CN117695384A - Nanometer immune adjuvant for treating endophyte-related infection in orthopedics department - Google Patents
Nanometer immune adjuvant for treating endophyte-related infection in orthopedics department Download PDFInfo
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Abstract
The invention relates to a nanometer immune adjuvant for orthopedic treatment of endophyte related infection, which is prepared by preparing a covalent organic framework RT-COF-1, preparing an iron-based covalent organic framework COF-Fe by using the obtained RT-COF-1, preparing a curcumin-carrying iron-based covalent organic framework COF-Fe@Cur by using the obtained COF-Fe to load curcumin, and finally preparing the hybridized monoatomic platinum by using the obtained COF-Fe@Cur. The nano-immune adjuvant has ideal biocompatibility, excellent inherent sterilization capability and strong immune activation effect, can effectively promote the healing of related infection of endophytes, solves the problems of poor inherent sterilization capability, weak immune regulation activity and the like of the existing biomedical materials, and has extremely wide clinical application potential.
Description
Technical Field
The invention belongs to the technical field of preparation, and particularly relates to a biomedical material, in particular to a nano-immune adjuvant for treating endophyte-related infection by orthopedics.
Background
Endophyte-related infections are one of the disastrous postoperative complications of orthopedic surgery such as joint replacement surgery. Bacteria adhere to the surfaces of endophytes to form biofilms, which are important causes of intractable and recurrent infections associated with endophytes. Specifically, the dense surface structure of the biological film can prevent the penetration of the bactericide through a physical barrier, and on the other hand, the inert biological film structure can hide the bacteria related antigen from being exposed, so that the immune system of the organism cannot effectively identify an infection focus and activate antibacterial immune response. Thus, disruption of biofilm structure and induction of an antibacterial immune response in the body are potentially effective strategies for treating endophyte-related infections.
With the rapid development of biomedical nano-medicine, designing nano-materials with biomedical functions for endophyte-related infections has attracted many researchers' interests and related samples have been developed. However, the prior art has the problems of high biotoxicity, low bactericidal activity, poor immunoregulation effect, difficult clinical transformation and the like. So far, no ideal biological nano material is applied to clinic, and the prevention and the treatment of the endophyte related infection are still mainly performed by antibiotics and revision surgery. Based on this, the present application was developed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a nano-immune adjuvant for treating endophyte-related infection, and solves the problems of poor inherent sterilization capability, weak immunoregulatory activity and the like of the existing biomedical material. The nano-immune adjuvant has ideal biocompatibility, excellent inherent sterilization capability and strong immune activation effect, can effectively promote the cure of related infection of endophytes, and has extremely wide clinical application potential.
The invention also provides a preparation method of the nano-immune adjuvant for treating the endophyte-related infection.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a nanoimmunoadjuvant for treating endophyte-related infections comprising an iron-based covalent organic framework supporting curcumin as a pharmaceutical carrier, and monoatomic platinum doped to enhance the hemodynamic effect of said curcumin. In particular to a preparation method of a nanometer immune adjuvant for orthopedic treatment of endophyte related infection, which comprises the following preparation steps:
1) Preparation of covalent organic frameworks RT-COF-1:
mixing methanol and ethanol, stirring at room temperature for 10-15 min, adding 1,3, 5-tris (4-aminophenyl) benzene and trimesic aldehyde, obtaining a reaction system A after complete dissolution (can be assisted by low-temperature ultrasonic treatment at 2-8 ℃ for 5-10 min), adding glacial acetic acid, stirring at room temperature for 15-30min, sequentially centrifugally washing by methanol, acetone and deionized water, and drying in vacuum to obtain solid RT-COF-1;
2) Preparing an iron-based covalent organic framework COF-Fe by using the obtained RT-COF-1:
dispersing the solid RT-COF-1 in deionized water, then adding an aqueous solution of ferric trichloride hexahydrate to obtain a reaction system B, stirring at room temperature for 20-28h, washing with deionized water, and drying in vacuum to obtain solid COF-Fe;
3) Preparing a curcumin-carrying iron-based covalent organic framework COF-Fe@Cur by using the obtained COF-Fe loaded curcumin:
dispersing solid COF-Fe in deionized water, then adding curcumin ethanol solution, stirring for 20-28h at room temperature, washing with deionized water, and vacuum drying to obtain solid COF-Fe@Cur;
4) Preparing a final nano-immune adjuvant COF-Fe@Cur@Pt by using the obtained COF-Fe@Cur hybridized monoatomic platinum:
dispersing solid COF-Fe@Cur in deionized water, then adding a hexahydrated chloroplatinic acid aqueous solution, stirring for 1-2 hours at a low temperature, then adding sodium borohydride, reacting for 3-5 hours at a low temperature, washing with deionized water, and drying in vacuum to obtain the final nano immune adjuvant COF-Fe@Cur@Pt.
Specifically, in the step 1), the mass ratio of the 1,3, 5-tri (4-aminophenyl) benzene to the trimellitic aldehyde is 2-3:1, and the final concentration of the 1,3, 5-tri (4-aminophenyl) benzene in the reaction system A is 3-4 mg/ml. Methanol and ethanol may be mixed in a volume of 1:1-3, preferably methanol and ethanol are mixed in equal volumes.
In the further step 1), glacial acetic acid is added in an amount of 1/9 to 1/11 of the volume of the reaction system A.
Specifically, in step 2), RT-COF-1 is dispersed in deionized water according to the concentration of 2-3 mg/ml; the final concentration of ferric trichloride hexahydrate in the reaction system B is 0.4-0.6 mg/ml.
Specifically, in the step 3), the mass ratio of the solid COF-Fe to the curcumin is 8-10:1. further preferably, the solid COF-Fe may be dispersed in deionized water at a concentration of 2-3 mg/ml, and the concentration of the curcumin ethanol solution may be 1-2mg/ml.
Specifically, in the step 4), the mass ratio of the solid COF-Fe@Cur to the hexahydrated chloroplatinic acid is 1:0.8-1.2. Further preferably, the solid COF-fe@cur, which is added to the chloroplatinic acid hexahydrate in mass, may be dispersed in deionized water at a concentration of 2 to 3 mg/ml.
Further, in the step 4), the addition amount of the sodium borohydride is 4-5 times of the mass of the chloroplatinic acid hexahydrate. The low temperature refers to a temperature of 2-8 ℃.
In the preparation process of the nano-immune adjuvant, the related centrifugation and vacuum drying time is achieved by adopting the conventional technology in the field. If centrifugation is carried out at 10000-13000 rpm for 20-30 min, the vacuum drying time can be 4-6 hr.
The invention provides the nano-immune adjuvant for treating the endophyte-related infection, which is prepared by adopting the preparation method.
The invention also provides application of the nano-immune adjuvant in preparing medicaments for treating endophyte-related infection in orthopedics.
Compared with the prior art, the invention has the following beneficial effects:
the application of endophytes such as steel plates, screws, artificial joints and the like is an important means for solving bone diseases such as fracture, end-stage arthritis and the like. However, 1% -3% of patients develop bacterial infections, collectively referred to as endophyte-related infections, after endophyte implantation. The difficulty in curing the endophyte-related infection is great due to the formation of bacterial biofilm and the destruction of the immune system of the organism, and the operation is often failed and even life is threatened. The Fe-COF@Cur Pt nano-immunoadjuvant provided by the invention plays a role in a mode of local injection in a plant-related infection focus. Under ultrasonic irradiation, the natural sound sensitizer curcumin catalyzes hydrogen peroxide to generate singlet oxygen, damages the biomembrane structure and kills bacteria. Whereas monoatomic platinum exerts catalase activity, catalyzes the production of oxygen, enhancing the hemodynamic effect of curcumin.
The nanometer immune adjuvant Fe-COF@Cur@Pt+ ultrasonic group treatment mode can effectively kill 93+/-5.5% of staphylococcus aureus and 88+/-2.9% of escherichia coli, and the curative effect of the nanometer immune adjuvant is even better than that of vancomycin antibiotics when the nanometer immune adjuvant is used for treating escherichia coli infection. And the ferric ions are used as classical immune activators to improve the activation rate of dendritic cells and neutrophils, strengthen the antibacterial immune response of organisms and further accelerate the elimination of infection. Compared with the existing preparation, the nano immune adjuvant COF-Fe@Cur@Pt has ideal biocompatibility, excellent inherent sterilization capability and strong immune activation effect, can effectively promote the healing of related infection of endophytes, and has extremely wide clinical application potential.
Drawings
FIG. 1 is a transmission electron microscope photograph of a COF-Fe (a), a COF-Fe@Cur (b) and a COF-Fe@Cur@Pt (c) in the preparation of the Fe-COF@Cur@Pt nano immunoadjuvant of the invention;
fig. 2 is a graph showing wound infection area curves of a blank group (a), a vancomycin group (b), a COF-fe@cur@pt group (c), and a COF-fe@cur pt+ultrasound group (d) under different treatment modes of a plant-related infection model in a mouse.
Detailed Description
The following describes the technical scheme of the present invention in further detail with reference to examples, but the scope of the present invention is not limited thereto.
In the following examples, unless otherwise indicated, all the materials used were commercially available products or were prepared by methods conventional in the art. Room temperature refers to 25±5 ℃.
The steps, test methods, and procedures not mentioned or described in detail may be performed by a technique generally known in the art.
Example 1
The invention relates to a preparation method of a nano-immune adjuvant for treating endophyte-related infection, which comprises the following steps:
step 1, preparing a covalent organic framework RT-COF-1:
30 mL methanol and 30 mL ethanol were mixed and stirred at room temperature for 10 minutes. 205 mg of 1,3, 5-tris (4-aminophenyl) benzene and 95 mg and trimellitic aldehyde were then added and sonicated at around 4℃for 5 min to aid dissolution. After complete dissolution, 6 mL glacial acetic acid was added and stirred at room temperature for 15 min. The resulting product was washed sequentially with methanol, acetone and deionized water by centrifugation (13000 rpm,20 minutes) and dried under vacuum for 6 h to give RT-COF-1 for later use.
Step 2, preparing an iron-based covalent organic framework COF-Fe:
10mg solid RT-COF-1 was dispersed in 5 mL deionized water (2 mg/mL) and then 0.1 mL of 20 mg mL was added -1 The reaction system B was obtained from an aqueous solution of ferric trichloride hexahydrate. After stirring at room temperature for 24 hours, the resulting product was washed by centrifugation with deionized water (13000 rpm,20 minutes) and dried in vacuo 6 h to give solid COF-Fe.
Step 3, loading of curcumin:
10mg solid COF-Fe was dispersed in 5 mL deionized water, then 1 mg mL of 1mL was added -1 Curcumin ethanol solution. After stirring at room temperature for 24 hours, the resulting product was washed by centrifugation with deionized water (13000 rpm,20 minutes) and dried in vacuo at 6 h to give a solid COF-fe@cur.
Step 4, preparing a nano immune adjuvant COF-Fe@Cur@Pt:
10mg solid COF-fe@cur was dispersed in 5 mL deionized water. Subsequently, 1mL of a 20 mM aqueous chloroplatinic acid hexahydrate solution (the mass of chloroplatinic acid hexahydrate was about 10 mg) was added thereto, and the mixture was stirred at a low temperature of 4℃for 1 hour. Then 10 mL concentration 4 mg mL was added -1 Sodium borohydride water-solubleThe reaction was carried out at 4℃for 3 hours. The obtained product is centrifugally washed by deionized water (13000 rpm,20 minutes), and is dried in vacuum for 6 h, so that the final nano immune adjuvant COF-Fe@Cur@Pt is obtained.
The appearance and the size of the products in each step are observed by adopting a transmission electron microscope, the model of the electron microscope is FEI Tecnai F20, and the voltage is 200 kV. FIG. 1 shows transmission electron micrographs of COF-Fe, COF-Fe@Cur and COF-Fe@Cur@Pt in the process of preparing the Fe-COF@Cur@Pt nanoimmunoadjuvant in the embodiment. As can be seen from fig. 1: the three nano particles of the COF-Fe, the COF-Fe@Cur and the COF-Fe@Cur@Pt are in regular spherical structures, and the particle sizes are uniformly distributed in a range of 50-100nm, so that the loading of curcumin and monoatomic Pt has no obvious influence on the morphological structure of the nano particles. In addition, a dispersed dot-like structure can be observed on the surface of the COF-Fe@Cur@Pt nanospheres, and successful loading of single-atom Pt is demonstrated.
Example 2
A method for preparing a nano-immunoadjuvant for orthopedic treatment of endophyte-related infections, which is different from example 1 in that: in the step 3, the mass ratio of the solid COF-Fe to the curcumin is 8:1.
a related performance test was performed on the nanoimmunoadjuvant COF-Fe@Cur Pt prepared in example 1 as follows.
Test:
the sterilization efficiency of the nano immune adjuvant COF-Fe@Cur@Pt is detected by adopting a plating method. Staphylococcus aureus and Escherichia coli are selected as representatives of gram positive bacteria and gram negative bacteria, and prepared at a concentration of 1×10 6 CFU/mL bacterial suspension. The bacterial suspension was then divided into four groups (1 mL each) of a blank, a COF-Fe@Cur@Pt group, a COF-Fe@Cur@Pt+ ultrasound group and a vancomycin antibiotic group, respectively. The blank group was not subjected to any treatment, the COF-Fe@Cur@Pt group was added with a nanoimmunoadjuvant COF-Fe@Cur@Pt with a final concentration of 100ug/ml, the COF-Fe@Cur@Pt+the ultrasonic group was added with a nanoimmunoadjuvant COF-Fe@Cur@Pt with a final concentration of 100ug/ml and subjected to ultrasonic treatment for 8 min (ultrasonic frequency of 1 MHZ, power of 1W/cm, duty ratio of 50%), and the vancomycin antibiotic group was subjected to vancomycin with a final concentration of 5 ug/ml. Culturing at 37deg.C 6 h, followingAnd quantifying the surviving bacteria of each treatment group by adopting a plating method, and calculating the respective sterilization rate by taking a blank control group as a reference. The results are shown in Table 1.
TABLE 1 Sterilization rates of Staphylococcus aureus and Escherichia coli by different in vitro treatments
As shown in Table 1, the treatment mode of the nano immune adjuvant COF-Fe@Cur@Pt+ ultrasonic group can effectively kill 93+/-5.5% of staphylococcus aureus and 88+/-2.9% of escherichia coli, and the curative effect of the nano immune adjuvant is even better than that of vancomycin antibiotics when the nano immune adjuvant is used for treating escherichia coli infection. In contrast, the sterilization rates of staphylococcus aureus and escherichia coli were only 58±6.4% and 52±3.1% for COF-fe@cur@pt groups without ultrasonic irradiation. This verifies our hypothesis that ultrasonic irradiation can excite the catalase activity of single-atom Pt, and finally greatly improves the sterilization efficiency of the COF-Fe@Cur@Pt nano immunoadjuvant by generating an oxygen enhanced curcumin acoustic kinetic effect.
The immune activation efficiency of the nano immune adjuvant COF-Fe@Cur@Pt is detected by adopting a flow cytometry. Dendritic cells and neutrophils were selected as representative of immune cells. The cells were divided into three groups, a blank control group, a COF-Fe@Cur@Pt group and a COF-Fe@Cur@Pt+ultrasound group. The blank control group is not treated at all, the nano immune adjuvant COF-Fe@Cur@Pt group with the final concentration of 100ug/ml is added, the nano immune adjuvant COF-Fe@Cur@Pt with the final concentration of 100ug/ml is added, and the nano immune adjuvant COF-Fe@Cur@Pt with the final concentration of 100ug/ml is added into the ultrasonic group and subjected to ultrasonic treatment for 8 min (ultrasonic frequency of 1 MHZ, power of 1W/cm and duty ratio of 50%). Incubation at 37℃6 h was followed by flow cytometry to detect the activation rates of both cells. The results are shown in Table 2.
TABLE 2 activation rates of dendritic cells and neutrophils by different in vitro treatments
As shown in Table 2, the therapeutic mode of the nano-immune adjuvant COF-Fe@Cur@Pt+ ultrasonic group provided by the invention increases the activation rates of dendritic cells and neutrophils from 16.7% and 39.6% to 32.24% and 57.73% respectively. Dendritic cells are the most critical antigen presenting cells of the body and are responsible for recognition, phagocytosis and presentation of bacterially related antigens. Neutrophils are the first line of defense of the body against bacterial infection. However, in the endophyte-associated infectious microenvironment, dendritic cell and neutrophil activation is hindered, resulting in inefficient immune and antibacterial. The therapeutic mode of COF-Fe@Cur@Pt+ ultrasound can effectively activate dendritic cells and neutrophils, which tends to accelerate the clearance of the immune system to bacteria.
A mouse subcutaneous endophyte infection model is constructed to detect the in-vivo curative effect of the nano-immune adjuvant COF-Fe@Cur@Pt. 48 male ICR mice (8-10 weeks old) were selected and modeled. First, mice were anesthetized by intraperitoneal injection of 1ml of 1% pentobarbital per 100g body weight, followed by shaving the back hair of the mice and disinfecting with 75% alcohol. A transverse incision of 1 cm length was made in the back of the mice, and a sterile titanium plate (8 mm diameter) was implanted subcutaneously through the incision. Around the titanium plate 100. Mu.L of Staphylococcus aureus suspension (1X 10) 6 CFU mL -1 ) And suturing the incision. Mice were randomly divided into 4 groups (12 per group). Wound injection treatment on postoperative day 3 and day 5: blank control group (without treatment), vancomycin antibiotic group (2 mg/kg vancomycin), COF-Fe@Cur@Pt group (2 mg/kg COF-Fe@Cur@Pt), COF-Fe@Cur@Pt+ ultrasonic group (2 mg/kg COF-Fe@Cur@Pt+ frequency of 1 MHZ, power of 1W cm -2 Sonication for 8 min with 50% duty cycle). Mice were periodically monitored for back wound infection after surgery and the infection areas were measured on days 1,3,5, 7, 10 using calipers and infection area curves were drawn. The results are shown in FIG. 2.
Fig. 2 shows the wound infection area curves of different treatment modes (blank, antibiotic, COF-fe@cur pt, COF-fe@cur pt+ultrasound) of the plant-related infection model in mice. As can be seen from fig. 2: the area of dorsal infection was continuously increased over time in mice from the blank group that did not receive any treatment. In contrast, vancomycin and COF-fe@cur pt group infection was inhibited to some extent, but there was still a large area of infection at day 10. In contrast, in the COF-Fe@Cur@Pt+ ultrasound mice, the infection area was continuously decreased after treatment, and almost no infection was observed on day 10. From the result, the effect of the combination mode of the COF-Fe@Cur@Pt+ultrasound on treating the endophyte related infection is obviously superior to that of vancomycin and the COF-Fe@Cur@Pt.
Claims (10)
1. The preparation method of the nano-immune adjuvant for treating the endophyte-related infection is characterized by comprising the following steps:
1) Preparation of covalent organic frameworks RT-COF-1: mixing methanol with ethanol, adding 1,3, 5-tri (4-aminophenyl) benzene and trimesic aldehyde, obtaining a reaction system A after complete dissolution, adding glacial acetic acid, stirring for 15-30min at room temperature, washing, and drying in vacuum to obtain solid RT-COF-1;
2) Preparation of an iron-based covalent organic framework COF-Fe: dispersing the solid RT-COF-1 in deionized water, then adding an aqueous solution of ferric trichloride hexahydrate to obtain a reaction system B, stirring at room temperature for 20-28h, washing, and drying in vacuum to obtain the solid COF-Fe;
3) Preparing a curcumin-carrying iron-based covalent organic framework COF-Fe@Cur: dispersing solid COF-Fe in deionized water, then adding curcumin ethanol solution, stirring for 20-28h at room temperature, washing, and vacuum drying to obtain solid COF-Fe@Cur;
4) Preparing a nano immune adjuvant COF-Fe@Cur@Pt: dispersing solid COF-Fe@Cur in deionized water, then adding a hexa-hydrated chloroplatinic acid aqueous solution, stirring for 1-2h at a low temperature, then adding sodium borohydride, reacting for 3-5h at a low temperature, washing, and drying in vacuum to obtain the finished product.
2. The method for preparing the nano-immunoadjuvant for the orthopedic treatment of endophyte-related infections according to claim 1, wherein in the step 1), the mass ratio of the 1,3, 5-tris (4-aminophenyl) benzene to the trimellitic aldehyde is 2-3:1, and the final concentration of the 1,3, 5-tris (4-aminophenyl) benzene in the reaction system a is 3-4 mg/ml.
3. The method for preparing a nano-immunoadjuvant for orthopedic treatment of endophyte-related infection as claimed in claim 1, wherein in the step 1), glacial acetic acid is added in an amount of 1/9 to 1/11 of the volume of the reaction system a.
4. The method of preparing a nanoimmunoadjuvant for the orthopedic treatment of endophyte-related infections according to claim 1, wherein in step 2), RT-COF-1 is dispersed in deionized water at a concentration of 2-3 mg/ml; the final concentration of ferric trichloride hexahydrate in the reaction system B is 0.4-0.6 mg/ml.
5. The method for preparing a nanoimmunoadjuvant for orthopedic treatment of endophyte-related infections according to claim 1, wherein in step 3), the mass ratio of solid COF-Fe to curcumin is 8-10:1.
6. the method for preparing a nanoimmunoadjuvant for orthopedic treatment of endophyte-related infections according to claim 1, wherein in step 4), the mass ratio of solid COF-fe@cur to chloroplatinic acid hexahydrate is 1:0.8-1.2.
7. The method for preparing a nano-immunoadjuvant for orthopedic treatment of endophyte-related infections according to claim 1, wherein in step 4), the added amount of sodium borohydride is 4-5 times the mass of chloroplatinic acid hexahydrate.
8. The method for preparing a nanoimmunoadjuvant for the orthopedic treatment of endophyte-related infections according to claim 1, wherein in step 4), the low temperature is referred to as a temperature of 2-8 ℃.
9. A nanoimmunoadjuvant for the treatment of endophyte-related infections prepared by the method of any one of claims 1 to 8.
10. Use of the nanoimmunoadjuvant according to claim 9 for the preparation of a medicament for the orthopedic treatment of endophyte-related infections.
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