Nanoparticles with both antibacterial and anti-inflammatory effects, preparation method and application thereof

Abstract

The present invention provides a nanoparticle having both antibacterial and anti-inflammatory effects, a preparation method and application thereof, wherein the nanoparticle comprises tannic acid, manganese ions and antibiotics; the mass ratio of the tannic acid, manganese ions and antibiotics is 1:(0.1-50):(0.1-50). The nanoparticle has excellent antibacterial and anti-inflammatory properties, and can effectively treat pneumonia and sepsis by simultaneously removing infection sources and multiple inflammatory mediators, controlling excessive immune responses in the host body.

Description

Technical Field

[0001] The present invention belongs to the technical field of new biomedical materials, and in particular relates to a nanoparticle having both antibacterial and anti-inflammatory effects, and a preparation method and application thereof. Background Art

[0002] Sepsis is the leading cause of death in hospital intensive care units. Despite the great efforts of the scientific community and medical workers to improve its mortality rate, the successful treatment of sepsis remains a huge challenge worldwide, and more than 11 million people die from sepsis each year (see Rudd, KE; Johnson, SC; Agesa, KM; Shackelford, KA; Tsoi, D.; Kievlan, DR; Colombara, DV; Ikuta, KS; Kissoon, N.; Finfer, S.; Fleischmann-Struzek, C.; Machado, FR; Reinhart, KK; Rowan, K.; Seymour, CW; Watson, RS; West, TE; Marinho, F.; Hay, SI; Lozano, R.; Lopez, AD; Angus, DC; Murray, CJL; Naghavi, M. The Lancet 2020, 395, 200). During the onset of sepsis, the main invading pathogens include bacteria, viruses, and fungi. When the host is infected, the immune system is overactivated, leading to the production and release of a variety of inflammatory mediators including cell-free DNA (cfDNA), reactive oxygen species (ROS), nitric oxide, histones, and proinflammatory cytokines. If pathogens and inflammatory mediators cannot be cleared promptly and effectively, the host will enter a vicious cycle of excessive inflammation and compensatory immunosuppression. Therefore, timely removal of pathogens and elimination of inflammatory mediators are crucial for the treatment of sepsis (see van der Poll, T.; Shankar-Hari, M.; Wiersinga, WJ Immunity 2021, 54, 2450). The co-delivery of antibiotics and anti-inflammatory drugs to the site of infection can not only quickly eliminate pathogens but also suppress the over-activated immune system, which is an effective strategy for treating sepsis (see Shi, C.; Wang, X.; Wang, L.; Meng, Q.; Guo, D.; Chen, L.; Dai, M.; Wang, G.; Cooney, R.; Luo, J. Nat Commun 2020, 11, 3384, Zhang, C. Y.; Gao, J.; Wang, Z. Adv Mater 2018, 30, e1803618). Therefore, the development of multifunctional nanomaterials with both antibacterial and anti-inflammatory activities has good prospects and important significance. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a nanoparticle having both antibacterial and anti-inflammatory effects, and a preparation method and application thereof. The nanoparticle can timely remove the source of infection through antibacterial action, remove various inflammatory mediators through anti-inflammatory action, and control the excessive immune response in the host, thereby achieving effective treatment of pneumonia and sepsis.

[0004] The present invention provides a nanoparticle having both antibacterial and anti-inflammatory effects, comprising tannic acid, manganese ions and antibiotics;

[0005] The mass ratio of the tannic acid, manganese ions and antibiotics is 1:(0.1-50):(0.1-50).

[0006] In the present invention, the antibiotic is selected from one or more of gentamicin, gatifloxacin, polymyxin E and polymyxin B.

[0007] In the present invention, the tannic acid has certain antioxidant and anti-inflammatory properties, can form stable hydrogen bonds with nucleic acids, can eliminate active oxygen and free nucleic acids generated during the inflammation process, and achieve anti-inflammatory properties.

[0008] In the present invention, the particle size of the nanoparticles having both antibacterial and anti-inflammatory effects is 100-200 nm.

[0009] The present invention provides a method for preparing nanoparticles having both antibacterial and anti-inflammatory effects as described in the above technical solution, comprising the following steps:

[0010] The tannic acid aqueous solution, the manganese chloride aqueous solution and the antibiotic aqueous solution are mixed, a pH adjuster is added, particles are precipitated, and nanoparticles having both antibacterial and anti-inflammatory effects are obtained.

[0011] The method is simple, easy to operate and has low preparation cost. The prepared nanoparticles have excellent antibacterial and anti-inflammatory properties, and can effectively treat pneumonia and sepsis by simultaneously clearing the source of infection and multiple inflammatory mediators, controlling the excessive immune response in the host body.

[0012] In the present invention, the mass ratio of tannic acid, manganese chloride and antibiotic is 1:(0.1-50):(0.1-50); in a specific embodiment, the mass ratio of tannic acid, manganese chloride and antibiotic is 1:0.1:0.5.

[0013] In the present invention, the pH adjuster is selected from ammonia water, sodium hydroxide or sodium hydroxide solution.

[0014] In the present invention, the pH value of the system is 7-9 after adding the pH adjuster.

[0015] In the present invention, the mixing time of the tannic acid aqueous solution, the manganese chloride aqueous solution and the antibiotic aqueous solution is 0.1 to 5 hours, preferably 0.1 to 0.2 hours;

[0016] After adding the pH adjuster, stirring is carried out for 0.1 to 5 hours, preferably 0.2 to 0.5 hours.

[0017] In the present invention, after the particles are precipitated, they are centrifuged, washed and concentrated to obtain nanoparticles with both antibacterial and anti-inflammatory effects; the centrifugal speed is 5000-20000 rpm, and the centrifugal time is 5-30 minutes.

[0018] The present invention provides an application of the nanoparticles having both antibacterial and anti-inflammatory effects described in the above technical solution in the preparation of a drug for treating pneumonia or sepsis.

[0019] The present invention is preferably carried out as follows:

[0020] 1) Cell culture: In the present invention, RAW264.7 and other cell lines are selected, and the cell culture method is a method well known to those skilled in the art and is not particularly limited. In the present invention, cells are preferably cultured in a DMEM medium containing 10% fetal bovine serum, and the culture conditions are preferably continuous culture in an incubator with a volume fraction of 5% carbon dioxide, and the culture temperature is preferably 37°C.

[0021] 2) Free nucleic acid detection: In the present invention, the Picogreen dsDNA quantitative kit is used to detect the adsorption of free nucleic acids by the material. First, 12.5 μL PicoGreen concentrate and 20 μL 2.5 mg / mL DNA standard solution are added to 10 mL TE buffer and mixed evenly. Then, different concentrations of materials (sample group) or Milli-Q water (blank group) and 100 μL of the above mixed solution are added to a 96-well plate, and the fluorescence intensity of the Picogreen-DNA complex is measured with a multifunctional microplate reader after incubation at 37°C for 30 minutes (λex=480nm, λem=520nm). DNA affinity is calculated according to the following formula.

[0022]

[0023] Among them, Fb and Fs are the fluorescence intensities of the treated sample group and the blank group, respectively.

[0024] 3) Reactive oxygen detection: RAW264.7 cell line was used for reactive oxygen evaluation. Cells were plated at 8×10 3The cells were seeded at a density of 100 μg / mL in a 96-well plate and cultured overnight. The cells were then treated with 50 μM tert-butyl hydroperoxide (TBHP) and 60 μg / mL of the material, where TBHP can induce cells to produce reactive oxygen species. After 24 hours, the cells were treated with a reactive oxygen species detection kit and imaged using a fluorescence microscope.

[0025] 4) Cell-level anti-inflammatory assay: RAW264.7 cell line was used for cell-level anti-inflammatory assay. Cells were plated at 2×10 4 The density of cells was planted in a 96-well plate. 25ng / mL of lipopolysaccharide (LPS) and samples of different concentrations were mixed in Milli-Q water, and the mixture was incubated at room temperature for 1 hour and then centrifuged. The role of LPS is to induce inflammation in cells. Then, 20μL of the supernatant obtained by centrifugation was added to a 96-well plate. After 24 hours of culture, the TNF-α content in the culture medium was detected using a tumor necrosis factor-α (TNF-α) ELISA kit. TNF-α is an inflammatory cytokine produced by macrophages, and its content is positively correlated with the level of cellular inflammation.

[0026] 5) Cytotoxicity: Cell lines such as RAW264.7 were used for cytotoxicity evaluation. Cells were plated at 1×10 4 The cells were seeded in a 96-well plate at a density of 100 μg / mL and cultured overnight. After 24 h of co-culture with the cells, the liquid in the 96-well plate was aspirated and added to a culture medium containing 10% CCK-8. After co-incubation, the absorbance of each well at 450 nm and 600 nm was detected by an ELISA reader. The cell survival rate was calculated by the following formula.

[0027] ΔA=A 450 / A 600

[0028] Cell survival rate (%) = (ΔA 样品 / ΔA 空白 )×100

[0029] 6) Bacterial culture: In the present invention, E. coli bacteria are selected, and the bacterial culture method is a method well known to those skilled in the art, and there is no special limitation. In the present invention, preferably Luria-Bertani liquid medium is used to culture the bacteria, and the culture condition is preferably continuous culture in an incubator at a culture temperature of 37°C.

[0030] 7) Animal model: In the present invention, Balb / c mice were used for pneumonia model experiments, and C57 mice were used for sepsis model experiments. Balb / c mice were anesthetized by intraperitoneal injection of 7% pentobarbital, and LPS aqueous solution was administered to the mouse lungs through the trachea to establish a pneumonia model. The treatment experiment was started 2 hours after the model was established. C57 mice were anesthetized using an isoflurane anesthesia system, and the abdomen was shaved and disinfected. An opening of about 1 cm was made in the midline of the mouse abdomen, the cecum was gently removed, and ligated with 4-0 silk thread at about 1.5 cm from the tail end of the cecum. The cecum was punctured with a No. 21 needle, and after squeezing the contents of the cecum, the cecum was returned to the abdominal cavity, and the abdominal incision was sutured to establish a sepsis model. The treatment experiment was started 1 hour after the model was established.

[0031] 8) In vivo inflammation detection: When detecting in vivo inflammation in the pneumonia model, the mouse lung tissue was dissected out and a 10% homogenate medium was prepared with PBS. The supernatant was collected by centrifugation at 3000rpm and 4°C for 20 minutes. The levels of inflammatory factors TNF-A and interleukin 6 (IL-6) in the serum and lung tissue homogenate supernatant of the pneumonia model mice were determined using an ELISA kit. When detecting in vivo inflammation in the pneumonia model, the levels of inflammatory factors TNF-A and IL-6 in the serum and peritoneal lavage fluid of the sepsis model mice were determined using an ELISA kit.

[0032] The present invention provides a nanoparticle having both antibacterial and anti-inflammatory effects, comprising tannic acid, manganese ions and antibiotics; the mass ratio of the tannic acid, manganese ions and antibiotics is 1:(0.1-50):(0.1-50). The nanoparticles utilize the coordination effect between tannic acid, manganese ions and antibiotics to have excellent antibacterial and anti-inflammatory properties, and can simultaneously remove infection sources and multiple inflammatory mediators, control the excessive immune response in the host body, thereby achieving effective treatment of pneumonia and sepsis. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a hydrodynamic diameter diagram of nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention;

[0034] Figure 2 This is a transmission electron microscopy image of the nanoparticles having both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention;

[0035] Figure 3 This is a scanning electron microscope image of the nanoparticles having both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention;

[0036] Figure 4 This is a scanning electron microscope image of the nanoparticles having both antibacterial and anti-inflammatory effects obtained in Example 2 of the present invention;

[0037] Figure 5This is a scanning electron microscope image of the nanoparticles having both antibacterial and anti-inflammatory effects obtained in Example 3 of the present invention;

[0038] Figure 6 This is a scanning electron microscope image of the nanoparticles having both antibacterial and anti-inflammatory effects obtained in Example 4 of the present invention;

[0039] Figure 7 The adsorption capacity of the nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention on free nucleic acids;

[0040] Figure 8 The scavenging ability of the nanoparticles with both antibacterial and anti-inflammatory effects on or against active oxygen obtained in Example 1 of the present invention;

[0041] Fig. 9 The antibacterial ability of the nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention;

[0042] Fig.10 The cytotoxicity evaluation of the nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention;

[0043] Fig.11 The anti-inflammatory ability of the nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention at the cellular level;

[0044] Fig.12 The anti-inflammatory therapeutic effect of the nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention in a pneumonia model;

[0045] Fig.13 The anti-inflammatory therapeutic effect of the nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention in a sepsis model;

[0046] Fig.14 The antibacterial therapeutic effect of the nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention in a sepsis model;

[0047] Fig.15 The nanoparticles with both antibacterial and anti-inflammatory effects obtained in Example 1 of the present invention improve the survival rate of mice in a sepsis model. DETAILED DESCRIPTION

[0048] To further illustrate the present invention, the nanoparticles with both antibacterial and anti-inflammatory effects provided by the present invention, as well as their preparation method and application are described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] Preparation process of nanoparticles: tannic acid, manganese chloride and antibiotics are first reacted, then sodium hydroxide aqueous solution is added to adjust the pH to 7-8, and nanoparticles are obtained by centrifugation, washing and concentration. In the present invention, the mass ratio of tannic acid, manganese chloride and antibiotics is shown in Table 1:

[0051] Table 1 Types of antibiotics in Examples 1 to 4 and ratios of raw materials for preparing nanoparticles

[0052] Example Types of antibiotic compounds Tannic acid, manganese chloride, antibiotic mass ratio 1 Polymyxin B 1：0.1：0.5 2 Polymyxin E 1：0.1：0.5 3 Gentamycin 1：0.1：0.5 4 Gatifloxacin 1：0.1：0.5

[0053] The present invention uses a nanoparticle size analyzer and a transmission electron microscope to analyze the particle size of the nanoparticles prepared in Example 1, and obtains the hydraulic diameter and the transmission electron microscope image, as shown in FIG. Figure 1 and Figure 2 As shown, the results show that the average size of the nanoparticles prepared in Example 1 is 50-200 nm. The present invention uses a scanning electron microscope to analyze the morphology of the nanoparticles prepared in Examples 1-4, and the obtained scanning electron microscope images are as follows: Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown, the results show that the nanoparticles of Examples 1 to 4 have similar morphologies.

[0054] Example 5

[0055] 12.5 μL PicoGreen concentrate and 20 μL 2.5 mg / mL DNA standard solution were added to 10 mL TE buffer and mixed evenly to prepare free nucleic acid detection solution. Then, 100 μL of Example 1 or Milli-Q water of different concentrations and 100 μL of the above free nucleic acid detection solution were added to a 96-well plate, incubated at 37°C for 30 minutes, and the fluorescence intensity of each group in the well plate was measured with a multifunctional microplate reader to calculate the affinity of the material for DNA. Figure 7 As shown, as the concentration of Example 1 increases, the affinity for DNA gradually increases, and when the concentration of Example 1 is 62.5 μg / mL, it has nearly 100% DNA adsorption capacity. The experimental results show that Example 1 retains the DNA affinity of tannic acid and polymyxin B, and because more hydrogen bonds are generated after coordination, its free nucleic acid adsorption capacity is stronger than tannic acid and polymyxin B.

[0056] Example 6

[0057] The cells were treated with 50 μM TBHP and 60 μg / mL material, wherein Example 1 and tannic acid have antioxidant properties and can eliminate the reactive oxygen species produced by cells induced by TBHP, such as Figure 8 shown.

[0058] Example 7

[0059] After treating E. coli with 12.5 μg / mL of the material for 10 hours, the mixture was spread on an agar plate and cultured at 37°C for 12 hours to observe the growth of the bacteria. Fig. 9 The results showed that Example 1 and polymyxin had excellent antibacterial properties.

[0060] Example 8

[0061] RAW264.7 cells were plated at 1×10 4 The cells were seeded at a density of 100 μg / cm2 in a 96-well plate and cultured overnight. After co-culturing the cells with the nanoparticles obtained in Example 1 at different concentrations for 24 h, the original culture medium was aspirated, and a culture medium containing 10% CCK8 was added. After co-incubation for 1 h, the cell survival rate was determined. Fig.10 The experimental results show that when the concentration of Example 1 is 100ug / mL, the cell survival rate can still reach 100%. Due to the stability and high charge density of the prepared nanomaterial, the effects of tannic acid and manganese chloride on cell proliferation are effectively avoided, the cytotoxicity is lower, and the cell compatibility is better, which is conducive to its application in vivo.

[0062] Example 9

[0063] 25 ng / mL lipopolysaccharide (LPS) was mixed with samples of different concentrations and centrifuged. The supernatant obtained by centrifugation was added to the cells. After culturing for 24 hours, the TNF-α content in the culture medium was detected. Fig.11 As shown, Example 1 has good anti-inflammatory ability and can reduce the content of inflammatory factors in macrophages.

[0064] Example 10

[0065] The nanoparticles obtained in Example 1 were mixed with PVP with a molecular weight of 40k at a mass ratio of 1:8, and the mixed nanoparticles were administered to mice with pneumonia through organs. The mice were killed after 22 hours of treatment, and the blood of the mice was collected. The main organs (heart, liver, spleen, lung, and kidney) were removed by dissection. The lung tissue was ground with PBS, and the TNF-α content in the grinding liquid supernatant and serum was determined using an ELISA kit.

[0066] The experimental results showed that the levels of inflammatory factors in the blood and lungs of the nanoparticle treatment group were lower than those in the positive control group, and were similar to the levels of inflammatory factors in normal mice. Fig.12 As shown, after treatment, the content of TNF-α in the lung inflammatory factor of mice was reduced from 450pg / mL to the normal level of 53pg / mL, which shows that Example 1 can effectively inhibit lung inflammation caused by LPS, showing a good treatment effect for pneumonia. In addition, the nanoparticles did not cause obvious toxic side effects on major organs (heart, liver, spleen, lung, and kidney), indicating that the application of nanomaterials in vivo is safe.

[0067] Example 7

[0068] The nanoparticles obtained in Example 1 were mixed with PVP with a molecular weight of 40k at a mass ratio of 1:8, and the mixed nanoparticles were injected into mice through the peritoneal cavity at 1h and 12h. After 24h, the blood and ascites of the mice were collected, and the main organs (heart, liver, spleen, lung, and kidney) were removed by dissection. The remaining mice were observed for 7 consecutive days, and the weight and body temperature of the mice were measured every 12h. The content of TNF-α in ascites and serum was determined by ELISA kit, and the bacteria in the peritoneal cavity were cultured on agar plates, such as Fig.13 and Fig.14 . Fig.13 The results showed that after treatment, the content of inflammatory factors in the abdominal cavity of mice decreased from 405pg / mL to 62pg / mL, and the inflammation in the abdominal cavity was alleviated. Fig.14 The results showed that the number of bacteria in the abdominal cavity of mice decreased significantly after treatment.

[0069] The experimental results showed that the survival rate of mice was improved after treatment with nanoparticles ( Fig.15 ), the survival period was significantly prolonged, from all deaths within 48 hours to 80% survival within 48 hours, and the lives of nearly 60% of mice were saved. In addition, after treatment with Example 1, the number of bacteria in the abdominal cavity of mice was significantly reduced, and the content of inflammatory factors in the abdominal cavity and blood was reduced to the same level as that of healthy mice, showing a good treatment effect for sepsis. In addition, the nanoparticles did not cause obvious toxic side effects on major organs (heart, liver, spleen, lungs, and kidneys), indicating that the application of nanomaterials in vivo is safe.

[0070] As can be seen from the above embodiments, the present invention provides a nanoparticle having both antibacterial and anti-inflammatory effects, comprising tannic acid, manganese ions and antibiotics; the mass ratio of the tannic acid, manganese ions and antibiotics is 1:(0.1-50):(0.1-50). The nanoparticle has excellent antibacterial and anti-inflammatory properties, and can effectively treat pneumonia and sepsis by simultaneously removing the infection source and removing multiple inflammatory mediators, controlling the excessive immune response in the host body.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of nanoparticles with both antibacterial and anti-inflammatory effects in the preparation of a drug for treating pneumonia; the pneumonia is a lung inflammation caused by lipopolysaccharide; The nanoparticles having both antibacterial and anti-inflammatory effects include tannic acid, manganese chloride and antibiotics; The mass ratio of the tannic acid, manganese chloride and antibiotic is 1:0.1:0.5; The antibiotic is selected from polymyxin E or polymyxin B.

2. The use according to claim 1, characterized in that: The particle size of the nanoparticles having both antibacterial and anti-inflammatory effects is 100-200 nm.

3. The use according to claim 1, characterized in that: The method for preparing the nanoparticles having both antibacterial and anti-inflammatory effects comprises the following steps: The tannic acid aqueous solution, the manganese chloride aqueous solution and the antibiotic aqueous solution are mixed, a pH adjuster is added, particles are precipitated, and nanoparticles having both antibacterial and anti-inflammatory effects are obtained.

4. The use according to claim 3, characterized in that: The pH regulator is selected from ammonia water or sodium hydroxide solution.

5. The use according to claim 3, characterized in that: After adding the pH adjuster, the pH value of the system is 7~9.

6. The use according to claim 3, characterized in that: The mixing time of the tannic acid aqueous solution, the manganese chloride aqueous solution and the antibiotic aqueous solution is 0.1 to 5 hours; After adding the pH adjuster, stir for 0.1~5h.

7. The use according to claim 3, characterized in that: After the particles are precipitated, they are centrifuged, washed and concentrated to obtain nanoparticles with both antibacterial and anti-inflammatory effects; The centrifugal speed is 5000-20000 rpm, and the centrifugal time is 5-30 min.

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