A chitosan antibacterial film loaded with polymer microspheres and its preparation method

Abstract

The present invention provides a chitosan antibacterial film loaded with polymer microspheres and a preparation method thereof. The preparation steps are as follows: S1: Preparation of a chitosan derivative solution; S2: Preparation of polymer microspheres; S3: Preparation of the antibacterial film. In the present invention, chitosan is derivatized to obtain benzoylated chitosan, which improves the solubility and antibacterial properties of chitosan; the organic ligand poly(lactic-co-glycolic acid) is complexed with magnesium sulfate to prepare polymer microspheres, and the carboxyl group in poly(lactic-co-glycolic acid) is covalently coupled with the primary amino group on chitosan, realizing the loading of polymer microspheres on chitosan and enhancing the mechanical properties of the antibacterial film. At the same time, the synergistic effect of metal magnesium ions and benzoylated chitosan makes the antibacterial properties of the antibacterial film more prominent, which has important significance for the application of chitosan in the fields of food, medicine, etc.

Description

Technical Field

[0001] The present invention relates to the field of preparation of antibacterial composite materials, and particularly relates to a chitosan antibacterial film loaded with polymer microspheres and a preparation method thereof. Background Art

[0002] Chitosan is the deacetylated product of chitin, the second largest natural organic matter in nature. It has the advantages of non-toxicity, biocompatibility, antioxidant property, antibacterial property, and biodegradability, and has great application potential in the fields of food, medicine, agriculture, textile, cosmetics, and water treatment. The strong hydrogen bonds between chitosan molecules make the solubility of chitosan extremely poor, which greatly limits the application of chitosan. Therefore, it is necessary to modify it. Usually, modification methods such as acylation, alkylation, etherification, quaternization, graft copolymerization, and crosslinking are used to improve the application value. In the modification process, aromatic substances represented by benzene can improve the solubility of chitosan. More importantly, aromatic substances themselves have characteristics such as antioxidant property, fluorescence property, and photosensitivity, which will endow chitosan with some new functions. That is, after the aromatic substances are grafted onto chitosan and through process optimization, the advantages of the new materials become more obvious, which greatly promotes the research and application of chitosan materials.

[0003] Due to the special physical and mechanical antibacterial effects and excellent properties such as easy modification, multi-functionality, and controllable properties, the research and development of antibacterial materials have developed rapidly, mainly focusing on the following four aspects: (1) carbon nanomaterials, including carbon nanotubes and graphene; (2) metal materials, including gold, silver, magnesium, aluminum, zinc, etc., and various metal hybrid enhanced types; (3) organic polymer materials, including microsphere layers formed by self-assembly of porphyrin molecules, triblock copolymer colloids, natural or chemically synthesized polycationic polymers, etc.; (4) metal and organic molecule hybrid materials, new metal-organic polymers.

[0004] Although the derivatives obtained by modifying chitosan have antibacterial and antioxidant effects, their mechanical properties are weak. Therefore, an attempt is made to combine metal-organic polymers with modified chitosan to prepare a new type of antibacterial agent to improve its mechanical properties and enhance the antibacterial effect, so that chitosan can be more widely applied in the fields of food, medicine, etc. Summary of the Invention

[0005] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a chitosan antibacterial film loaded with polymer microspheres and a preparation method thereof: reacting chitosan with benzoic acid to prepare a chitosan derivative solution, then preparing polymer microspheres by complexing polylactic acid-glycolic acid with magnesium sulfate, and then loading the polymer microspheres on the derivatized chitosan to prepare a new type of antibacterial film, which can not only enhance the antibacterial effect of chitosan, but also improve the mechanical properties of the antibacterial film.

[0006] Technical solution: A preparation method of a chitosan antibacterial film loaded with polymer microspheres, comprising the following steps:

[0007] S1. Preparation of chitosan derivative solution: Weigh chitosan and place it in a reactor, add acetic acid solution and stir until the chitosan is completely dissolved. Then add benzoic acid and vitamin C to the reactor, introduce high-purity nitrogen to remove air, add H2O2 solution and react for 12 - 20h, and then use a dialysis bag for dialysis to obtain a benzoylated chitosan solution;

[0008] S2. Preparation of polymer microspheres: Add 40 - 60 mL of polylactic acid-glycolic acid to 10 - 20 mL of dichloromethane and stir for 5 - 8h, and electrostatically spray into microspheres under high pressure. Immerse the obtained microspheres in an ethanol solution for activation for 10 - 20 min, wash with deionized water 3 - 5 times, then immerse in 10 - 20 mL of magnesium sulfate solution for 3 - 5h, take out, wash with deionized water 3 - 5 times, and freeze-dry to obtain polymer microspheres;

[0009] S3. Preparation of antibacterial film: Immerse the polymer microspheres in the benzoylated chitosan solution, add glycerol to obtain a film-forming solution, and use the casting method to uniformly coat the film-forming solution on a polytetrafluoroethylene plate and dry to obtain a chitosan antibacterial film loaded with polymer microspheres.

[0010] Further, the components in step S1 are as follows: The mass-volume ratio of chitosan to benzoic acid is 1g:0.2 - 0.7 mL, 80 - 100 mL of 2% acetic acid solution, 0.2 - 0.4 g of vitamin C, and 1 - 2 mL of 30% H2O2 solution.

[0011] Further, the stirring speed in step S1 is 300 - 700 r / min, and the stirring time is 5 - 10 min.

[0012] Further, the molecular weight cut-off of the dialysis bag in step S1 is 8 - 14 kDa, the dialysis time is 60 - 72h, and the water is changed every 8h.

[0013] Further, the concentration of the magnesium sulfate solution in step S2 is 0.05 - 0.1 mol / L.

[0014] Further, the high-pressure condition in step S2 is 28 - 40 kV.

[0015] Further, the addition amount of the polymer microspheres in step S3 is 1 - 2 wt.%, and the addition amount of glycerol is 1 - 1.5 vt.%.

[0016] Further, the drying temperature in step S3 is 50 - 60 °C, and the drying time is 2 - 3h.

[0017] Beneficial effects:

[0018] 1. The present invention prepares benzoylated chitosan by derivatizing chitosan with benzoic acid. Among them, benzoic acid is coupled with chitosan through an amide bond, which improves the solubility of chitosan. At the same time, the antibacterial property of benzoic acid itself makes the antibacterial effect of the derivatized chitosan more prominent.

[0019] 2. The present invention prepares polymer microspheres by complexing the organic ligand poly (lactic - co - glycolic acid) with magnesium sulfate. The carboxyl group in poly (lactic - co - glycolic acid) is covalently coupled with the primary amino group on chitosan, thereby realizing the loading of polymer microspheres on chitosan. At the same time, the microspheres prepared by complexing poly (lactic - co - glycolic acid) with magnesium sulfate also avoid the possible reduction of the antibacterial activity of chitosan by directly adding metal ions to the chitosan solution. When the polymer microspheres are loaded on benzoylated chitosan, magnesium ions and benzoylated chitosan have a synergistic effect, enhancing the antibacterial effect.

[0020] 3. The mechanical properties of the self - formed film of the benzoylated chitosan prepared in the present invention are weak. Since poly (lactic - co - glycolic acid) in the microspheres itself has a certain mechanical strength, after introducing the microspheres into the benzoylated chitosan solution, the microspheres can be evenly dispersed in the solution and tightly combined with chitosan by chemical bonds, effectively connecting the microspheres and chitosan. Therefore, when the antibacterial film is stretched by an external force, the chemical bonds can serve as the main stress points, effectively improving the mechanical properties of the antibacterial film. Detailed implementation mode

[0021] The present invention provides a chitosan antibacterial film loaded with polymer microspheres and a preparation method thereof. To make the purpose, technical solution and effect of the present invention clearer and more definite, the following will further elaborate on the present invention in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] Example 1

[0023] A preparation method of a chitosan antibacterial film loaded with polymer microspheres includes the following steps:

[0024] S1. Preparation of chitosan derivative solution: Weigh 1 g of chitosan and place it in a reactor, add 80 mL of 2% acetic acid solution, stir at 400 r / min for 6 min until the chitosan is completely dissolved. Then add 0.2 mL of benzoic acid and 0.2 g of vitamin C to the reactor. After purging the air with high - purity nitrogen, add 1 mL of 30% H2O2 solution and react for 12 h. Dialyze with an 8 - 14 kDa dialysis bag for 60 h, changing the water every 8 h to obtain the benzoylated chitosan solution;

[0025] S2. Preparation of polymer microspheres: Add 40 mL of poly (lactic-co-glycolic acid) to 10 mL of dichloromethane and stir for 5 h. Electrospray to form microspheres under the condition of a high voltage of 30 kV. Immerse the obtained microspheres in an ethanol solution for activation for 10 min, wash them 3 times with deionized water, then soak them in 10 mL of 0.05 mol / L magnesium sulfate solution for 3 h, take them out, wash them 3 times with deionized water, and freeze-dry to obtain polymer microspheres;

[0026] S3. Preparation of antibacterial film: Immerse 0.8 g of polymer microspheres in a benzoic acid-modified chitosan solution, add 0.8 mL of glycerol to obtain a film-forming solution, and uniformly coat the film-forming solution on a polytetrafluoroethylene plate by the casting method, and dry it at 50 °C for 3 h to obtain a chitosan antibacterial film loaded with polymer microspheres.

[0027] Example 2

[0028] A preparation method of a chitosan antibacterial film loaded with polymer microspheres includes the following steps:

[0029] S1. Preparation of chitosan derivative solution: Weigh 1 g of chitosan and place it in a reactor, add 100 mL of 2% acetic acid solution, stir at 700 r / min for 5 min until the chitosan is completely dissolved, then add 0.7 mL of benzoic acid and 0.4 g of vitamin C to the reactor. After purging the air by introducing high-purity nitrogen, add 2 mL of 30% H2O2 solution, react for 20 h, and dialyze with an 8-14 kDa dialysis bag for 70 h, changing the water every 8 h to obtain a benzoic acid-modified chitosan solution;

[0030] S2. Preparation of polymer microspheres: Add 60 mL of poly (lactic-co-glycolic acid) to 20 mL of dichloromethane solution and stir for 8 h. Electrospray to form microspheres under the condition of a high voltage of 38 kV. Immerse the obtained microspheres in an ethanol solution for activation for 20 min, wash them 5 times with deionized water, then soak them in 20 mL of 0.07 mol / L magnesium sulfate solution for 3 h, take them out, wash them 5 times with deionized water, and freeze-dry to obtain polymer microspheres;

[0031] S3. Preparation of antibacterial film: Immerse 1 g of polymer microspheres in a benzoic acid-modified chitosan solution, add 1.6 mL of glycerol to obtain a film-forming solution, and uniformly coat the film-forming solution on a polytetrafluoroethylene plate by the casting method, and dry it at 60 °C for 2 h to obtain a chitosan antibacterial film loaded with polymer microspheres.

[0032] Example 3

[0033] A preparation method of a chitosan antibacterial film loaded with polymer microspheres includes the following steps:

[0034] S1. Preparation of chitosan derivative solution: Weigh 1 g of chitosan and place it in a reactor. Add 90 mL of 2% acetic acid solution and stir at 500 r / min for 7 min until the chitosan is completely dissolved. Then add 0.5 mL of benzoic acid and 0.3 g of vitamin C to the reactor. After purging the air with high-purity nitrogen, add 1.5 mL of 30% H2O2 solution and react for 15 h. Dialyze with an 8 - 14 kDa dialysis bag for 70 h, changing the water every 8 h to obtain the benzoylated chitosan solution;

[0035] S2. Preparation of polymer microspheres: Add 50 mL of polylactic-co-glycolic acid to 15 mL of dichloromethane solution and stir for 6 h. Electrospray to form microspheres under a high voltage of 35 kV. Immerse the obtained microspheres in an ethanol solution for activation for 15 min, wash them 3 times with deionized water, then soak them in 15 mL of 0.1 mol / L magnesium sulfate solution for 5 h, take them out, wash them 3 times with deionized water, and freeze-dry to obtain polymer microspheres;

[0036] S3. Preparation of antibacterial film: Immerse 1.4 g of polymer microspheres in the benzoylated chitosan solution, add 0.9 mL of glycerol to obtain a film-forming solution. Use the casting method to evenly spread the film-forming solution on a polytetrafluoroethylene plate and dry it at 55 °C for 2.5 h to obtain the chitosan antibacterial film loaded with polymer microspheres.

[0037] Example 4

[0038] A preparation method of a chitosan antibacterial film loaded with polymer microspheres, comprising the following steps:

[0039] S1. Preparation of chitosan derivative solution: Weigh 1 g of chitosan and place it in a reactor. Add 80 mL of 2% acetic acid solution and stir at 400 r / min for 8 min until the chitosan is completely dissolved. Then add 0.5 mL of benzoic acid and 0.4 g of vitamin C to the reactor. After purging the air with high-purity nitrogen, add 1 mL of 30% H2O2 solution and react for 14 h. Dialyze with an 8 - 14 kDa dialysis bag for 60 h, changing the water every 8 h to obtain the benzoylated chitosan solution;

[0040] S2. Preparation of polymer microspheres: Add 50 mL of polylactic-co-glycolic acid to 20 mL of dichloromethane solution and stir for 5 - 8 h. Electrospray to form microspheres under a high voltage of 38 kV. Immerse the obtained microspheres in an ethanol solution for activation for 10 min, wash them 3 times with deionized water, then soak them in 10 mL of 0.1 mol / L magnesium sulfate solution for 3 h, take them out, wash them 3 times with deionized water, and freeze-dry to obtain polymer microspheres; S3. Preparation of antibacterial film: Immerse 1.7 g of polymer microspheres in the benzoylated chitosan solution, add 0.8 mL of glycerol to obtain a film-forming solution. Use the casting method to evenly spread the film-forming solution on a polytetrafluoroethylene plate and dry it at 60 °C for 2 h to obtain the chitosan antibacterial film loaded with polymer microspheres.

[0041] Comparative Example 1

[0042] The difference between this comparative example and Example 3 lies in the use of undenatured chitosan. Specifically:

[0043] A method for preparing a chitosan antibacterial film loaded with polymer microspheres includes the following steps:

[0044] S1. Preparation of chitosan solution: Weigh 1 g of chitosan and place it in a reactor. Add 90 mL of 2% acetic acid solution and stir at 500 r / min for 7 min until the chitosan is completely dissolved, thus obtaining the chitosan solution.

[0045] S2. Preparation of polymer microspheres: Add 50 mL of polylactic-co-glycolic acid to 15 mL of dichloromethane solution and stir for 6 h. Electrospray into microspheres under the condition of a high voltage of 35 kV. Immerse the obtained microspheres in an ethanol solution for activation for 15 min, wash 3 times with deionized water, then immerse them in 15 mL of 0.1 mol / L magnesium sulfate solution for 5 h, take them out, wash 3 times with deionized water, and freeze-dry to obtain polymer microspheres.

[0046] S3. Preparation of antibacterial film: Immerse 1.4 g of polymer microspheres in the chitosan solution, add 0.9 mL of glycerol to obtain a film-forming solution. Use the casting method to evenly spread the film-forming solution on a polytetrafluoroethylene plate and dry it at 55 °C for 2.5 h, thus obtaining the chitosan antibacterial film loaded with polymer microspheres.

[0047] Comparative Example 2

[0048] The difference between this comparative example and Example 3 lies in directly adding metal magnesium ions without preparing them into polymer microspheres. Specifically:

[0049] A method for preparing a chitosan antibacterial film loaded with polymer microspheres includes the following steps:

[0050] S1. Preparation of chitosan derivative solution: Weigh 1 g of chitosan and place it in a reactor. Add 90 mL of 2% acetic acid solution and stir at 500 r / min for 7 min until the chitosan is completely dissolved. Then add 0.5 mL of benzoic acid and 0.3 g of vitamin C to the reactor. After purging the air with high-purity nitrogen, add 1.5 mL of 30% H2O2 solution and react for 15 h. Dialyze with an 8-14 kDa dialysis bag for 70 h, changing the water every 8 h, thus obtaining the benzoylated chitosan solution.

[0051] S2. Preparation of antibacterial film: Add 15 mL of 0.1 mol / L magnesium sulfate solution to the benzoylated chitosan solution, add 1.1 mL of glycerol to obtain a film-forming solution. Use the casting method to evenly spread the film-forming solution on a polytetrafluoroethylene plate and dry it at 55 °C for 2.5 h, thus obtaining the chitosan antibacterial film loaded with polymer microspheres on the shell.

[0052] Comparative Example 3

[0053] The difference between this comparative example and Example 3 is that EDTA is used as the organic ligand to replace poly(lactic-co-glycolic acid). Specifically:

[0054] A method for preparing a chitosan antibacterial film loaded with polymer microspheres, comprising the following steps:

[0055] S1. Preparation of chitosan derivative solution: Weigh 1 g of chitosan and place it in a reactor. Add 90 mL of 2% acetic acid solution, stir at 500 r / min for 7 min until the chitosan is completely dissolved. Then add 0.5 mL of benzoic acid and 0.3 g of vitamin C to the reactor. After purging the air with high-purity nitrogen, add 1.5 mL of 30% H2O2 solution and react for 15 h. Dialyze with an 8-14 kDa dialysis bag for 70 h, changing the water every 8 h to obtain a benzoylated chitosan solution;

[0056] S2. Preparation of polymer microspheres: Add 50 mL of EDTA to 15 mL of dichloromethane solution and stir for 6 h. Electrospray into microspheres under a high voltage of 35 kV. Immerse the obtained microspheres in an ethanol solution for 15 min, wash 3 times with deionized water, then soak in 15 mL of 0.1 mol / L magnesium sulfate solution for 5 h, take out, wash 3 times with deionized water, and freeze-dry to obtain polymer microspheres;

[0057] S3. Preparation of antibacterial film: Immerse 1.4 g of polymer microspheres in the benzoylated chitosan solution, add 0.9 mL of glycerol to obtain a film-forming solution. Use the casting method to evenly spread the film-forming solution on a polytetrafluoroethylene plate and dry at 55 °C for 2.5 h to obtain a chitosan antibacterial film loaded with polymer microspheres.

[0058] Performance test:

[0059] (1) Mechanical properties

[0060] Cut the antibacterial film prepared in the present invention into specimens of 4 cm × 1.5 cm, and use a texture analyzer to measure the tensile strength (TS) and elongation at break (EAB). The probe is AT / G, the distance between the test parts is 3 cm, and the stretching speed is 0.5 mm / s. The calculation formulas for tensile strength and elongation at break are as follows:

[0061] TS = F / S

[0062] Where: TS is the tensile strength (MPa); F is the maximum tensile force received when the film breaks (N); S is the cross-sectional area of the film (m 2 )

[0063] EAB = (L0 - L) / L * 100%

[0064] Where: EAB is the elongation at break (%), L0 is the length at film break (cm), and L is the initial spacing (cm) of the test component of the film, which is 3 cm.

[0065] Table 1 Mechanical properties of each example

[0066] Tensile strength (MPa) Elongation at break (%) Example 1 56.2 82.5 Example 2 63.1 83.2 Example 3 65.8 85.3 Example 4 56.6 84.7 Comparative Example 1 60.5 80.3 Comparative Example 2 47.3 69.9 Comparative Example 3 53.9 78.4

[0067] As can be seen from Table 1, in Comparative Examples 1-3, undenatured chitosan was used, or magnesium metal ions were directly added, without preparing polymer microspheres, or after replacing poly(lactic-co-glycolic acid) with EDTA, the mechanical properties of the antibacterial films prepared were relatively poor compared with those of the antibacterial films prepared in Examples 1-4. The elongation at break of the antibacterial films prepared in the same examples was higher than that of the comparative examples. It can be seen that the chitosan derivative loaded with polymer microspheres prepared by the present invention can improve the mechanical properties of the antibacterial film.

[0068] (2) Antibacterial performance

[0069] The antibacterial diameter was used as the standard for evaluating the antibacterial performance of the antibacterial film. The experimental strains used were Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. The two strains were added to LB broth and cultured at 37 °C for 24 h. The bacterial solution was diluted 10 times with sterile water to obtain the initial bacterial solution. 0.1 mL of the initial bacterial solution was dropped on the PCA agar solid medium and spread evenly on the plate. The antibacterial film prepared by the present invention was cut into a circle with a diameter of 1 cm and pasted on the agar plate, and cultured at 37 °C for 24 h. The diameter of the antibacterial zone was observed and measured.

[0070] Table 2 Antibacterial zone diameters (mm) of each example

[0071]

[0072] As can be seen from Table 2, the antibacterial films prepared in the examples had stronger antibacterial properties than those prepared in the comparative examples, indicating that the loading of polymer microspheres on benzoylated chitosan enhanced the antibacterial activity of the antibacterial film. The direct addition of magnesium sulfate solution reduced the antibacterial activity of chitosan, and the effect on Gram-negative bacteria was greater than that on Gram-positive bacteria, resulting in relatively poor antibacterial effects of the antibacterial films prepared in Comparative Example 2. In addition, the antibacterial film prepared by the present invention had higher antibacterial activity against Staphylococcus aureus than against Escherichia coli, which may be related to the fact that the lipopolysaccharide layer on the cell membrane of Gram-negative bacteria can effectively prevent antibacterial substances from penetrating the cell membrane, thus having a certain drug resistance.

Claims

1. A method for preparing a chitosan antibacterial film loaded with polymer microspheres, characterized in that: The following steps are involved: S1. Preparation of chitosan derivative solution: weigh chitosan and place it in a reactor, add acetic acid solution and stir until chitosan is completely dissolved, then add benzoic acid and vitamin C to the reactor, pass high-purity nitrogen to exclude air, add H2O2 solution and react for 12-20h, then dialyze using a dialysis bag to obtain a benzoated chitosan solution; S2. Preparation of polymer microspheres: 40-60 mL of polylactic acid-co-glycolic acid was added to 10-20 mL of dichloromethane and stirred for 5-8 h, and electrostatically sprayed into microspheres under high pressure, and the obtained microspheres were immersed in an ethanol solution for activation for 10-20 min, washed with deionized water for 3-5 times, and then immersed in 10-20 mL of magnesium sulfate solution for 3-5 h, taken out, washed with deionized water for 3-5 times, and freeze-dried to obtain polymer microspheres; S3. Preparation of antibacterial film: immerse the polymer microspheres in a benzoated chitosan solution, add glycerol to obtain a film-forming solution, and use a casting method to evenly spread the film-forming solution on a polytetrafluoroethylene plate, and dry it to obtain a chitosan antibacterial film loaded with polymer microspheres; in the step S3, the amount of polymer microspheres added is 1-2wt.%, and the amount of glycerol added is 1-1.5vt.%.

2. The method for preparing a chitosan antibacterial film loaded with polymer microspheres according to claim 1, characterized in that: The components in step S1 are as follows: the mass volume ratio of chitosan to benzoic acid is 1 g: 0.2-0.7 mL, 80-100 mL 2% acetic acid solution, 0.2-0.4 g vitamin C, and 1-2 mL 30% H2O2 solution.

3. The method for preparing a chitosan antibacterial film loaded with polymer microspheres according to claim 1, characterized in that: In step S1, the stirring speed is 300-700 r / min, and the stirring time is 5-10 min.

4. The method for preparing a chitosan antibacterial film loaded with polymer microspheres according to claim 1, characterized in that: In step S1, the molecular weight cut-off of the dialysis bag is 8-14 kDa, the dialysis time is 60-72 hours, and the water is changed every 8 hours.

5. The method for preparing a chitosan antibacterial film loaded with polymer microspheres according to claim 1, characterized in that: The concentration of the magnesium sulfate solution in step S2 is 0.05-0.1 mol / L.

6. The method for preparing a chitosan antibacterial film loaded with polymer microspheres according to claim 1, characterized in that: The high voltage condition in step S2 is 28-40 kV.

7. The method for preparing a chitosan antibacterial film loaded with polymer microspheres according to claim 1, characterized in that: In step S3, the drying temperature is 50-60° C. and the drying time is 2-3 hours.

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