CN116942794B - Efficient corrosion inhibition bactericide and preparation method thereof - Google Patents
Efficient corrosion inhibition bactericide and preparation method thereof Download PDFInfo
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- 230000000844 anti-bacterial effect Effects 0.000 title claims abstract description 26
- 239000003899 bactericide agent Substances 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 230000007797 corrosion Effects 0.000 title abstract description 52
- 238000005260 corrosion Methods 0.000 title abstract description 52
- 230000005764 inhibitory process Effects 0.000 title abstract description 50
- 229920001661 Chitosan Polymers 0.000 claims abstract description 96
- 239000004005 microsphere Substances 0.000 claims abstract description 77
- 101800000245 Antibacterial peptide LL-37 Proteins 0.000 claims abstract description 70
- 102400000669 Antibacterial peptide LL-37 Human genes 0.000 claims abstract description 70
- 125000002091 cationic group Chemical group 0.000 claims abstract description 68
- 235000019832 sodium triphosphate Nutrition 0.000 claims abstract description 48
- 102100038608 Cathelicidin antimicrobial peptide Human genes 0.000 claims abstract description 40
- 101000741320 Homo sapiens Cathelicidin antimicrobial peptide Proteins 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000000243 solution Substances 0.000 claims description 100
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- 238000003760 magnetic stirring Methods 0.000 claims description 32
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- 229940057995 liquid paraffin Drugs 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 16
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 16
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 16
- 239000012154 double-distilled water Substances 0.000 claims description 16
- 238000001914 filtration Methods 0.000 claims description 16
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- 230000008569 process Effects 0.000 claims description 16
- 239000004094 surface-active agent Substances 0.000 claims description 16
- 229960000583 acetic acid Drugs 0.000 claims description 8
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- 238000004108 freeze drying Methods 0.000 claims description 8
- 239000012362 glacial acetic acid Substances 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 8
- 229920000053 polysorbate 80 Polymers 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 241000191967 Staphylococcus aureus Species 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims 5
- 230000000855 fungicidal effect Effects 0.000 claims 2
- 239000000417 fungicide Substances 0.000 claims 2
- 238000013268 sustained release Methods 0.000 claims 2
- 239000012730 sustained-release form Substances 0.000 claims 2
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Abstract
The invention provides a high-efficiency corrosion inhibition bactericide and a preparation method thereof. The LL37 variant 1-3 is obtained by modifying amino acid based on LL37 antibacterial peptide for the first time, and antibacterial activity analysis proves that the LL37 variant 1-3 prepared by the method has good antibacterial activity. The microsphere corrosion inhibition system prepared by the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated cationic antibacterial peptide LL37 variant preparation method has a spherical structure, high balling rate and no obvious adhesion aggregation between microspheres; the microsphere corrosion inhibition system has good corrosion inhibition effect, uniform microsphere particle size and high drug loading rate.
Description
Technical Field
The invention relates to the technical field of bactericides, in particular to a high-efficiency corrosion inhibition bactericide and a preparation method thereof.
Background
The nanomaterial is widely applied to the biomedical field due to the advantages of the nanomaterial such as surface effect and small-size effect. The nano drug-carrying system refers to a polymer as a carrier, and small-molecule drugs are combined in the polymer in a physical or chemical mode in an encapsulation or adsorption mode to form the drugs with the size of nano level. The nano-drug-carrying carrier is generally selected from polymers with high biocompatibility or which can be modified. Such vectors may be synthetic or natural. Unlike traditional medicine treating means, nanometer medicine carrying has the advantages of improving medicine solubility and raising medicine stability,
the loss of the drug is reduced as much as possible before the drug reaches the treatment site; secondly, the tissue permeability of the medicine is improved, the membrane permeability is improved, and the administration route is increased; thirdly, the targeting of the medicine is increased, the utilization rate of the medicine is improved, and after the nano medicine carrying system is modified, the nano medicine can release effective medicine molecules after reaching the lesion part of the organism, so that the medicine effect is exerted; and fourthly, the corrosion inhibition control release regulating effect is achieved.
LL37 is the only cationic antimicrobial peptide found in humans to date. Because LL37 has a killing effect on a variety of gram-positive and gram-negative bacteria, LL37 becomes an important component of the innate immunity of the body. LL37 has broad prospects for antimicrobial activity, while possessing multiple biological activities, and is considered a new hope for resistance to multi-drug resistant bacterial infections. However, at present, the antibacterial peptide has been deactivated in vitro, so that the antibacterial performance of the antibacterial peptide is reduced, and the application of the antibacterial peptide is greatly limited.
Disclosure of Invention
Aiming at the technical problems existing in the prior art, the invention provides a high-efficiency corrosion inhibition bactericide and a preparation method thereof. The bactericide takes the LL37 variant as a core antibacterial active substance, and realizes the efficient corrosion inhibition of the antibacterial peptide by constructing the LL37 variant embedded with chitosan/sodium tripolyphosphate corrosion inhibition microspheres, thereby overcoming the defect that the in vitro activity of the antibacterial peptide is lost in the prior art.
The invention aims to provide a preparation method of a chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated cationic antibacterial peptide LL37 variant, which comprises the following steps:
1) Dissolving 400-600mg of chitosan in 20-40mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 4-6mg BSA, 3-8mg DTT and 50-250 mug cationic antibacterial peptide LL37 or a variant thereof in 10-20mL sterile double distilled water, fully dissolving the solution under the magnetic stirring of 250r/min for 1, standing for 12 hours, and filtering to remove impurities to obtain a transparent cationic antibacterial peptide LL37 solution;
3) Slowly adding the cationic antibacterial peptide LL37 or the variant solution thereof in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL 37;
4) Slowly dissolving 10-20mL of Tween-80 in 200-300mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 or variants thereof is obtained;
6) Dropwise adding 10-25% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate corrosion inhibition microsphere emulsion coated with the cationic antibacterial peptide LL37 can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 3-5 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated with the cationic antibacterial peptide LL37, and storing the chitosan/sodium tripolyphosphate corrosion inhibition microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
The invention also aims to provide a high-efficiency corrosion inhibition bactericide which is prepared by the preparation method.
The invention further aims at providing the application of the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated cationic antibacterial peptide LL37 variant in preparing a high-efficiency corrosion inhibition bactericide.
Preferably, the bactericide is one that kills or inhibits escherichia coli or staphylococcus aureus.
It is another object of the present invention to provide a variant of cationic antibacterial peptide LL37, the amino acid sequence of which is shown in SEQ ID NO. 2-4.
Another object of the invention is to provide a use of a cationic antimicrobial peptide LL37 variant in the preparation of a highly effective corrosion inhibiting bactericide.
The invention has the following advantages: the LL37 variant 1-3 is obtained by modifying amino acid based on LL37 antibacterial peptide for the first time, and antibacterial activity analysis proves that the LL37 variant 1-3 prepared by the method has good antibacterial activity. The microsphere corrosion inhibition system prepared by the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated cationic antibacterial peptide LL37 variant preparation method has a spherical structure, high balling rate and no obvious adhesion aggregation between microspheres; the microsphere corrosion inhibition system has good corrosion inhibition effect, uniform microsphere particle size and high drug loading rate.
Drawings
FIG. 1 is a graph showing the results of in vitro antibacterial activity analysis of LL37 antibacterial peptide according to the present invention;
FIG. 2 is a graph showing the corrosion inhibition results of chitosan/sodium tripolyphosphate corrosion inhibition microspheres coated with cationic antimicrobial peptide LL37 variant microspheres according to the invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples so as to more clearly understand the present invention by those skilled in the art.
The following examples are given by way of illustration of the invention and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art without creative efforts are within the protection scope of the present invention based on the specific embodiments of the present invention.
In the examples of the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise; in the embodiments of the present invention, unless specifically indicated, all technical means used are conventional means well known to those skilled in the art.
Example 1
A method for preparing a cationic antibacterial peptide LL37 variant comprising the steps of:
taking the amino acid sequence (SEQ ID NO. 1) of the cationic antibacterial peptide LL37 as a starting antibacterial peptide, obtaining cationic antibacterial peptide LL37 variants by substituting amino acids at specific sites, and obtaining cationic antibacterial peptide LL37 variants 1 (SEQ ID NO. 1), cationic antibacterial peptide LL37 variants 2 (SEQ ID NO. 2) and cationic antibacterial peptide LL37 variants 3 (SEQ ID NO. 3) in a total.
The antibacterial performance of the cationic antibacterial peptide LL37 variant was verified with E.coli and Staphylococcus aureus as targets. The results are shown in FIG. 1: compared with LL37 antibacterial peptide, the antibacterial activity of LL37 variant 1-3 is effectively improved, and especially the antibacterial performance of LL37 variant 3 can reach 94.2+/-2.7% which is far higher than that of LL37 antibacterial peptide. In conclusion, the LL37 variant 1-3 prepared by the invention has good antibacterial activity.
Example 2
The preparation method of the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated cationic antibacterial peptide LL37 variant comprises the following steps:
1) Dissolving 400mg of chitosan in 20mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 4mg BSA, 3mg DTT and 50 mug cationic antimicrobial peptide LL37 variant 1, 2 or 3 in 10mL sterile double distilled water, fully dissolving the solution under the magnetic stirring of 250r/min for 1, standing for 12 hours, and filtering to remove impurities to obtain a transparent cationic antimicrobial peptide LL37 variant solution;
3) Slowly adding the cationic antibacterial peptide LL37 variant solution in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL37 variant;
4) Slowly dissolving 10mL of Tween-80 in 200mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 variant obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 variant is obtained;
6) Dropwise adding 10% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate corrosion inhibition microsphere emulsion coated with the cationic antimicrobial peptide LL37 variant can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 3 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated with the cationic antimicrobial peptide LL37 variant, and storing the chitosan/sodium tripolyphosphate corrosion inhibition microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
Example 3
The preparation method of the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated cationic antibacterial peptide LL37 variant comprises the following steps:
1) Dissolving 500mg of chitosan in 30mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 5mg BSA, 6mg DTT and 100 mug 50 mug cationic antimicrobial peptide LL37 variant 1, 2 or 3 in 15mL sterile double distilled water, placing the solution under magnetic stirring of 250r/min for fully dissolving 1, standing for 12 hours, and filtering to remove impurities to obtain transparent cationic antimicrobial peptide LL37 variant solution;
3) Slowly adding the cationic antibacterial peptide LL37 variant solution in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL37 variant;
4) Slowly dissolving 15mL of Tween-80 in 250mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing the surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 variant obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 variant is obtained;
6) Dropwise adding 10-25% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate corrosion inhibition microsphere emulsion coated with the cationic antimicrobial peptide LL37 variant can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 4 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated with the cationic antimicrobial peptide LL37 variant, and storing the chitosan/sodium tripolyphosphate corrosion inhibition microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
Example 4
The preparation method of the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated cationic antibacterial peptide LL37 variant comprises the following steps:
1) Dissolving 600mg of chitosan in 40mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 6mg BSA, 8mg DTT and 250 mug of 50 mug cationic antimicrobial peptide LL37 variant 1, 2 or 3 in 20mL sterile double distilled water, placing the solution under magnetic stirring of 250r/min for fully dissolving 1, standing for 12 hours, and filtering to remove impurities to obtain a transparent cationic antimicrobial peptide LL37 variant solution;
3) Slowly adding the cationic antibacterial peptide LL37 variant solution in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL37 variant;
4) Slowly dissolving 20mL of Tween-80 in 300mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing the surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 variant obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 variant is obtained;
6) Dropwise adding 25% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate corrosion inhibition microsphere emulsion coated with the cationic antimicrobial peptide LL37 variant can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 5 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated with the cationic antimicrobial peptide LL37 variant, and storing the chitosan/sodium tripolyphosphate corrosion inhibition microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
Example 5
To evaluate the corrosion inhibition efficacy of the chitosan/sodium tripolyphosphate corrosion inhibiting microspheres coated with the cationic antimicrobial peptide LL37 variant. 20mg of the chitosan/sodium tripolyphosphate corrosion inhibition microspheres coated with the cationic antibacterial peptide LL37 variant 1-3 obtained in the example 2 are respectively dissolved in 10mL of PBS solution, placed in a constant temperature shaking table at 37 ℃ and shaken at a constant speed of 120r/min, and 200-300 mu l of corrosion inhibition liquid is sucked at intervals for ELISA analysis to detect the content of the cationic antibacterial peptide LL37 variant in the solution. As shown in FIG. 2, the corrosion inhibition rates of the antibacterial peptide LL37 variant 1-3 of the corrosion inhibition microsphere reach 6%,8% and 10% respectively at 1 day, and then release at a relatively slow speed, and the corrosion inhibition rates of the antibacterial peptide LL37 variant 1-3 reach 90+/-2.4%, 94+/-1.3% and 98+/-1.2% respectively at 8 days, which indicates that the microsphere prepared by the invention has good corrosion inhibition effect.
Example 6
Aiming at the chitosan/sodium tripolyphosphate corrosion inhibition microspheres coated with the cationic antibacterial peptide LL37 variant prepared in the embodiment 2, detecting particle sizes by adopting a Zetasizer Nano ZS-90 laser particle sizer, measuring absorbance of the LL37 antibacterial peptide variant in the corrosion inhibition microspheres by adopting a high performance liquid chromatography, calculating drug loading rate of the chitosan/sodium tripolyphosphate corrosion inhibition microspheres to the LL37 antibacterial peptide variant according to a formula, co-culturing the corrosion inhibition microspheres with escherichia coli, and analyzing release rate of the cationic antibacterial peptide LL37 variant on the 8 th day by an ELISA method.
TABLE 1 particle size and drug loading ratio of nanoparticles
The result shows that the chitosan/sodium tripolyphosphate corrosion inhibition microsphere coated with the cationic antibacterial peptide LL37 variant prepared by the invention has uniform particle size, high drug loading rate and good corrosion inhibition rate.
It should be noted that the above examples are only for further illustrating and describing the technical solution of the present invention, and are not intended to limit the technical solution of the present invention, and the method of the present invention is only a preferred embodiment and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for preparing a chitosan/sodium tripolyphosphate slow-release microsphere coated cationic antimicrobial peptide LL37 variant, which is characterized by comprising the following steps:
1) Dissolving 400-600mg of chitosan in 20-40mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 4-6mg BSA, 3-8mg DTT and 50-250 mug cationic antimicrobial peptide LL37 variant 1, 2 or 3 in 10-20mL sterile double distilled water, fully dissolving the solution under the magnetic stirring of 250r/min for 1, standing for 12 hours, and filtering to remove impurities to obtain a transparent cationic antimicrobial peptide LL37 variant solution; wherein the amino acid sequence of the cationic antibacterial peptide LL37 variant 1, 2 or 3 is shown in SEQ ID NO. 2-4;
3) Slowly adding the cationic antibacterial peptide LL37 variant solution in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL37 variant;
4) Slowly dissolving 10-20mL of Tween-80 in 200-300mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 variant obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 variant is obtained;
6) Dropwise adding 10-25% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate slow-release microsphere emulsion coated with the cationic antibacterial peptide LL37 can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 3-5 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate slow-release microsphere coated with the cationic antibacterial peptide LL37 variant, and storing the chitosan/sodium tripolyphosphate slow-release microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
2. The method of claim 1, wherein the steps of:
1) Dissolving 400mg of chitosan in 20mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 4mg BSA, 3mg DTT and 50 mug cationic antimicrobial peptide LL37 variant 1, 2 or 3 in 10mL sterile double distilled water, fully dissolving the solution under the magnetic stirring of 250r/min for 1, standing for 12 hours, and filtering to remove impurities to obtain a transparent cationic antimicrobial peptide LL37 variant solution; wherein the amino acid sequence of the cationic antibacterial peptide LL37 variant 1, 2 or 3 is shown in SEQ ID NO. 2-4;
3) Slowly adding the cationic antibacterial peptide LL37 variant solution in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL37 variant;
4) Slowly dissolving 10mL of Tween-80 in 200mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 variant obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 variant is obtained;
6) Dropwise adding 10% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate slow-release microsphere emulsion coated with the cationic antimicrobial peptide LL37 variant can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 3 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate slow release microsphere coated with the cationic antibacterial peptide LL37 variant, and storing the chitosan/sodium tripolyphosphate slow release microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
3. The method of claim 1, wherein the steps of:
1) Dissolving 500mg of chitosan in 30mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 5mg BSA, 6mg DTT and 100 mug 50 mug cationic antimicrobial peptide LL37 variant 1, 2 or 3 in 15mL sterile double distilled water, placing the solution under magnetic stirring of 250r/min for fully dissolving 1, standing for 12 hours, and filtering to remove impurities to obtain transparent cationic antimicrobial peptide LL37 variant solution; wherein the amino acid sequence of the cationic antibacterial peptide LL37 variant 1, 2 or 3 is shown in SEQ ID NO. 2-4;
3) Slowly adding the cationic antibacterial peptide LL37 variant solution in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL37 variant;
4) Slowly dissolving 15mL of Tween-80 in 250mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing the surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 variant obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 variant is obtained;
6) Dropwise adding 10-25% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate slow-release microsphere emulsion coated with the cationic antimicrobial peptide LL37 variant can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 4 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate slow-release microsphere coated with the cationic antibacterial peptide LL37 variant, and storing the chitosan/sodium tripolyphosphate slow-release microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
4. The method of claim 1, wherein the steps of:
1) Dissolving 600mg of chitosan in 40mL of glacial acetic acid water solution, fully dissolving the solution under the magnetic stirring of 250r/min, standing for 12h, and filtering to remove impurities to obtain a transparent chitosan solution;
2) Dissolving 6mg BSA, 8mg DTT and 250 mug of 50 mug cationic antimicrobial peptide LL37 variant 1, 2 or 3 in 20mL sterile double distilled water, placing the solution under magnetic stirring of 250r/min for fully dissolving 1, standing for 12 hours, and filtering to remove impurities to obtain a transparent cationic antimicrobial peptide LL37 variant solution; wherein the amino acid sequence of the cationic antibacterial peptide LL37 variant 1, 2 or 3 is shown in SEQ ID NO. 2-4;
3) Slowly adding the cationic antibacterial peptide LL37 variant solution in the step 2) into the chitosan solution in the step 1), and fully and uniformly mixing to obtain a chitosan solution containing the cationic antibacterial peptide LL37 variant;
4) Slowly dissolving 20mL of Tween-80 in 300mL of liquid paraffin oil solution to obtain liquid paraffin oil solution containing the surfactant;
5) Dropwise adding the chitosan solution containing the cationic antibacterial peptide LL37 variant obtained in the step 3) into the liquid paraffin oil solution containing the surfactant obtained in the step 4), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan microsphere emulsion embedded with the cationic antibacterial peptide LL37 variant is obtained;
6) Dropwise adding 25% by volume of sodium tripolyphosphate aqueous solution into the chitosan microsphere emulsion obtained in the step 5), wherein the dropwise adding process is carried out under the magnetic stirring of 250r/min, and after the dropwise adding is finished, the chitosan/sodium tripolyphosphate slow-release microsphere emulsion coated with the cationic antimicrobial peptide LL37 variant can be obtained;
7) Repeatedly cleaning the microsphere emulsion obtained in the step 6) for 5 times by sequentially using petroleum ether, isopropanol and double distilled water to finally obtain the chitosan/sodium tripolyphosphate slow-release microsphere coated with the cationic antibacterial peptide LL37 variant, and storing the chitosan/sodium tripolyphosphate slow-release microsphere in a refrigerator at the temperature of minus 20 ℃ for standby after freeze drying.
5. A high-efficiency slow-release bactericide, which is obtained by the preparation method according to any one of claims 1 to 4, wherein the bactericide kills or inhibits escherichia coli or staphylococcus aureus.
6. Use of a chitosan/sodium tripolyphosphate sustained release microsphere coated cationic antibacterial peptide LL37 variant according to any one of claims 1 to 4 for preparing a high-efficiency sustained release bactericide, characterized in that the bactericide kills or inhibits escherichia coli or staphylococcus aureus.
7. A cationic antibacterial peptide LL37 variant, characterized in that the amino acid sequence of said variant is shown in SEQ ID No. 2-4.
8. Use of a cationic antibacterial peptide LL37 variant according to claim 7 for the preparation of a highly effective slow release fungicide, wherein the fungicide kills or inhibits escherichia coli or staphylococcus aureus.
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