Antibacterial gelatin microspheres with photodynamic effect as well as preparation method and application of antibacterial gelatin microspheres

Abstract

The invention discloses an antibacterial gelatin microsphere with a photodynamic effect and a preparation method and application of the antibacterial gelatin microsphere, and belongs to the technical field of biomedical materials and hemostasis. An olefinic unsaturated carboxylic acid monomer is grafted to a gelatin side chain through amidation reaction to obtain modified gelatin; the modified gelatin and a cross-linking agent free radical are subjected to a copolymerization reaction to form carboxyl functionalized gelatin microspheres, and a photosensitizer is covalently immobilized on the surfaces of the carboxyl functionalized gelatin microspheres through an esterification reaction to prepare the antibacterial gelatin microspheres with the photodynamic effect. According to the antibacterial gelatin microspheres with the photodynamic effect and the preparation method and application of the antibacterial gelatin microspheres, the efficiency and stability of subsequent photosensitizer loading can be remarkably improved, the postoperative infection risk is effectively solved, and the limitation that a traditional hemostatic material only pays attention to physical plugging is broken through.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical materials and hemostatic technology, in particular to an antibacterial gelatin microsphere with a photodynamic effect and a preparation method and application thereof. BACKGROUND

[0002] Globally, more than 5.8 million people die from severe trauma every year, about 40% of which is directly caused by uncontrolled bleeding or its complications. Therefore, the development of efficient hemostatic materials has great clinical value in reducing trauma mortality.

[0003] Current hemostatic microspheres mainly include natural polymer microspheres such as gelatin microspheres and chitosan microspheres, and synthetic polymer microspheres such as polyvinyl alcohol microspheres and polylactic acid microspheres. The microspheres are usually regular spherical or spherical, the surface has rough texture or porous structure, and the inside forms interconnected pores, which can adsorb blood components, concentrate clotting factors, and accelerate the hemostatic process through physical barrier effect.

[0004] Among the current commercial hemostatic microspheres, natural polymer microspheres have excellent biocompatibility and dominate the market, among which gelatin hemostatic microspheres have become the core category due to their degradability and clotting adaptability. In addition to the gelatin sponge embolization microspheres of Hainan Baimaik Medical Technology Co., Ltd., there are several clinical application products suitable for surgical superficial wounds and intracavitary hemorrhage. Although these commercial gelatin hemostatic microspheres are widely used in clinical hemostasis, they still have the limitation of single hemostasis function.

[0005] The existing commercial hemostatic microspheres generally lack antibacterial properties. Some natural polymer microspheres such as chitosan microspheres have certain antibacterial ability due to their cationic properties, but they have obvious limitations such as weak antibacterial universality and cationic interference with hemostatic effect. Photodynamic antibacterial technology can efficiently kill various bacteria without environmental restrictions by generating singlet oxygen to kill bacteria under specific light sources. Moreover, photodynamic reaction is mild and does not destroy the structure of microspheres and the function of coagulation. In addition, photodynamic microspheres can achieve on-demand and repeated antibacterial effect by adjusting the light source.

[0006] In view of the defects of the existing hemostatic microspheres without antibacterial properties and the unique advantages of photodynamic antibacterial technology, the combination of photodynamic antibacterial technology and hemostatic microspheres can develop antibacterial hemostatic microspheres with photodynamic effect, which can simultaneously achieve efficient hemostasis and antibacterial function, solve the problem of postoperative infection, and meet the clinical demand for multifunctional hemostatic materials. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of the prior art, provide an antibacterial gelatin microsphere with a photodynamic effect and a preparation method and application thereof, which can significantly improve the efficiency and stability of subsequent photosensitizer loading, effectively solve the risk of postoperative infection, and break through the limitation of traditional hemostatic materials focusing only on physical plugging.

[0008] To achieve the above object, the present application is implemented by using the following technical scheme:

[0009] In a first aspect, the present application provides an antibacterial gelatin microsphere with a photodynamic effect, which is prepared by grafting an olefinic unsaturated carboxylic acid monomer to the side chain of gelatin through an amidation reaction to obtain modified gelatin, copolymerizing the modified gelatin with a crosslinking agent through a free radical copolymerization reaction to form carboxyl-functionalized gelatin microspheres, and covalently fixing a photosensitizer on the surface of the carboxyl-functionalized gelatin microspheres through an esterification reaction to obtain the antibacterial gelatin microsphere with the photodynamic effect.

[0010] The olefinically modified gelatin contains polymerizable double bonds, and the acrylic ester monomer is one or more of glycidyl methacrylate, butyl acrylate, methacrylic acid and methacrylic acid derivatives.

[0011] Further, the free radical copolymerization is carried out under a hydrogen peroxide and ascorbic acid initiation system.

[0012] Further, the photosensitizer is one or more of phthalocyanine compounds, porphyrin compounds and derivatives thereof.

[0013] In a second aspect, the present application provides a preparation method of the antibacterial gelatin microsphere with the photodynamic effect, which comprises:

[0014] The gelatin is dissolved in a dilute acetic acid solution with a pH value of 2-4, the acrylic ester monomer is added, and an olefinically modified gelatin precursor solution is formed by reacting at 40-65℃.

[0015] The olefinically modified precursor solution is injected into a n-hexane oil phase, and a W / O emulsion is formed by stirring at a speed of 300-500 rpm.

[0016] The crosslinking agent is added to the W / O emulsion, and the free radical copolymerization is completed under the condition of an oxidation-reduction initiator by continuously stirring at 50-60℃ and 400-800 rpm for 4-8 h, and then the amino-functionalized gelatin microspheres are obtained by ethanol washing and vacuum drying.

[0017] The carboxyl-functionalized gelatin microspheres are shaken with the photosensitizer under light-proof conditions for 12-48 hours to obtain the final antibacterial gelatin microspheres.

[0018] Further, the molar ratio of the gelatin to the acrylic ester monomer is 1:(0.4-1.2).

[0019] Further, the mass concentration of acetic acid in the dilute acetic acid solution is 1-15%, and the mass concentration of gelatin in the olefinically modified precursor solution is 15-40%.

[0020] Further, the volume ratio of the oil phase to the water phase in the W / O emulsion is 5:1.

[0021] Further, the crosslinking agent is an acrylic acid derivative of ethylenediamine or hexanediamine, the mass ratio of the crosslinking agent to the W / O emulsion is 0.5-2wt%; the redox initiator comprises hydrogen peroxide and ascorbic acid, the mass ratio of the total amount of the hydrogen peroxide and the ascorbic acid to the W / O emulsion is 1-2.5wt%.

[0022] In a second aspect, the present application provides an application of the antibacterial gelatin microspheres with a photodynamic effect in preparing a hemostatic material product.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The antibacterial gelatin microspheres with a photodynamic effect provided by the present application directly introduce carboxyl groups through copolymerization, significantly improve the efficiency and stability of subsequent photosensitizer loading, and the traditional photosensitizer loading depends on physical adsorption or non-specific binding. The present application realizes efficient and directional fixation of the photosensitizer through chemical bonding, avoids the risk of leakage in the body application, realizes the antibacterial function through the photodynamic effect, effectively solves the risk of postoperative infection, breaks through the limitation of traditional hemostatic materials that only focus on physical plugging, and is especially suitable for complex wounds infected with drug-resistant bacteria. The antibacterial gelatin microspheres with a photodynamic effect have good biocompatibility, and thus have great application potential in the field of hemostatic materials.

[0025] The preparation method of the antibacterial gelatin microspheres with a photodynamic effect provided by the present application has the advantages that the prepared microspheres are uniform in size, good in dispersity, can fill complex wound surfaces, form a continuous hemostatic and antibacterial barrier, the synthesis method is simple and conducive to large-scale production, the used equipment has low requirements, the used reagents are common reagents, and the method is environment-friendly, non-toxic and harmless. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a photomicrograph of the photodynamic antibacterial gelatin microspheres prepared in Example 1 of the present application;

[0027] Figure 2 It is a water absorption rate graph of the photodynamic antibacterial gelatin microspheres prepared in Example 1 of the present application;

[0028] Figure 3 It is a biocompatibility result graph of the photodynamic antibacterial gelatin microspheres prepared in Example 1 of the present application;

[0029] Figure 4 It is a rat tail hemostasis result graph of the photodynamic antibacterial gelatin microspheres prepared in Example 2 of the present application;

[0030] Figure 5 It is an electron scanning microscope graph of the photodynamic antibacterial gelatin microspheres prepared in Example 2 of the present application after freeze-drying;

[0031] Figure 6 UV-visible spectrophotometer chart of the photodynamic antibacterial gelatin microspheres prepared for the embodiment 2 of the present application;

[0032] Figure 7 Chart for evaluating the ROS generation ability of the photodynamic antibacterial gelatin microspheres prepared for the embodiment 3 of the present application under laser irradiation;

[0033] Figure 8 Chart for the bactericidal results of the photodynamic antibacterial gelatin microspheres prepared for the embodiment 3 of the present application and its control group under laser irradiation; DETAILED DESCRIPTION

[0034] The present application will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application.

[0035] Embodiment 1

[0036] The present embodiment provides an antibacterial gelatin microsphere with a photodynamic effect and a preparation method thereof, and the preparation method comprises:

[0037] Step 1: Preparation of double bond functionalized gelatin precursor

[0038] (1) Take 10 g of gelatin (Araldine, biotechnology grade) and slowly add it to a 10% by mass dilute acetic acid (Guojia) solution. Under the condition of 300 rpm and 60 degrees Celsius stirring, make it fully dissolved.

[0039] (2) According to the molar ratio of 1:1 of acrylate monomer to gelatin, add 12.5 mmol of methyl methacrylate (Araldine) to the above solution.

[0040] (3) Then transfer the mixed solution to a reaction container and place it in a water bath environment of 45-65°C for reaction. Continue to stir during the reaction to promote the reaction to proceed fully, until a transparent double bond functionalized gelatin precursor solution is formed, and the mass concentration of gelatin in the solution is controlled to reach 30%. After the reaction is completed, stop heating and naturally cool to room temperature.

[0041] Step 2: Emulsion preparation

[0042] (1) Take 0.8 g of polyoxyethylene sorbitan fatty acid ester (Araldine) and add it to a certain amount of deionized water, stir and dissolve to prepare an aqueous phase with an emulsifier concentration of 2.5wt%. The amount of emulsifier added is determined according to the mass of gelatin in step 1, at a ratio of 4-12wt%.

[0043] (2) Mix the prepared double bond functionalized gelatin precursor solution with the above aqueous phase containing emulsifier thoroughly to ensure uniform mixing.

[0044] (3) Slowly inject the mixed solution into the n-hexane oil phase (the volume ratio of n-hexane oil phase to water phase is 5:1), and stir at a speed of 500 rpm to form a stable W / O emulsion. During stirring, observe the emulsion state to ensure uniformity and stability.

[0045] Step 3: Preparation of amino-functionalized gelatin microspheres

[0046] (1) Add 1.5 g of ethylenediamine acrylate derivative as a crosslinking agent (mass ratio of crosslinking agent to W / O emulsion is 1.5 wt%) to the W / O emulsion. When adding, slowly add the crosslinking agent to the emulsion and continue stirring (600 rpm) to ensure uniform dispersion in the emulsion system.

[0047] (2) Add a free radical initiator system composed of hydrogen peroxide and ascorbic acid to the emulsion according to a molar ratio of 1:1 and a total amount of 1.2% of the mass of the W / O emulsion. During the addition process, accurately control the amount of initiator to ensure smooth initiation of the reaction.

[0048] (3) Place the reaction system in a water bath at 45°C and continue stirring at a speed of 600 rpm for 5 hours to complete the free radical copolymerization reaction.

[0049] (4) After the reaction is completed, wash the product with acetone several times to remove unreacted substances and impurities. After washing is completed, transfer the product to a freeze-drying machine for freeze-drying treatment to obtain amino-functionalized gelatin microspheres.

[0050] Step 4: Preparation of photodynamic antibacterial gelatin microspheres

[0051] (1) Photosensitizer activation: Select the carboxylic acid derivative Ce6 (Aladdin) of dihydrogen porphyrin as the photosensitizer, and activate it using N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Dissolve the photosensitizer, NHS and EDC in 20 mL of DMSO according to a mass ratio of 1.5:1:1, and react in the dark at room temperature for 24 hours to obtain the activated photosensitizer solution.

[0052] (2) Coupling reaction: Add the amino-functionalized gelatin microspheres to the activated photosensitizer solution, adjust the pH of the reaction system to 7.0, and oscillate at 25°C for 24 hours to allow the photosensitizer to fully couple with the microspheres. During the reaction process, regularly detect the pH value of the reaction system to ensure that the reaction is carried out under appropriate conditions.

[0053] (3) Product post-processing: After the reaction is completed, the product is washed multiple times with 0.1M PBS to remove the un-coupled photosensitizer. After washing is complete, the product is transferred to a freeze dryer for freeze-drying treatment, and the final photodynamic antibacterial gelatin microspheres are obtained.

[0054] Principle flow:

[0055] Functional modification of gelatin molecular chain:

[0056] The gelatin is modified using an acrylic ester monomer containing a double bond. In step 1, the gelatin is dissolved in a dilute acetic acid solution and reacts with the acrylic ester monomer at a certain temperature to introduce polymerizable double bonds on the gelatin molecular chain. The introduction of these double bonds provides active sites for subsequent crosslinking reactions and also lays the foundation for the immobilization of photosensitizers.

[0057] The specific steps for double bond functionalization of the gelatin molecular chain by acrylic ester are as follows:

[0058] 1. Raw material pretreatment:

[0059] Dissolve the gelatin (Aladdin, biotechnology grade) in a dilute acetic acid solution with a concentration of 5-15% w / v, heat to 60°C and continuously stir until completely dissolved to obtain a gelatin aqueous solution.

[0060] 2. Double bond introduction

[0061] According to the molar ratio of acrylic ester monomer to gelatin of 1:1, add 12.5 mmol of methyl methacrylate to the above solution. Under nitrogen protection, react at 45-65°C for 4-8 hours with continuous stirring (300-500 rpm).

[0062] Microsphere crosslinking and solidification:

[0063] W / O emulsion template construction: Weigh 0.8g of polyoxyethylene sorbitan fatty acid ester (Aladdin) and add it to a certain amount of deionized water, stir to dissolve, and prepare a water phase with an emulsifier concentration of 2.5wt%. The amount of emulsifier added is determined according to the mass of gelatin in step 1, at a ratio of 4-12wt%. After mixing the double bond functionalized gelatin precursor solution with the emulsifier-containing water phase, inject the n-hexane oil phase (volume ratio of n-hexane oil phase to water phase is 5:1) and stir at 500rpm to form a W / O emulsion.

[0064] Crosslinking reaction: In step 3, crosslinking agent and free radical initiation system are added to the W / O emulsion. Under the action of the initiator, the crosslinking agent and the double bond functionalized gelatin undergo free radical copolymerization reaction. The functional groups in the crosslinking agent interact with the double bonds on the gelatin molecular chain, allowing the gelatin molecules to be connected by chemical bonds, thereby achieving crosslinking and solidification of the microspheres and forming amino-functionalized gelatin microspheres.

[0065] Example 2

[0066] This embodiment provides an antibacterial gelatin microsphere with photodynamic effect and its preparation method, the preparation method comprising:

[0067] Step 1: Preparation of double-bond functionalized gelatin precursor

[0068] (1) Weigh 6g of gelatin (Aladdin, biotechnology grade) and slowly add it to a 6% dilute acetic acid (Chinese medicine) solution. Stir at 200 rpm and 50°C until fully dissolved.

[0069] (2) Add 6 mmol of methyl methacrylate (Aladdin) to the above solution according to the molar ratio of acrylate monomer to gelatin of 1:0.8.

[0070] (3) Transfer the mixed solution to the reaction vessel and place it in a water bath at 45°C to react. Stir continuously to form a double-bond functionalized gelatin precursor solution. Control the mass concentration of gelatin in the solution to reach 22%. After the reaction is completed, allow it to cool naturally to room temperature.

[0071] Step 2: Emulsion Preparation

[0072] (1) Weigh 0.48g of polyoxyethylene sorbitan fatty acid ester (Aladdin) and add it to deionized water to prepare an aqueous phase with an emulsifier concentration of 1.8wt%.

[0073] (2) After the precursor solution is mixed evenly with the aqueous phase, the n-hexane oil phase is injected (the volume ratio of n-hexane oil phase to aqueous phase is 5:1), and the mixture is stirred at 300 rpm to form a W / O emulsion.

[0074] Step 3: Preparation of amino-functionalized gelatin microspheres

[0075] (1) Add 0.9g of ethylenediamine acrylic acid derivative to the W / O emulsion as a crosslinking agent (the mass ratio of crosslinking agent to W / O emulsion is 1.0wt%).

[0076] (2) Add hydrogen peroxide to the initiation system at a molar ratio of 1:0.8 and a total amount of 0.8% of the W / O emulsion mass.

[0077] (3) Free radical copolymerization was completed by stirring at 40°C water bath and 500 rpm for 4 hours. The product was washed with acetone and then freeze-dried to obtain amino-functionalized gelatin microspheres.

[0078] Step 4: Preparation of photodynamic antibacterial gelatin microspheres

[0079] (1) Naphthalene phthalocyanine carboxylic acid derivative (Aladdin) was selected as the photosensitizer. The photosensitizer, NHS and EDC were dissolved in 12 mL of DMSO at a mass ratio of 1.2:1:1 and activated at room temperature in the dark for 18 h.

[0080] (2) Add amino-functionalized gelatin microspheres to the activated solution, adjust the pH to 6.8, and shake at 23°C for 20 h.

[0081] (3) After washing with 0.1MPBS, the gelatin microspheres were freeze-dried to obtain photodynamic antibacterial gelatin microspheres.

[0082] In this embodiment, due to the reduced amount of gelatin and the lower reaction temperature, the degree of double bond functionalization is reduced, and the cross-linking density of the microspheres decreases accordingly, resulting in a larger microsphere particle size of 500-700 micrometers and weaker mechanical strength.

[0083] Example 3

[0084] This embodiment provides an antibacterial gelatin microsphere with photodynamic effect and its preparation method, the preparation method comprising:

[0085] Step 1: Preparation of double-bond functionalized gelatin precursor

[0086] (1) Weigh 15g of gelatin (Aladdin, biotechnology grade), add it to a 15% dilute acetic acid (Chinese medicine) solution, and stir at 400rpm and 65℃ until completely dissolved.

[0087] (2) Add 22.5 mmol of butyl acrylate (Aladdin) at a molar ratio of 1:1.5 of acrylate monomer to gelatin.

[0088] (3) React in a 65°C water bath with continuous stirring until the gelatin mass concentration reaches 40%, and then cool after the reaction is complete.

[0089] Step 2: Emulsion Preparation

[0090] (1) Weigh 1.8g of polyoxyethylene castor oil (Aladdin) and prepare an aqueous phase with an emulsifier concentration of 3.2wt%.

[0091] (2) Inject the mixture into the hexane oil phase and stir at 800 rpm to form a W / O emulsion.

[0092] Step 3: Preparation of amino-functionalized gelatin microspheres

[0093] (1) Add 2.4g of hexamethylenediamine acrylic acid derivative as a crosslinking agent (the mass ratio of crosslinking agent to W / O emulsion is 2.0wt%).

[0094] (2) Add hydrogen peroxide to the initiation system at a molar ratio of 1:1.2 to ascorbic acid, and the total amount accounts for 1.6% of the W / O emulsion mass.

[0095] (3) The mixture was stirred for 7 hours in a water bath at 55°C and 800 rpm, and then washed with acetone and freeze-dried to obtain amino-functionalized gelatin microspheres.

[0096] Step 4: Preparation of photodynamic antibacterial gelatin microspheres

[0097] (1) A dihydroporphyrin carboxylic acid derivative (Aladdin) was selected as a photosensitizer. The photosensitizer, NHS and EDC were dissolved in 28 mL of DMSO at a mass ratio of 1.8:1:1 and activated in the dark for 28 h.

[0098] (2) Add the microspheres to the activation solution, adjust the pH to 7.5, and shake at 28°C for 32 hours.

[0099] (3) After washing with 0.1MPBS, freeze-dry to obtain photodynamic antibacterial gelatin microspheres.

[0100] In this embodiment, the high concentration of gelatin and monomer dosage fully functionalizes the double bonds, resulting in a vigorous cross-linking reaction, increased cross-linking density of the microspheres, smaller particle size (200-400 micrometers), and enhanced mechanical strength. However, excessively high cross-linking degree affects the swelling properties of the microspheres, reducing the swelling rate by 50%.

[0101] Example 4

[0102] This embodiment provides an antibacterial gelatin microsphere with photodynamic effect and its preparation method, the preparation method comprising:

[0103] Step 1: Preparation of double-bond functionalized gelatin precursor

[0104] (1) Weigh 12g of gelatin (Aladdin, biotechnology grade), add it to a 12% dilute acetic acid (Chinese medicine) solution, and stir at 350rpm and 60℃ to dissolve.

[0105] (2) Add 18 mmol of a mixture of butyl acrylate and glycidyl methacrylate (molar ratio 1:1) at a ratio of 1:1.2 of acrylate monomer to gelatin.

[0106] (3) React in a 60°C water bath until the gelatin mass concentration reaches 35%, and then cool after the reaction is complete.

[0107] Step 2: Emulsion Preparation

[0108] (1) Weigh 1.44g of a mixture of polyoxyethylene dehydrated sorbitan fatty acid ester and polyoxyethylene castor oil (mass ratio 1:1) and prepare an aqueous phase with an emulsifier concentration of 3.0wt%.

[0109] (2) After injecting the n-hexane oil phase, stir at 650 rpm to form a W / O emulsion.

[0110] Step 3: Preparation of amino-functionalized gelatin microspheres

[0111] (1) Add 1.8g of hexamethylenediamine acrylic acid derivative as a crosslinking agent (the mass ratio of crosslinking agent to W / O emulsion is 1.5wt%).

[0112] (2) Add hydrogen peroxide to the initiation system at a molar ratio of 1:1.1 and a total amount of 1.3% of the W / O emulsion mass.

[0113] (3) The mixture was stirred for 5.5 hours in a water bath at 52°C and 700 rpm, and then washed and freeze-dried to obtain amino-functionalized gelatin microspheres.

[0114] Step 4: Preparation of photodynamic antibacterial gelatin microspheres

[0115] (1) A dihydroporphyrin carboxylic acid derivative (Aladdin) was selected as a photosensitizer and dissolved in 24 mL of DMSO at a mass ratio of 1.6:1:1. The mixture was activated in the dark for 26 h.

[0116] (2) Add the microspheres to the activation solution, adjust the pH to 7.3, and shake at 27°C for 28 hours.

[0117] (3) After washing with 0.1MPBS, freeze-dry to obtain photodynamic antibacterial gelatin microspheres.

[0118] In this embodiment, the higher reaction temperature and monomer dosage increased the degree of cross-linking of the microspheres, reduced the particle size to 100-300 micrometers, and increased the mechanical strength by 1.5 times. However, the increased degree of cross-linking resulted in a denser structure and a 50% reduction in water absorption compared to Example 1.

[0119] Example 5

[0120] This embodiment provides an antibacterial gelatin microsphere with photodynamic effect and its preparation method, the preparation method comprising:

[0121] Step 1: Preparation of double-bond functionalized gelatin precursor

[0122] (1) Weigh 10g of gelatin (Aladdin, biotechnology grade), add it to a 10% dilute acetic acid (Chinese medicine) solution, and stir at 300rpm and 60℃ to dissolve.

[0123] (2) Add 17.5 mmol of a mixture of methyl methacrylate, butyl acrylate and glycidyl methacrylate (molar ratio 1:1:1) at a ratio of 1:1.4 of acrylate monomers to gelatin.

[0124] (3) React in a water bath at 58°C until the gelatin mass concentration reaches 32%, and then cool after the reaction is complete.

[0125] Step 2: Emulsion Preparation

[0126] (1) Weigh 1.2g of a mixture of polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oil and dodecyl polyoxyethylene ether (mass ratio 1:1:1) and prepare an aqueous phase with an emulsifier concentration of 2.8wt%.

[0127] (2) After injecting the n-hexane oil phase, stir at 700 rpm to form a W / O emulsion.

[0128] Step 3: Preparation of amino-functionalized gelatin microspheres

[0129] (1) Add 1.7g of ethylenediamine acrylic acid derivative as a crosslinking agent (the mass ratio of crosslinking agent to W / O emulsion is 1.6wt%).

[0130] (2) Add hydrogen peroxide to the initiation system at a molar ratio of 1:1.1 and a total amount of 1.4% of the W / O emulsion mass.

[0131] (3) The mixture was stirred for 5 hours in a water bath at 48°C and at 650 rpm, and then washed and freeze-dried to obtain amino-functionalized gelatin microspheres.

[0132] Step 4: Preparation of photodynamic antibacterial gelatin microspheres

[0133] (1) Naphthalene phthalocyanine carboxylic acid derivative (Aladdin) was selected as a photosensitizer and dissolved in 22 mL DMSO at a mass ratio of 1.5:1:1 and activated in the dark for 48 h.

[0134] (2) Add the microspheres to the activation solution, adjust the pH to 7.2, and shake the reaction at 26°C for 48 hours.

[0135] (3) After washing with 0.1M PBS, the gelatin microspheres were freeze-dried to obtain photodynamic antibacterial gelatin microspheres.

[0136] This embodiment extends the activation and reaction time of the photosensitizer, which is beneficial for photosensitizer binding. The photosensitizer content is increased by 1.5 times, resulting in excellent photodynamic antibacterial performance and better performance in complex antibacterial scenarios.

[0137] A series of tests were conducted on the products provided in Examples 1-5 to verify their effectiveness, specifically including:

[0138] (1) The antibacterial gelatin microspheres prepared in Example 1 were observed and photographed under a microscope, and the results are as follows: Figure 1 As shown, the microspheres have a good spherical structure and uniform size, specifically 200-400 μm.

[0139] (2) The water absorption of the antibacterial gelatin microspheres prepared in Example 1 was tested. The weight of the antibacterial gelatin microspheres prepared in Example 1 when dry was recorded as follows: The dried gelatin microspheres were then immersed in 25 mL of PBS solution. Every 10 minutes, the microspheres were removed and the surface moisture was gently blotted with filter paper. The mass after water absorption was quickly weighed and recorded. The microspheres were then immediately returned to the solution for further immersion. When the mass change was less than 5% in two consecutive weighings, the microspheres were considered to have reached water saturation, and this mass was recorded as [value missing]. Calculate the final saturated water absorption rate. The formula for calculating the water absorption rate is:

[0140]

[0141] The results are as follows Figure 2 As shown, Figure 2 In the diagram, YNBY represents the Yunnan Baiyao experimental group, Gel-Sponge represents the gelatin sponge experimental group, ChitoGauze represents the chitosan gauze experimental group, Gel-Ms represents the microsphere experimental group prepared under water freeze-drying conditions, Gel-Ms-EtoH represents the microsphere experimental group prepared under ethanol freeze-drying conditions, and Gel-Ms-EtoH / H2O represents the microsphere experimental group prepared under water and ethanol freeze-drying conditions. Figure 2 As shown, the water absorption rate of the antibacterial gelatin microspheres prepared in Example 1 is about 900%.

[0142] (3) The biocompatibility of the photodynamic antibacterial gelatin microspheres prepared in Example 1 is as follows: Figure 3 As shown, the cells used in the experiment were mouse fibroblasts, purchased from Nanjing Kaiji Biotechnology Co., Ltd. After passage in DMEM medium containing 10% FBS, the old medium was discarded, and the cells were washed with PBS. Trypsin was added, and after 3 minutes, an equal volume of DMEM complete culture medium containing 10% FBS was added to terminate digestion. The cells were centrifuged at 800-1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in complete culture medium and counted. Cells were plated at an appropriate number (1×10⁶ cells / year). 4 Cells / mL), approximately 100 μL of cell suspension per well, with 5 replicates per group. Cells were incubated at 37°C for 12 h. After entering the logarithmic growth phase, different concentrations of extract were added. The 100% microsphere extract was diluted with culture medium to prepare 75%, 50%, 25%, and 12.5% ​​extracts, respectively. The negative control group consisted of 100 μL of pure culture medium. The positive control group consisted of 100 μL of culture medium containing 10% DMSO. The experimental groups consisted of 100 μL of extracts at different concentrations. After incubation for another 12 h, 10 μL of CCK-8 reagent was added and incubated for 2 hours. The OD value at 450 nm was then measured using a microplate reader.

[0143] Blood compatibility was assessed by centrifuging anticoagulated whole blood from rat veins to obtain erythrocytes, which were then washed three times with PBS to prepare a 2% erythrocyte suspension. Photodynamic antibacterial gelatin hemostatic microspheres were dispersed in PBS at four concentration gradients of 16, 8, 4, and 2 mg / mL to form experimental group solutions. Negative controls (PBS) and positive controls (deionized water) were also included. For each group, 200 μL of erythrocyte suspension was mixed with 800 μL of the corresponding solution and incubated for 1 hour before centrifugation. 200 μL of the supernatant was then used to measure the absorbance (OD value) at 540 nm using a microplate reader. Each experiment was repeated five times to evaluate the effect of different concentrations of microspheres on erythrocytes.

[0144] The results are as follows Figure 3 As shown, Figure 3 Image a is an optical photograph of the hemolysis experiment. Figure 3 Figure b shows the hemolysis test data. The results indicate that the photodynamic antibacterial gelatin hemostatic microspheres have a hemolysis rate of less than 5%. Figure 3 In the figure, c represents the statistical data on cell compatibility. The results show that after co-incubation with photodynamic antibacterial gelatin hemostatic microspheres for 48 hours, the cell survival rate was still above 90%, indicating good cell compatibility. Figure 3 Image d shows a cell compatibility micrograph, which clearly demonstrates that the photodynamic antibacterial gelatin hemostatic microspheres have good biocompatibility.

[0145] (4) The photodynamic antibacterial gelatin microspheres prepared in Example 2 of this invention were subjected to a rat tail amputation hemostasis test, and the results are as follows: Figure 4 As shown, Figure 4 Image a shows the hemostatic effect of rat tail amputation. Blank represents an optical photograph of tail amputation bleeding in rats without hemostatic material treatment, ChitoGauze represents an optical photograph of tail amputation bleeding in rats treated with chitosan gauze material, Gel-Ms represents an optical photograph of tail amputation bleeding in rats treated with gelatin microsphere material, and Gel-Ms-Ce6 represents an optical photograph of tail amputation bleeding in rats treated with photodynamic antibacterial gelatin hemostatic microsphere material. Figure 4 In Figure b, the bleeding quality is determined by different materials used in the treatment. Figure 4 In the figure, c represents the bleeding time after treatment with different materials. The results show that photodynamic gelatin hemostatic microspheres have a better hemostatic effect than commercially available hemostatic materials.

[0146] (5) Figure 5 The image shown is an electron scanning microscope image of the photodynamic antibacterial gelatin microspheres prepared in Example 2 of this invention after lyophilization. The results show that the photodynamic antibacterial gelatin microspheres have a rough surface morphology.

[0147] (6) Figure 6The image shows a UV-Vis spectrophotometer image of the photodynamic antibacterial gelatin microspheres prepared in Example 3 of this invention. The results show that the photodynamic embolization microspheres have a UV absorption peak of photosensitizer Ce6 at 420 nm, indicating that the photosensitizer was successfully loaded.

[0148] (7) Figure 7 This image shows the ROS generation capability of the photodynamic antibacterial gelatin microspheres prepared in Example 3 of this invention under laser irradiation. The results indicate that the photodynamic antibacterial gelatin hemostatic microspheres have a good reactive oxygen species generation effect.

[0149] (8) The photodynamic antibacterial gelatin microspheres prepared in Example 3 of this invention and their control group were subjected to an experiment on bacterial killing under laser irradiation. 48-well plates were prepared, MRSA was cultured to a density of 1×10⁵ CFU / mL, and photodynamic antibacterial gelatin microspheres were added for further culture for 6 h. Optical photographs were taken, and the absorbance of the bacterial solution was measured using a multi-functional microplate reader. Each group was tested three times. After culture, a 660 nm laser (0.5 W / cm²) was used. 2 After irradiation for 2 minutes, the supernatant was aspirated and PBS was added. The absorbance of the bacterial solution was then measured using a spectrophotometer to calculate the bacterial survival rate. Figure 8 The results of the photodynamic antibacterial gelatin microspheres prepared in Example 3 of this invention and its control group were obtained from the bacterial killing experiment under laser irradiation. Figure 8 Figure 'a' shows the survival rate of E. coli. Figure 8 Figure b shows the survival rate of methicillin-resistant Staphylococcus aureus. Control represents the control group, Gel-MS represents the gelatin microsphere experimental group, and Gel-MS-Ce6 represents the photodynamic antibacterial gelatin hemostatic microsphere experimental group. The results show that after laser irradiation, the antibacterial effect of the photodynamic antibacterial gelatin hemostatic microspheres is above 95%.

[0150] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. An antibacterial gelatin microsphere having a photodynamic effect, characterized by, The gelatin is dissolved in a dilute acetic acid solution with pH value of 2-4, acrylic ester monomers are added, and the reaction is carried out at 40-65 DEG C to form an alkenyl-modified gelatin precursor solution; the alkenyl-modified gelatin precursor solution is injected into a n-hexane oil phase, and a W / O emulsion is formed by stirring at a rotation speed of 300-500 rpm; a crosslinking agent is added to the W / O emulsion, and a radical copolymerization is carried out under the condition of an oxidation-reduction initiator at 50-60 DEG C and continuous stirring at 400-800 rpm for 4-8 h; the amino-functionalized gelatin microspheres are obtained by ethanol washing and vacuum drying; the carboxyl-functionalized gelatin microspheres are reacted with a photosensitizer under the condition of light shielding for 12-48 h to obtain the final antibacterial gelatin microspheres. The alkenyl-modified gelatin comprises polymerizable double bonds, and the acrylic ester monomers are one or more of glycidyl methacrylate, butyl acrylate, methacrylic acid and methacrylic acid derivatives.

2. The antibacterial gelatin microspheres having a photodynamic effect according to claim 1, wherein, The radical copolymerization is carried out under a hydrogen peroxide and ascorbic acid initiation system.

3. The antibacterial gelatin microspheres having a photodynamic effect according to claim 1, wherein the photosensitizer is a porphyrin compound. The photosensitizer is one or more of phthalocyanine compounds, porphyrin compounds and derivatives thereof.

4. A method for preparing the antibacterial gelatin microspheres having a photodynamic effect according to any one of claims 1 to 3, characterized by, The method comprises: The gelatin is dissolved in a dilute acetic acid solution with pH value of 2-4, acrylic ester monomers are added, and the reaction is carried out at 40-65 DEG C to form an alkenyl-modified gelatin precursor solution; the alkenyl-modified gelatin precursor solution is injected into a n-hexane oil phase, and a W / O emulsion is formed by stirring at a rotation speed of 300-500 rpm; a crosslinking agent is added to the W / O emulsion, and a radical copolymerization is carried out under the condition of an oxidation-reduction initiator at 50-60 DEG C and continuous stirring at 400-800 rpm for 4-8 h; the amino-functionalized gelatin microspheres are obtained by ethanol washing and vacuum drying; the carboxyl-functionalized gelatin microspheres are reacted with a photosensitizer under the condition of light shielding for 12-48 h to obtain the final antibacterial gelatin microspheres. The molar ratio of the gelatin to the acrylic ester monomers is 1:(0.4-1.2). The acetic acid in the dilute acetic acid solution has a mass concentration of 1-15%, and the gelatin in the alkenyl-modified gelatin precursor solution has a mass concentration of 15-40%. The volume ratio of the oil phase to the water phase in the W / O emulsion is 5:

1.

5. The method of claim 4, wherein: The crosslinking agent is an acrylic acid derivative of ethylenediamine or hexanediamine, and the mass ratio of the crosslinking agent to the W / O emulsion is 0.5-2 wt%; the oxidation-reduction initiator comprises hydrogen peroxide and ascorbic acid, and the mass ratio of the total amount of the hydrogen peroxide and the ascorbic acid to the W / O emulsion is 1-2.5 wt%.

6. The method of claim 4, wherein: The antibacterial gelatin microspheres are the antibacterial gelatin microspheres according to any one of claims 1-3 or prepared by the preparation method according to any one of claims 4-8.

7. The method of claim 4, wherein: ​ 8. The method of claim 4, wherein: ​ 9. Use of an antibacterial gelatin microsphere having a photodynamic effect in the preparation of a hemostatic material product, characterized by: ​

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