CN114129594B

CN114129594B - Porous organic polymer loaded with nano silver and preparation method and application thereof

Info

Publication number: CN114129594B
Application number: CN202111476004.3A
Authority: CN
Inventors: 吕镗烽; 周宝龙; 谭薇; 宋勇; 王栋; 朱素华; 罗昊天
Original assignee: Eastern Theater General Hospital of PLA
Current assignee: Eastern Theater General Hospital of PLA
Priority date: 2021-12-06
Filing date: 2021-12-06
Publication date: 2023-09-12
Anticipated expiration: 2041-12-06
Also published as: CN114129594A

Abstract

To solve the problems of the prior art that most of the prepared materials are Ag ⁺ The invention provides a preparation method of an antibacterial agent for accelerating wound healing, which comprises the steps of S1, preparing an imidazolyl organic porous polymer, S2 and preparing a nano silver loaded porous organic polymer. The invention also provides an antibacterial agent obtained by the preparation method and application thereof in preparing antibacterial drugs for accelerating wound healing. The preparation method can effectively prevent silver from gathering, greatly enhance the antibacterial effect and has the antibacterial activity of a porous host. The prepared IM-POP-Ag has low toxicity, good biocompatibility and excellent antibacterial activity, and can remarkably kill infectious bacteria in wounds and promote wound healing.

Description

Porous organic polymer loaded with nano silver and preparation method and application thereof

Technical Field

The invention relates to the technical field of medical treatment, in particular to a nano-silver loaded porous organic polymer, and a preparation method and application thereof.

Background

The skin serves as the largest organ and natural barrier for humans and can effectively prevent and protect us from invasion and infection by external bacteria and viruses. However, the skin is easily broken in any accident or operation. Once injured, wounds, such as war wounds and caesarean wounds, result in skin that is vulnerable to external pathogens, causing wound infections. However, the difficulty in wound healing due to bacterial infection has been a serious threat to human health. For performing potent bactericidal effects, clinical antibiotic therapy always requires high doses of antibiotics for a sustained, long period of time, which not only increases the likelihood of antibiotic resistance, but also causes serious side effects to the human body. Therefore, development of new antibacterial agents is imperative to achieve effective treatment of bacterial infections.

The rapid development of nanotechnology has greatly accelerated the development of metallic nanoantimicrobial agents. To date, various metal antibacterial materials such as metal nanoparticles, metal oxides, carbon nanomaterials, and composite materials thereof have been developed. The nanotechnology which is rising in the eighties of the twentieth century opens up a new direction for the structural design and property exploration of novel materials. The application of various nano materials in biomedicine has been receiving increasing attention, particularly nano antibacterial materials such as nano gold, nano silver, nano copper, nano zinc oxide, nano titanium dioxide, etc. as antibiotic substitutes. Among those metal-based bactericides, nanosilver-based materials having broad-spectrum antibacterial activity have attracted significant attention.

However, ag of most of the prepared materials ⁺ The load efficiency is low. Moreover, as a typical heavy metal element, the naked AgNPs particles are easier to interact with irrelevant proteins, cause serious self-aggregation and greatly damage the AgNPs particlesTheir antibacterial activity. All of these hinder Ag ⁺ The nanoparticle antibacterial agent is widely applied. Therefore, it is very necessary to develop a novel AgNPs sterilization system with high Ag loading efficiency, which can not only reduce the biotoxicity of AgNPs, but also enhance the therapeutic effect at the same time.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a porous organic polymer loaded with nano silver, and a preparation method and application thereof.

A first object of the present invention is to provide a method for preparing a nano-silver loaded porous organic polymer, the method comprising the steps of:

s1, preparing an imidazolyl organic porous polymer: mixing imidazole-2-formaldehyde and melamine in DMSO, heating for reaction, collecting a solid product after the reaction is finished, removing unreacted components, purifying and drying to obtain an imidazolyl organic porous polymer;

s2, preparing a nano silver loaded porous organic polymer:

s2-1, mixing and grinding the imidazolyl organic porous polymer obtained in the S1 with a small amount of methanol, and adding the methanol after finishing grinding to ensure that the concentration of the imidazolyl organic porous polymer in the methanol is 1-2 mg/mL, thereby obtaining a reaction system; in a particular embodiment, the milling time is from 30 minutes to the nanometer scale. The concentration of the imidazolyl organic porous polymer in methanol was 1.6mg/mL.

S2-2, agNO is added into the reaction system obtained in the S2-1 ₃ Solution of AgNO ₃ The mass ratio of the organic porous polymer to the imidazolyl is 1.2:1-1.4:1, and preferably the AgNO ₃ The concentration of the solution is 8.75mg/mL; removal of unreacted AgNO from the reaction product ₃ And after the free nano silver, obtaining a silver ion-loaded porous organic polymer;

s2-3, reducing silver ions in the silver ion loaded porous organic polymer obtained in the step S2-2 into nano silver, sealing and stirring, cleaning with distilled water, centrifuging, and collecting precipitate to obtain the nano silver loaded porous organic polymer.

Further, the ratio of the amount of the imidazole-2-formaldehyde to the amount of the melamine in S1 is 3:1-3.5:1; preferably, the ratio of the amount of the imidazole-2-formaldehyde to the melamine in S1 is 3:1.

Further, the concentration of the imidazole-2-formaldehyde in the DMSO is 0.068g/mL, and the concentration of the melamine in the DMSO is 0.03g/mL.

And further, the mixed heating reaction in the step S1 is carried out for 3-4 days under the protection of inert gas at 180 ℃. In a specific embodiment, the reaction conditions of the mixed heating reaction are 180 ℃ under the protection of inert gas and stirring for 3 days.

Further, the removal of unreacted components as described in S1 is to add the collected solid product to acetone and stir at 30-60℃for 3-9 h, in a particular embodiment, at 45℃for 6h.

Further, the purification of S1 is carried out by stirring the solid product with tetrahydrofuran at 55℃for 3 hours, followed by stirring with dichloromethane at 60℃for 6 to 9 hours.

Further, the drying in S1 is vacuum drying at 80 ℃.

S2-2 removal of unreacted AgNO ₃ And the specific operations of the free nano silver are as follows: removing solvent from the reaction product, washing with distilled water, centrifuging, and collecting precipitate to obtain porous organic polymer loaded with silver ions; preferably, the centrifugation conditions are 9000rpm,5min.

Further, in the reduction of S2-3, adding the silver ion loaded porous organic polymer obtained in S2-2 into a mixed solvent of methanol and water, adding a sodium borohydride methanol solution to reduce silver ions in the silver ion loaded porous organic polymer into nano silver, wherein the mixed solvent is a mixture of methanol and water in a volume ratio of 3:2, the concentration of the silver ion loaded porous organic polymer in the mixed solvent is 3-4 mg/mL, the concentration of the sodium borohydride methanol solution is 15-16 mg/mL, the concentration of the silver ion loaded porous organic polymer in the sodium borohydride methanol solution is 6-8 mg/mL, the stirring time is 1-2 days, and the washing is water and methanol washing.

The second object of the invention is to provide the nano-silver loaded porous organic polymer prepared by the preparation method of the nano-silver loaded porous organic polymer.

In a particular embodiment, the nano-silver loaded porous organic polymer has a particle size of: 500nm.

The third object of the present invention is to provide an application of the nano-silver loaded porous organic polymer in preparation of an antibacterial drug, preferably an antibacterial drug capable of accelerating wound healing.

Further, the antibacterial drug is an anti-gram-negative bacterial drug and/or a gram-positive bacterial drug, preferably, the antibacterial drug is an anti-escherichia coli drug and/or a staphylococcus aureus drug.

The IM of the invention refers to: imidazolyl;

the POP refers to: an organic porous polymer;

the IM-POP refers to: imidazolyl organic porous polymers;

the IM-POP-Ag refers to: loading nano silver porous organic polymer;

the AgNPs of the invention refer to: nano silver.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the preparation method adopts a chemical reduction method to simply prepare the nano silver loaded porous organic polymer (IM-POP-Ag) which has low cost and can be produced in batch. IM-POP is prepared easily by copolymerization of imidazole-2-formaldehyde and melamine in the absence of catalyst through Schiff base chemistry, which is inexpensive, readily available and industrially produced. The unique imidazolyl POP skeleton has N content as high as 30.43at%, provides rich N coordination sites for Ag+, ensures high Ag loading capacity of 26wt% through coordination self-assembly, and can anchor Ag in the porous skeleton effectively to avoid self aggregation of nanometer Ag. On the one hand, the anchoring of Ag at the N position (30.43 at%) can effectively prevent the aggregation of silver and greatly enhance the antibacterial effect. On the other hand, the coordination of silver changes the imidazole framework into imidazolium, which imparts antibacterial activity to the carrier.

Compared with pure Ag nano-particles, the prepared IM-POP-Ag not only has low toxicity and good biocompatibility, but also has excellent antibacterial activity on gram-negative bacteria (escherichia coli) and gram-positive bacteria (staphylococcus aureus) in vitro. At the same silver content, the antibacterial activity of IM-POP-Ag is significantly higher than that of AgNPs. Coli and staphylococcus aureus against 200 μg mL ^-1 IM-POP-Ag (corresponding to 50. Mu.g mL) ^-1 AgNPs) was 3.44% and 9.62%, however, E.coli and Staphylococcus aureus against 50. Mu.g mL ^-1 The survival rates of AgNPs were 12.19% and 20.20%.

In addition, in vivo mouse experiments prove that IM-POP-Ag can obviously kill infectious bacteria in wounds and promote wound healing. The new strategy can greatly reduce the use amount of Ag and save the cost. Meanwhile, a theoretical basis is provided for applying the silver nano particles with higher efficacy and biocompatibility in the near future.

Drawings

Fig. 1. A schematic synthesis of Ag nanoparticle-encapsulated porous organic polymer (IM-POP) for antibacterial and wound healing.

FIG. 2 a) Fourier Transform Infrared (FTIR) spectra; b) Solid state IM-POP ¹³ CNMR spectroscopy.

Fig. 3. Physical characterization of the prepared samples: a) FTIR spectra of IM-POP-Ag and IM-POP samples; b) Preparing XRD spectrogram of IM-POP-Ag; c) Preparing TGA spectrograms of the IM-POP and the IM-POP-Ag; d) Low temperature N of IM-POP and IM-POP-Ag at 77K ₂ An absorption isotherm spectrogram; e) Pore size distribution curve of sample IM-POP; f) Pore size distribution curve of sample IM-POP-Ag.

FIG. 4 a) particle size distribution of AgNPs; b) Particle size distribution of IM-POP-Ag; c) Zate potential of IM-POP, agNPs, IM-POP-Ag.

SEM and TEM of IM-POP-Ag. a. b) SEM at IM-POP-Ag is 5 μm and 500nm scale respectively; c. d, e) the proportion scales of the IM-POP-Ag are 1000 nm, 100nm and 50nm respectively; f) The HRTEM scale of IM-POP-Ag is 10nm; g) Element diagram of IM-POP-Ag in 100nm scale, h) carbon element distribution diagram, i) nitrogen element distribution diagram, j) silver element distribution diagram, h) oxygen element distribution diagram.

IM-POP-Ag of XPS. a) XPS measurement spectrum of IM-POP-Ag; b) N1s spectrum of IM-POP-Ag c) Ag 3d spectrum of IM-POP-Ag.

FIG. 7 in vitro bacterial growth inhibition assay. a) Photographs of bacterial colonies formed by E.coli and d) Staphylococcus aureus IM-POP-Ag and AgNPs treatments were based on plate counting (left to right concentrations: IM-POP-ag=0, 50, 100, 200 μg mL ^-1 ,AgNPs＝0、12.5、25、50μg mL ^-1 ). b) The survival rate of the AgNPs to the bacteria after the treatment of the escherichia coli; c) The survival rate of the bacteria after the IM-POP-Ag is treated with escherichia coli; e) Is the bacterial survival rate of AgNPs after the treatment of staphylococcus aureus; and f) is the bacterial survival rate of IM-POP-Ag after treatment with Staphylococcus aureus.

FIG. 8 antibacterial activity against E.coli and Staphylococcus aureus pellets. a) Coliform bacteria and b) staphylococcus aureus. c) Coliform and d) staphylococcus aureus.

Fig. 9 fluorescence images of staphylococcus aureus and escherichia coli incubated with PBS, IM-POP, agNPs, IM-POP-Ag co-stained with pi and SYTO 9 (scale bar = 100 μm).

Fig. 10 TEM images of bacteria. a-d) after PBS, IM-POP, agNPs and IM-POP-Ag treatment, obtaining staphylococcus aureus; e-h) PBS, IM-POP, agNPs and IM-POP-Ag to obtain the E.coli.

FIG. 11 hemolysis assay and cell viability assay. Hemolysis test with different concentrations of a) IM-POP, b) AgNPs, c) IM-POP-Ag. Survival (%) of L929 cells incubated with different concentrations of d) IM-POP, e) AgNPs, f) IM-POP-Ag (p <0.05 compared to control).

Figure 12 in vivo antibacterial efficacy of im-POP, agNPs, IM-POP-Ag. a) Representative wound photographs of KM mice back staphylococcus aureus infected wound with IM-POP, agNPs, IM-POP-Ag at each time point and corresponding control group PBS; b) Corresponding wound shrinkage versus time curve; c) Changes in mouse body weight during dosing d) 4 groups day 14 wound tissue H & E and Masson trichromatic staining images. The scale bar is 200 μm.

Fig. 13. Bacterial amounts at each set of wounds on the first day and tenth day of treatment were modeled.

FIG. 14H & E staining (scale bar 200 μm) of major organs (heart, liver, spleen, lung and kidney) of mice after different treatments.

Fig. 15 blood convention after 14 days of treatment for each group of mice. Fig. 15 a) Red Blood Cells (RBC), fig. 15 b) White Blood Cells (WBC), fig. 15 c) mean red blood cell volume (MCV), fig. 15 d) mean red blood cell hemoglobin concentration (MCHC), fig. 15 e) Hemoglobin (HGB), fig. 15 f) Hematocrit (HCT), fig. 15 j) mean red blood cell hemoglobin amount (MCH), fig. 15 h) Platelets (PLT).

Detailed Description

EXAMPLE 1 preparation of the antimicrobial agent IM-POP-Ag of the present invention

1. Preparation of imidazolyl organic porous polymer IM-POP

To a round bottom flask was added imidazole-2-carbaldehyde (3.42 g,35.7 mmol) and melamine (1.50 g,11.9 mmol) and 50mL DMSO was added as solvent. The mixture was then stirred at 180℃for 3 days under argon protection, and after the heating reaction was completed, the solid product was collected.

To remove unreacted components, the collected solid was added to 100mL of acetone and stirred at 45℃for 6h.

Then, the solid was washed with tetrahydrofuran (100 mL, 3h at 55 ℃ C.) followed by dichloromethane (100 mL, 6h at 60 ℃ C.) with stirring.

Finally, the solid was collected and dried in a vacuum oven at 80℃to give 3.1g of an imidazolyl organic porous polymer IM-POP.

2. Preparation of porous organic polymer IM-POP-Ag loaded with nano silver

S2-1, accurately weighing the imidazolyl organic porous polymer IM-POP (75.0 mg), placing in an agate mortar in methanol (1 mL), and grinding for 30min to the nano-scale. The milled IM-POP was rinsed from the mortar with methanol (45 mL) and collected in a 100mL round bottom flask to give a concentration of 1.6mg/mL of imidazolyl organic porous polymer in methanol, which was well dispersed to give a reaction system.

S2-2, injecting AgNO into the reaction system obtained by the S2-1 ₃ The solution (12 mL,8.75 mg/mL) was added to the round bottom flask, the AgNO ₃ With imidazolyl organic porous polymerizationThe mass ratio is 1.4:1.

unreacted AgNO is then removed ₃ And free nano silver, which comprises the following specific operations: the reaction product was passed through a rotary evaporator to remove the solvent at 50 ℃. After complete removal of the solvent, the resulting solid was washed 5 times with distilled water (30 mL), centrifuged at 9000rpm for 5min, and the precipitate was collected to obtain 110mg of silver ion-loaded porous organic polymer.

S2-3 the silver ion-loaded porous organic polymer obtained in S2-2 was dissolved in a mixed solvent (30 mL) of methanol and water in a volume ratio of 3:2, followed by slowly adding 15mL of NaHB with magnetic stirring ₄ (227 mg,6.0 mmol) in methanol. The beaker was sealed with filter paper and stirring was continued for 2 days. The sample was washed 3 times with water and methanol to remove free AgNPs and other impurities. Finally, the sample was dried with a vacuum oven. The porous organic polymer IM-POP-Ag with nano silver load prepared in the embodiment has the particle size of: 500nm.

Comparative example 1 Synthesis of AgNPs

AgNPs were prepared according to the experimental procedure described above. AgNO was added to 30mL of water ₃ (105.0 mg,0.618 mmol). After 1min of vortexing, 15mL NaBH was slowly added to the solution under magnetic stirring ₄ Is stirred for 2 days by sealing the beaker with filter paper (227 mg NaHB4). The product was washed 3 times with water and methanol and dried in a vacuum oven.

Experimental example 1 characterization

The IM-POP-Ag composite material is synthesized by a two-step method. Initially, POP supports were prepared by the catalyst-free schiff base chemical method by copolymerization of imidazole-2-carbaldehyde and melamine, which is readily available and industrially mass produced. Then Ag ⁺ Chelate to the N-rich POP by coordinated self-assembly. Subsequently, ag ⁺ The chelate POP is subjected to in-situ chemical reduction by NaBH4 to obtain an AgNPs-coated IM-POP composite material (figure 1).

To verify that AgNPs were successfully loaded into porous IM-POP, a series of characterizations were performed, including X-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR), elemental Analysis (EA), inductively coupled plasma emission spectroscopy (ICP-OES), thermogravimetric analysis (TGA), specific surface area testing (BET), and X-ray photoelectron spectroscopy (XPS).

(1) The particle size distribution of IM-POP-Ag and AgNPs was measured with a Zetasizer nanoZS90 (Malvern Instruments, malvern, UK) at a temperature of 25 ℃.

The results show that: as can be seen from the Particle Size Distribution (PSD) and Zeta potential values of the prepared material by Dynamic Light Scattering (DLS) measurement using data acquisition software provided by zetasizer nanozs90, the PSD of AgNPs was found to be mainly around 200nm (fig. 4 a), whereas after loading of AgNPs, the PSD was concentrated around 500nm (fig. 4 b). In FIG. 4, the Zeta potentials of the IM-POP and AgNPs are about +20 and-40 mV, respectively. Such a potential difference facilitates the loading of silver nanoparticles. After encapsulation of AgNPs, the potential of the IM-POP was reduced from +20mV to-20 mV, demonstrating that negatively charged AgNPs were successfully loaded into the backbone of the IM-POP (FIG. 4 c). The AgNPs loading content of IM-POP-Ag, accurately measured by ICP-OES, was 26wt% much higher than the porous loading reported previously summarized in Table 1.

TABLE 1 comparison of silver loading levels of other porous antimicrobial Agents

(2) The morphology of IM-POP-Ag was observed by Transmission Electron Microscopy (TEM) and scanning electron microscopy (SEM, JSM-6510) (FIG. 5).

The results show that: as is clear from SEM (fig. 5a and 5 b), IM-POP-Ag consists of irregular spherical particles. The TEM shown in fig. 5c-e verifies the presence of AgNPs in the porous scaffold, whereby widely distributed nanoparticles (black dots) assigned to the nanoscale Ag particles can be clearly observed. Moreover, similar to the XRD results, the crystal structure of AgNPs in IM-POP-Ag can be further verified by HRTEM (FIG. 5 f), wherein a clear lattice spacing of 0.24nm can be observed for the (111) plates assigned to AgNPs. The elemental map of IM-POP-Ag shows that Ag, C, O and N are uniformly distributed on the porous backbone (FIG. 5 g).

(3) Infrared data were obtained by fourier transform infrared spectroscopy (FT-IR, VERTEX 70) (fig. 2a and 3 a).

The results show that: it can be clearly seen thatQuadrants of the triazine ring (1549 cm ^-1 ) And semicircle extension (1481 cm) ^-1 ) The corresponding distinct bands, and characteristic vibrational peaks in the imidazole region between 3124-2540 (NH-N), 1543 (N-H) and 1448 (triazine ring extension). At the same time, due to the-NH of the respective melamine and imidazole monomers ₂ (3465, 3419 and 1651 cm) ^-1 ) and-CHO (1641 and 2842 cm) ^-1 ) Is characterized by almost weakening the stretching vibration. In contrast, due to-CHO and-NH ₂ Is at 2910cm ^-1 There is a new vibrational band of methylene (-CH-) groups. All these results indicate that melamine and imidazole were successfully incorporated into the porous backbone of IM-POP. As shown in FIG. 2a, except for 1000-1600 cm due to the coordination of Ag with the N-rich backbone ^-1 Outside of the slight red shift, the FTIR of IM-POP-Ag almost overlapped that of pure IM-POP. Such results indicate that the structure of the POP is well preserved after loading the AgNPs.

(4) Determination of carbon spectra of IM-POP by solid state nuclear magnetism

The results show that: from IM-POP ¹³ Clear carbon signals of melamine (164 ppm) and imidazole (130.9, 136.6 and 150.6) ppm were observed simultaneously in CNMR. At the same time, a clear carbon signal of 55ppm can also be detected, which is due to the newly formed methylene group (FIG. 2 b).

(5) The silver element content in IM-POP-Ag was determined by Inductively Coupled Plasma (ICP) atomic emission spectrometry (ICP-OES 730, agilent Technologies, japan).

The results show that: the silver content of IM-POP-Ag was 26wt%.

(6) The valence state of elemental silver was measured by scientific X-ray diffractometer (XRD) (fig. 3 b).

The results show that: XRD of IM-POP in FIG. 3b showed no characteristic peaks, which demonstrated an amorphous framework structure. However, at 38.12 °,44.19 °,64.35 °,77.19 °,81.53 °, clear diffraction peaks assigned to the (111), (200), (220), (331), (222) planes of AgNPs were detected from the XRD pattern of IM-POP-Ag, demonstrating successful encapsulation of AgNPs into N-rich IM-POP networks.

(7) The specific surface area and pore distribution of IM-POP and IM-POP-Ag were measured using physical gas adsorption Performance test (BET, ASAP 2460, US) (FIGS. 3e, 3 f).

The results show that: IM-POP (FIG. 3 d) followed a type II reversible adsorption isotherm, indicating the broad presence of macropores, favoring the loading of AgNPs. 28 calculated Brunauer-Emmett-Teller (BET) surface area is 31.55m 2g for IM-POP-Ag, much smaller than IM-POP (137.69 m2 g), further confirming the nano-silver loading.

(8) The thermal stability of IM-POP and IM-POP-Ag was checked by a thermo gravimetric analyzer (TG, TA Discover, US) (FIG. 3 c).

The results show that: IM-POP-Ag maintained 66.5% higher than impu by 36.5% at 800 ℃.

(9) Coexistence of N, C, ag and O in IM-POP-Ag was detected using X-ray photoelectron spectroscopy (XPS) (FIG. 6 a).

The results show that: the atomic contents of N, C, ag and O in IM-POP-Ag were 30.43, 55.23, 7.44 and 6.91 (%), respectively (Table 2).

TABLE 2 IM-POP-Ag surface element content calculated from XPS spectra

(9) High resolution XPS spectroscopy detected IM-POP-Ag (FIGS. 6b and 6 c).

The results show that: peak values at 367.4 and 373.4eV, spin energy separation of 6.0eV, indicate the presence of metallic Ag in IM-POP-Ag ⁰ Successful assembly of AgNPs on an N-rich porous scaffold was revealed. The peak at 397.8eV demonstrated the presence of Ag-N, demonstrating that AgNPs were immobilized on the IM-POP backbone by the N element as a binding site.

All the above characterizations indicate that AgNPs were successfully loaded into IM-POP.

Experimental example 2 in vitro bacterial growth inhibition test

Single colonies of E.coli (ATCC 8739) and Staphylococcus aureus (ATCC 25922) as gram negative and gram positive bacterial models, respectively, were transferred to 3mL of Luria-Bertani (LB) medium (containing tryptone 10g/L, yeast extract 5g/L, naCl g/L, PH =7.4), respectively, and the medium was shaken at 37℃in an incubatorGrown overnight at 230 rpm. Fresh strains were diluted in LB medium with phosphate buffer (PBS, ph=7.4) to obtain the desired concentration. Inoculated into 900. Mu.L AgNPs (200, 100, 50. Mu.g mL) ^-1 ) Or IM-POP-Ag (50, 25, 12.5. Mu.g mL) ^-1 ) Bacteria in LB medium treated differently (100. Mu.L 10) ⁵ CFU mL ^-1 ) The cells were cultured in a shaking incubator at 37℃for 12 hours. 70 mu L of the bacterial liquid treated by each sample is uniformly coated on an LB plate. Incubate in an incubator inverted at 37℃for 16 hours. The number of colonies grown on the plates was counted. (FIG. 7).

FIG. 7 a) is a photograph of bacterial colonies formed by the treatment of E.coli with IM-POP-Ag prepared in example 1 and AgNPs prepared in comparative example 1, wherein the first row is treated with AgNPs of 0, 12.5, 25, 50. Mu.g/mL from left to right and the second row is treated with IM-POP-Ag of 0, 50, 100, 200. Mu.g/mL from left to right, respectively.

FIG. 7 b) shows bacterial viability of AgNPs prepared in comparative example 1 at various concentrations (0, 12.5, 25, 50. Mu.g/mL) after E.coli treatment.

FIG. 7 c) shows bacterial viability of IM-POP-Ag prepared in example 1 after E.coli treatment at various concentrations (0, 50, 100, 200. Mu.g/mL).

FIG. 7 d) is a photograph of bacterial colonies formed by the treatment of Staphylococcus aureus with IM-POP-Ag prepared in example 1 and with AgNPs prepared in comparative example 1, wherein the first row is treated with AgNPs of 0, 12.5, 25, 50. Mu.g/mL from left to right, and the second row is treated with IM-POP-Ag of 0, 50, 100, 200. Mu.g/mL from left to right, respectively.

FIG. 7 e) shows bacterial viability of AgNPs prepared in comparative example 1 after treatment with Staphylococcus aureus at various concentrations (0, 12.5, 25, 50. Mu.g/mL).

FIG. 7 f) shows bacterial viability of IM-POP-Ag prepared in example 1 after treatment with Staphylococcus aureus at various concentrations (0, 50, 100, 200. Mu.g/mL).

The results show that: the antibacterial properties of the synthesized IM-POP-Ag prepared in example 1 and AgNPs prepared in comparative example 1 were evaluated by plate counting.

FIGS. 7a-c show the antibacterial results on E.coli plates. Control set at 0 μg mL ^-1 Concentration, it can clearly be seen that with increasing concentration the antibacterial effect of AgNPs increases slightly. However, due to the severe aggregation of pure AgNPs during use, even at 50. Mu.g mL ^-1 At a concentration of (2) bacteria still showed a survival rate of 12.19%, which is far from satisfactory. Under the condition of the same silver concentration, the antibacterial effect of the IM-POP-Ag group is far better than that of the AgNPs group. When the concentration of IM-POP-Ag was 200. Mu.g/mL (corresponding to 50. Mu.g/mL AgNPs), the E.coli survival rate was only 3.44%.

The antibacterial results of the prepared samples against Staphylococcus aureus are shown in FIGS. 7d-f. Consistent with the results for E.coli, the antibacterial effect of AgNPs on Staphylococcus aureus increased slightly with increasing concentration. The bacterial viability was 20.20% at a concentration of AgNPs of 50. Mu.g/mL. And the survival rate of the IM-POP-Ag staphylococcus aureus with 200 mug/mL is 9.62 percent under the same silver-containing concentration.

These conclusions confirm that AgNPs can effectively enhance the antibacterial effect against E.coli and Staphylococcus aureus after immobilization on IM-POP.

Experimental example 3 antibacterial zone test

Bacterial suspensions of Staphylococcus aureus and Escherichia coli (100. Mu.L 10 ⁶ CFU mL ^-1 ) Uniformly coating on the surface of an agar plate. Then, a sterile filter paper having a diameter of 6mm was placed in the center of each culture plate. Then, IM-POP-Ag prepared in example 1 (8. Mu.L, 200. Mu.g mL) was completely dissolved in PBS ^-1 Equivalent to 50. Mu.g mL ^-1 AgNPs) and bare AgNPs prepared in comparative example 1 (8. Mu.L, 50. Mu.g mL ^-1 ) Added to the corresponding filter paper. PBS (8. Mu.L) and IM-POP (8. Mu.L, 50. Mu.g mL) prepared in step (1) of example 1 ^-1 ) Used as a negative control group. All plates were placed in an incubator at 37℃for 24h. The average diameter around the zone of inhibition measured with a ruler represents the bacteriostatic activity against staphylococcus aureus and escherichia coli. PBS and IM-POP prepared in step (1) of example 1 were used as negative control groups. All plates were placed in an incubator at 37℃for 24h. Potential activity against staphylococcus aureus and escherichia coli was indicated by zone diameter (fig. 8).

FIG. 8 a) is a photograph of a zone of inhibition formed by E.coli treated with PBS, IM-POP prepared in step (1) of example 1, IM-POP-Ag prepared in example 1, and AgNPs prepared in comparative example 1.

FIG. 8 b) is a photograph of a zone of inhibition formed by treatment of Staphylococcus aureus with PBS, IM-POP prepared in step (1) of example 1, IM-POP-Ag prepared in example 1, and AgNPs prepared in comparative example 1.

FIG. 8 c) is a histogram of the diameter of the zone of inhibition of E.coli treated with PBS, IM-POP prepared in step (1) of example 1, IM-POP-Ag prepared in example 1 and AgNPs prepared in comparative example 1.

FIG. 8 d) is a histogram of the diameter of the inhibition zone of PBS, IM-POP prepared in step (1) of example 1, IM-POP-Ag prepared in example 1, and AgNPs treated Staphylococcus aureus prepared in comparative example 1.

The results show that: the in vitro antibacterial activity of IM-POP-Ag, IM-POP and AgNPs against Staphylococcus aureus and Escherichia coli was further determined by a paper sheet antibacterial method, the PBS group provided a negative control, and the size of ZOI (Zone of inhibition ) reflects the ability of the sample to grow bacteria, or the sensitivity of the bacteria to the reagents, as shown in FIGS. 8a and 8b, respectively. The ZOI of the IM-POP-Ag group was significantly better than the PBS, IM-POP and AgNPs groups.

FIGS. 8c and 8d show ZOI diameters of PBS, IM-POP, agNPs, and IM-POP-Ag on E.coli and Staphylococcus aureus, respectively, after 24h incubation. After 24h with E.coli, the ZOI of IM-POP-Ag was 12.37.+ -. 0.41mm, which is much larger than that of IM-POP group (0.3.+ -. 0.28 mm) and AgNPs group (7.39.+ -. 0.37 mm) (FIG. 6 c). The same phenomenon also occurs in Staphylococcus aureus, where the ZOI (11.46.+ -. 0.21 mm) of IM-POP-Ag is significantly stronger than the IM-POP (0.3.+ -. 0.28 mm) and AgNPs (6.82.+ -. 0.1 mm) groups after 24h of Staphylococcus aureus treatment. The IM-POP-Ag is sensitive to bacteria, and has stronger antibacterial activity than IM-POP and AgNPs. It can be seen that wrapping AgNPs in IM-POP can effectively improve antibacterial ability, which is consistent with the conclusion of bacterial plate counting.

Experimental example 4 bacterial live/dead staining

The effects of Propidium Iodide (PI) and the green fluorescent nucleic acid dye SYTO-9 are used to distinguish live bacteria from dead bacteria. Live/dead bacterial cell staining was performed using the mixed dyes iodine PI and SYTO-9. SYTO-9 is a green dye that can mark bacterial cells as green, since all bacterial cell membranes (intact and damaged) are penetrated and both live and dead bacteria can be stained simultaneously. PI is a red dye that can only penetrate the damaged cell membrane to mark cells as red and stain only dead bacteria with damaged cell membranes. If the bacteria survive more, the more bacteria are stained green after staining by the live/dead dye. If fewer bacteria survive, the more bacteria stain red after staining with live/dead dye.

This experiment was conducted with 100. Mu.L of E.coli or Staphylococcus aureus treated with PBS, IM-POP (150. Mu.g/mL), agNPs (50. Mu.g/mL), IM-POP-Ag (200. Mu.g/mL) (10 ⁸ CFU mL ^-1 ) And 1. Mu.L PI (1.0X10) ^-3 Mu M) and 2 mu LSYTO-9 (1.5X10) ^-3 μM) was treated at 37℃in the absence of light for 15min for live/dead staining. After staining, the samples were centrifuged with PBS to remove excess SYTO-9 and PI. The stained staphylococcus aureus and large intestine were captured using confocal fluorescence microscopy (fig. 9).

The results show that: the IM-POP group is similar to the PBS control group, almost no red fluorescence is seen, and the effect of killing staphylococcus aureus and large intestine by the IM-POP group is not great. And a large amount of red fluorescence appears in AgNPs and IM-POP-Ag, which shows that AgNPs and IM-POP-Ag can effectively kill bacteria. IM-POP-Ag has little green fluorescence, while AgNPs have little green fluorescence, which indicates that the sterilization effect of IM-POP-Ag is better than AgNPs.

Experimental example 5 Transmission Electron microscope scanning of bacteria

Transmission Electron Microscopy (TEM) has made a deeper observation of the internal structural changes of bacteria, and in order to understand the antibacterial mechanism of IM-POP-Ag, we acquired transmission electron microscopy images of bacteria. Bacteria were collected after centrifugation (5000 rpm,3 min). Bacteria were fixed with 2.5% glutaraldehyde solution (4 ℃,24 h), washed with PBS, embedded with agar, blocked, osmium acid fixed, dehydrated for 10min at room temperature with gradient ethanol solution (30%, 50%, 70%, 90%, 95%, 100%), dealcoholized with acetone at room temperature for 3h, and penetrated by an embedding medium gradient, counterstained, sectioned and placed on a nickel screen for TEM observation (fig. 10).

FIGS. 10 a-d) are sequentially PBS, IM-POP, agNPs, and IM-POP-Ag to obtain Staphylococcus aureus;

FIGS. 10 e-h) E.coli were obtained after treatment with PBS, IM-POP, agNPs and IM-POP-Ag in this order.

The results show that: IM-POP was run at 150 μg mL ^-1 Is similar to the result of PBS (control) group after 12h of concentration treatment of staphylococcus aureus, and well retains the smooth surface of staphylococcus aureus. In contrast, with naked AgNPs (50. Mu.g mL ^-1 ) After 12h incubation, the cell surface of staphylococcus aureus became rough. With IM-POP-Ag (200. Mu.g mL) ^-1 ) After 12h incubation, staphylococcus aureus lost the integrity of the cell membrane. At the same time, many blisters form on the bacterial surface, which can be attributed to the penetration of positively charged polymers into negatively charged cell membranes by electrostatic interactions. Similarly, E.coli showed a smooth surface after treatment with PBS (control) and IM-POP. While the morphology of the IM-POP-Ag and AgNPs treated E.coli changed significantly, and the cell membrane was incomplete and collapsed. Thus, the synergistic effect of silver nanoparticles and positively charged imidazolyl polymer backbones together damage bacterial cell membranes, not only resulting in leakage of intracellular components from the cytoplasm, but also disrupting bacterial function, ultimately leading to bacterial death.

Experimental example 6 hemolysis test

The hemolysis test is one of the important indicators for evaluating the biocompatibility of materials. If the material is less compatible with erythrocytes, the structure of the erythrocytes is destroyed, causing hemolysis. According to international organization for standardization (ISO) regulations, a hemolysis ratio exceeding 5% may be regarded as hemolysis. Thus, a hemolysis test was performed to examine whether IM-POP, agNPs and IM-POP-Ag prepared in step (1) of example 1 have a damaging effect on erythrocytes. Fresh blood was obtained from KM mice (derived from Swiss mice). Red Blood Cells (RBCs) were collected by centrifugation at 1500rpm for 20min and then washed 3 times with PBS. RBC (4% w/w) was then combined with different concentrations (12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL) of IM-POP, agNPs and IM-POP-Ag at 1: the 9v/v ratio was incubated at 37℃for 3h and then centrifuged at 12000rpm for 20min. Then, it was measured at 540nm by ultraviolet-visible spectrometry. Distilled water was used as positive control and PBS as negative control. Hemolysis was calculated using the formula.

Hemolysis ratio (%) = (As-An/(Ap-An) ×100%

Where "As" is the absorbance generated by the addition of IM-POP, agNPs and IM-POP-Ag to the red blood cell suspension. "An" represents the absorbance generated by the addition of PBS to the red blood cell suspension (negative control). "Ap" refers to the absorbance generated by adding distilled water to a red blood cell suspension (positive control) (fig. 11).

FIG. 11 a) is a histogram and picture of the hemolysis rate of PBS, water, IM-POP (12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL) prepared in step (1) of example 1.

FIG. 11 b) is a histogram of the hemolysis rate of PBS, water, agNPs prepared in comparative example 1 (12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL).

FIG. 11 c) is a histogram of the hemolysis ratio of PBS, water, and IM-POP-Ag (12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL) prepared in example 1.

The results show that: with the increase of the concentration, the hemolysis rate of the IM-POP and the IM-POP-Ag is slightly increased, and the total level in the concentration range of 12.5-200 mug/mL is kept below 4%, which shows that the IM-POP-Ag and the IM-POP have good biocompatibility. However, as the concentration increases, the rate of hemolysis of the bare AgNPs group gradually increases. Even at a low concentration of 50. Mu.g/mL, the hemolysis rate of the naked AgNPs group was 9.35%. When the concentration was increased to 200. Mu.g/mL, the hemolysis rate reached 23.14%, indicating that naked AgNPs were toxic to erythrocytes. And is mainly due to direct contact of bare AgNPs in solution with the cell membrane of RBCs, leading to hemolysis. IM-POP-Ag can effectively reduce direct contact of anchored Ag and erythrocytes, thereby reducing the hemolysis rate.

Experimental example 7 cytotoxicity test

Cytotoxicity is yet another important indicator for evaluating the biocompatibility of materials. We selected mouse fibroblasts (L929 cells from the american ATCC cell bank) to test cytotoxicity of IM-POP, agNPs and IM-POP-Ag. MTT Activity assay cytotoxicity of the IM-POP material prepared in step (1) of comparative example 1, agNPs prepared in comparative example 1 and IM-POP-Ag prepared in example 1 on L929 cells in bovine blood containing 10% (v/v)Culture medium of 1% penicillin/streptomycin at 37deg.C in 5% CO ₂ Culturing in atmosphere. Cells (8X 10 per well) ³ ) Inoculated in 96-well plates, incubated for 24h, after removal of the medium, fresh medium containing different concentrations (6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL) of IM-POP, agNPs, IM-POP-Ag was added. After incubation for 20h, 10. Mu.L of MTT solution (5 mg/mL) was added, the cells were incubated for another 4h, and then the medium in each well was replaced with 150. Mu.L of dimethyl sulfoxide (DMSO). The absorbance at 490nm wavelength was determined by a Bio-TekELx800 microplate reader (BioTek, US). Cell viability (%) was calculated. In the in vitro biocompatibility study, cells were incubated with different concentrations of IM-POP, agNPs, IM-POP-Ag for 24h, and cell viability was calculated by the standard MTT method.

FIG. 11 d) is a histogram of cell viability of PBS, water, IM-POP prepared from step (1) of example 1 (6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL).

FIG. 11 e) is a histogram of cell viability of PBS, water, agNPs prepared in comparative example 1 (6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL).

FIG. 11 f) is a histogram of cell viability of PBS, water, IM-POP-Ag prepared in example 1 (6.25. Mu.g/mL, 12.5. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL).

The results show that: with the increase of the concentration of the IM-POP and the IM-POP-Ag, the cell survival rate is basically unchanged and can be maintained above 85 percent. While it can be seen from fig. 11e that the cell viability is largely related to the concentration of the AgNPs solution, the concentration of the AgNPs solution decreases significantly as the concentration of AgNPs increases. At an Ag concentration of 50. Mu.g mL ^-1 When naked AgNPs were the lowest in cytotoxicity, the cell viability was 73.86% (93.95% for IM-POP-Ag). When AgNPs concentration is 200 mug mL ^-1 At this time, the temperature was reduced to 63.93% (FIG. 11 e). Such results reveal that encapsulation of AgNPs into IM-POPs can significantly improve biocompatibility, enabling its use in vivo.

Experimental example 8 in vivo wound healing test

To further demonstrate the usefulness of this antimicrobial strategy, we studied to promote golden yellowCircular MC of Staphylococcus-infected mice circular wound healing ability of mice circular wound was treated with IM-POP, agNPs, IM-POP-Ag and PBS (control). Male KM mice (derived from Switzerland mice) of 5 weeks of age (n=5, 25-30g per group) were used and divided into PBS group, IM-POP group, agNPs group and IM-POP-Ag group. The back hair of each mouse was shaved preoperatively, resulting in a circular wound with d=6 mm. With Staphylococcus aureus (1X 10) ⁶ CFU mL ^-1 ) Skin wounds were pretreated for 24h. The following solution IM-POP-Ag (200. Mu.g mL) was used ^-1 )、AgNPs(50μg mL ^-1 )、IM-POP(100μg mL ^-1 ) And PBS treatment of the infected wound area. Subsequently, the wound surface was photographed on days 0, 4, 7 and 14, the progress of wound healing was recorded with photographs at different time intervals (fig. 12 a), the wound area was counted (fig. 12 b) and the mice were weighed (fig. 12 c).

To further evaluate the skin regeneration capacity of IM-POP-Ag, agNPs and IM-POP in mice, histological analysis of wound healing tissue of staphylococcus aureus infection was evaluated at 14 days of surgery according to H & E and Masson's trichromatic staining. Image analysis program (imag.j) was used. Mice were humane sacrificed on days 4, 7 and 14. Wound skin, heart, liver, spleen, lung and kidney were excised, fixed with 10% formalin, then assessed by H & E staining and Masson trichrome staining, excised and fixed in 10% formalin (fig. 12 d).

Toxicity studies were performed on IM-POP-Ag, agNPs and IM-POP, and the main organs (heart, liver, spleen, kidney and lung) eliminated by treatment with IM-POP-Ag, IM-POP and AgNPs were H & E stained on day 14 to determine the damage (FIG. 14).

The wound of KM mice infected with Staphylococcus aureus for 24 hours was sampled with a sterile cotton swab, and the sampled bacteria were cultured in a solid medium to develop a large number of bacterial colonies. Indicating that staphylococcus aureus has proliferated on the wound surface. On day 10 after treatment, wound bacteria were harvested and cultured again (fig. 13).

The blood of the fundus artery of the mouse is sampled by 1-2 mL. 200. Mu.L of each blood sample was taken for routine blood analysis. Hematology includes a) Red Blood Cells (RBC), b) White Blood Cells (WBC), c) average red blood cell volume (MCV), d) average red blood cell hemoglobin concentration (MCHC), e) Hemoglobin (HGB), f) Hematocrit (HCT), j) average red blood cell hemoglobin amount (MCH), h) Platelets (PLT) (fig. 15).

Figure 12 in vivo antibacterial efficacy of im-POP, agNPs, IM-POP-Ag.

Fig. 12 a) representative wound photographs of KM mice back staphylococcus aureus infected wounds at time points IM-POP, agNPs, IM-POP-Ag and corresponding control PBS.

Fig. 12 b) corresponding wound shrinkage versus time curve.

Figure 12 c) change in body weight of mice during dosing.

Fig. 12 d) trichromatic images of wound tissue H & E and Masson's on day 14 of 4 groups. The scale bar is 200 μm.

Figure 13 models bacterial numbers at wounds one day and 10 days of treatment.

Fig. 15 blood is conventional after 14 days of treatment for each group of mice.

Fig. 15 a) Red Blood Cells (RBC), fig. 15 b) White Blood Cells (WBC), fig. 15 c) mean red blood cell volume (MCV), fig. 15 d) mean red blood cell hemoglobin concentration (MCHC), fig. 15 e) Hemoglobin (HGB), fig. 15 f) Hematocrit (HCT), fig. 15 j) mean red blood cell hemoglobin amount (MCH), fig. 15 h) Platelets (PLT).

The results show that: all wounds showed severe bacterial infection with untreated wounds (PBS group) slightly edematous, shrinking 9.87% on day 4. Whereas IM-POP, agNPs and IM-POP-Ag treated wounds showed significant healing evidence, approximately 14.98%, 18.85% and 20.87% wound shrinkage, respectively. IM-POP-Ag treated wounds had begun to scab by day 4. On day 7, there was still edema in the PBS group with a large amount of yellow secretions, whereas the wounds treated by AgNPs and IM-POP-Ag had crusted in comparison to the IM-POP and PBS covered wounds, which contracted 27.81% (PBS), 28.31% (IM-POP), 33.45% (AgNPs) and 41.24% (IM-POP-Ag), respectively. Wound shrinkage on day 14 was 66.19% and 69.19% for PBS and IM-POP groups, respectively. However, IM-POP-Ag treated wounds almost completely healed on day 14. The wound shrinkage after 14d treatment with AgNPs was only 86.53%. It was demonstrated that IM-POP-Ag can reduce bacterial invasion at the wound and accelerate the wound healing process (fig. 12a and 12 b).

Meanwhile, during the 14-day dosing period, animals had no significant abnormalities in behavior and no significant changes in body weight (fig. 12 c).

Wound healing was estimated for different materials after 14 days using hematoxylin and eosin (H & E) trichromatic staining of Masson's. Histological analysis of staphylococcus aureus infected wounds showed that the AgNPs and IM-POP groups had new capillary and skin growth, whereas the control and IM-POP groups did not find epidermal structures. The IM-POP-Ag group exhibited fewer inflammatory cells and more newly generated glands, capillaries and collagen deposition than the AgNPs group. Low levels of inflammation can accelerate wound healing, especially at early stages. Thus, IM-POP-Ag can accelerate wound remodeling thanks to the integrated properties of low biotoxicity and high antimicrobial properties (fig. 12 d).

On day 10 after treatment, the wound surface was again cultured with bacteria. As a result, it was found that a large amount of golden bacteria was present in the wounds of the control group and the IM-POP group, while the bacteria in AgNPs and IM-POP-Ag were significantly reduced. Interestingly, there was little bacterial growth in the wound site by IM-POP-Ag, and the results indicate that AgNPs and IM-POP-Ag did significantly reduce bacteria in the wound site, continuing to kill bacteria with high efficiency (fig. 13).

Toxicological studies demonstrated that IM-POP-Ag, IM-POP and AgNPs had no apparent inflammatory or morphological lesions to the major viscera (heart, liver, spleen, kidney and lung) (fig. 14).

There was no significant difference in all blood routine analyses between groups (fig. 15).

Claims

1. A method for preparing a nano-silver loaded porous organic polymer, which is characterized by comprising the following steps:

s1, preparing an imidazolyl organic porous polymer: mixing imidazole-2-formaldehyde and melamine in DMSO, heating for reaction, collecting a solid product after the reaction is finished, removing unreacted components, purifying and drying to obtain an imidazolyl organic porous polymer; the mass ratio of the imidazole-2-formaldehyde to the melamine is 3:1-3.5:1;

s2, preparing a nano silver loaded porous organic polymer:

s2-1, mixing and grinding the imidazolyl organic porous polymer obtained in the S1 with a small amount of methanol to a nano level, and adding methanol after finishing grinding to ensure that the concentration of the imidazolyl organic porous polymer in the methanol is 1-2 mg/mL, and fully dispersing to obtain a reaction system;

s2-2, agNO is added into the reaction system obtained in the S2-1 ₃ Solution, agNO ₃ The mass ratio of the imidazole-based organic porous polymer to the imidazole-based organic porous polymer is 1.2:1-1.4:1; removal of unreacted AgNO from the reaction product ₃ And free nano silver to obtain a silver ion-loaded porous organic polymer;

s2-3, reducing silver ions in the silver ion loaded porous organic polymer obtained in the step S2-2 into nano silver, sealing and stirring, cleaning with distilled water, centrifuging, and collecting precipitate to obtain the nano silver loaded porous organic polymer;

the mixed heating reaction condition is that stirring is carried out for 3-4 days at 180 ℃ under the protection of inert gas;

and S2-3, adding the silver ion-loaded porous organic polymer obtained in the step S2-2 into a mixed solvent of methanol and water, adding a sodium borohydride methanol solution, continuously stirring, washing to reduce silver ions in the silver ion-loaded porous organic polymer into nano silver, wherein the mixed solvent is a mixture of methanol and water in a volume ratio of 3:2, the concentration of the silver ion-loaded porous organic polymer in the mixed solvent is 3-4 mg/mL, the concentration of the sodium borohydride methanol solution is 15-16 mg/mL, the concentration of the silver ion-loaded porous organic polymer in the sodium borohydride methanol solution is 6-8 mg/mL, the stirring time is 1-2 days, and the washing is water and methanol washing.

2. The method for preparing the nano-silver loaded porous organic polymer according to claim 1, wherein the mass ratio of the imidazole-2-formaldehyde to the melamine is 3:1; s1, wherein the concentration of imidazole-2-formaldehyde in DMSO is 0.068g/mL, and the concentration of melamine in DMSO is 0.03g/mL.

3. The method for preparing the nano-silver loaded porous organic polymer according to claim 1, wherein the step of removing unreacted components in the step S1 is to add the collected solid product into acetone, and stir the mixture at 30-60 ℃ for 3-9 h.

4. The method for preparing the nano-silver loaded porous organic polymer according to claim 1, wherein the removing of the unreacted components in S1 is to add the collected solid product into acetone and stir at 45 ℃ for 6 hours.

5. The method for preparing the nano-silver loaded porous organic polymer according to claim 1, wherein the purification of S1 is performed by stirring the solid product with tetrahydrofuran at 55 ℃ for 3 hours, followed by stirring with dichloromethane at 60 ℃ for 6-9 hours.

6. The method for preparing the nano-silver loaded porous organic polymer according to claim 1, wherein the concentration of the S2-1 imidazolyl porous organic polymer in methanol is 1.6mg/mL.

7. The method for preparing nano-silver loaded porous organic polymer according to claim 1, wherein S2-2 is AgNO ₃ The concentration of the solution was 8.75 mg/mL.

8. The method for preparing nano-silver loaded porous organic polymer according to claim 1, wherein S2-2 removes unreacted AgNO ₃ And the specific operations of the free nano silver are as follows: and removing the solvent from the reaction product, washing with distilled water, centrifuging, and collecting the precipitate to obtain the silver ion-loaded porous organic polymer.

9. The method for preparing a nano-silver loaded porous organic polymer according to claim 8, wherein the centrifugation condition is 9000rpm for 5min.

10. The nano-silver loaded porous organic polymer prepared by the preparation method of the nano-silver loaded porous organic polymer according to claim 1.

11. The use of the nano-silver loaded porous organic polymer according to claim 10 for preparing antibacterial drugs.

12. The use according to claim 11, wherein the antibacterial agent is an antibacterial agent capable of accelerating wound healing.

13. The use according to claim 11, wherein the antibacterial agent is an anti-gram-negative and/or gram-positive bacterial agent.

14. The use according to claim 13, wherein the antibacterial agent is an anti-escherichia coli agent and/or a staphylococcus aureus agent.