A kind of magnetic nanoparticle of Fe3O4 modified by saltheterocyclic cationic polymer and its preparation method and application

Abstract

The application belongs to the technical field of medical biomaterials, and relates to a sultonium cationic polymer modified Fe3O4 magnetic nanoparticle, a sultonium cationic polymer (formula I) as shown in formula I is modified on the surface of the Fe3O4 magnetic nanoparticle through electrostatic interaction to obtain a sultonium cationic polymer modified Fe3O4 magnetic nanoparticle (formula II) as shown in formula II. The application also provides a preparation method and application of the sultonium cationic polymer modified Fe3O4 magnetic nanoparticle. The sultonium cationic polymer modified Fe3O4 magnetic nanoparticle provided by the application can be used for bacterial / fungal enrichment, sterilization and biofilm removal, and has good biocompatibility.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical biomaterials, and relates to a kind of sump salt cationic polymer modified Fe3O4 Magnetic nanoparticles and its preparation method and application. BACKGROUND

[0002] Bacterial infection and adhesion is one of the main reasons that threaten human life and health. In addition to pathogenic bacteria hazards, bacteria adhering to the surface layer are prone to stacking to form biofilms, leading to wound infection and causing various infectious diseases. To address the problem of bacterial pathogenicity, the production of antibiotics has greatly reduced the pathogenicity hazards, but the over-reliance and misuse of antibiotics have led to the evolution of drug resistance in bacteria, and even the emergence of multi-drug resistant bacteria. In recent years, there has been a lack of new antibiotics to combat the problem of drug resistance caused by existing antibiotics, and if the use of antibiotics is not controlled, infectious diseases will again become a life-threatening disease. Cationic polymers are designed to mimic natural antimicrobial peptides, and due to their spectrum of membrane-destroying antibacterial activity, they have a lower chance of developing drug resistance. However, the slow bactericidal rate of some cationic polymers is a major factor limiting their widespread application.

[0003] In addition, diseases related to biofilm infection, such as colitis, gingivitis, and otitis media, are numerous and numerous. According to statistics, 80% of all microbial infections in humans are caused by biofilms. Most free bacteria can attach to the surface and grow into multicellular colonies, which are the extracellular polymeric substance (EPS) of biofilms. EPS is composed of extracellular polysaccharides, nucleic acids, proteins, and lipids, which form a protective microenvironment for bacteria and promote their strong resistance to external antimicrobial drugs. Therefore, the therapeutic effect of traditional antibiotics on bacterial biofilms is significantly reduced, in other words, antibiotics have problems such as poor penetration and high concentration for biofilms.

[0004] In recent years, advances in nanotechnology have brought new hope for the treatment of bacterial infections, but its limited penetration is still a major problem, so developing new antibacterial nanomaterials for effective treatment of biofilm infections remains a challenge. Fe3O4 Magnetic nanoparticles are used as antibacterial agent carriers due to their superparamagnetism, high specific surface area, and biocompatibility, and have shown excellent bactericidal effect when used with various antibacterial agents. Under the action of an external magnetic field, Fe3O4 Magnetic nanoparticles can achieve strong penetration and target into biofilms. Webster et al. used Fe3O4 Magnetic nanoparticles for the treatment of medical device-related infections, which can penetrate 20 μm thick biofilms with the help of an external magnetic field, thereby completely eliminating bacteria in the biofilm. These studies show that Fe3O4 Magnetic nanoparticles have great potential in combating biofilm infections. SUMMARY

[0005] The present application provides a kind of sutton salt cationic polymer modified Fe3O4 Magnetic nanoparticles, which can be used for bacteria / fungus enrichment, sterilization and biofilm removal, and also has good biocompatibility.

[0006] To solve the above technical problems, the purpose of the present application is realized by the following technical solutions:

[0007] A kind of sutton salt cationic polymer modified Fe3O4 Magnetic nanoparticles,

[0008]

[0009] The sutton salt cationic polymer as described in formula I is modified on the surface of Fe3O4 Magnetic nanoparticles by electrostatic interaction, to obtain the sutton salt cationic polymer modified Fe3O4 Magnetic nanoparticles as shown in formula II.

[0010] The present application also provides a preparation method of the above-mentioned sutton salt cationic polymer modified Fe3O4 Magnetic nanoparticles, which comprises the following steps:

[0011] (1) 1,6-hexanedithiol and 1,5-hexadiene are subjected to ultraviolet light initiation under the addition of a photoinitiator to obtain a precursor polymer; the precursor polymer is subjected to sulfonium saltization by adding iodomethane to obtain the sutton salt cationic polymer as shown in formula I;

[0012] (2) the sutton salt cationic polymer as shown in formula I is combined with the magnetic Fe3O4 nanoparticles with negative charges on the surface through electrostatic interaction to obtain the sutton salt cationic polymer modified magnetic Fe3O4 nanoparticles as shown in formula II.

[0013] In the above-mentioned preparation method of the sutton salt cationic polymer modified Fe3O4 Magnetic nanoparticles, the preparation method of the magnetic Fe3O4 nanoparticles with negative charges on the surface is as follows:

[0014]

[0015] The magnetic Fe3O4 nanoparticles as shown in formula III are prepared by co-precipitation of ferrous ions and ferric ions under alkaline conditions and at a certain temperature; the magnetic Fe3O4 nanoparticles are modified by sodium citrate to obtain the magnetic Fe3O4 nanoparticles with negative charges on the surface as shown in formula IV.

[0016] Preferably, the ferrous ions are ferrous sulfate heptahydrate, and the ferric ions are ferric chloride hexahydrate. The alkaline conditions are 25%-28% ammonia solution, and the certain temperature is 60-80℃.

[0017] In the preparation method of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle, the step (2) comprises: dispersing the sodium citrate modified magnetic Fe3O4 nanoparticle and the sulfonium salt cationic polymer in an aqueous solution, combining through electrostatic interaction to obtain the sulfonium salt cationic polymer modified magnetic Fe3O4 nanoparticle; the mass ratio of the sodium citrate modified magnetic Fe3O4 nanoparticle and the sulfonium salt cationic polymer is 1:1; preferably, the concentration of the sodium citrate modified magnetic Fe3O4 nanoparticle and the sulfonium salt cationic polymer is 5 mg / mL.

[0018] In the preparation method of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle, the method for modifying the magnetic Fe3O4 nanoparticle by sodium citrate is: ultrasonic dispersion of the magnetic Fe3O4 nanoparticle in ultrapure water, addition of sodium citrate, and continuous ultrasonic to generate negatively charged sodium citrate modified magnetic Fe3O4 nanoparticles; the molar ratio of the sodium citrate and the magnetic Fe3O4 nanoparticle is 2:3.

[0019] The application further provides the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle.

[0020] The sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle is applied to rapid enrichment and sterilization of bacteria / fungi. The method and principle are as follows: the surface of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle is provided with a large number of positive charges, and the cell membrane of bacteria / fungi is negative, when the two are contacted, they are rapidly combined together through electrostatic interaction. Under the action of an external magnetic field, the magnetic Fe3O4 nanoparticles are attracted, and the bacteria are rapidly enriched and separated from the solution; meanwhile, the cationic polymer with positive charges destroys the cell membrane of bacteria / fungi, so that the sterilization effect is achieved.

[0021] The sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle is applied to removal of biofilm. The method and principle are as follows: a bacterial / fungal suspension with a certain concentration is cultured in a 96-well plate, the liquid is changed every other day, and a mature biofilm is generated after 72 hours of culture. After removing the upper solution, a PBS solution of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle with different concentrations is added. Under the action of a magnetic field, the nanoparticles move left and right, excavate channels in the biofilm, deepen the penetration depth of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticle in the biofilm, and accelerate the penetration speed. The sulfonium salt cationic polymer is delivered faster and deeper into the biofilm, so that a better biofilm removal effect is achieved.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] 1、The Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer are obtained by the electrostatic interaction after the Fe3O4 nanoparticles are synthesized by the coprecipitation method and then modified by sodium citrate, so that the structure of the sulfonium salt cationic polymer is not damaged, and the method is environment-friendly and easy to operate, and has a good application prospect.

[0024] 2、The Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer can realize rapid enrichment of bacteria / fungi, and the bacteria / fungi can be effectively adsorbed by using a lower particle concentration, and the bacteria / fungi can be rapidly enriched in the moment when the Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer contact the bacteria / fungi.

[0025] 3、The Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer can effectively remove biofilms, and has an ideal penetration depth and penetration speed, so that the sulfonium salt cationic polymer can be delivered to the inside of the biofilm faster and deeper, so as to achieve a better biofilm removal effect.

[0026] 4、The Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer have good biocompatibility and low toxicity, and are an ideal new type of antibacterial agent. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a schematic diagram of the synthesis of the Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer. Figure 1 Figure 2 is the bacterial enrichment kinetics result of the Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer in Example 5.

[0028] Figure 3 is the hemolysis performance of the Fe3O4 magnetic nanoparticles modified by the sulfonium salt cationic polymer in Example 7. Figure 2 DETAILED DESCRIPTION

[0029] The present application is further described below by the description of the specific embodiments, but this is not a limitation of the present application, and those skilled in the art can make various modifications or improvements according to the basic idea of the present application, as long as the modifications or improvements do not deviate from the basic idea of the present application, and are within the scope of the present application.

[0030] Example 1

[0031] The preparation of the sulfonium salt cationic polymer is as follows:

[0032] ​Take 1,6-hexanedithiol (2.5 mmol) and 1,5-hexadiene (2.5 mmol), dissolve in dichloromethane, then add phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide (0.01625 mmol), room temperature, ultraviolet lamp irradiation reaction 2h, the resulting polymer intermediate is added to 4mL of N,N-dimethylformamide as solvent, to the system added excess iodomethane (10 mmol), room temperature reaction 48h, after the reaction, the reaction liquid is first with sodium chloride solution (0.1M) and deionized water dialysis 2 days, the resulting solution freeze-drying, to get the sulfonium salt cationic polymer, the yield is 80%.

[0033] Example 2

[0034] The preparation of sodium citrate modified Fe3O4 magnetic nanoparticles, the specific operation steps are as follows:

[0035] a, take 6mmol of ferric chloride hexahydrate and 3mmol of ferrous sulfate heptahydrate in the reaction bottle, add 150mL of deionized water, stirring under nitrogen atmosphere for 30min. The reaction temperature is raised to 80℃, continue to keep N2 environment, add 10mL of ammonia water (25%-28%), stirring for 1h after cooling to room temperature. The generated precipitate is separated by magnet, washed with ethanol and water, centrifuged, redispersed, repeated for several times, dried in vacuum oven at 60℃, to get Fe3O4 nanoparticles.

[0036] b, take 1.2mmol of dried Fe3O4 nanoparticles and ultrasonic dispersion in 80mL of ultrapure water, add 1.8mmol of sodium citrate, continue to ultrasonic for 1h. The product after reaction is washed with ultrapure water, centrifuged, redispersed, repeated for several times, dispersed in ultrapure water. Finally, the sodium citrate modified Fe3O4 nanoparticles have negative charge on the surface, and the particle size and zeta potential are shown in table 1.

[0037] Table 1 properties of sodium citrate modified Fe3O4 nanoparticles

[0038] Performance Particle size (nm) PDI Zeta potential (mV) Example 2 129.5 0.383 -24.9

[0039] From the test results in table 1, it can be seen that the sodium citrate modified Fe3O4 nanoparticles prepared in example 2 of the present application have a particle size of about 129.5nm and a zeta potential of-24.9mV, indicating that the negatively charged Fe3O4 magnetic nanoparticles are successfully synthesized.

[0040] Example 3

[0041] The preparation of sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles, the specific steps are as follows:

[0042] The sodium citrate modified Fe3O4 nanoparticles dispersed in ultrapure water in Example 2 were diluted to 5 mg / mL, and 5 mg of the sulfonium salt cationic polymer was dissolved in 1 mL of ultrapure water. After mixing and stirring, sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles were obtained. After the reaction was completed, the nanoparticles were washed with ultrapure water, centrifuged, and redispersed several times, and finally dispersed in PBS buffer (pH = 7.4). The particle size and zeta potential of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles are shown in Table 2.

[0043] Table 2 Properties of sulfonium salt cationic polymer modified Fe3O4 nanoparticles

[0044] Performance Particle size (nm) PDI Zeta potential (mV) Example 3 235.8 0.485 29.9

[0045] As can be seen from the test results in Table 2, the sulfonium salt cationic polymer modified Fe3O4 nanoparticles prepared in Example 3 have a particle size of about 235.8 nm and a zeta potential of 29.9 mV. The change in surface potential indicates that the Fe3O4 magnetic nanoparticles modified with sulfonium salt cationic polymer are successfully synthesized.

[0046] Example 4

[0047] The bacterial / fungal enrichment and rapid sterilization experiment of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles was performed as follows:

[0048] 1) The high concentration PBS solution of the sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles in Example 3 was diluted to different concentrations and placed in a 96-well plate for use.

[0049] 2) The bacterial / fungal solution cultured overnight was centrifuged to remove TSB / SDB, and diluted with PBS to the target concentration.

[0050] 3) The diluted bacterial solution was added to the 96-well plate containing Fe3O4 nanoparticles, and left to stand at room temperature for 5 min. The bottom of the plate was magnetically attracted for 30 s, and the upper clear solution was taken and coated on a TSB / SDB agar plate. The pure bacterial / fungal solution was coated on a plate as a control group, and all samples and the control group were incubated in a 37°C / 28°C water incubator for 24 h. The bottom nanoparticles magnetically attracted were washed twice with PBS, redispersed, and coated on an agar plate, which was also placed in a 37°C / 28°C water incubator for 24 h. After 24 h, the number of bacterial / fungal colonies on the agar plate was counted.

[0051] In this example, three types of bacteria were selected, namely Gram-positive bacteria (S. aureus), Gram-negative bacteria (E. coli), and fungi (C. albicans). The results of the minimum particle concentration at which the number of colonies in the upper clear solution and the lower precipitate was ≤0.1% are shown in Table 3.

[0052] Table 3 Bacteria / fungus enrichment and sterilization results of the Fe304 magnetic nanoparticles modified by the cationic polymer of the sulfonium salt

[0053]

[0054]

[0055] As can be seen from the experimental results in Table 3, when the concentration of the Fe304 magnetic nanoparticles modified by the cationic polymer of the sulfonium salt is 256 μg / mL, the experimental group can achieve 99.9% adsorption of the bacteria in the solution compared with the control group, and the rapid enrichment of Gram-positive bacteria (S. aureus), Gram-negative bacteria (E. coli) and fungi (C. albicans) can be achieved in 5 min, and the adsorbed bacteria / fungi can be effectively killed at the same concentration.

[0056] Example 5

[0057] The bacteria / fungus enrichment kinetics of the Fe304 magnetic nanoparticles modified by the cationic polymer of the sulfonium salt, taken S. aureus as an example, the specific steps are as follows:

[0058] 1) From Example 3, the minimum particle concentration for S. aureus sterilization rate ≥ 99.9% is 256 μg / mL, so the Fe304 magnetic nanoparticles modified by the cationic polymer of the sulfonium salt is diluted to 256 μg / mL with PBS;

[0059] 2) The S. aureus bacteria solution cultured overnight is centrifuged to remove TSB and diluted to the target concentration with PBS;

[0060] 3) The diluted bacteria solution is added to the Fe304 nanoparticle PBS solution, the time just after adding is set as 0 min, six experimental groups are set for 0-5 min, and the initial bacteria solution is used as the control group. The upper clear liquid is taken at the bottom of the hole plate by the magnet at six time points of 0, 1, 2, 3, 4 and 5 min, and the number of bacteria on the agar plate is calculated after 24 h of culture.

[0061] The specific experimental results are shown in Figure 1 .

[0062] As can be seen from the experimental results, Figure 1 the Fe304 magnetic nanoparticles modified by the cationic polymer of the sulfonium salt can achieve rapid enrichment of bacteria (enrichment rate ≥ 99.9%) at the moment of mixing with S. aureus, that is, the moment of contacting with the bacteria. This result shows that the Fe304 magnetic nanoparticles modified by the cationic polymer of the sulfonium salt can achieve rapid enrichment of bacteria in the solution under the environment of an external magnetic field.

[0063] Example 6

[0064] The application of the Sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles in mature biofilm removal is as follows:

[0065] 1) Dilute the overnight cultured bacteria / fungi solution to the target concentration, place it in a 96-well plate, and incubate it in a water incubator at 37°C / 28°C for 72 h, replace the culture medium every 24 h to form mature biofilm at the bottom of the 96-well plate;

[0066] 2) Remove the upper solution of the cultured mature biofilm, add different concentrations of Sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles PBS solution, and use a magnet to move the nanoparticles left and right at the bottom, which lasts for 5 min. After gentle blowing and uniformity, stand for 10 min, and repeat the 5 min magnetic attraction movement, repeat the above operation, and the total time is 30 min. Among them, the PBS buffer without nanoparticles is used as the control group;

[0067] 3) After 30 min, remove the upper suspension, wash twice with PBS, add a certain amount of PBS, and ultrasonic dispersion for 15 min to make the bottom biofilm fall off;

[0068] 4) Blow the fallen biofilm solution evenly, take a certain amount of solution and coat it on the agar plate, and count the number of colonies after 24 h.

[0069] When the biofilm removal rate is ≥99.9% in this example, the minimum concentration of the Sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles is shown in Table 4.

[0070] Table 4 Sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles for bacterial / fungal biofilm removal effect

[0071]

[0072]

[0073] As can be seen from Table 4, the Sulfonium salt cationic polymer modified Fe3O4 magnetic nanoparticles prepared in the example have a certain degree of removal effect on bacterial / fungal biofilm. Among them, for E. coli biofilm, the particle concentration of 256 μg / mL can achieve 99.9% removal effect, and S. aureus and C. albicans biofilm also achieve ideal removal effect at 512 μg / mL and 2048 μg / mL, respectively.

[0074] Example 7

[0075] The Fe3O4 magnetic nanoparticles modified with sulfonium salt cationic polymers in Example 3 were subjected to a hemolysis test on erythrocytes. The specific procedures are as follows:

[0076] 1) Take 2 mL of sheep red blood cells, centrifuge, wash with PBS, centrifuge again, and repeat the above operation 4 times;

[0077] 2) The washed red blood cells were redispersed in PBS buffer, and the Fe3O4 magnetic nanoparticles modified with sulfonium salt cationic polymer were diluted with PBS to different concentrations;

[0078] 3) Red blood cells and Fe3O4 nanoparticles in PBS solution were mixed at a ratio of 1:49. Red blood cells mixed with PBS buffer served as a negative control, and red blood cells mixed with ultrapure water served as a positive control. After co-culturing at room temperature for 2 hours, the mixture was centrifuged, and the supernatant was placed in a 96-well plate. The absorbance at 540 nm was measured. The hemolysis rate was calculated using the following formula:

[0079]

[0080] Hemolysis results as follows Figure 2 As shown.

[0081] A higher rate of hemolysis in red blood cells indicates poorer biocompatibility and higher toxicity of the substance. From Figure 2 The experimental results show that the hemolysis rate of the sulfonium salt cationic polymer-modified Fe3O4 magnetic nanoparticles prepared in the embodiments of the present invention is still less than 50% at a concentration of 1000 μg / mL. This indicates that the sulfonium salt cationic polymer-modified Fe3O4 magnetic nanoparticles have good biocompatibility and meet the requirements for use as antibacterial agents.

Claims

1. A sulton cationic polymer modified Fe3O4 magnetic nanoparticle, characterized in that, the sulton cationic polymer as shown in Formula I is modified on the surface of Fe3O4 magnetic nanoparticles by electrostatic interaction to obtain the sulton cationic polymer modified Fe3O4 magnetic nanoparticle as shown in Formula II; the sulton cationic polymer modified Fe3O4 magnetic nanoparticle is prepared by the following method: (1) 1,6-hexanedithiol and 1,5-hexadiene are subjected to ultraviolet light irradiation under the addition of a photoinitiator to obtain a precursor polymer; the precursor polymer is subjected to sultonization by adding iodomethane to obtain the sulton cationic polymer as shown in Formula I; (2) the sulton cationic polymer as shown in Formula I is combined with the magnetic Fe3O4 nanoparticle with a negative charge on the surface by electrostatic interaction to obtain the sulton cationic polymer modified magnetic Fe3O4 nanoparticle as shown in Formula II; the preparation method of the magnetic Fe3O4 nanoparticle with a negative charge on the surface is: Formula IV; the magnetic Fe3O4 nanoparticle as shown in Formula III is prepared by co-precipitation of ferrous iron and ferric iron under alkaline conditions and at a certain temperature; the magnetic Fe3O4 nanoparticle is modified by sodium citrate to obtain the magnetic Fe3O4 nanoparticle with a negative charge on the surface as shown in Formula IV.

2. The method of claim 1, wherein the method is characterized by the steps of: the step (2) comprises: the magnetic Fe3O4 nanoparticle modified by sodium citrate and the sulton cationic polymer are dispersed in an aqueous solution and combined by electrostatic interaction to obtain the sulton cationic polymer modified magnetic Fe3O4 nanoparticle; the mass ratio of the magnetic Fe3O4 nanoparticle modified by sodium citrate and the sulton cationic polymer is 1:1; the concentration of the magnetic Fe3O4 nanoparticle modified by sodium citrate and the sulton cationic polymer is both 5 mg / mL.

3. The method for preparing Fe3O4 magnetic nanoparticles modified with sulfonium salt cationic polymers according to claim 1, characterized in that, the method for modifying the magnetic Fe3O4 nanoparticle by sodium citrate is: the magnetic Fe3O4 nanoparticle is ultrasonically dispersed in ultrapure water, sodium citrate is added, and ultrasonic is continued to generate the magnetic Fe3O4 nanoparticle modified by sodium citrate with a negative charge; the molar ratio of sodium citrate to the magnetic Fe3O4 nanoparticle is 2:3.

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