CN117257987A - Bionic antibacterial peptide delivery system and preparation method and application thereof - Google Patents
Bionic antibacterial peptide delivery system and preparation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Proteomics, Peptides & Aminoacids (AREA)
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- Gastroenterology & Hepatology (AREA)
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Abstract
The invention relates to a bionic antibacterial peptide delivery system, a preparation method and application thereof. A biomimetic antimicrobial peptide delivery system comprising gelatin nanoparticles loaded with antimicrobial peptides, the surface of which is modified with dextran. The invention also provides a preparation method of the bionic antibacterial peptide delivery system, which comprises the steps of stirring gelatin solution at 50 ℃ until the gelatin solution is clear, and filtering to obtain filtrate; and adding acetone, glutaraldehyde and sodium metabisulfite into the filtrate, centrifuging to obtain gelatin nano-particles, adding the gelatin nano-particles into an antibacterial peptide solution, standing to obtain gelatin nano-particles loaded with the antibacterial peptide, and then adding the gelatin nano-particles into a dextran solution to obtain the bionic antibacterial peptide delivery system. The invention also provides application of the bionic antibacterial peptide delivery system in medicines for killing bacterial biofilms. The invention solves the problems of low stability, poor penetrability, random release, high dosage of antibacterial peptide, low sterilization efficiency, large toxic and side effects and the like of the traditional nano antibacterial peptide delivery system.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a bionic antibacterial peptide delivery system, a preparation method and application thereof.
Background
The treatment of chronic wound surfaces of skin caused by the causes of burns, wounds, diabetic foot ulcers and the like is one of the clinical difficulties. The chronic wound brings long-lasting physical and psychological pain to the patient and also brings great medical burden to society. Recent studies have shown that bacterial biofilms are the primary cause of recurrent infections and long lasting healing of skin wounds. Statistics data show that 60% -90% of chronic wounds have bacterial biofilms formed, and continuous or repeated infection is caused by continuous release of bacteria from the chronic wounds, excessive inflammatory reaction is caused, and finally, the wounds are prolonged and not healed. Biofilms are mainly composed of extracellular matrices (extracellular polymeric substances, EPS) secreted by bacteria themselves, including polysaccharides, proteins, DNA, etc. Bacteria are wrapped in the biological film, on one hand, the killing of medicines and the phagocytosis of immune cells of an organism can be effectively resisted through the barrier effect of the biological film, and the dosage of antibiotics required for killing the bacteria of the biological film is more than 1000 times of that of free bacteria; on the other hand, drug resistance genes can be mutually transmitted through quorum sensing, so that the problem of bacterial drug resistance is further aggravated. Therefore, effective removal of bacterial biofilm is of great significance for clinical wound treatment.
The main methods currently used for treating bacterial biofilms are: (1) Surgical debridement can initially effectively clear the biofilm from the wound surface, but studies have shown that biofilm reappears within 48 hours after debridement; (2) Photothermal treatment (the common temperature is more than or equal to 60 ℃), but the biological membrane is killed and the surrounding normal tissues are damaged to some extent; (3) Bleach and surfactant, but have poor patient compliance and some cytotoxicity. Thus, there is an urgent need to develop more efficient and safe methods to remove bacterial biofilm.
The antibacterial peptide is used as a natural immune defensive substance, and has been widely paid attention to the prevention and treatment of multi-drug resistant bacteria infection due to the broad-spectrum antibacterial activity and good biocompatibility. Our earlier-stage research results show that the antibacterial peptide KR-12 has good killing effect on escherichia coli, staphylococcus aureus and methicillin-resistant staphylococcus aureus (MRSA). However, further research results indicate that KR-12 has poor therapeutic effects on bacterial biofilms. The analysis of the cause is mainly in two aspects: (1) The barrier action (physical barrier, efflux pump, enzymatic hydrolysis reaction, etc.) of the bacterial biofilm impedes the passage of the antimicrobial peptide KR-12; (2) Antibacterial peptide KR-12 is easy to degrade and inactivate, so that the antibacterial peptide cannot stably reach a bacteria enrichment area to play a role. These causes ultimately lead to inefficient killing of the biofilm by the antimicrobial peptide, thereby limiting the clinical immediate use of the antimicrobial peptide. Therefore, to achieve effective killing of bacterial biofilms by the antimicrobial peptide KR-12, it is highly desirable to solve the following problems: (1) penetrability; (2) stability.
Nano-drug delivery systems have attracted considerable attention in recent years, which have the following advantages: 1) The antibacterial peptide is loaded to prevent degradation and inactivation of the antibacterial peptide, so that the stability is improved; 2) The particle size is small (100 nm), and the physical barrier (pore diameter: 125nm-1000 nm); 3) The specific surface area is large, and the bacteria can be fully contacted to play a role. In recent years, scholars use gold nanoparticles, chitosan nanoparticles and the like to load antibacterial peptide to construct a nano delivery system, so that the stability of the antibacterial peptide is effectively improved. However, the penetrability of the nano system is still not ideal, and the release of the antibacterial peptide has randomness, can not be concentrated on bacterial infection sites to play a role, and has the defects of high dosage of the antibacterial peptide, low sterilization efficiency, large toxic and side effects and the like, and has a great gap from the clinical actual demands.
Disclosure of Invention
The invention aims to provide a bionic antibacterial peptide delivery system, a preparation method and application thereof, and aims to solve the problems of high antibacterial peptide dosage, low sterilization efficiency, large toxic and side effects and the like caused by low stability, poor penetrability and random release of the existing nanometer antibacterial peptide delivery system, so that the clinical actual requirements are difficult to meet.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a biomimetic antimicrobial peptide delivery system comprising gelatin nanoparticles loaded with antimicrobial peptide, the surface of the gelatin nanoparticles being modified with dextran.
According to the technical means, as the bacteria in the biological film are metabolized and rich in gelatin hydrolase, gelatin nano particles (Gelatin Nanoparticles, GNPs) are used for loading the antibacterial peptide to construct an enzyme response antibacterial peptide delivery system, so that the stability of the antibacterial peptide is effectively improved, the antibacterial peptide delivery system can be released on demand in a bacteria enrichment area due to gelatin hydrolysis, the sterilization efficiency is improved, and the toxic and side effects are reduced; meanwhile, energy is provided for bacterial metabolism, and dextran which can enter the biological membrane under the action of glucosyltransferase to participate in the synthesis of extracellular polysaccharide is used as a modifier, and nutrient substances required by bionic bacteria are used for forming a Trojan horse effect, so that the penetrability of a nano delivery system penetrating through the bacterial biological membrane is effectively promoted.
Among them, the present inventors found during the course of the study that the inside of the biofilm is rich in gelatin hydrolase due to bacterial metabolic activity. Inspired by this phenomenon, the selection of gelatin nanoparticles to load the antibacterial peptide KR-12 is envisaged to construct an enzyme-responsive antibacterial peptide delivery system, which not only can improve the stability of KR-12, but also can enable the antibacterial peptide to be released on demand in a bacteria enrichment area due to gelatin hydrolysis, thereby increasing the sterilization efficiency and reducing the toxic and side effects. However, in practical experiments, gelatin nanoparticles were found to have less than ideal ability to penetrate bacterial biofilms.
The inventor also finds that the barrier effect of the biological membrane is realized not only through physical barrier, but also has a molecular sieve function, and can prevent the passage of non-self substances such as antibacterial drugs and the like through an efflux pump, electrostatic effect and enzymolysis reaction. Thus, the antibacterial peptide-loaded gelatin nanoparticle cannot effectively penetrate the biological membrane. However, bacteria can selectively allow the desired nutrients to penetrate the biological membrane barrier in order to maintain their metabolism. Dextran (Dex) is one of the main nutrients of bacteria, and not only provides energy for bacterial metabolism, but also can enter the inside of a biological membrane to participate in the synthesis of extracellular polysaccharide under the action of glucosyltransferase. Thus, dextran has the property of penetrating bacterial biofilms. Accordingly, it is speculated that the dextran surface modification may simulate the nutrients required by bacteria, forming a "trojan horse" effect, facilitating the penetration of the nano-delivery system through the bacterial biofilm by camouflage.
Therefore, the application surrounds the key problem that the antibacterial peptide KR-12 has poor penetrability and stability to the bacterial biofilm, builds a dextran/gelatin/KR-12 nanometer delivery system (Dex/GNPs/KR-12) based on a bionic strategy, and can effectively penetrate and kill the bacterial biofilm by camouflage into bacterial nutrient substances.
The invention has novel design thought and construction strategy, based on the micro-environmental characteristics of the bacterial biofilm, adopts a bionic strategy, namely, the dextran surface modification to make the nano material camouflage as a self-needed substance of the bacteria, actively enters the inside of the biofilm (the prior research mainly forcedly breaks the barrier of the biofilm by a physicochemical method and the like), then utilizes the characteristics of the bacteria to generate gelatinase, intelligently hydrolyzes gelatin nano particles in a bacteria enrichment area to release antibacterial peptide for in-situ sterilization, and finally thoroughly eliminates the biofilm.
Preferably, the antibacterial peptide is selected from antibacterial peptide KR-12.
Wherein the amino acid sequence of the antibacterial peptide KR-12 is CKRIVKRIKKWLR, and the sequence is a bioactive fragment derived from human antibacterial peptide LL-37. The antibacterial peptide KR-12 has broad-spectrum antibacterial capability and low toxicity.
Preferably, the mass ratio of the gelatin nanoparticle to the antibacterial peptide KR-12 is 10:1.
Preferably, the mass ratio of the gelatin nanoparticle loaded with the antibacterial peptide to the dextran is 1:2.
The invention also provides a preparation method of the bionic antibacterial peptide delivery system, which comprises the following steps:
s1, adjusting the pH value of a gelatin solution to 5.0-7.0, stirring the gelatin solution at 37-60 ℃ until the gelatin solution is clear, cooling, and filtering to obtain filtrate; adding acetone and glutaraldehyde solution into the filtrate for crosslinking reaction; adding sodium metabisulfite solution, centrifuging to obtain precipitate, and washing the precipitate to obtain gelatin nano particles;
s2, adding gelatin nano-particles into the antibacterial peptide solution, standing, and dialyzing to obtain gelatin nano-particles loaded with the antibacterial peptide;
s3, removing oxygen in the dextran solution by using inert gas under ice bath condition, then adding gelatin nano particles loaded with the antibacterial peptide, stirring, reacting, and dialyzing to obtain the bionic antibacterial peptide delivery system.
Preferably, in the S1, the gelatin solution is a Type B gelatin solution, and the mass percentage of Type B gelatin in the Type B gelatin solution is 0.5%; the glutaraldehyde content of the glutaraldehyde solution is 25% by volume; the mass percentage content of sodium metabisulfite in the sodium metabisulfite solution is 0.4 percent.
Preferably, in the S1, the volume ratio of the gelatin solution, the acetone, the glutaraldehyde solution and the sodium metabisulfite solution is 25-125 ml: 25-125 mL:0.5 to 2.5mL:1 to 5mL.
Preferably, in S1, the pH of the gelatin solution is adjusted to 6.0, and the gelatin solution is stirred at 50 ℃ until the gelatin solution is clear, cooled, and filtered to obtain a filtrate.
Preferably, in the step S2, the concentration of the antibacterial peptide in the antibacterial peptide solution is 1mg/mL.
Preferably, in the step S3, the concentration of the dextran in the dextran solution is 0.5g/mL.
Preferably, in S3, the molecular weight of the dextran in the dextran solution is 5kD, 10kD, 20kD, 40kD or 70kD.
The invention also provides application of the bionic antibacterial peptide delivery system, which is applied to medicines for killing bacterial biofilms.
Preferably, the biomimetic antimicrobial peptide delivery system is applied to a medicament for killing methicillin-resistant staphylococcus aureus (MRSA) biofilms.
The invention has the beneficial effects that:
the bionic antibacterial peptide delivery system provided by the invention is based on the characteristics of bacterial biofilm microenvironment and adopts a bionic strategy pertinence design, so that a Trojan horse effect can be formed to actively enter a biofilm and release antibacterial peptide as required, and the problem of insufficient penetrability and stability of the existing antibacterial peptide drug to the biofilm is solved. The design principle and clinical application meaning of the invention are as follows: because bacteria in the biological film are metabolized and rich in gelatin hydrolase, gelatin nano particles (Gelatin Nanoparticles, GNPs) are used for loading the antibacterial peptide to construct an enzyme response antibacterial peptide delivery system, so that the stability of the antibacterial peptide is effectively improved, the antibacterial peptide delivery system can be released on demand in a bacteria enrichment area due to gelatin hydrolysis, the sterilization efficiency is improved, and the toxic and side effects are reduced; meanwhile, energy is provided for bacterial metabolism, and dextran which can enter the biological membrane under the action of glucosyltransferase to participate in the synthesis of extracellular polysaccharide is used as a modifier, so that nutrient substances required by the bionic bacteria are used for 'self' to form a 'Trojan horse' effect, and the penetrability of the nano delivery system to the bacterial biological membrane is effectively promoted. In addition, the core components gelatin, dextran and the antibacterial peptide of the bionic antibacterial peptide delivery system have good biocompatibility, and are suitable for clinical practical application. Therefore, the bionic antibacterial peptide delivery system solves the problems of insufficient penetrability and stability of the traditional antibacterial peptide drug to the biological membrane, has the release characteristic of 'on demand', realizes high-efficiency killing of the bacterial biological membrane, provides a new thought and a new direction for clinically treating bacterial biological membrane infection, and has popularization and application values in the technical field of medicines.
According to the preparation method of the bionic antibacterial peptide delivery system, gelatin nanoparticles are obtained by heating, stirring and crosslinking reaction of gelatin, then the antibacterial peptide is loaded by the expansion and electrostatic action of the gelatin nanoparticles, and dextran is modified on the gelatin nanoparticles loaded with the antibacterial peptide under the low temperature and inert conditions, so that the bionic antibacterial peptide delivery system can be prepared, and has the advantages of simple preparation process, mild reaction conditions, strong operability and suitability for large-scale mass production.
Drawings
FIG. 1 is a flow chart (top) of the synthesis of a biomimetic antimicrobial peptide delivery system of the present invention, and a schematic diagram (bottom) of the biomimetic antimicrobial peptide delivery system killing bacterial biofilms;
FIG. 2 is a transmission electron microscope image of the biomimetic antimicrobial peptide delivery system prepared in example 1;
FIG. 3 is a graph showing the particle size distribution of the bionic antimicrobial peptide delivery system prepared in example 1;
FIG. 4 is a Zeta potential diagram of the biomimetic antimicrobial peptide delivery system prepared in example 1;
FIG. 5 is a graph showing the results of fluorescent microscopy;
FIG. 6 is a graph of fluorescence intensity test results;
FIG. 7 is a graphical representation of bacterial colony numbers;
FIG. 8 is a graph comparing the results of bacterial counts;
FIG. 9 is a morphological image of cells under a microscope;
FIG. 10 is a graph showing the results of absorbance measurement.
Detailed Description
Further advantages and effects of the present invention will become readily apparent to those skilled in the art from the disclosure herein, by referring to the accompanying drawings and the preferred embodiments. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be understood that the preferred embodiments are presented by way of illustration only and not by way of limitation.
It should be noted that the illustrations provided in the following embodiments merely illustrate the basic concept of the present invention by way of illustration, and only the components related to the present invention are shown in the drawings and are not drawn according to the number, shape and size of the components in actual implementation, and the form, number and proportion of the components in actual implementation may be arbitrarily changed, and the layout of the components may be more complicated.
Example 1
As shown in fig. 1, a method for preparing a bionic antimicrobial peptide delivery system comprises the following steps:
s1, synthesizing Gelatin Nano Particles (GNPs): 50mL of Type B gelatin solution (0.5% w/v) was prepared and the pH of the gelatin solution was adjusted to 6.0 with sodium hydroxide; stirring gelatin solution at 50deg.C and rotation speed of 100rpm until it is clear, cooling, and filtering to obtain filtrate; adding 50mL of acetone to the filtrate until the filtrate is slightly turbid, then adding 1mL of glutaraldehyde solution (25% v/v), and stirring for 2h at room temperature and 1000rpm to perform a crosslinking reaction; then adding 2mL of sodium metabisulfite solution (0.4% w/v) to terminate the crosslinking reaction, centrifuging for 10min by using a centrifugal machine with the rotating speed of 10000rpm to obtain a precipitate, washing the precipitate with deionized water for 3 times to obtain gelatin nano particles, and freeze-drying to obtain gelatin nano particle powder;
s2, synthesizing gelatin nano-particles (GNPs/KR-12) loaded with antibacterial peptide KR-12: dissolving antibacterial peptide by using PBS (pH=7.4), preparing antibacterial peptide KR-12 into antibacterial peptide KR-12 solution with a final concentration of 1mg/mL, wherein the pH value of the antibacterial peptide KR-12 solution is 7.4, and then the mass ratio of gelatin nano particles to the antibacterial peptide KR-12 is 10:1 adding gelatin nano particles into the antibacterial peptide KR-12 solution in proportion, standing for 6 hours at room temperature, loading the antibacterial peptide KR-12 by expanding and electrostatic attraction of the gelatin nano particles to obtain a mixed solution, and dialyzing the mixed solution by a dialysis method to remove free antibacterial peptide KR-12 to obtain the gelatin nano particles loaded with the antibacterial peptide KR-12;
s3, synthesizing a bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12): dissolving 12.5g of dextran with molecular weight of 10kD in 25mL of deionized water, and stirring to fully dissolve the dextran to obtain a dextran solution; putting the dextran solution into ice bath, purifying with nitrogen to remove oxygen in the dextran solution, adding gelatin nanoparticles loaded with the antibacterial peptide KR-12 into the dextran solution according to the mass ratio of the gelatin nanoparticles loaded with the antibacterial peptide KR-12 (GNPs/KR-12) to the dextran (Dex) of 1:2, stirring at 4 ℃ for reaction for 2 hours, collecting the reaction solution, removing free dextran in the reaction solution by a dialysis method to obtain the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12), and preserving at 4 ℃ for later use.
Example 2
As shown in fig. 1, a method for preparing a bionic antimicrobial peptide delivery system comprises the following steps:
s1, synthesizing Gelatin Nano Particles (GNPs): 50mL of Type B gelatin solution (0.5% w/v) was prepared and the pH of the gelatin solution was adjusted to 6.0 with sodium hydroxide; stirring gelatin solution at 50deg.C and rotation speed of 100rpm until it is clear, cooling, and filtering to obtain filtrate; adding 50mL of acetone to the filtrate until the filtrate is slightly turbid, then adding 1mL of glutaraldehyde solution (25% v/v), and stirring for 2h at room temperature and 1000rpm to perform a crosslinking reaction; then adding 2mL of sodium metabisulfite solution (0.4% w/v) to terminate the crosslinking reaction, centrifuging for 10min by using a centrifugal machine with the rotating speed of 10000rpm to obtain a precipitate, washing the precipitate with deionized water for 3 times to obtain gelatin nano particles, and freeze-drying to obtain gelatin nano particle powder;
s2, synthesizing gelatin nano-particles (GNPs/KR-12) loaded with antibacterial peptide KR-12: dissolving antibacterial peptide by using PBS (pH=7.4), preparing antibacterial peptide KR-12 into antibacterial peptide KR-12 solution with a final concentration of 1mg/mL, wherein the pH value of the antibacterial peptide KR-12 solution is 7.4, and then the mass ratio of gelatin nano particles to the antibacterial peptide KR-12 is 10:1 adding gelatin nano particles into the antibacterial peptide KR-12 solution in proportion, standing for 6 hours at room temperature, loading the antibacterial peptide KR-12 by expanding and electrostatic attraction of the gelatin nano particles to obtain a mixed solution, and dialyzing the mixed solution by a dialysis method to remove free antibacterial peptide KR-12 to obtain the gelatin nano particles loaded with the antibacterial peptide KR-12;
s3, synthesizing a bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12): dissolving 12.5g of dextran with a molecular weight of 5kD in 25mL of deionized water, and stirring to fully dissolve the dextran to obtain a dextran solution; putting the dextran solution into ice bath, purifying with nitrogen to remove oxygen in the dextran solution, adding gelatin nanoparticles loaded with the antibacterial peptide KR-12 into the dextran solution according to the mass ratio of the gelatin nanoparticles loaded with the antibacterial peptide KR-12 (GNPs/KR-12) to the dextran (Dex) of 1:2, stirring at 4 ℃ for reaction for 2 hours, collecting the reaction solution, removing free dextran in the reaction solution by a dialysis method to obtain the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12), and preserving at 4 ℃ for later use.
Example 3
As shown in fig. 1, a method for preparing a bionic antimicrobial peptide delivery system comprises the following steps:
s1, synthesizing Gelatin Nano Particles (GNPs): 75mL of Type B gelatin solution (0.5% w/v) was prepared and the pH of the gelatin solution was adjusted to 6.0 with sodium hydroxide; stirring gelatin solution at 50deg.C and rotation speed of 100rpm until it is clear, cooling, and filtering to obtain filtrate; adding 75mL of acetone to the filtrate until the filtrate is slightly turbid, then adding 1.5mL of glutaraldehyde solution (25% v/v), and stirring for 2h at room temperature and 1000rpm to perform a crosslinking reaction; then adding 3mL of sodium metabisulfite solution (0.4% w/v) to terminate the crosslinking reaction, centrifuging for 10min by using a centrifugal machine with the rotating speed of 10000rpm to obtain a precipitate, washing the precipitate with deionized water for 3 times to obtain gelatin nano particles, and freeze-drying to obtain gelatin nano particle powder;
s2, synthesizing gelatin nano-particles (GNPs/KR-12) loaded with antibacterial peptide KR-12: dissolving antibacterial peptide by using PBS (pH=7.4), preparing antibacterial peptide KR-12 into antibacterial peptide KR-12 solution with a final concentration of 1mg/mL, wherein the pH value of the antibacterial peptide KR-12 solution is 7.4, and then the mass ratio of gelatin nano particles to the antibacterial peptide KR-12 is 10:1 adding gelatin nano particles into the antibacterial peptide KR-12 solution in proportion, standing for 6 hours at room temperature, loading the antibacterial peptide KR-12 by expanding and electrostatic attraction of the gelatin nano particles to obtain a mixed solution, and dialyzing the mixed solution by a dialysis method to remove free antibacterial peptide KR-12 to obtain the gelatin nano particles loaded with the antibacterial peptide KR-12;
s3, synthesizing a bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12): dissolving 12.5g of dextran with molecular weight of 20kD in 25mL of deionized water, and stirring to fully dissolve the dextran to obtain a dextran solution; putting the dextran solution into ice bath, purifying with nitrogen to remove oxygen in the dextran solution, adding gelatin nanoparticles loaded with the antibacterial peptide KR-12 into the dextran solution according to the mass ratio of the gelatin nanoparticles loaded with the antibacterial peptide KR-12 (GNPs/KR-12) to the dextran (Dex) of 1:2, stirring at 4 ℃ for reaction for 2 hours, collecting the reaction solution, removing free dextran in the reaction solution by a dialysis method to obtain the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12), and preserving at 4 ℃ for later use.
Example 4
As shown in fig. 1, a method for preparing a bionic antimicrobial peptide delivery system comprises the following steps:
s1, synthesizing Gelatin Nano Particles (GNPs): 75mL of Type B gelatin solution (0.5% w/v) was prepared and the pH of the gelatin solution was adjusted to 6.0 with sodium hydroxide; stirring gelatin solution at 50deg.C and rotation speed of 100rpm until it is clear, cooling, and filtering to obtain filtrate; adding 75mL of acetone to the filtrate until the filtrate is slightly turbid, then adding 1.5mL of glutaraldehyde solution (25% v/v), and stirring for 2h at room temperature and 1000rpm to perform a crosslinking reaction; then adding 3mL of sodium metabisulfite solution (0.4% w/v) to terminate the crosslinking reaction, centrifuging for 10min by using a centrifugal machine with the rotating speed of 10000rpm to obtain a precipitate, washing the precipitate with deionized water for 3 times to obtain gelatin nano particles, and freeze-drying to obtain gelatin nano particle powder;
s2, synthesizing gelatin nano-particles (GNPs/KR-12) loaded with antibacterial peptide KR-12: dissolving antibacterial peptide by using PBS (pH=7.4), preparing antibacterial peptide KR-12 into antibacterial peptide KR-12 solution with a final concentration of 1mg/mL, wherein the pH value of the antibacterial peptide KR-12 solution is 7.4, and then the mass ratio of gelatin nano particles to the antibacterial peptide KR-12 is 10:1 adding gelatin nano particles into the antibacterial peptide KR-12 solution in proportion, standing for 6 hours at room temperature, loading the antibacterial peptide KR-12 by expanding and electrostatic attraction of the gelatin nano particles to obtain a mixed solution, and dialyzing the mixed solution by a dialysis method to remove free antibacterial peptide KR-12 to obtain the gelatin nano particles loaded with the antibacterial peptide KR-12;
s3, synthesizing a bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12): dissolving 12.5g of dextran with a molecular weight of 40kD in 25mL of deionized water, and stirring to fully dissolve the dextran to obtain a dextran solution; putting the dextran solution into ice bath, purifying with nitrogen to remove oxygen in the dextran solution, adding gelatin nanoparticles loaded with the antibacterial peptide KR-12 into the dextran solution according to the mass ratio of the gelatin nanoparticles loaded with the antibacterial peptide KR-12 (GNPs/KR-12) to the dextran (Dex) of 1:2, stirring at 4 ℃ for reaction for 2 hours, collecting the reaction solution, removing free dextran in the reaction solution by a dialysis method to obtain the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12), and preserving at 4 ℃ for later use.
Example 5
As shown in fig. 1, a method for preparing a bionic antimicrobial peptide delivery system comprises the following steps:
s1, synthesizing Gelatin Nano Particles (GNPs): 100mL of Type B gelatin solution (0.5% w/v) was prepared and the pH of the gelatin solution was adjusted to 6.0 with sodium hydroxide; stirring gelatin solution at 50deg.C and rotation speed of 100rpm until it is clear, cooling, and filtering to obtain filtrate; 100mL of acetone was added to the filtrate until the filtrate was slightly turbid, then 2mL of glutaraldehyde solution (25% v/v) was added, and the mixture was stirred at room temperature at 1000rpm for 2 hours to perform a crosslinking reaction; then adding 4mL of sodium metabisulfite solution (0.4% w/v) to terminate the crosslinking reaction, centrifuging for 10min by using a centrifugal machine with the rotating speed of 10000rpm to obtain a precipitate, washing the precipitate with deionized water for 3 times to obtain gelatin nano particles, and freeze-drying to obtain gelatin nano particle powder;
s2, synthesizing gelatin nano-particles (GNPs/KR-12) loaded with antibacterial peptide KR-12: dissolving antibacterial peptide by using PBS (pH=7.4), preparing antibacterial peptide KR-12 into antibacterial peptide KR-12 solution with a final concentration of 1mg/mL, wherein the pH value of the antibacterial peptide KR-12 solution is 7.4, and then the mass ratio of gelatin nano particles to the antibacterial peptide KR-12 is 10:1 adding gelatin nano particles into the antibacterial peptide KR-12 solution in proportion, standing for 6 hours at room temperature, loading the antibacterial peptide KR-12 by expanding and electrostatic attraction of the gelatin nano particles to obtain a mixed solution, and dialyzing the mixed solution by a dialysis method to remove free antibacterial peptide KR-12 to obtain the gelatin nano particles loaded with the antibacterial peptide KR-12;
s3, synthesizing a bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12): dissolving 12.5g of dextran with a molecular weight of 70kD in 25mL of deionized water, and stirring to fully dissolve the dextran to obtain a dextran solution; putting the dextran solution into ice bath, purifying with nitrogen to remove oxygen in the dextran solution, adding gelatin nanoparticles loaded with the antibacterial peptide KR-12 into the dextran solution according to the mass ratio of the gelatin nanoparticles loaded with the antibacterial peptide KR-12 (GNPs/KR-12) to the dextran (Dex) of 1:2, stirring at 4 ℃ for reaction for 2 hours, collecting the reaction solution, removing free dextran in the reaction solution by a dialysis method to obtain the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12), and preserving at 4 ℃ for later use.
Detection analysis
1) Characterization analysis
Dripping the bionic antibacterial peptide delivery system prepared in the embodiment 1 into a copper mesh, observing by adopting a transmission electron microscope, and calculating the diameter of the nano-particles by using Image J software; the hydrated particle size distribution of the nanoparticles and the Zeta potential of the surface of the nanoparticles during synthesis were measured using a nanoparticle sizer, and the results are shown in fig. 2 to 4.
FIG. 2 is a transmission electron microscope image of a bionic antimicrobial peptide delivery system, and as can be seen from FIG. 2, the bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12) is in the shape of a sphere with an average particle size of about 108nm; FIG. 3 is a graph showing particle size distribution, and as can be seen from the analysis in FIG. 3, the particle size of the bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12) is normally distributed and concentrated around 100 nm; fig. 4 is a Zeta potential diagram, and analysis in fig. 4 shows that the surface of the gelatin nanoparticle is negative, the surface of the antibacterial peptide KR-12 is positive, and the gelatin nanoparticle and the antibacterial peptide are negative, so that the antibacterial peptide is successfully loaded into the gelatin nanoparticle, and after the dextran is modified on the surface, the surface of the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12) is reversed to positive, so that the successful loading of the dextran on the surface of the gelatin nanoparticle loaded with the antibacterial peptide is demonstrated. The results show that the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12) is successfully prepared, has the particle size smaller than that of the biomembrane pores, and can effectively penetrate the biomembrane barrier to further play a role in sterilization.
2) Sterilization test
The method specifically comprises the following steps:
culture of methicillin-resistant staphylococcus aureus (MRSA) biofilms: single MRSA colony is added into TSB culture medium, a shaking table (200 rpm) at 37 ℃ is used for overnight acquisition of logarithmic phase bacterial suspension, then 100 mu L of bacterial liquid is added into a hole of a 96-well plate, and the culture is carried out for 72 hours at 37 ℃ to obtain MRSA biomembrane model;
experimental grouping: control group: adding only PBS solution; experimental group: only the biomimetic antimicrobial peptide delivery system (Dex/GNPs/KR-12) prepared in example 1 was added;
mu.L of PBS solution and Dex/GNPs/KR-12 were added to MRSA biofilm, respectively, and co-cultured at 37℃for 6 hours, and then examined by various methods: i, adopting a live/dead bacteria staining kit to stain an MRSA biological film for 15 minutes at normal temperature in a dark place, using a fluorescence microscope to observe after PBS (phosphate buffered saline) cleaning, wherein green fluorescence represents live bacteria, red fluorescence represents dead bacteria, and measuring fluorescence intensity by adopting Image J software; II, removing supernatant, adding 100 mu LPBS solution into each hole, performing ultrasonic reaction for 15 minutes, repeatedly blowing, then performing gradient dilution by using PBS solution, uniformly coating 20 mu L of final diluent on the surface of a nutrient agar plate, incubating at 37 ℃ for 24 hours, and photographing and recording, wherein the results are shown in figures 5-8 (the ". Times" represent P <0.05, and the ". Times" represent P < 0.01).
Fig. 5 is a graph showing the results of fluorescent microscopy, green fluorescence represents living bacteria, red fluorescence represents dead bacteria, fig. 6 is a graph showing the results of fluorescent intensity test, fig. 7 is a graph showing the results of comparing the numbers of bacterial colonies grown on the surface of a plate, fig. 8 is a graph showing that the comparison of the numbers of bacteria is that the comparison group is almost all green fluorescence, which indicates that the comparison group is almost all living bacteria, the green fluorescence in the experimental group is remarkably reduced, and the red fluorescence representing dead bacteria is remarkably enhanced, which indicates that the experimental group is almost all dead bacteria, and meanwhile, the results of the comparison of fig. 7 and fig. 8 are consistent with the results of fig. 5 and fig. 6, the number of bacterial colonies grown on the surface of a plate in the experimental group is remarkably lower than that in the comparison group, thereby proving that the bionic antimicrobial peptide delivery system (Dex/GNPs/KR-12) of the invention can effectively kill methicillin-resistant staphylococcus aureus (MRSA) biological film.
3) Cell compatibility test
The method specifically comprises the following steps:
NIH3T3 cells were seeded into 96-well plates at a density of 3000 per well, and the cell culture broth was DMEM medium containing 10% fetal bovine serum;
experimental grouping: control group: adding only PBS solution; experimental group: only the biomimetic antimicrobial peptide delivery system (Dex/GNPs/KR-12) prepared in example 1 was added;
after co-culturing 50. Mu.L of PBS and Dex/GNPs/KR-12 with cells for 1 day, 2 days and 3 days, respectively, the following method was used for detection: i, placing the cells under a microscope for morphological observation; II, detecting the activity of the cells by adopting a CCK-8 method: after the original medium was discarded, 100. Mu.L of fresh medium and 10. Mu.L of CCK-8 reagent were added to each well and incubated with the cells at 37℃for 2 hours in a incubator, the absorbance of the solution at 450nm was measured by using a microplate reader, and the results are shown in FIGS. 9 and 10.
FIG. 9 is a morphological image of cells under a microscope, and as can be seen from the analysis in FIG. 9, the morphology and number of cells in the experimental group are substantially identical to those in the control group, and no significant difference is seen; fig. 10 is a graph showing the absorbance test results, and from the analysis in fig. 10, the absorbance of the control group and the absorbance of the experimental group have no significant difference on days 1, 2 and 3, which indicates that the cell activity of the experimental group has no significant effect. Therefore, the bionic antibacterial peptide delivery system (Dex/GNPs/KR-12) has no obvious cytotoxicity to eukaryotic cells, has good cell compatibility, and can be applied to clinical treatment.
In summary, the bionic antibacterial peptide delivery system of the invention is rich in gelatin hydrolase due to bacterial metabolic activity in the biological membrane, so that the antibacterial peptide is loaded by gelatin nanoparticles (Gelatin Nanoparticles, GNPs) to construct an enzyme-responsive antibacterial peptide delivery system, which not only effectively improves the stability of the antibacterial peptide, but also enables the antibacterial peptide delivery system to be released on demand in a bacterial enrichment area due to gelatin hydrolysis, thereby improving the sterilization efficiency and reducing the toxic and side effects; meanwhile, energy is provided for bacterial metabolism, and dextran which can enter the biological membrane under the action of glucosyltransferase to participate in the synthesis of extracellular polysaccharide is used as a modifier, and nutrient substances required by the bionic bacteria are used to form a Trojan horse effect, so that the penetrability of a nano delivery system penetrating through the bacterial biological membrane is effectively promoted.
According to the preparation method of the bionic antibacterial peptide delivery system, gelatin nanoparticles are obtained by heating, stirring and crosslinking reaction of gelatin, then the antibacterial peptide is loaded by the expansion and electrostatic action of the gelatin nanoparticles, and dextran is modified on the gelatin nanoparticles loaded with the antibacterial peptide under the low temperature and inert conditions, so that the bionic antibacterial peptide delivery system can be prepared, and the preparation method has the advantages of simple preparation process, strong operability and suitability for large-scale mass production.
The above embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention.
Claims (10)
1. A biomimetic antimicrobial peptide delivery system comprising gelatin nanoparticles loaded with antimicrobial peptide, the surface of the gelatin nanoparticles being modified with dextran.
2. The biomimetic antimicrobial peptide delivery system of claim 1, wherein the antimicrobial peptide is selected from the group consisting of antimicrobial peptide KR-12.
3. The biomimetic antimicrobial peptide delivery system of claim 2, wherein the mass ratio of the gelatin nanoparticle to the antimicrobial peptide KR-12 is 10:1.
4. The biomimetic antimicrobial peptide delivery system of claim 1, wherein the mass ratio of the antimicrobial peptide loaded gelatin nanoparticle to the dextran is 1:2.
5. The method of preparing a biomimetic antimicrobial peptide delivery system according to any one of claims 1 to 4, comprising the steps of:
s1, adjusting the pH value of a gelatin solution to 5.0-7.0, stirring the gelatin solution at 37-60 ℃ until the gelatin solution is clear, cooling, and filtering to obtain filtrate; adding acetone and glutaraldehyde solution into the filtrate for crosslinking reaction; adding sodium metabisulfite solution, centrifuging to obtain precipitate, and washing the precipitate to obtain gelatin nano particles;
s2, adding gelatin nano-particles into the antibacterial peptide solution, standing, and dialyzing to obtain gelatin nano-particles loaded with the antibacterial peptide;
s3, removing oxygen in the dextran solution by using inert gas under ice bath condition, then adding gelatin nano particles loaded with the antibacterial peptide, stirring, reacting, and dialyzing to obtain the bionic antibacterial peptide delivery system.
6. The method for preparing a biomimetic antimicrobial peptide delivery system according to claim 5, wherein in S1, the gelatin solution is a Type B gelatin solution, and the mass percentage of Type B gelatin in the Type B gelatin solution is 0.5%; the glutaraldehyde content of the glutaraldehyde solution is 25% by volume; the mass percentage content of sodium metabisulfite in the sodium metabisulfite solution is 0.4 percent.
7. The method for preparing a bionic antibacterial peptide delivery system according to claim 5, wherein in S1, a volume ratio of gelatin solution, acetone, glutaraldehyde solution and sodium metabisulfite solution is added is 25-125 ml: 25-125 mL:0.5 to 2.5mL:1 to 5mL.
8. The method of claim 5, wherein the concentration of the antimicrobial peptide in the antimicrobial peptide solution in S2 is 1mg/mL.
9. The method of claim 5, wherein in S3, the concentration of dextran in the dextran solution is 0.5g/mL; in S3, the molecular weight of the dextran in the dextran solution is 5kD, 10kD, 20kD, 40kD or 70kD.
10. The use of a biomimetic antimicrobial peptide delivery system according to any one of claims 1 to 4, wherein the biomimetic antimicrobial peptide delivery system is used in a medicament for killing bacterial biofilms.
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