CN114984243A - Magnetic nano-antibiotic composite particle and preparation method and application thereof - Google Patents

Magnetic nano-antibiotic composite particle and preparation method and application thereof Download PDF

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CN114984243A
CN114984243A CN202210599081.6A CN202210599081A CN114984243A CN 114984243 A CN114984243 A CN 114984243A CN 202210599081 A CN202210599081 A CN 202210599081A CN 114984243 A CN114984243 A CN 114984243A
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nano
magnetic
composite particles
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paa
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汪熙
臧依桐
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Changzhou Vocational Institute of Light Industry
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Abstract

The invention belongs to the field of medical biomaterials, and particularly relates to magnetic nano-antibiotic composite particles and a preparation method and application thereof; the process comprises the following steps: polyacrylic acid modified magnetic Fe 3 O 4 Nanoparticle to obtain Fe 3 O 4 @ PAA magnetic nanoparticles; alternately assembling gentamicin (Gen) as cation assembling liquid and Hyaluronic Acid (HA) as anion assembling liquid to Fe 3 O 4 Obtaining Fe on the surface of the @ PAA magnetic nano-particle 3 O 4 @(Gen/HA) n (ii) a The antibacterial nano particles obtained by the invention have the antibacterial effect of dual response of pH and hyaluronidase, can be effectively applied to reducing intractable bacterial infection caused by a biological membrane, and can also reduce the occurrence of drug resistance. The magnetic nano antibiotic composite particle prepared by the invention can be used as an additive to be added into some medical composite materials.

Description

Magnetic nano-antibiotic composite particle and preparation method and application thereof
Technical Field
The invention relates to a magnetic nano-antibiotic composite particle, a preparation method and application thereof, belonging to the field of medical biomaterials.
Background
Broad-spectrum antibiotics are often used clinically to treat secondary infections caused by pathogenic bacteria. However, studies have shown that: more than 65% of secondary infections are not caused by free pathogenic bacteria living in the environment, but are associated with the formation of bacterial biofilms. Biofilm is approximately 10-1000 times less sensitive to antibiotics than planktonic bacteria, and thus conventional antibiotic therapy tends to be ineffective against biofilm infection, while the problem of resistance that can arise from abuse of antibiotics is much more non-negligible. An evaluation study of the impact of antibiotic resistance in 204 countries and regions worldwide was reported in the authoritative journal "lancet" 2022, 1/19. The results show that: antibiotic resistance has become a major cause of death worldwide, and the severity of antibiotic resistance is exacerbated by the continuing outbreak of new coronary epidemics. Therefore, how to improve the drug utilization rate of the traditional antibiotics (such as gentamicin and the like) in the antibacterial biomembrane and reduce the risk of drug resistance becomes an important problem to be solved urgently.
The application of the nano material in a novel drug delivery system provides a new idea for solving the problem. The common design concept is to utilize nanoparticles, and CN104524587A adopts mesoporous silica as a drug delivery carrier to realize controlled release, so as to improve the release utilization rate and local drug concentration of the drug. These nano-drug delivery systems show better antibacterial ability against infections caused by free pathogenic bacteria. However, the antibacterial ability exhibited in the face of bacterial biofilms is greatly diminished. On one hand, the nano-drug delivery system can release effective concentration of antibiotics under the stimulation of a bacterial microenvironment, but the nano-drug delivery system is also accompanied with the disordered release of a certain amount of antibiotics in a normal physiological environment without bacterial infection, so that the drug utilization rate is obviously reduced, and the risk of bacterial drug resistance is existed; on the other hand, the above-mentioned nano-drug delivery systems often do not penetrate deep into the interior of the biofilm, preventing effective delivery of antibiotics and thus are not effective against biofilm-induced infections.
In recent years, magnetic Fe 3 O 4 The nano particles have a plurality of excellent properties such as good chemical stability, biocompatibility, superparamagnetism, larger specific surface area and the like, and great research interest of researchers is aroused. Especially in the field of biological medicine which is concerned, the application potential is huge. CN109463381B reports a Fe-based alloy 3 O 4 The quaternary ammonium salt-nano silver type nano antibacterial composite particle shows good contact-release dual antibacterial performance when resisting free bacteria. However, the nano-particles continuously release the nano-silver antibacterial agent in the bacterial liquid, and the controllable response slow-release performance is lacked, so that the further application of the nano-silver antibacterial agent is limited; in addition, whether it is effective against bacterial biofilm infections that are more recalcitrant than free bacteria has not been reported.
Disclosure of Invention
In order to solve the defect that the existing nano material cannot effectively cope with the problem of bacterial biofilm infection when being used as an antibacterial drug delivery system, the invention aims to provide a magnetic nano antibiotic composite particle, a preparation method and application thereof in the aspect of treating bacterial biofilm infection.
The technical scheme for realizing the purpose of the invention is as follows: a preparation method of magnetic nano-antibiotic composite particles is characterized by comprising the following steps:
(1) magnetic Fe modified by polyacrylic acid by solvothermal method 3 O 4 Nanoparticle to obtain Fe 3 O 4 @ PAA magnetic nanoparticles;
(2) adopting a layer-by-layer electrostatic self-assembly technology to alternately assemble the Fe obtained in the step (1) by taking gentamicin (Gen) as a cation assembly liquid and Hyaluronic Acid (HA) as an anion assembly liquid 3 O 4 The surface of the @ PAA magnetic nano particle is coated with Fe with a multilayer antibacterial film 3 O 4 @(Gen/HA) n The nano particles are characterized in that n ranges from 1 to 8 and is a positive integer.
The step (1) of the invention is specifically as follows: FeCl is added 3 ·6H 2 Mixing O, ethylene glycol and diethylene glycol according to a certain proportion, stirring until the solid is completely dissolved, and adding FeCl 3 ·6H 2 Adding quantitative anhydrous sodium acetate and polyacrylic acid into the amount of O, performing ultrasonic treatment to fully dissolve and uniformly mix reactants, transferring the solution into a reaction kettle for heating reaction, after the reaction kettle is cooled to room temperature after full reaction, removing the reaction mixture, adding deionized water, washing to remove unreacted raw materials, and obtaining Fe 3 O 4 @ PAA magnetic nanoparticles.
In the step (1), the magnetic nanoparticles are prepared by a solvothermal method in a double-solvent system, wherein the double-solvent system is a reaction system consisting of two solvents, namely Ethylene Glycol (EG) and diethylene glycol (DEG), and based on the basic rule of crystal growth, the adopted double-solvent system can provide a high-reduction atmosphere with rapid convection of the solvents and effective diffusion of solutes, so that perfect ferroferric oxide crystals with few defects and low thermal stress can be generated.
As a further improvement of the invention: in the step (1): FeCl 3 ·6H 2 The proportion of O, glycol and diglycol is as follows: FeCl 3 ·6H 2 The molar ratio of O to ethylene glycol is 1:50, and the volume ratio of ethylene glycol to diethylene glycol is 1: 1-5.
In the solvothermal method in the step (1), all reactants are fully dissolved and uniformly mixed by adopting ultrasound, the ultrasound time is 10-40 min, and the subsequent sedimentation in a hydrothermal reaction kettle is avoided.
Preferably, the Fe 3 O 4 The average particle size of the @ PAA magnetic nanoparticles is 30-50 nm. In the present invention, Fe 3 O 4 @ PAA magnetic fieldThe particle size of the nano particles has obvious influence on the antibacterial effect, and the smaller the particle size, the larger the specific surface area of the particles, so that more antibiotics can be loaded.
In the step (1), the heating reaction temperature of the reaction kettle is 200 ℃, and the reaction time is 12 h. The heating temperature is controlled to be 200 ℃ in the system, which is favorable for the growth and crystallization of magnetic nuclei and superparamagnetism on one hand, and belongs to low-temperature solvothermal at 200 ℃ on the other hand, so that the system is more suitable for future industrial production.
In the invention, the step (2) is specifically as follows: mixing Fe 3 O 4 Ultrasonically stirring and dispersing the @ PAA magnetic nanoparticles into deionized water to form a dispersion liquid, dropwise adding the dispersion liquid into gentamicin cation assembly liquid, violently stirring by using ultrasound, and dispersing the dispersion liquid into the deionized water again after the assembly is finished through external magnetic field separation and water washing; then dropwise adding the dispersion into hyaluronic acid anion assembly liquid, continuing ultrasonic vigorous stirring, and dispersing again in deionized water after assembly through external magnetic field separation and water washing; repeating the above assembly process with the same anion and cation assembly solution for n times, washing with deionized water, and vacuum drying at 40 deg.C to obtain Fe 3 O 4 @(Gen/HA) n Nanoparticles; wherein n is a positive integer not less than 1.
As a preferred embodiment of the present invention: the repeated assembly process of the anion and cation assembly liquid is preferably n-2-3, and the subsequent dispersibility of the nanoparticles is reduced along with the increase of the assembly times, so that the efficiency is reduced.
As a preferred embodiment of the present invention: the mass concentration ratio of the gentamicin cation assembly liquid to the hyaluronic acid anion assembly liquid is 1:1, assembling liquid to Fe 3 O 4 @ PAA magnetic nanoparticle excess; through research, Fe is obtained under the conditions of different mass concentration ratios 3 O 4 The @ PAA magnetic nanoparticles have uneven charge distribution, so that the influence of different mass concentration ratios on assembled particles is larger; when the concentration ratio of the two assembling liquids is enlarged or reduced, the dispersing performance of the assembled nano particles is obviously reduced, and obvious aggregation is generated to be unfavorable for subsequent assembling.
In the invention, the assembling process in the step (2) is carried out under the condition that the pH value is 5-6, and the pH value of an assembling liquid is adjusted by adopting glacial acetic acid; glacial acetic acid is a unitary organic weak acid, and compared with strong acid such as hydrochloric acid, ionization of hydrogen ions is more moderate, so that control of pH is facilitated.
Fe can be obtained according to the preparation method of the magnetic nano-antibiotic composite particles 3 O 4 Nano antibiotic composite particles as a carrier.
And the above with Fe 3 O 4 The nano antibiotic composite particle as carrier may be used as additive for medical composite material.
The invention has the following remarkable advantages: 1. the assembly technology of the invention has simple process and mild condition, the experimental process does not relate to organic reagent basically, and the invention is green and environment-friendly and is beneficial to popularization and application.
2. The invention creatively uses the characteristic of high-efficiency water locking of hyaluronic acid for packaging the medicine, is beneficial to reducing the disordered release of antibiotics and simultaneously improves the biocompatibility of the material.
3.Fe 3 O 4 The superparamagnetism of the antibacterial material can realize targeted recovery of the antibacterial material, and can penetrate through the biological membrane under a certain external magnetic field, so that antibiotics can be more effectively carried to permeate into the biological membrane to release medicaments for sterilization.
4. The results of the antibacterial biofilm study show that: under the external magnetic field, the nano antibiotic composite particles can completely destroy the structure of the biological membrane and kill bacteria, and have good application prospect of resisting biofilm infection.
According to the invention, the type of the assembling liquid is skillfully screened to construct an intelligent response antibacterial nano particle, so that the bacterial biofilm infection which is more stubborn than free bacteria can be effectively resisted. The invention overcomes the defects of CN109463381B by improving an antibacterial mechanism, and the CN109463381B assembly liquid is chitosan and trisodium citrate, so that the assembly layer has no response performance. The assembly liquid is hyaluronic acid and antibiotics, particularly the hyaluronic acid is skillfully and novel in application, the high moisture-preserving and water-locking function of the hyaluronic acid (commonly called hyaluronic acid) and the acid responsiveness of aminoglycoside antibiotics are fully utilized, and the controllable slow release of the antibiotics is facilitated, so that the antibiotics can be continuously released to kill bacteria in stubborn biofilms after entering the biofilms.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 shows a-Fe in example 1 of the present invention 3 O 4 @PAA，b:Fe 3 O 4 @Gent，c：Fe 3 O 4 @(Gent/HA) 3 And (4) an infrared spectrum.
FIG. 2 shows a-Fe in example 1 of the present invention 3 O 4 @PAA，b:Fe 3 O 4 @(Gent/HA) 3 A TEM image of (a).
FIG. 3 shows a-Fe in example 1 of the present invention 3 O 4 @PAA，b:Fe 3 O 4 @(Gent/HA) 3 XRD pattern of (a). FIG. 4 shows a-Fe in example 1 of the present invention 3 O 4 @PAA，b:
Fe 3 O 4 @(Gent/HA) 3 Thermogravimetric plot.
FIG. 5 shows Fe in example 1 of the present invention 3 O 4 @(Gent/HA) 3 Graph of Zeta potential change during assembly.
FIG. 6 shows a-Fe in example 1 of the present invention 3 O 4 @PAA，b:Fe 3 O 4 @(Gent/HA) 3 The magnetic hysteresis curve of VSM and the dispersion condition under the action of an external magnetic field.
Fig. 7 is a graph showing the response release kinetics of gentamicin in example 2 of the present invention.
FIG. 8 shows a in example 3 of the present invention wherein a is no sample; b is Fe 3 O 4 @(Gent/HA) 3 At pH 5.5; c is Fe 3 O 4 @(Gent/HA) 3 Photograph of inhibition zone of S.aureus under the condition of pH5.5/HA enzyme.
FIG. 9 shows Fe in example 4 of the present invention 3 O 4 @(Gent/HA) 3 Plot of antimicrobial kinetics for a: s.
FIG. 10 shows Fe in example 5 of the present invention 3 O 4 @(Gent/HA) 3 A laser confocal electron microscope 3D overlay and cross-sectional scan which are destructive to S.aureus biological membranes.
Detailed Description
The magnetic nano-antibiotic composite particles, the preparation method and the application thereof are described in detail below with reference to examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
Example 1
The invention provides a preparation method of magnetic nano-antibiotic composite particles, which mainly comprises the following steps:
step (1) adding 0.54g FeCl to a 50mL beaker 3 ·6H 2 O, 5mL of ethylene glycol and 15mL of diethylene glycol were mixed and stirred until the solid was completely dissolved, then 50. mu.L of polyacrylic acid and 1.5g of anhydrous sodium acetate were added rapidly under sonication, and the mixture was sonicated and mechanically stirred for 30min to ensure adequate dissolution. Finally transferring the uniformly dispersed mixed solution into a polytetrafluoroethylene high-pressure kettle, heating to 200 ℃, and reacting for 12 hours at the temperature; then cooling the reaction kettle to room temperature, removing the reaction kettle, and repeatedly washing the reaction kettle with deionized water and absolute ethyl alcohol to obtain Fe 3 O 4 @ PAA magnetic nanoparticles; wherein, the addition amount of anhydrous sodium acetate and polyacrylic acid is determined by FeCl 3 ·6H 2 The dosage of O is proportioned according to the proportion;
taking Fe 3 O 4 The @ PAA magnetic nano particles are dispersed into deionized water by ultrasonic stirring to prepare Fe with the concentration of 0.3mg/ml 3 O 4 @ PAA magnetic nanoparticle dispersion liquid for later use;
step (2): 150mg of gentamicin and 150mg of hyaluronic acid are weighed and respectively dissolved in 300mL of deionized water to prepare 0.5mg/mL gentamicin cation assembly liquid (Gent) and 0.5mg/mL hyaluronic acid anion assembly liquid (HA), and the pH value is adjusted to be 5.5; taking 50mL of Fe obtained in the step (1) 3 O 4 The @ PAA magnetic nanoparticle dispersion liquid is gradually dropped into 50mL of cation assembly liquid, stirred vigorously by ultrasound for 20min, after the assembly is finished, the nanoparticles are separated by an external magnetic field, washed three times by deionized water (the pH value of the assembly liquid is consistent), and then dispersed in 50mL of deionized water again; then, 50mL of the dispersion is added into 50mL of hyaluronic acid anion assembly liquid drop by drop, ultrasonic vigorous stirring is continued for 20min, and assembly is finishedThen, separating by an external magnetic field, washing for three times by deionized water (the pH value of the assembly liquid is consistent with that of the assembly liquid), and dispersing in 50mL of deionized water again; repeating the above assembly process for 2 times to obtain Fe 3 O 4 @(Gent/HA) 3 Nanoparticles.
In the present invention, the dispersion is oily for a certain period of time after the assembly is completed.
The product obtained after vacuum drying and extensive grinding was characterized by FTIR, TEM, XRD, TG, Zeta potential, VSM and compared to the nanoparticles at different stages in the preparation process, as shown in fig. 1-6.
1. Analysis of infrared spectra
In fig. 1, two more distinct peaks appear in curve a: 537cm -1 Stretching vibration of 1710cm under Fe-O -1 The existence of a peak Fe-O indicates that Fe is present in PAA due to stretching vibration of C ═ O 3 O 4 Nanoparticles were successfully prepared and the presence of C ═ O indicated that PAA was successfully coated with magnetic Fe 3 O 4 The surface of the nanoparticles.
In fig. 1, curve b shows two more distinct peaks: 537cm -1 The position is the stretching vibration of Fe-O, 1500 cm-1600 cm -1 The deformation vibration peak of N-H on gentamicin indicates that the antibiotic has been successfully assembled to Fe 3 O 4 @ PAA magnetic nanoparticle surface.
1710cm in curve c compared to a and b in FIG. 1 -1 Has an N-H stretching vibration double peak at 1300cm -1 An O-H bending vibration peak appears, and the two characteristic peaks jointly indicate that the hyaluronic acid is in Fe 3 O 4 The surface of the @ PAA magnetic nanoparticle is successfully assembled. These results together indicate that the antibiotic-loaded hyaluronic acid magnetic nanoparticles have been successfully prepared.
2. Transmission Electron microscopy analysis
FIG. 2, a shows that the solvent-thermal method is used to prepare Fe 3 O 4 The @ PAA magnetic nano particles are relatively uniform in distribution, relatively uniform in particle size and relatively good in dispersity.
As can be seen from the b diagram in FIG. 2, the surface of the magnetic nanoparticles is coated with a distinct layer, which is illustrated by Fe 3 O 4 The surface of @ PAA is indeed coated with organic matter, demonstrating the successful assembly of HA with gentamicin. Comparing the a diagram and the b diagram in fig. 2, it can be seen that the coating of the polymer has little influence on the particle size of the magnetic nanoparticle particles, so that the nanoparticles can be ensured to have a high specific surface area after surface coating. In addition, in the b diagram, the gaps between the nanoparticles are significantly smaller than in the a diagram, which indicates that the dispersibility of the assembled system is reduced. The phenomena jointly illustrate that the surface properties of the magnetic nanoparticles have great influence on the dispersion performance of the magnetic nanoparticles; the dispersibility of the nanoparticles in the solution directly affects the assembly process and the antibacterial process, so the dispersibility is an important index.
XRD spectrum analysis
Comparing the two spectra of spectrum a and spectrum b in fig. 3, it can be found that: six diffraction peaks appear at 2 theta of 30.1 degrees, 35.6 degrees, 43.1 degrees, 54.2 degrees, 57.0 degrees and 63.1 degrees in both maps, and compared with JCPDS cards (NO.19-629), the six diffraction peaks correspond to Fe with a face-centered cubic structure 3 O 4 The crystal planes (220), (311), (400), (422), (511) and (440) of the nano-particle show that the crystal form of the nano-particle is not obviously changed in the assembling process. But the intensity of the peaks in the b spectrum is reduced. Therefore, the magnetic nano-particles have no obvious crystal form change before and after assembly, but the coating of hyaluronic acid and gentamicin is used for coating Fe 3 O 4 The diffraction peak of the @ PAA magnetic nano particle generates a shielding effect, so that the diffraction peak is strongly weakened. The HA and the gentamicin are successfully coated on the surface of the magnetic nanoparticles.
4. Thermogravimetric analysis
In FIG. 4, Fe can be seen from the lines a and b 3 O 4 Before and after the modification of the @ PAA magnetic nanoparticles, the mass change is small below 200 ℃, and the change is mainly expressed as volatilization of residual small molecular substances such as water and the like; at the stage of 200-600 ℃, the weight loss of the nano particles is obvious, and Fe 3 O 4 The weight loss of the @ PAA magnetic nanoparticles is caused by the thermal decomposition of PAA on the surfaces of the nanoparticles, and Fe 3 O 4 @(Gent/HA) 3 The weight loss is the result of thermal decomposition of the polymeric layer on the surface of the nanoparticles; above 600 ℃, the sample weight hardly changes. PAA in magnetic Fe 3 O 4 The coating amount on the surface of the nanoparticles is about 9 percent (Gent/HA) 3 In Fe 3 O 4 The coating amount of the surface of the @ PAA magnetic nanoparticles is about 23%, and the coating rate is obviously increased after modification, so that hyaluronic acid and gentamicin are successfully coated on the surface of the nanoparticles. And the pH value, concentration and assembly time of the assembly liquid can be regulated and controlled to improve the coating effect.
Zeta potential map analysis
In FIG. 5, line a is Fe 3 O 4 @ PAA, b line Fe 3 O 4 @ Gent, line c: fe 3 O 4 @ HA, d line Fe 3 O 4 @ HA @ Gent, line e Fe 3 O 4 @(Gent/HA) 3 About-37 mV, +20mV, -23mV, +15mV, -20mV, respectively, indicate that PAA and HA, which have negative charges on the surface, and gentamicin, which HAs positive charges on the surface, have indeed successfully coated and magnetized the nanoparticle surface. And the higher the absolute (positive and negative) Zeta potential, the more stable the system and the less likely the sample to aggregate in solution. Comparing the potentials of the assembly layers can obtain that the dispersibility of the anionic polymer nanoparticle system is superior to that of the cationic nanoparticle system.
VSM hysteresis loop plot analysis
In FIG. 6, lines a and b in A1 are the hysteresis loops at room temperature of the samples, all of which exhibit ferromagnetism, and graph A2 shows the dispersion under the action of an applied magnetic field. Comparing line a, line b in graph A1, Fe 3 O 4 @ PAA saturation magnetization of 71.2emu/g, Fe 3 O 4 @(Gent/HA) 3 The surface was coated with HA and gentamicin, and the magnetization was reduced to 39.2 emu/g. In addition, it can be seen that the magnetization curves are "S" shaped, indicating Fe before and after assembly 3 O 4 The nanoparticles all have superparamagnetism.
Example 2: responsive release test for gentamicin
Three equal portions of each 5mg Fe are weighed 3 O 4 @(Gent/HA) 3 The nanoparticles were dispersed in 10mL solutions of different release media (pH 7.4, pH5.5/HA enzyme, and pH 7.4/HA enzyme, without significant decrease in enzyme activity under mildly acidic conditions)And in turn, into a dialysis bag. Then, the dialysis bag was immersed in the same medium, and finally the whole system was placed in a constant temperature shaker at 37 ℃ for sustained release. At selected time intervals, the buffer (2mL) outside the dialysis bag was removed for UV-Vis spectroscopy and supplemented with the same volume of fresh release medium solution. The absorbance of the samples was tested at 247nm to calculate the amount of gentamicin released according to a standard curve, measured three times per sample.
As can be seen from the graph in FIG. 7, under all three conditions, the release amount of gentamicin in the sample reaches the maximum within 12h, and almost no antibiotic is released after 12 h; at 12h, the release of gentamicin was maximal at pH5.5 in the presence of hyaluronidase, about 65%, the release of gentamicin was second to the release of gentamicin at pH 7.4 in the presence of hyaluronidase, about 38%, and the release of gentamicin at pH 7.4 in the absence of hyaluronidase was minimal, only about 19%. The experiment results show that the release amount of gentamicin in the sample is the largest in an acidic environment with hyaluronidase added, and the release mechanism is that hyaluronic acid on the outermost layer of the sample is decomposed by hyaluronidase so that gentamicin on the second outermost layer is released. When bacterial infection occurs on the surface containing the magnetic nano-antibiotic composite particles, many harmful microorganisms such as gram bacteria and fungi can secrete hyaluronidase, so that the hyaluronic acid on the surface of the nano-particles is decomposed, the antibacterial agent outside the nano-particles is released, and the harmful bacteria are inactivated. An enzyme-responsive release test shows that hyaluronic acid plays a response role in external bacterial infection in the antibacterial magnetic nanoparticle system, so that the antibacterial agent is released in a responsive manner.
Examples 3 to 5
Fe prepared in example 1 3 O 4 @(Gent/HA) 3 The nanoparticles were evaluated for antibacterial performance against the gram-negative bacterium escherichia coli (e.coli) and the gram-positive bacterium staphylococcus aureus (s.aureus), respectively. The invention respectively carries out bacteriostasis zone, antibacterial kinetics and anti-biofilm experiments.
The method comprises the following specific steps:
(1) preparation of LB liquid culture medium
5g of yeast extract, 10g of tryptone and 10g of sodium chloride are respectively weighed and added into a 1000mL beaker, and 950mL of deionized water is added and stirred for 10min by ultrasound so as to be completely dissolved. Then adjusting the pH value to 7.4 with 5mol/L NaOH solution, transferring the solution into a 1L volumetric flask, placing into an autoclave for sterilization, and sterilizing with high-pressure steam at 121 ℃ for 30 min.
(2) Preparation of LB solid culture medium
5g of yeast extract, 10g of tryptone, 10g of sodium chloride and 15g of agar were weighed respectively and added to a 1000mL beaker, and 950mL of deionized water was added and heated to be completely dissolved. Then adjusting the pH value to 7.4 by using 5mol/L NaOH solution, transferring the solution into a 1L volumetric flask, putting the volumetric flask into an autoclave for sterilization, and sterilizing the volumetric flask for 30min by using high-pressure steam at 121 ℃.
(3) Preparation of PBS buffer
0.27g of monopotassium phosphate, 1.42g of disodium hydrogen phosphate, 8g of sodium chloride and 0.2g of potassium chloride are respectively weighed and added into a 1000mL beaker, 800mL of ionized water is added, ultrasonic stirring is carried out for 10min to enable the materials to be fully dissolved, then concentrated hydrochloric acid is added dropwise to adjust the pH value of the solution to 7.4, the solution is transferred into a 1L volumetric flask, the volumetric flask is placed into an autoclave for sterilization, and the volumetric flask is sterilized by high-pressure steam at 121 ℃ for 30 min.
(4) Preparation of plate Medium
Heating and melting the LB solid culture medium into liquid, quickly adding about 10mL of LB culture medium into each sterile culture dish, horizontally placing on a sterile workbench, cooling and solidifying at room temperature to prepare the solid LB plate culture medium.
(5) Preparation of bacterial suspensions
The inoculating loop was burned on an alcohol burner flame, sterilized, and single colonies were picked from the plate on which the bacteria had been cultured with the inoculating loop, and added to a flask containing 100mL of LB liquid medium, and cultured for 16 to 24 hours in a shaker at 37 ℃ and 160 rpm.
(6) Cultivation of biofilms
Fresh TSBg tryptone soy broth is first prepared according to a fixed formulation and autoclaved for 15min in an autoclave at 121 ℃ for future use. The new culture medium is prepared by using TSBg culture solutionDiluting fresh staphylococcus aureus liquid to about 10% 6 –10 7 CFU/mL, then 100. mu.L of this broth was added to a 12-well plate previously loaded with a circular coverslip, and 900. mu. LTSBg of nutrient medium was added to each well. Statically culturing at 37 deg.C for 48h, and replacing fresh nutrient culture solution every 24 h. After the mature bacterial biofilm had formed, the surface of the coverslip was gently rinsed with a small amount of PBS buffer to remove surface planktonic bacteria.
Example 3 zone of inhibition test
Dispersing 1mg/mL of nanoparticles in different buffer media, fully soaking a disinfection filter paper sheet with the diameter of 6mm in supernatant liquid separated by magnetic attraction, taking out after a period of time, and naturally airing for later use. The concentration of 100 μ L is 10 6 The bacterial liquid of the staphylococcus aureus of CFU/mL is evenly coated on an LB solid culture medium, and a prepared filter paper sheet is fixed in the center of the culture medium coated with the bacteria and cultured for 24 hours at the temperature of 37 ℃. The bacteriostatic ability of the nano particles is measured by measuring the diameter of a bacteriostatic circle around the filter paper sheet.
As shown in FIG. 8, the width of the zone of inhibition around the circular filter paper sheet in panel b is about 5mm, which indicates that Fe is present at pH5.5 3 O 4 @(Gent/HA) 3 Can responsively release a certain amount of gentamicin, thereby generating an obvious zone of inhibition. The c diagram shows that the width of the zone of inhibition around the circular filter paper sheet is increased to 10mm, and Fe is added under the condition that the pH value is 5.5 3 O 4 @(Gent/HA) 3 The antibacterial ring generated under the action of hyaluronidase is more obvious because the hyaluronic acid on the surface of the nano-particle is decomposed, so that more antibiotics on the nano-particle are released. The above results show that, comparing the three graphs in FIG. 8, Fe was produced 3 O 4 @(Gent/HA) 3 The nano-particles can show stronger antibacterial property under hyaluronidase and acidic environment.
Example 4 antimicrobial kinetics testing
Taking 5mgFe 3 O 4 @(Gent/HA) 3 The nano particles are respectively dispersed in 4.5mL of different buffer media by ultrasonic, and 500 mu L of fresh bacterial liquid is added into the sample dispersion liquid. From the addition of bacterial liquidTiming is started, the initial time is recorded as 0min, the nanoparticles are quickly separated to the bottom of the container by using a magnet when the contact time is 30min, 60min, 90min, 120min and 150min respectively, 100 mu L of bacterial liquid and 900 mu L of PBS solution are taken and diluted step by step according to the proportion of 1: 10. 100 μ L of the appropriate dilution was applied to an LB agar plate, the plate was incubated at 37 ℃ for 20 hours, and the number of colonies formed on the plate was counted, i.e., the survival rate of bacteria was A/B, where A is the number of colonies after the sample was contacted and B is the number of colonies in the blank group. And calculating the viable count at different time and calculating the antibacterial efficiency of the sample, and repeating the operation for three times to obtain an average value.
As can be seen from fig. 9, starting from 0h, the survival rate of the experimental strain is 100%, after 30min, the survival rate of staphylococcus aureus is reduced to about 61%, and the survival rate of escherichia coli is reduced to about 70%, and after that, the survival rate of the strain is tested every 30min, the survival rate of the strain is reduced compared with the last test, at 120min, the survival rate of staphylococcus aureus is substantially 0, the survival rate of escherichia coli is about 8%, and at 150min, escherichia coli is almost completely killed. The experimental results show that: the nano-particles have good inhibition effect on two kinds of bacteria, can quickly and efficiently kill the bacteria, and has stronger inhibition effect on staphylococcus aureus.
Example 5 anti-biofilm test
The control group was a biofilm to which PBS buffer was added. Subsequently adding Fe 3 O 4 @(Gent/HA) 3 The nanoparticles are dispersed in the TSBg culture solution and added into 12-hole plates respectively for culturing for 24h, and an external magnetic field is introduced when needed. After the culture is finished, the cover glass is taken out and is slowly washed with PBS for three times, the biological membrane is dyed for 5min by AO and EB dyes under the environment of room temperature and light shielding, and the appearance of the dyed biological membrane is observed under a laser confocal microscope (the excitation wavelength is 488 nm; the emission wavelength is 535 nm).
As shown in FIG. 10, the biofilm growth in the blank control group of panels a and c is very dense, the bacterial accumulation layer is complete, and the growth state of the untreated biofilm is good. The biofilm treated by the magnetic field was significantly changed as shown in panels b and d. On one hand, the thickness of the biological membrane is obviously reduced, on the other hand, the surface structure of the biological membrane begins to split and collapse, and the whole structure is completely destroyed, which shows that the magnetic nano-antibiotic composite particles prepared by the invention have good application prospect in treating infection caused by the biological membrane under an external magnetic field.

Claims (10)

1. A preparation method of magnetic nano-antibiotic composite particles comprises the following steps:
(1) modifying magnetic Fe by polyacrylic acid by solvothermal method 3 O 4 Nanoparticle to obtain Fe 3 O 4 @ PAA magnetic nanoparticles;
(2) adopting a layer-by-layer electrostatic self-assembly technology to alternately assemble the Fe obtained in the step (1) by taking gentamicin (Gen) as a cation assembly liquid and Hyaluronic Acid (HA) as an anion assembly liquid 3 O 4 The surface of the @ PAA magnetic nano particle is coated with Fe with a plurality of antibacterial films 3 O 4 @(Gen/HA) n The nano particles are characterized in that n ranges from 1 to 8 and is a positive integer.
2. The method for preparing magnetic nano-antibiotic composite particles according to claim 1, wherein the method comprises the following steps: the step (1) is specifically as follows: FeCl 3 ·6H 2 Mixing O, ethylene glycol and diethylene glycol according to a certain proportion, stirring until the solid is completely dissolved, and adding FeCl 3 ·6H 2 Adding quantitative anhydrous sodium acetate and polyacrylic acid into the amount of O, performing ultrasonic treatment to fully dissolve and uniformly mix reactants, transferring the solution into a reaction kettle for heating reaction, after the reaction kettle is cooled to room temperature after full reaction, removing the reaction mixture, adding deionized water, washing to remove unreacted raw materials, and obtaining Fe 3 O 4 @ PAA magnetic nanoparticles.
3. The method for preparing magnetic nano-antibiotic composite particles according to claim 2, wherein the method comprises the following steps: in the step (1): FeCl 3 ·6H 2 O, ethylene glycol and a ketalThe diethylene glycol is prepared from the following components in proportion: FeCl 3 ·6H 2 The molar ratio of O to ethylene glycol is 1:50, and the volume ratio of ethylene glycol to diethylene glycol is 1: 1-5.
4. The method for preparing magnetic nano-antibiotic composite particles according to claim 1 or 2, wherein: said Fe 3 O 4 The average particle size of the @ PAA magnetic nanoparticles is 30-50 nm.
5. The method for preparing magnetic nano-antibiotic composite particles according to claim 2, wherein the method comprises the following steps: in the step (1), the heating reaction temperature is 200 ℃, and the reaction time is 12 h.
6. The method for preparing magnetic nano-antibiotic composite particles according to claim 1, wherein the method comprises the following steps: the step (2) is specifically to use the Fe prepared in the step (1) 3 O 4 The method comprises the following steps of ultrasonically stirring and dispersing @ PAA magnetic nanoparticles in deionized water to form dispersion liquid, dropwise adding the dispersion liquid into gentamicin cation assembly liquid, ultrasonically and violently stirring, separating the nanoparticles through an external magnetic field after assembly, washing with water, and dispersing the nanoparticles in the deionized water again; then dropwise adding the dispersion into hyaluronic acid anion assembly liquid, continuously and violently stirring by ultrasound, separating by an external magnetic field after assembly, washing, re-dispersing in deionized water, repeating the assembly process for n times, washing by deionized water, and drying in vacuum at 40 ℃ to obtain Fe 3 O 4 @(Gen/HA) n The nano particles, wherein n is a positive integer more than or equal to 1.
7. The method for preparing magnetic nano-antibiotic composite particles according to claim 6, wherein the method comprises the following steps: the mass concentration ratio of the gentamicin cation assembly liquid to the hyaluronic acid anion assembly liquid is 1: 1.
8. the method for preparing magnetic nano-antibiotic composite particles according to claim 6, wherein the method comprises the following steps: and (3) carrying out the assembling process in the step (2) under the condition that the pH value is 5-6, and adjusting the pH value by adopting glacial acetic acid.
9. Fe obtained by the production method according to any one of claims 1 to 8 3 O 4 Nano antibiotic composite particles as a carrier.
10. Use of the nano-antibiotic composite particles, according to claim 9, as a medical biomaterial.
CN202210599081.6A 2022-05-30 2022-05-30 Magnetic nano-antibiotic composite particle and preparation method and application thereof Withdrawn CN114984243A (en)

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