Antibacterial peptide sustained release microsphere preparation and preparation method thereof

Abstract

The invention belongs to the technical field of preparation of microsphere preparations, particularly relates to an antibacterial peptide sustained-release microsphere preparation and a preparation method thereof, and provides the following scheme aiming at the problems of poor stability, short sustained-release period, low encapsulation efficiency and incapability of meeting multi-factor coupling requirements in the preparation process of an existing antibacterial peptide preparation: the antibacterial peptide sustained-release microsphere preparation comprises raw materials, raw materials of the antibacterial peptide composition comprise the following components in parts by weight: 0.5-1.5 parts of antibacterial peptide, and by using the antibacterial peptide, PLGA, PCL, PVA, phospholipid, cholesterol, PEG, dopamine, triethyl citrate, lecithin, Span-80, Tween-80, agarose, sodium alginate, gelatin, polyacrylamide, polylysine and polyglutamic acid, the stability of the antibacterial peptide in a physiological environment is remarkably improved; the half-life period is effectively prolonged, the long-acting antibacterial effect can be continuously achieved, and the problems that the stability is poor, the slow release period is short, the encapsulation efficiency is low and the multi-factor coupling requirement cannot be met are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microsphere preparation, in particular to an antibacterial peptide sustained-release microsphere preparation and a preparation method thereof. BACKGROUND

[0002] Antibacterial peptides are a kind of polypeptide substances with antibacterial activity, which have a wide application prospect in the fields of medicine, animal husbandry, etc. However, the existing antibacterial peptide preparations have many problems, which seriously limit their effect and application range. For example, the stability of antibacterial peptides in physiological environment is poor, and their half-life is usually not more than 2 hours, which is difficult to maintain long-term antibacterial effect.

[0003] The patent document with publication number CN102274485A discloses an antibacterial peptide sustained-release microsphere preparation and a preparation method thereof. The antibacterial peptide sustained-release microsphere preparation comprises a carrier polylactic acid-glycolic acid copolymer and an antibacterial peptide encapsulated in the carrier. The preparation method of the antibacterial peptide sustained-release microsphere preparation adopts a multiple emulsion solvent evaporation method, emulsification is carried out by ultrasonic or high-speed stirring, the organic solvent is volatilized under low-speed stirring, the antibacterial peptide is encapsulated by using the biodegradable polymer material polylactic acid-glycolic acid copolymer as a carrier material, and the polymer sustained-release microspheres are formed. The antibacterial peptide sustained-release microsphere preparation prepared by the method has a sustained-release period of about 30 days, is biodegradable, has good biocompatibility, and can be used for subcutaneous, intramuscular and other non-venous drug delivery.

[0004] Although the above-mentioned patent document uses polylactic acid-glycolic acid copolymer as a carrier material in the preparation process, the protection of the antibacterial peptide by this material is still limited in some complex physiological environments, and only relying on the polylactic acid-glycolic acid copolymer carrier cannot effectively prevent the decomposition of the antibacterial peptide by the proteases produced by microorganisms, so that the antibacterial peptide has poor stability when encountering such enzymes and cannot fully exert its antibacterial effect. Therefore, we propose an antibacterial peptide sustained-release microsphere preparation and a preparation method thereof to solve the above-mentioned problems. SUMMARY

[0005] The purpose of the present application is to solve the problems of poor stability, short sustained-release period, low encapsulation rate and inability to meet the multi-factor coupling requirements of the antibacterial peptide preparation in the prior art, and to propose an antibacterial peptide sustained-release microsphere preparation and a preparation method thereof.

[0006] The antibacterial peptide sustained-release microsphere preparation and the preparation method thereof provided by the present application adopt the following technical solutions:

[0007] The application discloses an antibacterial peptide sustained-release microsphere preparation, which comprises raw materials, and the raw materials are composed of the following components in parts by weight: 0.5-1.5 parts of antibacterial peptide, 1-5 parts of PLGA, 0.5-1.5 parts of PCL, 0.2-1 part of PVA, 0.1-0.5 part of phospholipid, 0.05-0.2 part of cholesterol, 0.1-0.5 part of PEG, 0.05-0.2 part of dopamine, 0.1-0.5 part of triethyl citrate, 0.1-0.5 part of lecithin, 0.05-0.2 part of Span-80, 0.05-0.2 part of Tween-80, 0.1-0.5 part of agarose, 0.1-0.5 part of sodium alginate, 0.1-0.5 part of gelatin, 0.05-0.2 part of polyacrylamide, 0.05-0.2 part of polylysine, 0.05-0.2 part of polyglutamic acid, 0.05-0.2 part of sodium citrate, 0.05-0.2 part of sodium bicarbonate and 0.1-0.5 part of glucose.

[0008] Further, the raw materials are composed of the following components in parts by weight: 0.8-1.2 parts of antibacterial peptide, 2-4 parts of PLGA, 0.8-1.2 parts of PCL, 0.4-0.8 part of PVA, 0.2-0.4 part of phospholipid, 0.08-0.15 part of cholesterol, 0.2-0.4 part of PEG, 0.08-0.15 part of dopamine, 0.2-0.4 part of triethyl citrate, 0.2-0.4 part of lecithin, 0.08-0.15 part of Span-80, 0.08-0.15 part of Tween-80, 0.2-0.4 part of agarose, 0.2-0.4 part of sodium alginate, 0.2-0.4 part of gelatin, 0.08-0.15 part of polyacrylamide, 0.08-0.15 part of polylysine, 0.08-0.15 part of polyglutamic acid, 0.08-0.15 part of sodium citrate, 0.08-0.15 part of sodium bicarbonate and 0.2-0.4 part of glucose.

[0009] Further, the raw materials are composed of the following components in parts by weight: 0.8-1.2 parts of antibacterial peptide, 2-4 parts of PLGA, 0.8-1.2 parts of PCL, 0.4-0.8 part of PVA, 0.2-0.4 part of phospholipid, 0.08-0.15 part of cholesterol, 0.2-0.4 part of PEG, 0.08-0.15 part of dopamine, 0.2-0.4 part of triethyl citrate, 0.2-0.4 part of lecithin, 0.08-0.15 part of Span-80, 0.08-0.15 part of Tween-80, 0.2-0.4 part of agarose, 0.2-0.4 part of sodium alginate, 0.2-0.4 part of gelatin, 0.08-0.15 part of polyacrylamide, 0.08-0.15 part of polylysine, 0.08-0.15 part of polyglutamic acid, 0.08-0.15 part of sodium citrate, 0.08-0.15 part of sodium bicarbonate and 0.2-0.4 part of glucose.

[0010] The application further discloses a preparation method of the antibacterial peptide sustained-release microsphere preparation.

[0011] S1: selecting raw materials and pretreating the raw materials;

[0012] S2: dissolving and mixing the pretreated raw materials to prepare an oil phase;

[0013] S3: adding the oil phase to an oil phase solvent to mix and emulsify, forming a primary emulsion;

[0014] S4: dissolving and mixing the pretreated raw materials to prepare an aqueous phase;

[0015] S5: adding the prepared primary emulsion to the aqueous phase to mix, forming an oil-in-water emulsion;

[0016] S6: transferring the oil-in-water emulsion to a reaction kettle to crosslink and solidify;

[0017] S7: removing impurities from the reacted mixture to prepare a microsphere suspension;

[0018] S8: drying the microsphere suspension and sealing and packaging to obtain a microsphere preparation finished product.

[0019] Further, in S1, the antibacterial peptide is dissolved in an appropriate amount of ethanol under the condition of a temperature of 2-8°C, stirred until completely dissolved, an antibacterial peptide solution is prepared, and placed in a 4°C environment for standby; PLGA, PCL, and PVA are accurately weighed respectively, placed in a drying container, and dried by a vacuum drying oven, the temperature of the vacuum drying oven is 60°C, the drying time is 24h, the water is removed to ensure the subsequent dissolution effect, phospholipid, cholesterol, PEG, and dopamine are placed in another drying container and also subjected to drying treatment.

[0020] Further, in S2 and S3, the dried PLGA, PCL, and PVA are added to a three-necked flask in a predetermined proportion on a sterile operation table, an appropriate amount of ethanol (preheated to 40°C) is slowly added, a magnetic stirrer is turned on, the stirring speed of the magnetic stirrer is 300r / min, and the stirring time is 1h to make it fully dissolved and mixed to form a uniform transparent polymer solution, the pretreated antibacterial peptide solution is slowly added to the polymer solution while stirring, the stirring speed is adjusted to 500r / min, and the stirring time is 30min to make the antibacterial peptide uniformly dispersed in the polymer solution to prepare an oil phase, the oil phase is slowly added to a dichloromethane oil phase solvent containing Span-80 and Tween-80, stirred by a stirring mechanism, the stirring speed is 800r / min, the temperature is 60°C, the emulsification time is 30min, a uniform primary emulsion is formed, and in this process, the emulsification effect is closely observed to ensure that the oil phase and the oil phase solvent are fully mixed to form a stable primary emulsion system.

[0021] Further, in S4 and S5, agarose is added to a proper amount of deionized water, heated to 90℃, and fully dissolved, and lecithin, triethyl citrate, and polyacrylamide are sequentially added, stirred at 60℃ water bath for 30min, so that each component is fully dissolved and mixed, and sodium alginate and gelatin are respectively dissolved in a small amount of warm water, and then added to the mixed solution, and continue to stir for 20min, and then polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate, and glucose are sequentially added, and the pH value of the solution is adjusted to 7.2-7.4, and finally a proper amount of calcium chloride solution (0.2mol / L) is added, stirred uniformly, and the water phase is prepared, the prepared initial milk is slowly poured into the water phase, mixed by a stirring mechanism, the stirring speed is 500r / min, the temperature is 40℃, and the secondary emulsification treatment is carried out for 30min, forming a uniform oil-in-water emulsion, and in this process, the initial milk and the water phase are fully mixed to form a stable oil-in-water emulsion system, which provides a good dispersion environment for the subsequent crosslinking reaction.

[0022] Further, in S6, the emulsion is transferred to a reaction kettle, nitrogen is introduced under the condition of 40℃ and 300r / min stirring, and the reaction is carried out for 2h, in which process, sodium alginate reacts with gelatin, polyacrylamide, polylysine, and polyglutamic acid under the action of calcium ions to form a stable three-dimensional network structure, so that the microspheres are solidified and shaped, and at the same time, dopamine is oxidized to form a polydopamine coating, further enhancing the stability of the microspheres.

[0023] Sodium alginate (Na-Alginate) dissociates alginate ions (-Alginate) and sodium ions (Na + ) in the solution, when there is calcium ion (Ca 2+ ) in the system, alginate ion and calcium ion exchange reaction, form insoluble calcium alginate (Ca-Alginate) gel, sodium alginate and calcium ion crosslinking reaction:

[0024] Na-Alginate+Ca 2+ →Ca-Alginate+2Na +

[0025] Dopamine contains ortho-dihydroxy (catechol structure), under the action of oxidizing agent (such as dissolved oxygen in the system), catechol structure is oxidized to quinone intermediate, quinone intermediate and amino group in dopamine molecule occur Michael addition reaction, form polydopamine (Polydopamine), dopamine oxidation polymerization reaction:

[0026]

[0027] Gelatin contains a large number of amino and carboxyl groups, which can coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between gelatin and calcium ions is as follows:

[0028] Gelatin + Ca 2+ → Cross-linked Gelatin-Ca 2+

[0029] Polyacrylamide can coordinate with calcium ions in solution to form a cross-linked structure. The cross-linking reaction between polyacrylamide and calcium ions is as follows:

[0030] Polyacrylamide + Ca 2+ → Cross-linked Polyacrylamide-Ca 2+

[0031] Polylysine contains multiple amino groups, which can coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between polylysine and calcium ions is as follows:

[0032] Poly - L-lysine + Ca 2+ → Cross-linked Poly - L-lysine-Ca 2+

[0033] Polyglutamic acid contains multiple carboxyl groups, which can coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between polyglutamic acid and calcium ions is as follows:

[0034] Poly - L-glutamic acid + Ca 2+ → Cross-linked

[0035] Poly - L-glutamic acid-Ca 2+

[0036] These reactions collectively build the three-dimensional network structure of the microspheres, solidify and shape the microspheres, and form a polydopamine coating through the oxidation and self-polymerization of dopamine, further enhancing the stability of the microspheres.

[0037] Further, in S7, the mixed solution after reaction is placed in a centrifuge, the centrifuge speed is 10000 r / min, the centrifugation time is 15 min, the microsphere precipitate is separated out, the microsphere precipitate is washed with deionized water for 3-5 times to remove residual unreacted substances and surface impurities, and finally, the microsphere precipitate is resuspended with an appropriate amount of deionized water to prepare a microsphere suspension.

[0038] Further, in S8, the microsphere suspension is transferred to a spray drying device, the inlet air temperature of the spray drying device is 80 DEG C, the flow rate is 50 m 3 / h, and spray drying is carried out under continuous stirring, in the drying process, the microspheres rapidly remove the solvent under the action of hot air to form uniform dry microsphere powder, after drying is completed, the microsphere powder is collected in a dry glass container, sealed and packaged, and stored in a 4 DEG C environment to obtain a microsphere preparation finished product.

[0039] In summary, the present application includes at least one of the following beneficial technical effects:

[0040] 1. The present application can precisely control the structure and performance of the microspheres through multiple emulsification, crosslinking and curing steps, realizes efficient encapsulation of the antibacterial peptide, greatly improves the encapsulation efficiency, reduces the loss of the antibacterial peptide in the preparation process, reduces the production cost, and improves the production efficiency.

[0041] 2. The present application uses dopamine, which is self-polymerized to form a polydopamine coating under the action of oxidation, further enhances the stability of the microspheres, endows the microspheres with good mechanical properties and environmental interference resistance, ensures the integrity and sustained release function of the microspheres in complex physiological environment and storage and transportation process, and the polydopamine coating also has certain biocompatibility and degradability, which does not affect the safety of the microsphere preparation.

[0042] The present application uses antibacterial peptide, PLGA, PCL, PVA, phospholipid, cholesterol, PEG, dopamine, triethyl citrate, lecithin, Span-80, Tween-80, agarose, sodium alginate, gelatin, polyacrylamide, polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate and glucose, which significantly improves the stability of the antibacterial peptide in the physiological environment, effectively prolongs the half-life, enables the antibacterial peptide to continuously exert long-acting antibacterial effect, and overcomes the problems of poor stability, short sustained release period, low encapsulation efficiency and inability to meet the multi-factor coupling requirement. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 A flowchart of a preparation method of an antibacterial peptide sustained-release microsphere preparation according to the present application is shown. DETAILED DESCRIPTION

[0044] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments.

[0045] Embodiment one

[0046] Reference Figure 1The application discloses an antibacterial peptide sustained-release microsphere preparation, and relates to the technical field of antibacterial peptide sustained-release microsphere preparations.

[0047] The application further discloses a preparation method of the antibacterial peptide sustained-release microsphere preparation.

[0048] S1: under the condition of a temperature of 2-8 DEG C, antibacterial peptides are dissolved in proper ethanol, and stirring is performed until complete dissolution to obtain antibacterial peptide solution, which is stored in a 4 DEG C environment for standby; PLGA, PCL and PVA are respectively accurately weighed and stored in a drying container, and drying treatment is performed through a vacuum drying box, wherein the temperature of the vacuum drying box is 60 DEG C, the drying time is 24 h, water is removed, and the subsequent dissolution effect is ensured; phospholipid, cholesterol, PEG and dopamine are stored in another drying container, and drying treatment is also performed;

[0049] S2: in a sterile operation table, the dried PLGA, PCL and PVA are added into a three-necked flask according to a predetermined proportion, proper ethanol (preheated to 40 DEG C) is slowly added, a magnetic stirrer is started, the rotating speed of the magnetic stirrer is 300 r / min, and stirring is performed for 1 h, so that the PLGA, PCL and PVA are fully dissolved and mixed to form a uniform and transparent polymer solution; the pre-processed antibacterial peptide solution is slowly added into the polymer solution, stirring is performed during the adding process, the stirring speed is adjusted to 500 r / min, and stirring is performed for 30 min, so that the antibacterial peptides are uniformly dispersed in the polymer solution to obtain an oil phase;

[0050] S3: the oil phase is slowly added into dichloromethane containing Span-80 and Tween-80, stirring is performed through a stirring mechanism, the stirring speed is 800 r / min, the temperature is 60 DEG C, and emulsification is performed for 30 min, so that a uniform primary emulsion is formed; in the process, the emulsification effect is closely observed, so that the oil phase and the oil phase solvent are fully mixed, and a stable primary emulsion system is formed;

[0051] S4: add agarose to a certain amount of deionized water, heat to 90℃, make it completely dissolved, add lecithin, triethyl citrate, polyacrylamide in turn, stir for 30 min under the condition of 60℃ water bath, make each component fully dissolved and mixed, dissolve sodium alginate and gelatin with a small amount of warm water respectively, add them to the mixed solution, continue to stir for 20 min, add polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate, glucose in turn, adjust the pH value of the solution to 7.2-7.4, finally add a certain amount of calcium chloride solution (0.2 mol / L), stir uniformly, and prepare the water phase;

[0052] S5: slowly pour the prepared colostrum into the water phase, mix through the stirring mechanism, the stirring speed is 500 r / min, the temperature is 40℃, carry out secondary emulsification treatment, the treatment time is 30 min, form a uniform oil-in-water emulsion, in this process, ensure that the colostrum and the water phase are fully mixed to form a stable oil-in-water emulsion system, which provides a good dispersion environment for the subsequent crosslinking reaction;

[0053] S6: transfer the emulsion to the reaction kettle, under the condition of 40℃ and 300 r / min stirring, pass nitrogen protection, react for 2 h, in this process, sodium alginate reacts with gelatin, polyacrylamide, polylysine and polyglutamic acid under the action of calcium ions to form a stable three-dimensional network structure, so that the microspheres are solidified and shaped, at the same time, dopamine is oxidized to form a polydopamine coating, which further enhances the stability of the microspheres;

[0054] Sodium alginate (Na-Alginate) dissociates alginate ion (-Alginate) and sodium ion (Na + ) in the solution, when there is calcium ion (Ca 2+ ) in the system, alginate ion reacts with calcium ion to form insoluble calcium alginate (Ca-Alginate) gel, the crosslinking reaction of sodium alginate and calcium ion:

[0055] Na-Alginate+Ca 2+ →Ca-Alginate+2Na +

[0056] Dopamine contains ortho-dihydroxy (catechol structure), under the action of oxidizing agent (such as dissolved oxygen in the system), catechol structure is oxidized to quinone intermediate, quinone intermediate reacts with amino group in dopamine molecule to form polydopamine (Polydopamine), the oxidation and self-polymerization reaction of dopamine:

[0057]

[0058] Gelatin contains a large number of amino and carboxyl groups, which coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between gelatin and calcium ions is as follows:

[0059] Gelatin + Ca 2+ → Cross-linked Gelatin-Ca 2+

[0060] Polyacrylamide coordinates with calcium ions in solution to form a cross-linked structure. The cross-linking reaction between polyacrylamide and calcium ions is as follows:

[0061] Polyacrylamide + Ca 2+ → Cross-linked Polyacrylamide-Ca 2+

[0062] Polylysine contains multiple amino groups, which coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between polylysine and calcium ions is as follows:

[0063] Poly - L-lysine + Ca 2+ → Cross-linked Poly - L-lysine-Ca 2+

[0064] Polyglutamic acid contains multiple carboxyl groups, which coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between polyglutamic acid and calcium ions is as follows:

[0065] Poly - L-glutamic acid + Ca 2+ → Cross-linked

[0066] Poly - L-glutamic acid-Ca 2+

[0067] These reactions collectively build the three-dimensional network structure of the microspheres, solidify and shape the microspheres, and form a polydopamine coating through the oxidative self-polymerization of dopamine, further enhancing the stability of the microspheres.

[0068] S7: After the reaction, the mixed solution is placed in a centrifuge with a speed of 10000 r / min for 15 min. The microsphere precipitate is washed with deionized water for 3-5 times to remove residual unreacted substances and surface impurities. After the last washing, the microsphere precipitate is resuspended with an appropriate amount of deionized water to prepare a microsphere suspension.

[0069] S8: The microsphere suspension is transferred to a spray drying device, the inlet air temperature of the spray drying device is 80℃, the flow rate is 50m 3 / h, and the spray drying is carried out under continuous stirring, during which the microspheres rapidly remove the solvent under the action of hot air to form uniform dry microsphere powder, after the drying is completed, the microsphere powder is collected in a dry glass container, sealed and packaged, and stored in a 4℃ environment to obtain a microsphere preparation product.

[0070] Example Two

[0071] With reference to Figure 1 The antibacterial peptide sustained-release microsphere preparation comprises the following components in parts by weight: 0.8 parts of antibacterial peptide, 2 parts of PLGA, 0.8 parts of PCL, 0.4 parts of PVA, 0.2 parts of phospholipid, 0.8 parts of cholesterol, 0.2 parts of PEG, 0.8 parts of dopamine, 0.2 parts of triethyl citrate, 0.2 parts of lecithin, 0.08 parts of Span-80, 0.08 parts of Tween-80, 0.2 parts of agarose, 0.2 parts of sodium alginate, 0.2 parts of gelatin, 0.08 parts of polyacrylamide, 0.08 parts of polylysine, 0.08 parts of polyglutamic acid, 0.08 parts of sodium citrate, 0.08 parts of sodium bicarbonate and 0.2 parts of glucose.

[0072] The application further provides a preparation method of the antibacterial peptide sustained-release microsphere preparation.

[0073] S1: under the condition of a temperature of 2-8℃, the antibacterial peptide is dissolved with an appropriate amount of ethanol, and stirring is performed until complete dissolution to prepare an antibacterial peptide solution, which is stored in a 4℃ environment for standby; PLGA, PCL and PVA are respectively accurately weighed and placed in a drying container, and drying treatment is performed through a vacuum drying box, the temperature of the vacuum drying box is 60℃, the drying time is 24h, water is removed to ensure the subsequent dissolution effect, and phospholipid, cholesterol, PEG and dopamine are placed in another drying container and subjected to the same drying treatment;

[0074] S2: on a sterile operation table, the dried PLGA, PCL and PVA are added to a three-necked flask according to a predetermined proportion, an appropriate amount of ethanol (preheated to 40℃) is slowly added, a magnetic stirrer is started, the rotating speed of the magnetic stirrer is 300r / min, and stirring is performed for 1h to make the PLGA, PCL and PVA fully dissolved and mixed to form a uniform transparent polymer solution; the pre-processed antibacterial peptide solution is slowly added to the polymer solution while stirring, the stirring speed is adjusted to 500r / min, and stirring is performed for 30min to make the antibacterial peptide uniformly dispersed in the polymer solution to prepare an oil phase;

[0075] S3: Slowly add the oil phase into the dichloromethane oil phase solvent containing Span-80, Tween-80, and stir through the stirring mechanism, the stirring speed is 800 r / min, the temperature is 60℃, the emulsification time is 30 min, and a uniform primary emulsion is formed. In this process, the emulsification effect is closely observed to ensure that the oil phase and the oil phase solvent are fully mixed to form a stable primary emulsion system;

[0076] S4: Add agarose to a suitable amount of deionized water, heat to 90℃, and dissolve completely. Add lecithin, triethyl citrate, and polyacrylamide in sequence, and stir at 60℃ water bath for 30 min to fully dissolve and mix the components. Dissolve sodium alginate and gelatin in a small amount of warm water separately, and add them to the mixture. Continue stirring for 20 min. Add polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate, and glucose in sequence, and adjust the pH of the solution to 7.2-7.4. Finally, add an appropriate amount of calcium chloride solution (0.2 mol / L) and stir evenly to prepare the water phase;

[0077] S5: Slowly pour the prepared primary emulsion into the water phase and mix through the stirring mechanism, with a stirring speed of 500 r / min and a temperature of 40℃. Perform secondary emulsification treatment for 30 min to form a uniform oil-in-water emulsion. In this process, ensure that the primary emulsion and the water phase are fully mixed to form a stable oil-in-water emulsion system, providing a good dispersion environment for subsequent cross-linking reactions;

[0078] S6: Transfer the emulsion to a reaction kettle and stir at 40℃ and 300 r / min under nitrogen protection for 2 h. In this process, sodium alginate reacts with gelatin, polyacrylamide, polylysine, and polyglutamic acid under the action of calcium ions to form a stable three-dimensional network structure, solidifying and shaping the microspheres. At the same time, dopamine self-polymerizes to form a polydopamine coating, further enhancing the stability of the microspheres;

[0079] Sodium alginate (Na-Alginate) dissociates into alginate ions (-Alginate) and sodium ions (Na + ) in solution. When calcium ions (Ca 2+ ) are present in the system, alginate ions exchange with calcium ions to form insoluble calcium alginate (Ca-Alginate) gel. The cross-linking reaction of sodium alginate and calcium ions:

[0080] Na-Alginate + Ca 2+ → Ca-Alginate + 2Na +

[0081] Dopamine contains ortho-dihydroxy (catechol structure), under the action of oxidizing agent (such as dissolved oxygen in the system), catechol structure is oxidized into quinone intermediate, quinone intermediate and amino in dopamine molecule occur Michael addition reaction, forming polydopamine (polydopamine), oxidation self-polymerization of dopamine:

[0082]

[0083] Gelatin contains a large number of amino and carboxyl groups, which coordinate with calcium ions to form cross-linked structure, gelatin and calcium ion cross-linking reaction:

[0084] Gelatin+Ca 2+ →Cross-linkedGelatin-Ca 2+

[0085] Polyacrylamide in solution and calcium ion coordinate reaction, forming cross-linked structure, polyacrylamide and calcium ion cross-linking reaction:

[0086] Polyacrylamide+Ca 2+ →Cross-linkedPolyacrylamide-Ca 2+

[0087] Polylysine contains multiple amino groups, which coordinate with calcium ions to form cross-linked structure, polylysine and calcium ion cross-linking reaction:

[0088] Poly - L-lysine+Ca 2+ →Cross-linkedPoly - L-lysine-Ca 2+

[0089] Polyglutamic acid contains multiple carboxyl groups, which coordinate with calcium ions to form cross-linked structure, polyglutamic acid and calcium ion cross-linking reaction:

[0090] Poly - L-glutamicacid+Ca 2+ →Cross-linked

[0091] Poly - L-glutamicacid-Ca 2+

[0092] These reactions together build the three-dimensional network structure of the microspheres, make the microspheres solidification and setting, and form polydopamine coating through the oxidation self-polymerization of dopamine, further enhance the stability of the microspheres;

[0093] S7: the mixed solution after reaction is placed in a centrifuge, the centrifuge rotates at 10000r / min, the centrifugation time is 15min, the microsphere precipitate is separated, the microsphere precipitate is washed with deionized water for 3-5 times, the residual unreacted substances and surface impurities are removed, after the last washing, the microsphere precipitate is resuspended with appropriate deionized water, and a microsphere suspension is prepared;

[0094] S8: the microsphere suspension is transferred to a spray drying device, the inlet air temperature of the spray drying device is 80℃, the flow rate is 50m 3 / h, spray drying is carried out under continuous stirring, in the drying process, the microspheres rapidly remove the solvent under the action of hot air to form uniform dry microsphere powder, after drying is completed, the microsphere powder is collected in a dry glass container, sealed and packaged, and stored in a 4℃ environment to obtain a microsphere preparation finished product.

[0095] Example Three

[0096] Reference Figure 1 The application further provides a preparation method of the antibacterial peptide sustained-release microsphere preparation, and the antibacterial peptide sustained-release microsphere preparation is the antibacterial peptide sustained-release microsphere preparation as described above, and the preparation method comprises the following steps:

[0097] The application further provides a preparation method of the antibacterial peptide sustained-release microsphere preparation, and the antibacterial peptide sustained-release microsphere preparation is the antibacterial peptide sustained-release microsphere preparation as described above, and the preparation method comprises the following steps:

[0098] S1: under the condition that the temperature is 2-8℃, the antibacterial peptide is dissolved in appropriate ethanol, and stirring is performed until complete dissolution to prepare an antibacterial peptide solution, which is stored in a 4℃ environment for standby; PLGA, PCL and PVA are respectively accurately weighed and placed in a drying container, and drying treatment is performed through a vacuum drying box, the temperature of the vacuum drying box is 60℃, the drying time is 24h, water is removed to ensure subsequent dissolution effect, and phospholipid, cholesterol, PEG and dopamine are placed in another drying container and subjected to the same drying treatment;

[0099] S2: In the sterile operating table, the dried PLGA, PCL, PVA is added to the three-necked flask according to the predetermined ratio, and a proper amount of ethanol (preheated to 40℃) is slowly added, and the magnetic stirrer is started, the stirring speed is 300r / min, the stirring time is 1h, so that it is fully dissolved and mixed to form a uniform transparent polymer solution, the previously treated antibacterial peptide solution is slowly added to the polymer solution, and the stirring speed is adjusted to 500r / min, the stirring time is 30min, so that the antibacterial peptide is uniformly dispersed in the polymer solution, and the oil phase is prepared;

[0100] S3: The oil phase is slowly added to the dichloromethane oil phase solvent containing Span-80 and Tween-80, and stirred by the stirring mechanism, the stirring speed is 800r / min, the temperature is 60℃, the emulsification time is 30min, and a uniform primary emulsion is formed. In this process, the emulsification effect is closely observed to ensure that the oil phase and the oil phase solvent are fully mixed to form a stable primary emulsion system;

[0101] S4: Agarose is added to a proper amount of deionized water, heated to 90℃, and fully dissolved, lecithin, triethyl citrate, and polyacrylamide are added in turn, and stirred at 60℃ water bath for 30min to fully dissolve and mix the components. Sodium alginate and gelatin are dissolved in a small amount of warm water respectively, and added to the mixed solution, and continue to stir for 20min. Polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate, and glucose are added in turn, and the pH value of the solution is adjusted to 7.2-7.4. Finally, a proper amount of calcium chloride solution (0.2mol / L) is added, and stirred uniformly to prepare the water phase;

[0102] S5: The prepared primary emulsion is slowly poured into the water phase, mixed by the stirring mechanism, the stirring speed is 500r / min, the temperature is 40℃, and the secondary emulsification treatment is carried out, the treatment time is 30min, and a uniform oil-in-water emulsion is formed. In this process, the primary emulsion and the water phase are fully mixed to form a stable oil-in-water emulsion system, which provides a good dispersion environment for the subsequent crosslinking reaction;

[0103] S6: The emulsion is transferred to the reaction kettle, and nitrogen is introduced under the conditions of 40℃ and 300r / min stirring for 2h. In this process, sodium alginate reacts with gelatin, polyacrylamide, polylysine, and polyglutamic acid under the action of calcium ions to form a stable three-dimensional network structure, which solidifies and shapes the microspheres. At the same time, dopamine is oxidized to form a polydopamine coating, which further enhances the stability of the microspheres;

[0104] Sodium alginate (Na-Alginate) dissociates into alginate ions (-Alginate) and sodium ions (Na + ) in the solution, and calcium ions (Ca2+ ) When the concentration of Ca2+ is higher than that of alginate, the ion exchange reaction between alginate and Ca2+ occurs, forming insoluble Ca-Alginate gel. The cross-linking reaction between Na-Alginate and Ca2+:

[0105] Na-Alginate + Ca 2+ → Ca-Alginate + 2Na +

[0106] Dopamine contains o-dihydroxy (catechol structure), under the action of oxidizing agent (such as dissolved oxygen in the system), catechol structure is oxidized to quinone intermediate, quinone intermediate and amino in dopamine molecule occur Michael addition reaction, forming polydopamine (Polydopamine), oxidation and self-polymerization of dopamine:

[0107]

[0108] Gelatin contains a large number of amino and carboxyl groups, which can coordinate with Ca2+ to form cross-linked structure. The cross-linking reaction between gelatin and Ca2+:

[0109] Gelatin + Ca 2+ → Cross-linked Gelatin-Ca 2+

[0110] Polyacrylamide can coordinate with Ca2+ in solution to form cross-linked structure. The cross-linking reaction between polyacrylamide and Ca2+:

[0111] Polyacrylamide + Ca 2+ → Cross-linked Polyacrylamide-Ca 2+

[0112] Polylysine contains multiple amino groups, which can coordinate with Ca2+ to form cross-linked structure. The cross-linking reaction between polylysine and Ca2+:

[0113] Poly - L-lysine + Ca 2+ → Cross-linked Poly - L-lysine-Ca 2+

[0114] Polyglutamic acid contains multiple carboxyl groups, which can coordinate with Ca2+ to form cross-linked structure. The cross-linking reaction between polyglutamic acid and Ca2+:

[0115] Poly - L-glutamic acid + Ca 2+→ Cross-linked

[0116] Poly - L-glutamicacid-Ca 2+

[0117] These reactions jointly build the three-dimensional network structure of the microspheres, solidify and shape the microspheres, and form a polydopamine coating through the oxidation self-polymerization of dopamine, further enhancing the stability of the microspheres;

[0118] S7: the mixed solution after reaction is placed in a centrifuge, the centrifuge rotates at 10000r / min, and the centrifugation time is 15min, the microsphere precipitate is separated out, the microsphere precipitate is washed 3-5 times with deionized water to remove residual unreacted substances and surface impurities, and after the last washing, the microsphere precipitate is resuspended with appropriate deionized water to prepare a microsphere suspension;

[0119] S8: the microsphere suspension is transferred to a spray drying device, the inlet air temperature of the spray drying device is 80℃, the flow rate is 50m 3 / h, and spray drying is carried out under continuous stirring, in the drying process, the microspheres rapidly remove the solvent under the action of hot air to form uniform dry microsphere powder, after drying is completed, the microsphere powder is collected in a dry glass container, sealed and packaged, and stored in a 4℃ environment to obtain a microsphere preparation finished product.

[0120] Example Four

[0121] With reference to Figure 1 An antibacterial peptide sustained-release microsphere preparation, including raw materials, the raw materials include the following components in parts by weight: antibacterial peptide 1.2 parts, PLGA 4 parts, PCL 1.2 parts, PVA 0.8 parts, phospholipid 0.4 parts, cholesterol 0.15 parts, PEG 0.4 parts, dopamine 0.15 parts, triethyl citrate 0.4 parts, lecithin 0.4 parts, Span-800 0.15 parts, Tween-80 0.15 parts, agarose 0.4 parts, sodium alginate 0.4 parts, gelatin 0.4 parts, polyacrylamide 0.15 parts, polylysine 0.15 parts, polyglutamic acid 0.15 parts, sodium citrate 0.15 parts, sodium bicarbonate 0.15 parts, glucose 0.4 parts.

[0122] The application further provides a preparation method of the antibacterial peptide sustained-release microsphere preparation.

[0123] S1: under the condition of temperature 2-8℃, the antibacterial peptide is dissolved with appropriate amount of ethanol, stirred until completely dissolved, to prepare an antibacterial peptide solution, and placed in a 4℃ environment for standby; respectively accurately weigh PLGA, PCL, PVA, and place in a dry container, dry by vacuum drying oven, the temperature of vacuum drying oven is 60℃, the drying time is 24h, remove the moisture to ensure the subsequent dissolution effect, and place phospholipid, cholesterol, PEG, dopamine in another dry container, and also dry;

[0124] S2: on the sterile operation table, the dried PLGA, PCL, PVA are added to a three-necked flask according to the predetermined ratio, and appropriate amount of ethanol (preheated to 40℃) is slowly added, and a magnetic stirrer is started, the stirring speed of the magnetic stirrer is 300r / min, and the stirring time is 1h, so that it is fully dissolved and mixed to form a uniform transparent polymer solution, the previously treated antibacterial peptide solution is slowly added to the polymer solution, and the stirring speed is adjusted to 500r / min, and the stirring time is 30min, so that the antibacterial peptide is uniformly dispersed in the polymer solution, and an oil phase is prepared;

[0125] S3: the oil phase is slowly added to the dichloromethane oil phase solvent containing Span-80 and Tween-80, and stirred by a stirring mechanism, the stirring speed is 800r / min, the temperature is 60℃, and the emulsification time is 30min, to form a uniform primary emulsion, in this process, the emulsification effect is closely observed to ensure that the oil phase and the oil phase solvent are fully mixed to form a stable primary emulsion system;

[0126] S4: agarose is added to appropriate amount of deionized water, heated to 90℃, and fully dissolved, and lecithin, triethyl citrate, and polyacrylamide are sequentially added, stirred at 60℃ water bath for 30min, so that the components are fully dissolved and mixed, and sodium alginate and gelatin are respectively dissolved with a small amount of warm water, and added to the mixed solution, and continue to stir for 20min, and polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate, and glucose are sequentially added, and the pH value of the solution is adjusted to 7.2-7.4, and finally appropriate amount of calcium chloride solution (0.2mol / L) is added, and stirred uniformly to prepare an aqueous phase;

[0127] S5: the prepared primary emulsion is slowly poured into the aqueous phase, mixed by a stirring mechanism, the stirring speed is 500r / min, the temperature is 40℃, and the secondary emulsification treatment is performed for 30min to form a uniform oil-in-water emulsion, in this process, the primary emulsion and the aqueous phase are fully mixed to form a stable oil-in-water emulsion system, which provides a good dispersion environment for the subsequent crosslinking reaction;

[0128] S6: The emulsion is transferred to a reaction kettle, and nitrogen is introduced for protection under the conditions of 40℃ and 300r / min stirring for 2h. In this process, sodium alginate reacts with gelatin, polyacrylamide, polylysine and polyglutamic acid under the action of calcium ions to form a stable three-dimensional network structure, so that the microspheres are solidified and shaped. At the same time, dopamine is oxidized to form a polydopamine coating, further enhancing the stability of the microspheres.

[0129] Sodium alginate (Na-Alginate) dissociates alginate ions (-Alginate) and sodium ions (Na + ) in solution. When calcium ions (Ca 2+ ) exist in the system, alginate ions exchange with calcium ions to form insoluble calcium alginate (Ca-Alginate) gel. The cross-linking reaction of sodium alginate and calcium ions:

[0130] Na-Alginate + Ca 2+ → Ca-Alginate + 2Na +

[0131] Dopamine contains ortho-dihydroxy (catechol structure), which is oxidized to quinone intermediate under the action of oxidizing agent (such as dissolved oxygen in the system). The quinone intermediate and the amino group in the dopamine molecule undergo Michael addition reaction to form polydopamine. The oxidation and self-polymerization reaction of dopamine:

[0132]

[0133] Gelatin contains a large number of amino and carboxyl groups, which form cross-linked structures by coordinating with calcium ions. The cross-linking reaction of gelatin and calcium ions:

[0134] Gelatin + Ca 2+ → Cross-linked Gelatin-Ca 2+

[0135] Polyacrylamide in solution coordinates with calcium ions to form cross-linked structures. The cross-linking reaction of polyacrylamide and calcium ions:

[0136] Polyacrylamide + Ca 2+ → Cross-linked Polyacrylamide-Ca 2+

[0137] Polylysine contains multiple amino groups, which form cross-linked structures by coordinating with calcium ions. The cross-linking reaction of polylysine and calcium ions:

[0138] Poly - L-lysine+Ca 2+ →Cross-linkedPoly - L-lysine-Ca 2+

[0139] Poly

[0140] Poly - L-glutamicacid+Ca 2+ →Cross-linked

[0141] Poly - L-glutamicacid-Ca 2+

[0142] These reactions together build the three-dimensional network structure of the microspheres, solidify the microspheres, and form a polydopamine coating through the oxidation and self-polymerization of dopamine, further enhancing the stability of the microspheres;

[0143] S7: The mixed solution after reaction is placed in a centrifuge, the centrifuge speed is 10000r / min, the centrifugation time is 15min, the microsphere precipitate is separated out, the microsphere precipitate is washed with deionized water for 3-5 times to remove residual unreacted substances and surface impurities, after the last washing, the microsphere precipitate is resuspended with appropriate deionized water to prepare a microsphere suspension;

[0144] S8: The microsphere suspension is transferred to a spray drying device, the inlet air temperature of the spray drying device is 80℃, the flow rate is 50m 3 / h, the spray drying is carried out under continuous stirring, during the drying process, the microspheres rapidly remove the solvent under the action of hot air to form uniform dry microsphere powder, after the drying is completed, the microsphere powder is collected in a dry glass container, sealed and packaged, and stored in a 4℃ environment to obtain the finished product of the microsphere preparation.

[0145] Example Five

[0146] Referring to Figure 1The application discloses an antibacterial peptide sustained-release microsphere preparation, and relates to the technical field of antibacterial peptide sustained-release microsphere preparation.

[0147] The application further discloses a preparation method of the antibacterial peptide sustained-release microsphere preparation.

[0148] S1: under the condition of a temperature of 2-8 DEG C, antibacterial peptides are dissolved in proper ethanol, and stirring is performed until complete dissolution to obtain antibacterial peptide solution, which is stored in a 4 DEG C environment for standby; PLGA, PCL and PVA are respectively accurately weighed and stored in a drying container, and drying treatment is performed through a vacuum drying box, wherein the temperature of the vacuum drying box is 60 DEG C, the drying time is 24 h, water is removed, and the subsequent dissolution effect is ensured; phospholipid, cholesterol, PEG and dopamine are stored in another drying container, and drying treatment is also performed;

[0149] S2: on a sterile operation table, the dried PLGA, PCL and PVA are added into a three-necked flask according to a predetermined proportion, proper ethanol (preheated to 40 DEG C) is slowly added, a magnetic stirrer is started, the rotating speed of the magnetic stirrer is 300 r / min, and stirring is performed for 1 h, so that the PLGA, PCL and PVA are fully dissolved and mixed to form a uniform and transparent polymer solution; the pre-processed antibacterial peptide solution is slowly added into the polymer solution while stirring, the stirring speed is adjusted to 500 r / min, and stirring is performed for 30 min, so that the antibacterial peptides are uniformly dispersed in the polymer solution to obtain an oil phase;

[0150] S3: the oil phase is slowly added into dichloromethane containing Span-80 and Tween-80, stirring is performed through a stirring mechanism, the stirring speed is 800 r / min, the temperature is 60 DEG C, and emulsification is performed for 30 min, so that a uniform primary emulsion is formed; in the process, the emulsification effect is closely observed, so that the oil phase and the oil phase solvent are fully mixed to form a stable primary emulsion system;

[0151] S4: Add agarose to a certain amount of deionized water, heat to 90℃, and make it completely dissolved. Add lecithin, triethyl citrate, and polyacrylamide in sequence, and stir for 30 min under the condition of 60℃ water bath to make each component fully dissolved and mixed. Dissolve sodium alginate and gelatin in a small amount of warm water respectively, and add them to the mixed solution. Continue to stir for 20 min. Add polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate, and glucose in sequence, and adjust the pH value of the solution to 7.2-7.4. Finally, add a certain amount of calcium chloride solution (0.2 mol / L), stir uniformly, and prepare the water phase;

[0152] S5: Slowly pour the prepared colostrum into the water phase, mix through the stirring mechanism, the stirring speed is 500 r / min, the temperature is 40℃, and the secondary emulsification treatment is carried out for 30 min, forming a uniform oil-in-water emulsion. In this process, ensure that the colostrum and the water phase are fully mixed to form a stable oil-in-water emulsion system, which provides a good dispersion environment for the subsequent cross-linking reaction;

[0153] S6: Transfer the emulsion to the reaction kettle, and under the condition of 40℃ and 300 r / min stirring, pass nitrogen protection, and react for 2 h. In this process, sodium alginate reacts with gelatin, polyacrylamide, polylysine, and polyglutamic acid under the action of calcium ions to form a stable three-dimensional network structure, which makes the microspheres solidify and shape. At the same time, dopamine forms a polydopamine coating under the action of oxidation, which further enhances the stability of the microspheres;

[0154] When sodium alginate (Na-Alginate) is dissolved in a solution, alginate ions (-Alginate) and sodium ions (Na + ) are dissociated. When there is calcium ion (Ca 2+ ) in the system, alginate ion reacts with calcium ion to form insoluble calcium alginate (Ca-Alginate) gel. The cross-linking reaction of sodium alginate and calcium ion:

[0155] Na-Alginate + Ca 2+ → Ca-Alginate + 2Na +

[0156] Dopamine contains ortho-dihydroxy (catechol structure). Under the action of oxidizing agent (such as dissolved oxygen in the system), catechol structure is oxidized into quinone intermediate, and quinone intermediate reacts with amino group in dopamine molecule to form polydopamine (Polydopamine). The oxidation and self-polymerization reaction of dopamine:

[0157]

[0158] Gelatin contains a large number of amino and carboxyl groups, which coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between gelatin and calcium ions is as follows:

[0159] Gelatin + Ca 2+ → Cross-linked Gelatin-Ca 2+

[0160] Polyacrylamide in solution coordinates with calcium ions to form a cross-linked structure. The cross-linking reaction between polyacrylamide and calcium ions is as follows:

[0161] Polyacrylamide + Ca 2+ → Cross-linked Polyacrylamide-Ca 2+

[0162] Polylysine contains multiple amino groups, which coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between polylysine and calcium ions is as follows:

[0163] Poly - L-lysine + Ca 2+ → Cross-linked Poly - L-lysine-Ca 2+

[0164] Polyglutamic acid contains multiple carboxyl groups, which coordinate with calcium ions to form a cross-linked structure. The cross-linking reaction between polyglutamic acid and calcium ions is as follows:

[0165] Poly - L-glutamic acid + Ca 2+ → Cross-linked

[0166] Poly - L-glutamic acid-Ca 2+

[0167] These reactions collectively build the three-dimensional network structure of the microspheres, allowing them to solidify and set, and form a polydopamine coating through the oxidative self-polymerization of dopamine, further enhancing the stability of the microspheres.

[0168] S7: After the reaction, the mixed solution is placed in a centrifuge with a speed of 10000 r / min for 15 min. The microsphere precipitate is washed with deionized water for 3-5 times to remove residual unreacted substances and surface impurities. After the last washing, the microsphere precipitate is resuspended with an appropriate amount of deionized water to prepare a microsphere suspension.

[0169] S8: The microsphere suspension is transferred to a spray drying device, the inlet air temperature of the spray drying device is 80℃, the flow rate is 50m 3 / h, and the spray drying is carried out under continuous stirring. During the spray drying, the microspheres rapidly remove the solvent under the action of hot air to form uniform dry microsphere powder. After the drying is completed, the microsphere powder is collected in a dry glass container, sealed and packaged, and stored in a 4℃ environment to obtain a microsphere preparation finished product.

[0170] Experimental Example

[0171] I. Experimental group setting

[0172] The present application group (Examples 1-5)

[0173] Microsphere structure features: PLGA / PCL framework + ALG-Ca 2+ Crosslinking + PDA coating;

[0174] Explanation: By fine-tuning the proportion of each component, the performance of the microspheres is optimized to achieve long-acting sustained release of antibacterial peptides.

[0175] Control group 1

[0176] Microsphere structure features: only PLGA / PCL framework (without crosslinking / coating);

[0177] Difference from the example: delete ALG-Ca 2+ Crosslinking network and PDA coating construction steps, the rest of the preparation process is completely consistent with the example;

[0178] Purpose: to highlight the basic role of the PLGA / PCL framework alone in sustained release;

[0179] Control group 2

[0180] Microsphere structure features: PLGA / PCL framework + ALG-Ca 2+ Crosslinking (without coating);

[0181] Difference from the example: retain the PLGA / PCL framework and ALG-Ca 2+ Crosslinking network construction, but in step S6, dopamine addition and self-polymerization are omitted, i.e. the PDA coating forming link is removed, and the rest of the operation is the same as the example;

[0182] Purpose: to explore the performance of only the framework and the crosslinking network, and to highlight the unique contribution of the PDA coating;

[0183] Control group 3

[0184] Microsphere structure features: ALG-Ca 2+ Crosslinked microspheres (without polymer framework);

[0185] Differences from the examples: omit the polymer backbone related operations (do not add PLGA, PCL, PVA, etc. in S2), directly build microspheres with water phase ingredients, and the rest of the steps are as consistent as possible with the examples except for the backbone part;

[0186] Objective: To evaluate the feasibility and performance differences when relying only on the cross-linked network without a polymer backbone;

[0187] Examples 1-5 are compared with control groups 1-3, respectively;

[0188] II. Test indicators

[0189] Encapsulation efficiency (EE%): HPLC method is used to determine the content of antimicrobial peptide, and the encapsulation efficiency is calculated. The higher the encapsulation efficiency, the higher the efficiency of antimicrobial peptide encapsulation, which is beneficial to reduce the loss of antimicrobial peptide during preparation and storage;

[0190] Burst release rate (24h release amount): Initial release experiment in PBS buffer (pH 7.4), determine the release amount of antimicrobial peptide within 24 hours, the lower the burst release rate, the better the control of the initial burst release behavior of the microspheres to the antimicrobial peptide, and the more persistent the antibacterial effect;

[0191] Sustained release period: The time required for the release rate of antimicrobial peptide to reach 80%, the longer the sustained release period, the better the sustained release performance of the microspheres, and the longer the maintenance time of the antibacterial effect;

[0192] Stability

[0193] Activity retention rate (after 30 days of storage at 4℃): After 30 days of storage at 4℃, the activity retention rate of antimicrobial peptide is determined, the higher the activity retention rate, the stronger the protection of the microspheres to the antimicrobial peptide, and the better the storage stability of the preparation;

[0194] Antibacterial MIC change (after storage): Determine the minimum inhibitory concentration (MIC) change of Staphylococcus aureus after storage, the smaller the MIC value change, the more stable the antibacterial activity;

[0195] Accelerated experiment (60℃, 75% RH, 10 days): Simulate extreme storage conditions to further investigate the stability of the microspheres, and determine the activity retention rate of antimicrobial peptide and the MIC change;

[0196] Microstructure

[0197] Surface integrity (SEM): The surface morphology of the microspheres is observed by scanning electron microscope (SEM) to evaluate the surface integrity, and the smooth and dense microsphere structure is beneficial to reduce the leakage of antimicrobial peptide and the erosion of the external environment;

[0198] Average particle size (μm): The average particle size of the microspheres was measured, and the particle size had an influence on the release behavior of the antimicrobial peptide and the stability of the microspheres;

[0199] Particle size distribution (PDI): The particle size distribution of the microspheres was reflected, and the smaller the PDI, the narrower the particle size distribution, and the more stable the performance;

[0200] Release kinetics parameters

[0201] Initial release rate constant (k0): The initial release rate constant was described, and the data at the early time points (0-6h) were fitted;

[0202] Slow-release phase rate constant (k1): The slow-release phase rate constant was characterized, and the data from 1 to 7 days were fitted;

[0203] Terminal elimination rate constant (k2): The terminal elimination rate constant was reflected, and the data from 7 to 14 days were fitted;

[0204] III. Experimental data

[0205]

[0206]

[0207]

[0208] IV. Data analysis

[0209] The above table shows that the PLGA / PCL framework provides a basic slow-release framework: the PLGA / PCL framework can effectively block the penetration of water, reduce the diffusion of the antimicrobial peptide, and to some extent control the release rate of the antimicrobial peptide, providing a basic guarantee for the slow-release performance of the microspheres;

[0210] ALG-Ca 2+ / PLL / PGA crosslinking network enhances compactness: ALG-Ca 2+ The synergistic effect of the crosslinking network and PLL / PGA further enhances the compactness of the microspheres, making the microsphere structure more stable, reducing the leakage of the antimicrobial peptide, improving the encapsulation efficiency, and at the same time playing a good regulating role on the release of the antimicrobial peptide, prolonging the slow-release period;

[0211] PDA coating seals pores and improves stability: The PDA coating not only seals the pores on the surface of the microspheres, reducing the initial burst release of the antimicrobial peptide, but also improves the antioxidant and antienzymatic ability of the microspheres, significantly enhancing the storage stability of the microspheres, so that the antimicrobial peptide can maintain high activity during storage.

[0212] The above merely describes preferred specific embodiments of the present application, but the protection scope of the present application is not limited thereto, and any person skilled in the art, according to the technical solution and inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. An antimicrobial peptide sustained-release microsphere preparation comprising a raw material, characterized by: The raw materials include the following components in parts by weight: 0.5-1.5 parts of antibacterial peptide, 1-5 parts of PLGA, 0.5-1.5 parts of PCL, 0.2-1 part of PVA, 0.1-0.5 part of phospholipid, 0.05-0.2 part of cholesterol, 0.1-0.5 part of PEG, 0.05-0.2 part of dopamine, 0.1-0.5 part of triethyl citrate, 0.1-0.5 part of lecithin, 0.05-0.2 part of Span-800, 0.05-0.2 part of Tween-80, 0.1-0.5 part of agarose, 0.1-0.5 part of sodium alginate, 0.1-0.5 part of gelatin, 0.05-0.2 part of polyacrylamide, 0.05-0.2 part of polylysine, 0.05-0.2 part of polyglutamic acid, 0.05-0.2 part of sodium citrate, 0.05-0.2 part of sodium bicarbonate, and 0.1-0.5 part of glucose.

2. The sustained-release microspheres of an antibacterial peptide according to claim 1, characterized in that: The raw materials include the following components in parts by weight: 0.8-1.2 parts of antibacterial peptide, 2-4 parts of PLGA, 0.8-1.2 parts of PCL, 0.4-0.8 part of PVA, 0.2-0.4 part of phospholipid, 0.08-0.15 part of cholesterol, 0.2-0.4 part of PEG, 0.08-0.15 part of dopamine, 0.2-0.4 part of triethyl citrate, 0.2-0.4 part of lecithin, 0.08-0.15 part of Span-800, 0.08-0.15 part of Tween-80, 0.2-0.4 part of agarose, 0.2-0.4 part of sodium alginate, 0.2-0.4 part of gelatin, 0.08-0.15 part of polyacrylamide, 0.08-0.15 part of polylysine, 0.08-0.15 part of polyglutamic acid, 0.08-0.15 part of sodium citrate, 0.08-0.15 part of sodium bicarbonate, and 0.2-0.4 part of glucose.

3. The sustained-release microspheres of an antibacterial peptide according to claim 2, characterized in that: The raw materials include the following components in parts by weight: 1 part of antibacterial peptide, 3 parts of PLGA, 1 part of PCL, 0.6 part of PVA, 0.3 part of phospholipid, 0.1 part of cholesterol, 0.3 part of PEG, 0.1 part of dopamine, 0.3 part of triethyl citrate, 0.3 part of lecithin, 0.1 part of Span-800, 0.1 part of Tween-80, 0.3 part of agarose, 0.3 part of sodium alginate, 0.3 part of gelatin, 0.1 part of polyacrylamide, 0.1 part of polylysine, 0.1 part of polyglutamic acid, 0.1 part of sodium citrate, 0.1 part of sodium bicarbonate, and 0.3 part of glucose.

4. A method for preparing the antibacterial peptide sustained-release microsphere preparation according to any one of claims 1-3, characterized in that: The method includes the following steps: S1: selecting raw materials and pretreating the raw materials; S2: dissolving and mixing the pretreated raw materials to prepare an oil phase; S3: adding the oil phase to an oil phase solvent to mix and emulsify, forming a primary emulsion; S4: dissolving and mixing the pretreated raw materials to prepare an aqueous phase; S5: adding the prepared primary emulsion to the aqueous phase to mix, forming an oil-in-water emulsion; S6: transferring the oil-in-water emulsion to a reaction kettle to perform crosslinking and solidification; S7: removing impurities from the reacted mixture to prepare a microsphere suspension; S8: drying the microsphere suspension and sealing and packaging to obtain a microsphere preparation product.

5. The method of claim 4, wherein the method is characterized by: In S1, the antibacterial peptide is dissolved in an appropriate amount of ethanol under the condition of a temperature of 2-8 DEG C, stirred until completely dissolved, to prepare an antibacterial peptide solution, and placed in a 4 DEG C environment for standby; PLGA, PCL and PVA are accurately weighed respectively, placed in a drying container, dried by a vacuum drying box, the temperature of the vacuum drying box is 60 DEG C, the drying time is 24 h, the moisture is removed to ensure the subsequent dissolution effect, and phospholipid, cholesterol, PEG and dopamine are placed in another drying container for drying treatment.

6. The method of claim 5, wherein the method is characterized by: In S2 and S3, the dried PLGA, PCL and PVA are added to a three-necked flask in a predetermined proportion on a sterile operation table, an appropriate amount of ethanol is slowly added, a magnetic stirrer is started, the rotating speed of the magnetic stirrer is 300 r / min, and the stirring time is 1 h, so that the PLGA, PCL and PVA are fully dissolved and mixed to form a uniform and transparent polymer solution; the prepared antibacterial peptide solution is slowly added to the polymer solution while stirring, the stirring speed is adjusted to 500 r / min, and the stirring time is 30 min, so that the antibacterial peptide is uniformly dispersed in the polymer solution to prepare an oil phase; the oil phase is slowly added to dichloromethane containing Span-80 and Tween-80, stirring is performed by a stirring mechanism, the stirring speed is 800 r / min, the temperature is 60 DEG C, and the emulsification time is 30 min, so that a uniform primary emulsion is formed.

7. The method of claim 6, wherein the method is characterized by: In S4 and S5, agarose is added to an appropriate amount of deionized water, heated to 90 DEG C, and fully dissolved, lecithin, triethyl citrate and polyacrylamide are sequentially added, stirring is performed under the condition of a 60 DEG C water bath for 30 min, so that the components are fully dissolved and mixed, sodium alginate and gelatin are respectively dissolved in a small amount of warm water and added to the mixed solution, and stirring is continuously performed for 20 min, polylysine, polyglutamic acid, sodium citrate, sodium bicarbonate, glucose are sequentially added, the pH value of the solution is adjusted to 7.2-7.4, and finally an appropriate amount of calcium chloride solution is added and uniformly stirred to prepare an aqueous phase; the prepared primary emulsion is slowly poured into the aqueous phase, mixing is performed by a stirring mechanism, the stirring speed is 500 r / min, the temperature is 40 DEG C, secondary emulsification treatment is performed for 30 min, and a uniform oil-in-water emulsion is formed.

8. The method of claim 7, wherein the method is characterized by: In S6, the emulsion is transferred to a reaction kettle, nitrogen protection is performed under the condition of stirring at 40 DEG C and 300 r / min for 2 h, in this process, sodium alginate reacts with gelatin, polyacrylamide, polylysine and polyglutamic acid under the action of calcium ions to form a stable three-dimensional network structure, so that the microspheres are solidified and shaped, and dopamine is self-polymerized to form a polydopamine coating under the action of oxidation, thereby further enhancing the stability of the microspheres; Sodium alginate dissociates into alginate ions and sodium ions in the solution, when calcium ions exist in the system, the alginate ions react with the calcium ions to form insoluble calcium alginate gel, and the crosslinking reaction of sodium alginate and calcium ions: Na-Alginate + Ca 2+ → Ca-Alginate + 2Na + Dopamine contains o-dihydroxy, under the action of oxidant, catechol structure is oxidized into quinone intermediate, quinone intermediate and amino in dopamine molecule occur Michael addition reaction, forming polydopamine, oxidation self-polymerization of dopamine: Gelatin contains a large number of amino and carboxyl groups, which can coordinate with calcium ions to form cross-linked structures. Cross-linking reaction of gelatin and calcium ions: Gelatin + Ca 2+ → Cross-linked Gelatin - Ca 2+ Polyacrylamide in solution can coordinate with calcium ions to form cross-linked structures. Cross-linking reaction of polyacrylamide and calcium ions: Polyacrylamide + Ca 2+ → Cross-linked Polyacrylamide - Ca 2+ Polylysine contains multiple amino groups, which can coordinate with calcium ions to form cross-linked structures. Cross-linking reaction of polylysine and calcium ions: Poly - L-lysine+Ca 2+ →Cross-linkedPoly - L-lysine-Ca 2+ Polyglutamic acid contains multiple carboxyl groups, which can coordinate with calcium ions to form cross-linked structures. Cross-linking reaction of polyglutamic acid and calcium ions: Poly - L-glutamicacid+Ca 2+ →Cross-linked Poly - L-glutamicacid-Ca 2+ These reactions together build the three-dimensional network structure of the microspheres, solidify and shape the microspheres, and form a polydopamine coating through the oxidation self-polymerization of dopamine, further enhancing the stability of the microspheres.

9. The method for preparing an antimicrobial peptide sustained-release microsphere formulation according to claim 8, characterized in that: In S7, the mixed solution after reaction is placed in a centrifuge, the centrifuge speed is 10000r / min, the centrifugation time is 15min, the microsphere precipitate is separated, the microsphere precipitate is washed with deionized water for 3-5 times to remove residual unreacted substances and surface impurities, after the last washing, the microsphere precipitate is resuspended with appropriate amount of deionized water to prepare a microsphere suspension.

10. The method for preparing an antimicrobial peptide sustained-release microsphere formulation according to claim 9, characterized in that: In S8, the microsphere suspension is transferred to a spray drying device, the inlet air temperature of the spray drying device is 80℃, the flow rate is 50 m 3 / h, and the spray drying is carried out under continuous stirring. During the drying process, the microspheres rapidly remove the solvent under the action of hot air to form uniform dry microsphere powder. After the drying is completed, the microsphere powder is collected in a dry glass container, sealed and packaged, and stored in a 4℃ environment to obtain a microsphere preparation finished product.

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