore, the polycation is diethylaminoethyl dextran, and when the concentration is 1 mg / mL, the optimal concentration of the small molecule anion is 3-12 mM when the small molecule anion is adenosine triphosphate disodium salt; the optimal concentration of the polyanion is 0.5-1.7 mg / mL when the polyanion is heparin sodium; the optimal concentration of the polyanion is 0.8-2.0 mg / mL when the polyanion is polystyrene sulfonate sodium; and the optimal concentration of the polyanion is 0.6-2.0 mg / mL when the polyanion is dextran sulfate.

[0017] The beneficial effects of this invention are as follows:

[0018] 1) By using polyelectrolyte condensed droplets as a self-sacrificing soft template, the encapsulation process and template removal were carried out simultaneously, reducing the introduction of impurities during template removal and lowering the process complexity of capsule preparation.

[0019] 2) The size of the self-sacrificing soft template is controlled by the formation time of polyelectrolyte condensed droplets, which simplifies the process of traditional emulsion template / hard template preparation and provides spatiotemporal control capability for template design.

[0020] 3) Polyelectrolytes and small molecule anions have a wide range of applications, and the optimal concentration can be determined by titration curves, providing rich application scenarios and design possibilities for capsule preparation by this method. Attached Figure Description

[0021] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0022] Figure 1 The optical density titration curve of disodium adenosine triphosphate against protamine sulfate solution (hydroxyethylpiperazine ethanesulfonic acid-sodium hydroxide buffer system) is shown.

[0023] Figure 2 The optical density titration curve of heparin sodium against protamine sulfate buffer solution (hydroxyethylpiperazine ethanesulfonic acid-sodium hydroxide buffer system).

[0024] Figure 3 Microscopic images of the encapsulated droplets of protamine sulfate / adenosine triphosphate disodium salt phase separation after incubation for different times.

[0025] Figure 4 Box plot of size distribution of protamine sulfate / adenosine triphosphate disodium salt phase-separated droplet encapsulation products after incubation for different times.

[0026] Figure 5 The optical density titration curve of disodium adenosine triphosphate against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0027] Figure 6 The optical density titration curve of sodium polystyrene sulfonate to protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0028] Figure 7 The optical density titration curve of dextran sulfate-40 kDa against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0029] Figure 8 The optical density titration curve of disodium adenosine triphosphate against polydiallyl dimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0030] Figure 9 The optical density titration curve of sodium heparin against polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0031] Figure 10 The optical density titration curve of sodium polystyrene sulfonate to polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0032] Figure 11 The optical density titration curve of dextran sulfate-40 kDa against polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0033] Figure 12 The optical density titration curve of disodium adenosine triphosphate against polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0034] Figure 13 The optical density titration curve of sodium heparin on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0035] Figure 14 The optical density titration curve of sodium polystyrene sulfonate on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0036] Figure 15 The optical density titration curve of dextran sulfate-40 kDa against polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0037] Figure 16 The optical density titration curve of disodium adenosine triphosphate against diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0038] Figure 17 The optical density titration curve of heparin sodium against diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0039] Figure 18 The optical density titration curve of sodium polystyrene sulfonate against diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0040] Figure 19 The optical density titration curve of dextran sulfate-40 kDa versus diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution).

[0041] Figure 20 The optical density titration curve of pyromellitic acid against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0042] Figure 21 The optical density titration curve of sodium heparin on protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0043] Figure 22 The optical density titration curve of pyromellitic acid against polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0044] Figure 23 The optical density titration curve of pyromellitic acid on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown.

[0045] Figure 24 These are fluorescence micrographs of the encapsulated products from Examples 1-12.

[0046] Figure 25 Fluorescence micrographs of the encapsulated products from Examples 13-21. Detailed Implementation

[0047] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0048] Example 1

[0049] 1. The steps for heparin sodium-induced droplet encapsulation of protamine sulfate / adenosine triphosphate disodium salt phase separation are as follows:

[0050] 1) Titrate adenosine triphosphate disodium salt by gradually adding it to a hydroxyethylpiperazine ethanesulfonic acid-sodium hydroxide (pH 7.0, concentration 20 mM) buffer solution containing 1 mg / mL protamine sulfate, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 1 The optical density titration curve is shown for the dihydropyridine triphosphate disodium salt against a protamine sulfate solution (hydroxyethylpiperazine ethanesulfonic acid-sodium hydroxide buffer system). Figure 1 As can be seen from the curve, the concentration range of adenosine triphosphate disodium salt corresponding to an optical density greater than 0.1 is 4-7 mM, and this range is considered the optimal concentration range.

[0051] 2) Heparin sodium was added in a gradient to a hydroxyethylpiperazine ethanesulfonic acid-sodium hydroxide buffer solution (pH 7.0, concentration 20 mM) containing 1 mg / mL protamine sulfate, and the optical density-heparin sodium concentration curve was plotted. Figure 2 The optical density titration curve is shown for heparin sodium versus protamine sulfate buffer solution. Figure 2 As can be seen from the curve, the concentration range of heparin sodium corresponding to an optical density greater than 0.1 is 0.75-1.2 mg / mL, and this range is considered the optimal concentration range.

[0052] 3) Prepare a phase separation droplet containing 1 mg / mL protamine sulfate and 5 mM adenosine triphosphate disodium salt using a hydroxyethylpiperazine ethanesulfonic acid-sodium hydroxide buffer system (pH 7.0, concentration 20 mM). Use a 4.5 mm amplitude and a rotation speed of 500 rpm for 10 s in a circular vortex, and then let each droplet stand for 1-9 min.

[0053] 4) Add heparin sodium to the above dispersion to ensure the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2), then vortex at 500 rpm for 10 s with an amplitude of 4.5 mm, and centrifuge at 1000 rpm for 3 min to obtain the precipitate. Disperse the precipitate with hydroxyethylpiperazine ethanesulfonic acid-sodium hydroxide buffer solution (pH 7.0, concentration 20 mM) to obtain the encapsulated product.

[0054] 2. Fluorescence microscopy observation

[0055] The above product was stained with 0.1% by mass of fluorescein isothiocyanate (FITC)-labeled protamine sulfate and observed using a fluorescence microscope.

[0056] Figure 3 Microscopic images of the phase-separated droplet encapsulation products of protamine sulfate / adenosine triphosphate disodium salt after incubation for different times. From Figure 3 As can be seen, the size of the encapsulated product gradually increases with the increase of the incubation time of the phase-separated droplets from 1 min to 9 min. This is because the phase-separated droplets, acting as a soft template, fuse and grow with increasing incubation time, thereby controlling the capsule size through the size of the soft template. This indicates that the capsule size prepared by this method can be controlled over time.

[0057] Figure 4 Box plots showing the size distribution of the encapsulated products from the phase-separated droplets of protamine sulfate / adenosine triphosphate disodium salt after incubation for different times. Figure 4 As can be seen, with the increase of incubation time, the average area of ​​the encapsulated product increased from 7 μm to 77 μm, and its size was significantly positively correlated with the incubation time of the polyelectrolyte condensed droplets. This indicates that the size of the capsules prepared by this method can be controlled over time.

[0058] Example 2

[0059] 1. The specific steps for the encapsulation of protamine sulfate / adenosine triphosphate disodium salt phase separation droplets induced by sodium polystyrene sulfonate are as follows:

[0060] 1) Titrate adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 50 mM) containing 1 mg / mL protamine sulfate, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 5 The optical density titration curve is shown for the dihydrogen phosphate disodium salt against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution). From... Figure 5 As can be seen from the curve, the concentration range of adenosine triphosphate disodium salt corresponding to an optical density greater than 0.1 is 4-7 mM, and this range is considered the optimal concentration range.

[0061] 2) Add sodium polystyrene sulfonate in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 50 mM) containing 1 mg / mL protamine sulfate, and plot the optical density-sodium polystyrene sulfonate concentration curve. Figure 6 The optical density titration curve of sodium polystyrene sulfonate against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution). From Figure 6 As can be seen from the curve, the concentration range of sodium polystyrene sulfonate corresponding to an optical density greater than 0.1 is 0.8-1.8 mg / mL, and this range is considered the optimal concentration range.

[0062] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL protamine sulfate and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 50 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0063] 4) Add sodium polystyrene sulfonate to the protamine sulfate / adenosine triphosphate disodium salt phase separation droplet from step 3), ensuring the final concentration is 1.6 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and centrifuge at 1000 rpm for 3 min. The resulting precipitate is redispersed with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 7.5, concentration 10 mM) to obtain the encapsulated product.

[0064] 2. Fluorescence microscopy observation

[0065] Add 0.1% by mass of FITC-labeled protamine sulfate for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images show fluorescence micrographs of the encapsulated products from Examples 1-12. As can be seen from the images, the micrographs corresponding to Example 2 demonstrate the characteristics of empty capsules in the dispersion state.

[0066] Example 3

[0067] Dextran sulfate-40 kDa induced the encapsulation of protamine sulfate / adenosine triphosphate disodium salt phase-separated droplets, the specific steps of which are as follows:

[0068] 1) Titrate adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 50 mM) containing 1 mg / mL protamine sulfate, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 5 The optical density titration curve of adenosine triphosphate disodium salt against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of adenosine triphosphate disodium salt corresponding to an optical density greater than 0.1 is 4-7 mM, and this range is taken as the optimal concentration range.

[0069] 2) Add dextran sulfate-40 kDa in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 50 mM) containing 1 mg / mL protamine sulfate, and plot the optical density-dextran sulfate-40 kDa concentration curve. Figure 7 The optical density titration curve of dextran sulfate-40 kDa against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of dextran sulfate-40 kDa corresponding to an optical density greater than 0.1 is 1.6-2.0 mg / mL, and this range is taken as the optimal concentration range.

[0070] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL protamine sulfate and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 50 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0071] 4) Add 40 kDa of dextran sulfate to the above dispersion to ensure the final concentration is 1.8 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and centrifuge at 1000 rpm for 3 min. The precipitate is then redispersed with sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 50 mM) to obtain the encapsulated product.

[0072] 2. Fluorescence microscopy examination

[0073] Add 0.1% by mass of FITC-labeled protamine sulfate for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 3 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0074] Example 4

[0075] 1. The encapsulation of phase-separated droplets formed by heparin sodium-induced polydiallyl dimethylammonium chloride and disodium adenosine triphosphate is described in the following steps:

[0076] 1) Titrate adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and plot the optical density-disodium triphosphate disodium salt concentration curve. Figure 8 The optical density titration curve of disodium adenosine triphosphate (ATP) on polydiallyl dimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of disodium ATP corresponding to an optical density greater than 0.1 is 2.0-3.0 mM, and this range is considered the optimal concentration range.

[0077] 2) Heparin sodium was gradually added to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and the titration was performed. An optical density-heparin sodium concentration curve was plotted. Figure 9 The optical density titration curve of heparin sodium against polydiallyldimethylammonium chloride (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of heparin sodium corresponding to an optical density greater than 0.1 is 1.2-2.4 mg / mL, and this range is taken as the optimal concentration range.

[0078] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polydiallyldimethylammonium chloride and 2.5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min for later use.

[0079] 4) Add heparin sodium to the above dispersion to ensure that the final concentration is 1.8 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0080] 2. Fluorescence microscopy observation

[0081] Add 0.1% by mass of FITC-labeled polydiallyldimethylammonium chloride for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 4 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0082] Example 5

[0083] 1. The encapsulation of phase-separated droplets formed by polydiallyldimethylammonium chloride and disodium adenosine triphosphate induced by sodium polystyrene sulfonate is described in the following steps:

[0084] 1) Titrate adenosine triphosphate (ATP) disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and plot the optical density-ATP disodium salt concentration curve. Figure 8 The optical density titration curve of disodium adenosine triphosphate (ATP) on polydiallyl dimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of disodium ATP corresponding to an optical density greater than 0.1 is 2.0-3.0 mM, and this range is considered the optimal concentration range.

[0085] 2) Add sodium polystyrene sulfonate in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution containing 1 mg / mL polydiallyldimethylammonium chloride (pH 7.5, concentration 20 mM) and titrate. Plot the optical density-sodium polystyrene sulfonate concentration curve. Figure 10 The optical density titration curve of sodium polystyrene sulfonate to polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of sodium polystyrene sulfonate corresponding to an optical density greater than 0.1 is 0.6-1.4 mg / mL, and this range is taken as the optimal concentration range.

[0086] 3) Prepare a phase separation droplet containing 1.0 mg / mL polydiallyldimethylammonium chloride and 2.5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). The droplet should be 100 μL in total. Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0087] 4) Add sodium polystyrene sulfonate to the dispersion in step 3) to ensure that the final concentration is 1.0 mg / mL. Then, use a vortex mixer with an amplitude of 4.5 mm and a speed of 500 rpm for 10 s. Then, centrifuge at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0088] 2. Fluorescence microscopy observation

[0089] Add 0.1% by mass of FITC-labeled polydiallyldimethylammonium chloride for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 5 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0090] Example 6

[0091] 1. The encapsulation of phase-separated droplets formed by polydiallyldimethylammonium chloride and disodium adenosine triphosphate induced by dextran sulfate-40 kDa is as follows:

[0092] 1) Titrate with adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 8 The optical density titration curve of disodium adenosine triphosphate (ATP) on polydiallyl dimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of disodium ATP corresponding to an optical density greater than 0.1 is 2.0-3.0 mM, and this range is considered the optimal concentration range.

[0093] 2) Add dextran sulfate-40 kDa in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and perform titration. Plot the optical density-dextran sulfate-40 kDa concentration curve. Figure 11The optical density titration curve of dextran sulfate-40 kDa against polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of dextran sulfate-40 kDa corresponding to an optical density greater than 0.1 is 0.7-2.3 mg / mL, and this range is considered the optimal concentration range.

[0094] 3) Prepare a phase separation droplet containing 1 mg / mL polydiallyldimethylammonium chloride and 2.5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM), totaling 100 μL. Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0095] 4) Add 40 kDa of dextran sulfate to the above dispersion to ensure that the final concentration is 1.2 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0096] 2. Fluorescence microscopy observation

[0097] Add 0.1% by mass of FITC-labeled polydiallyldimethylammonium chloride for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 6 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0098] Example 7

[0099] 1. The encapsulation of phase-separated droplets formed by heparin sodium-induced polylysine and disodium adenosine triphosphate is carried out through the following steps:

[0100] 1) Titrate adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 12 The optical density titration curve of disodium adenosine triphosphate (ATP) on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of disodium ATP corresponding to an optical density greater than 0.1 is 3.0-6.0 mM, and this range is considered the optimal concentration range.

[0101] 2) Heparin sodium was added in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution containing 1 mg / mL polylysine (pH 7.5, concentration 20 mM) and titrated. The optical density-heparin sodium concentration curve was plotted. Figure 13 The optical density titration curve of heparin sodium on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of heparin sodium corresponding to an optical density greater than 0.1 is 0.8-1.4 mg / mL, and this range is taken as the optimal concentration range.

[0102] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polylysine and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a 4.5 mm amplitude and a rotation speed of 500 rpm for 10 s in a circular vortex, and then let it stand for 5 min.

[0103] 4) Add heparin sodium to the above dispersion to ensure the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2), then vortex at 500 rpm for 10 s with an amplitude of 4.5 mm, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0104] 2. Fluorescence microscopy observation

[0105] Add 0.1% (w / w) of FITC-labeled polylysine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 7 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0106] Example 8

[0107] 1. The encapsulation of phase-separated droplets formed by poly(lysine) and disodium adenosine triphosphate induced by sodium polystyrene sulfonate is described in the following steps:

[0108] 1) Titrate with adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 12The optical density titration curve of disodium adenosine triphosphate (ATP) on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of disodium ATP corresponding to an optical density greater than 0.1 is 3.0-6.0 mM, and this range is considered the optimal concentration range.

[0109] 2) Add sodium polystyrene sulfonate in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution containing 1 mg / mL polylysine (pH 7.5, concentration 20 mM) and titrate. Plot the optical density-sodium polystyrene sulfonate concentration curve. Figure 14 The optical density titration curve of sodium polystyrene sulfonate on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of sodium polystyrene sulfonate corresponding to an optical density greater than 0.1 is 0.7-1.7 mg / mL, and this range is taken as the optimal concentration range.

[0110] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polylysine and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min for later use.

[0111] 4) Add sodium polystyrene sulfonate to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0112] 2. Fluorescence microscopy observation

[0113] Add 0.1% (w / w) of FITC-labeled polylysine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 8 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0114] Example 9

[0115] 1. The encapsulation of phase-separated droplets formed by polylysine and disodium adenosine triphosphate induced by dextran sulfate-40 kDa is shown in the following steps:

[0116] 1) Titrate with adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 12 The optical density titration curve of disodium adenosine triphosphate (ATP) on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of disodium ATP corresponding to an optical density greater than 0.1 is 3.0-6.0 mM, and this range is considered the optimal concentration range.

[0117] 2) Add dextran sulfate-40 kDa in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine and titrate. Plot the optical density-dextran sulfate-40 kDa concentration curve. Figure 15 The optical density titration curve of dextran sulfate-40 kDa against polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of dextran sulfate-40 kDa corresponding to an optical density greater than 0.1 is 0.5-2.0 mg / mL, and this range is taken as the optimal concentration range.

[0118] 3) Prepare 100 μL of phase-separated droplets containing 1 mg / mL polylysine and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min to obtain polylysine / adenosine triphosphate disodium salt phase-separated droplets.

[0119] 4) Add 40 kDa of dextran sulfate to the above dispersion to ensure the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s. After that, centrifuge at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0120] 2. Fluorescence microscopy observation

[0121] Add 0.1% (w / w) of FITC-labeled polylysine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 9 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0122] Example 10

[0123] 1. The encapsulation of phase-separated droplets formed by heparin sodium-induced diethylaminoethyl dextran and disodium adenosine triphosphate is carried out through the following steps:

[0124] 1) Titrate adenosine triphosphate disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL diethylaminoethyl dextran, and plot the optical density-adenosine triphosphate disodium salt concentration curve. Figure 16 The optical density titration curve of adenosine triphosphate disodium salt against diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of adenosine triphosphate disodium salt corresponding to an optical density greater than 0.1 is 3.0-12.0 mM, and this range is taken as the optimal concentration range.

[0125] 2) Heparin sodium was added in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL diethylaminoethyl dextran and titrated. The optical density-heparin sodium concentration curve was plotted. Figure 17 The optical density titration curve of heparin sodium against diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of triheparin sodium corresponding to an optical density greater than 0.1 is 0.5-1.7 mg / mL, and this range is taken as the optimal concentration range.

[0126] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL diethylaminoethyl dextran and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min for later use.

[0127] 4) Add heparin sodium to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0128] 2. Fluorescence microscopy observation

[0129] Add 0.1% by mass of FITC-labeled diethylaminoethyl dextran for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24The images shown are fluorescence micrographs of the encapsulated products from Examples 1-12. The micrograph corresponding to Example 10 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0130] Example 11

[0131] 1. The encapsulation of phase-separated droplets formed by sodium polystyrene sulfonate-induced diethylaminoethyl dextran and disodium adenosine triphosphate is carried out through the following steps:

[0132] 1) Titrate adenosine triphosphate (ATP) disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL diethylaminoethyl dextran, and plot the optical density-ATP disodium salt concentration curve. Figure 16 The optical density titration curve of adenosine triphosphate disodium salt against diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of adenosine triphosphate disodium salt corresponding to an optical density greater than 0.1 is 3.0-12.0 mM, and this range is taken as the optimal concentration range.

[0133] 2) Add sodium polystyrene sulfonate in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL diethylaminoethyl dextran and titrate accordingly. Plot the optical density-sodium polystyrene sulfonate concentration curve. Figure 18 The optical density titration curve of sodium polystyrene sulfonate with diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of sodium polystyrene sulfonate corresponding to an optical density greater than 0.1 is 0.8-2.0 mg / mL, and this range is taken as the optimal concentration range.

[0134] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL diethylaminoethyl dextran and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a suitable vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0135] 4) Add sodium polystyrene sulfonate to the above dispersion to ensure that the final concentration is 1.3 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0136] 2. Fluorescence microscopy observation

[0137] Add 0.1% by mass of FITC-labeled diethylaminoethyl dextran for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products of Examples 1-12. The micrograph corresponding to Example 11 in the figures can demonstrate the characteristics of the empty capsules in the dispersion state.

[0138] Example 12

[0139] 1. The encapsulation of phase-separated droplets formed by diethylaminoethyl dextran and disodium adenosine triphosphate induced by 40 kDa sulfate dextran is as follows:

[0140] 1) Titrate adenosine triphosphate (ATP) disodium salt by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL diethylaminoethyl dextran, and plot the optical density-ATP disodium salt concentration curve. Figure 16 The optical density titration curve of adenosine triphosphate disodium salt against diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of adenosine triphosphate disodium salt corresponding to an optical density greater than 0.1 is 3.0-12.0 mM, and this range is taken as the optimal concentration range.

[0141] 2) Add dextran sulfate-40 kDa in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL diethylaminoethyl dextran and titrate accordingly. Plot the optical density-dextran sulfate-40 kDa concentration curve. Figure 19 The optical density titration curve of dextran sulfate-40 kDa versus diethylaminoethyl dextran (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of dextran sulfate-40 kDa corresponding to an optical density greater than 0.1 is 0.6-2.0 mg / mL, and this range is considered the optimal concentration range.

[0142] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL diethylaminoethyl dextran and 5 mM adenosine triphosphate disodium salt using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a suitable vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0143] 4) Add 40 kDa of dextran sulfate to the above dispersion to ensure that the final concentration is 1.1 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate obtained is redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration of 20 mM) to obtain the product.

[0144] 2. Fluorescence microscopy observation

[0145] Add 0.1% by mass of FITC-labeled diethylaminoethyl dextran for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 24 The images shown are fluorescence micrographs of the encapsulated products of Examples 1-12. The micrograph corresponding to Example 12 in the figures can demonstrate the characteristics of the empty capsules in the dispersion state.

[0146] Example 13

[0147] 1. The specific steps for the heparin sodium-induced phase separation and droplet encapsulation of protamine sulfate / pyromellitic acid are as follows:

[0148] 1) Titrate with pyromellitic acid by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL protamine, and plot the optical density-pyromellitic acid concentration curve. Figure 20 The optical density titration curve of pyromellitic acid on protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 0.7-1.2 mM, and this range is taken as the optimal concentration range.

[0149] 2) Heparin sodium was added in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL protamine, and the optical density-heparin sodium concentration curve was plotted. Figure 21 The optical density titration curve of heparin sodium on protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of heparin sodium corresponding to an optical density greater than 0.1 is 0.75-1.2 mg / mL, and this range is taken as the optimal concentration range.

[0150] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL protamine and 1.2 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0151] 4) Add heparin sodium to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0152] 2. Fluorescence microscopy observation

[0153] Add 0.1% (w / w) of FITC-labeled protamine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrographs corresponding to Example 13 in the figures demonstrate the characteristics of empty capsules in the dispersion state.

[0154] Example 14

[0155] 1. The specific steps for encapsulating sodium polystyrene sulfonate-induced protamine sulfate / pyromellitic acid phase separation droplets are as follows:

[0156] 1) Titrate with pyromellitic acid in a gradient of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL protamine, and plot the optical density-pyromellitic acid concentration curve. Figure 20 The optical density titration curve of pyromellitic acid on protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 0.7-1.2 mM, and this range is taken as the optimal concentration range.

[0157] 2) Sodium polystyrene sulfonate was titrated by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL protamine sulfate. An optical density-sodium polystyrene sulfonate concentration curve was plotted. Figure 6 The optical density titration curve of sodium polystyrene sulfonate on protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of sodium polystyrene sulfonate corresponding to an optical density greater than 0.1 is 0.8-1.8 mg / mL, and this range is taken as the optimal concentration range.

[0158] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL protamine and 1.2 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0159] 4) Add sodium polystyrene sulfonate to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0160] 2. Fluorescence microscopy observation

[0161] Add 0.1% (w / w) of FITC-labeled protamine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrograph corresponding to Example 14 in the figures demonstrates the characteristics of empty capsules in the dispersion state.

[0162] Example 15

[0163] 1. The encapsulation of protamine sulfate / pyromellitic acid phase-separated droplets induced by dextran sulfate-40 kDa is as follows:

[0164] 1) Titrate with pyromellitic acid in a gradient of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL protamine, and plot the optical density-pyromellitic acid concentration curve. Figure 20 The optical density titration curve of pyromellitic acid on protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 0.7-1.2 mM, and this range is taken as the optimal concentration range.

[0165] 2) Titrate with a gradient of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL protamine sulfate, adding dextran sulfate-40 kDa gradually, and plot the optical density-dextran sulfate-40 kDa concentration curve. Figure 7The optical density titration curve of dextran sulfate-40 kDa against protamine sulfate solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of dextran sulfate-40 kDa corresponding to an optical density greater than 0.1 is 1.6-2.0 mg / mL, and this range is taken as the optimal concentration range.

[0166] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL protamine and 1.2 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0167] 4) Add 40 kDa of dextran sulfate to the above dispersion to ensure that the final concentration is 1.6 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0168] 2. Fluorescence microscopy observation

[0169] Add 0.1% (w / w) of FITC-labeled protamine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrograph corresponding to Example 115 in the figures demonstrates the characteristics of empty capsules in the dispersion state.

[0170] Example 16

[0171] 1. The specific steps for the heparin sodium-induced polydiallyldimethylammonium chloride / pyromellitic acid phase separation droplet encapsulation are as follows:

[0172] 1) Add pyromellitic acid in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and plot the optical density-pyromellitic acid concentration curve. Figure 22 The optical density titration curve of pyromellitic acid on polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 1.6-3.0 mM, and this range is taken as the optimal concentration range.

[0173] 2) Heparin sodium was gradually added to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and titration was performed. The optical density-heparin sodium concentration curve was then plotted. Figure 9 The optical density titration curve of heparin sodium against polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of heparin sodium corresponding to an optical density greater than 0.1 is 1.2-2.4 mg / mL, and this range is taken as the optimal concentration range.

[0174] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polydiallyldimethylammonium chloride and 2.5 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0175] 4) Add heparin sodium to the above dispersion to ensure that the final concentration is 1.8 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0176] 2. Fluorescence microscopy observation

[0177] Add 0.1% by mass of FITC-labeled polydiallyldimethylammonium chloride for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrograph corresponding to Example 16 in the figures demonstrates the characteristics of empty capsules in the dispersion state.

[0178] Example 17

[0179] 1. The specific steps for encapsulating polydiallyldimethylammonium chloride / pyromellitic acid phase-separated droplets induced by sodium polystyrene sulfonate are as follows:

[0180] 1) Titrate with pyromellitic acid by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and plot the optical density-pyromellitic acid concentration curve. Figure 22The optical density titration curve of pyromellitic acid on polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 1.6-3.0 mM, and this range is taken as the optimal concentration range.

[0181] 2) Sodium polystyrene sulfonate was titrated by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and the optical density-sodium polystyrene sulfonate concentration curve was plotted. Figure 10 The optical density titration curve of sodium polystyrene sulfonate to polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of sodium polystyrene sulfonate corresponding to an optical density greater than 0.1 is 0.7-1.4 mg / mL, and this range is taken as the optimal concentration range.

[0182] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polydiallyldimethylammonium chloride and 2.5 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0183] 4) Add sodium polystyrene sulfonate to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0184] 2. Fluorescence microscopy examination

[0185] Add 0.1% by mass of FITC-labeled polydiallyldimethylammonium chloride for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrograph corresponding to Example 17 in the figures demonstrates the characteristics of empty capsules in the dispersion state.

[0186] Example 18

[0187] 1. The encapsulation of polydiallyldimethylammonium chloride / pyromellitic acid phase-separated droplets induced by dextran sulfate-40 kDa is shown in the following steps:

[0188] 1) Titrate with pyromellitic acid by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, and plot the optical density-pyromellitic acid concentration curve. Figure 22 The optical density titration curve of pyromellitic acid on polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 1.6-3.0 mM, and this range is taken as the optimal concentration range.

[0189] 2) Titrate with a gradient of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polydiallyldimethylammonium chloride, adding dextran sulfate-40 kDa in a gradient. Plot the optical density-dextran sulfate-40 kDa concentration curve. Figure 11 The optical density titration curve of dextran sulfate-40 kDa against polydiallyldimethylammonium chloride solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of dextran sulfate-40 kDa corresponding to an optical density greater than 0.1 is 0.7-2.3 mg / mL, and this range is considered the optimal concentration range.

[0190] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polydiallyldimethylammonium chloride and 2.5 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min for later use.

[0191] 4) Add 40 kDa of dextran sulfate to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0192] 2. Fluorescence microscopy observation

[0193] Add 0.1% by mass of FITC-labeled polydiallyldimethylammonium chloride for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrograph corresponding to Example 18 in the figures demonstrates the characteristics of empty capsules in the dispersion state.

[0194] Example 19

[0195] 1. The specific steps for the heparin sodium-induced polylysine / pyromellitic acid phase separation droplet encapsulation are as follows:

[0196] 1) Titrate with pyromellitic acid by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and plot the optical density-pyromellitic acid concentration curve. Figure 23 The optical density titration curve of pyromellitic acid on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 1.6-3.0 mM, and this range is taken as the optimal concentration range.

[0197] 2) Titrate with sodium heparin by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and plot the optical density-sodium heparin concentration curve. Figure 13 The optical density titration curve of heparin sodium on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of heparin sodium corresponding to an optical density greater than 0.1 is 0.8-1.4 mg / mL, and this range is taken as the optimal concentration range.

[0198] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polylysine and 2.5 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0199] 4) Add heparin sodium to the above powder to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s. Then, centrifuge at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0200] 2. Fluorescence microscopy observation

[0201] Add 0.1% (w / w) of FITC-labeled polylysine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrograph corresponding to Example 19 in the figures demonstrates the characteristics of the empty capsules in the dispersion state.

[0202] Example 20

[0203] 1. The specific steps for encapsulating polylysine / pyromellitic acid phase-separated droplets induced by sodium polystyrene sulfonate are as follows:

[0204] 1) Titrate with pyromellitic acid by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and plot the optical density-pyromellitic acid concentration curve. Figure 23 The optical density titration curve of pyromellitic acid on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 1.6-3.0 mM, and this range is taken as the optimal concentration range.

[0205] 2) Add sodium polystyrene sulfonate in a gradient to a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and titrate accordingly. Plot the optical density-sodium polystyrene sulfonate concentration curve. Figure 14 The optical density titration curve of sodium polystyrene sulfonate on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of sodium polystyrene sulfonate corresponding to an optical density greater than 0.1 is 0.7-1.7 mg / mL, and this range is taken as the optimal concentration range.

[0206] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polylysine and 2.5 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0207] 4) Add sodium polystyrene sulfonate to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0208] 2. Fluorescence microscopy examination

[0209] Add 0.1% (w / w) of FITC-labeled polylysine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25The images shown are fluorescence micrographs of the encapsulated products from Examples 13-21. The micrograph corresponding to Example 20 in the figures demonstrates the characteristics of empty capsules in the dispersion state.

[0210] Example 21

[0211] 1. The specific steps for encapsulating polylysine / pyromellitic acid phase-separated droplets induced by dextran sulfate-40 kDa are as follows:

[0212] 1) Titrate with pyromellitic acid by gradually adding sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, and plot the optical density-pyromellitic acid concentration curve. Figure 23 The optical density titration curve of pyromellitic acid on polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of pyromellitic acid corresponding to an optical density greater than 0.1 is 1.6-3.0 mM, and this range is taken as the optimal concentration range.

[0213] 2) Titrate with a gradient of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution (pH 7.5, concentration 20 mM) containing 1 mg / mL polylysine, adding dextran sulfate-40 kDa gradually, and plot the optical density-dextran sulfate-40 kDa concentration curve. Figure 15 The optical density titration curve of dextran sulfate-40 kDa against polylysine solution (sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution) is shown. The concentration range of dextran sulfate-40 kDa corresponding to an optical density greater than 0.1 is 0.5-2.0 mg / mL, and this range is taken as the optimal concentration range.

[0214] 3) Prepare 100 μL of phase separation droplets containing 1 mg / mL polylysine and 2.5 mM pyromellitic acid using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM). Use a vortexing method with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, and then let it stand for 5 min.

[0215] 4) Add 40 kDa of dextran sulfate to the above dispersion to ensure that the final concentration is 1.0 mg / mL (within the optimal concentration range of step 2). Then, use a vortex mixer with an amplitude of 4.5 mm and a rotation speed of 500 rpm for 10 s, followed by centrifugation at 1000 rpm for 3 min. The precipitate is then redispersed using a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.5, concentration 20 mM) to obtain the encapsulated product.

[0216] 2. Fluorescence microscopy observation

[0217] Add 0.1% (w / w) of FITC-labeled polylysine for staining. Take 20 μL of the stained sample onto a glass slide for observation under a fluorescence microscope. Figure 25 The images shown are fluorescence micrographs of the encapsulated products of Examples 13-21. The micrograph corresponding to Example 21 in the figures can demonstrate the characteristics of empty capsules in the dispersion state.

[0218] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing polyelectrolyte capsules using a self-sacrificing soft template, characterized in that, The preparation steps are as follows: 1) Fix the concentration of the polycationic solution, titrate with a small-molecule anionic solution, and plot the curve of optical density as a function of titration molecular concentration. The small-molecule anion concentration range corresponding to an optical density greater than 0.1 is taken as the optimal concentration range. The small-molecule anion is one or more of adenosine triphosphate disodium salt, pyromellitic acid, or sodium 1,2,3,4,5,6-cyclohexanehexacarboxylate. The polycation is one or more of protamine sulfate, polydiallyldimethylammonium chloride, polylysine, and diethylaminoethyl dextran. 2) Fix the concentration of the polycationic solution, titrate with the polyanionic solution, and plot the curve of optical density as a function of the concentration of titrated molecules. The concentration range of the polyanion corresponding to the optical density of the curve greater than 0.1 is taken as the optimal concentration range; the polyanion is heparin sodium, sodium polystyrene sulfonate, or dextran sulfate. 3) Mix the polycationic solution with the small molecule anion solution to bring the concentration of the small molecule anion to the optimal concentration range determined in step 1), forming phase-separated droplets; 4) Add the polyanion solution to the phase separation and coagulation droplets in step 3) to bring the concentration of polyanion to the optimal concentration range determined in step 2). After mixing evenly with a vortex mixer, centrifuge and precipitate and then disperse again to obtain polyelectrolyte capsules.

2. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: The method for determining the optimal concentration range of the small molecule anion solution in step 1) is as follows: Prepare a polycationic solution, titrate it using small molecule anions, measure the absorbance of the titrated sample at 600 nm using a UV-Vis spectrophotometer as the optical density, and plot the correlation curve between optical density and small molecule anion concentration. Take the portion of the curve located in the region where the optical density is greater than 0.1, and take the small molecule anion concentration range of the curve in this region as the optimal concentration range.

3. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: The method for determining the optimal concentration range of the polyanionic solution in step 2) is as follows: Prepare a polycationic solution, titrate with the polycationic solution, measure the absorbance of the titrated sample at 600 nm using a UV-Vis spectrophotometer as the optical density, and plot the correlation curve between optical density and polycationic concentration. Take the portion of the curve located in the region where the optical density is greater than 0.1, and take the polyanionic concentration range of the curve in this region as the optimal concentration range.

4. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: The preparation method of the phase-separated droplets in step 3) is as follows: prepare a polycationic solution and a small molecule anion solution, with the pH of the solution between the pKa of the small molecule anion and the polycation, and control the formation time of the phase-separated droplets to 0-10 min.

5. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: Step 4) The amplitude of the vortex generator is 4.5 mm, the speed is 200-3000 rpm, and the vortex time is 20 s.

6. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: The polycation is protamine sulfate. When the concentration is 1 mg / mL, the optimal concentration is 4-7 mM when the small molecule anion is disodium adenosine triphosphate, 0.7-1.2 mM when the small molecule anion is pyromellitic acid, 0.75-1.2 mg / mL when the polyanion is sodium heparin, 0.8-1.8 mg / mL when the polyanion is sodium polystyrene sulfonate, and 1.6-2.0 mg / mL when the polyanion is dextran sulfate.

7. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: The polycation is polydiallyldimethylammonium chloride. When the concentration is 1 mg / mL, the optimal concentration is 2.0-3 mM when the small molecule anion is adenosine triphosphate disodium salt, 1.6-3 mM when the small molecule anion is pyromellitic acid, 1.2-2.4 mg / mL when the polyanion is heparin sodium, 0.6-1.4 mg / mL when the polyanion is sodium polystyrene sulfonate, and 0.7-2.3 mg / mL when the polyanion is dextran sulfate.

8. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: The polycation is polylysine. When the concentration is 1 mg / mL, the optimal concentration is 3-6 mM when the small molecule anion is disodium adenosine triphosphate, 1.6-3.0 mM when the small molecule anion is pyromellitic acid, 0.8-1.4 mg / mL when the polyanion is sodium heparin, 0.7-1.7 mg / mL when the polyanion is sodium polystyrene sulfonate, and 0.5-2.0 mg / mL when the polyanion is dextran sulfate.

9. The method for preparing polyelectrolyte capsules using a self-sacrificing soft template according to claim 1, characterized in that: The polycation is diethylaminoethyl dextran, and the optimal concentration is 3-12 mM when the small molecule anion is adenosine triphosphate disodium salt; the optimal concentration is 0.5-1.7 mg / mL when the polyanion is heparin sodium; the optimal concentration is 0.8-2.0 mg / mL when the polyanion is sodium polystyrene sulfonate; and the optimal concentration is 0.6-2.0 mg / mL when the polyanion is dextran sulfate.

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