CN114984246B - Preparation method and application of mesoporous polydopamine NO nanoparticles with diagnosis and treatment integration - Google Patents

Abstract

The invention provides a preparation method of mesoporous polydopamine NO nano particles, which comprises the steps of connecting maltotriose to a PEG chain through a click reaction to synthesize PEG-maltotriose; the method comprises the following steps of (1) fixing maltotriose on the surface of mesoporous polydopamine through reaction with the mesoporous polydopamine to synthesize MPDA-PEG-Mal; and loading the nitric oxide donor into the mesoporous structure of the mesoporous polydopamine through a conjugate effect to obtain the mesoporous polydopamine NO nano-particle. The invention combines the bacterial targeted maltotriose molecule and the mesoporous dopamine which is loaded with NO and has photothermal effect, realizes the diagnosis and treatment of in vivo infected bacteria, can accurately target the infected part of the bacteria, introduces photothermal effect to carry out synergistic sterilization under the condition that the NO sterilization effect is not particularly good, improves the multiple drug resistance of the bacteria by dissipating a biomembrane on one hand, enhances the NO effect by photothermal effect on the other hand, can carry out continuous sterilization, has higher sterilization efficiency, and can be widely applied to clinic.

Description

A preparation method and application of mesoporous polydopamine NO nanoparticles with integrated diagnosis and treatment

technical field

The invention relates to the field of biomedical materials, in particular to a preparation method and application of mesoporous polydopamine NO nanoparticles with integrated diagnosis and treatment.

Background technique

The problem of bacterial infection has attracted more and more attention worldwide. In recent decades, due to the overuse of antibiotics, the number of drug-resistant bacteria has increased sharply. Methicillin-resistant Staphylococcus aureus and vancomycin-resistant bacteria have appeared successively. Enterococcus faecalis. The emergence of drug-resistant bacteria has seriously threatened human health. If the infection site can be detected in the early stage of bacterial infection and effective treatment can be taken, the complications and mortality caused by bacteria can be reduced. Bacterial infections on the surface of the body are easy to observe, but bacterial infections in the skin or deep in the body are difficult to diagnose. Common clinical diagnostic methods include computed tomography (CT) and emission computed tomography, such as single photon emission computed tomography ( SPECT) and positron emission tomography (PET), and magnetic resonance imaging (MRI). However, these diagnostic methods cannot determine whether the infection site is bacterial infection or non-infection (endotoxin or tumor). Therefore, it is urgent to develop a contrast agent for high-precision and specific imaging of bacteria.

Maltodextrin mainly includes maltotriose, maltotetraose, maltohexaose, and maltoheptaose, and has attracted widespread attention because of its targeting properties to many kinds of bacteria. Maltodextrin is the main source of bacterial glucose, which can enter the interior of bacteria through maltose transporter. By combining with fluorescent molecules or PET tracers, it can specifically target and image the bacterial infection site, so as to realize the detection of the bacterial infection site. position. Most Gram-negative bacteria and Gram-negative bacteria have a strong internalization effect on maltodextrin, and mammalian cells will not have an uptake effect on maltodextrin, which can greatly avoid human endogenous The interference of sex cells makes it more targeted. Maltodextrin can be combined with nano-mesoporous polydopamine, hollow silica and other carriers for the diagnosis and treatment of bacterial infections in vivo. Pang et al. targeted MRSA by photoacoustic therapy by linking maltotriose into liposomes, and then treated MRSA infection with the loaded drug. (ACS Nano 13(2)(2019) 2427-2438.) Xu et al. loaded maltohexaose into nano-hollow silica and combined with gadolinium (Gd) for specific imaging of bacterial infection sites (Small, 2021, 17 (44):2103627.). These works have proved that maltotriose has a very good targeting effect, can exist stably in the body, and has high targeting to bacteria, and has good biocompatibility and will not cause immunity reaction, but at present, a large number of researchers use maltodextrin as a specific imaging molecule or simply play a role in targeting bacteria, and have not combined the emerging nanotechnology. Nanoparticles have smaller The size can be well enriched in bacteria or cells, and maltotriose can also endow nanomaterials with targeted functions. However, how to combine maltotriose targeting molecules with nanoparticles and endow them with new functions is an open problem. At the same time, the site of bacterial infection in the body is difficult to diagnose and treat by conventional means. Maltotriose can be used to target bacterial infection in the body well, and then maltotriose is grafted onto nanoparticles with therapeutic functions through chemical reactions to realize the integration of diagnosis and treatment. Effectively removes bacteria from infected sites in the body.

In recent years, various nanoparticles with antibacterial ability emerge in an endless stream, but conventional antibacterial means will breed drug resistance of bacteria, leading to the emergence of drug-resistant bacteria. NO gas molecule has become a "star" in the antibacterial field. It can effectively dissipate the bacterial biofilm, weaken the multi-drug resistance of bacteria, and also have a certain bactericidal effect. However, the bactericidal ability of pure NO is limited, which greatly slows down the bactericidal effect and healing speed, resulting in a slowdown of the healing rate. The inventor's research group has long been committed to the development of NO carriers, and the use of chitosan to graft dendritic macromolecule PAMAM to achieve NO High-efficiency loading, while high-concentration NO has certain toxicity while sterilizing. (Chemical Engineering Journal 347(2018):923-931.) Therefore, it is necessary to enhance the therapeutic effect and controllable release of NO by combining with other means, such as photothermal therapy and photodynamic therapy, which can achieve a rapid bactericidal effect. The inventor's research group has also developed a photothermal controlled-release hydrogen antibacterial nano-platform, which uses photothermal release of hydrogen for antibacterial, which has good antibacterial effect and low toxicity. (Advanced Functional Materials 29.50(2019): 1905697.) The combination of photothermal and NO has a good bactericidal effect on bacteria infected on the surface of the body, but due to the lack of targeting function, it cannot be applied to deep bacterial infections in the body. its application in antibacterial aspects.

Contents of the invention

In view of the defects in the above-mentioned prior art, the purpose of the present invention is to propose a mesoporous polydopamine NO nanoparticle and its preparation method and application. Maltotriose is modified on the PEG chain by a click reaction, and then the PEG chain is modified on the mesoporous Then, the nitric oxide donor BNN6 was loaded on MPDA, and the mesoporous polydopamine NO nanoparticles with integrated diagnosis and treatment were obtained, which realized the localization and bactericidal effect on the bacterial infection site.

The purpose of the present invention will be achieved through the following technical solutions:

One of object of the present invention is to provide a kind of preparation method of mesoporous polydopamine NO nanoparticle, comprises the following steps:

S1. Linking maltotriose to the PEG chain through a click reaction to synthesize PEG-maltotriose;

S2. Immobilizing maltotriose on the surface of mesoporous polydopamine by reacting with mesoporous polydopamine to synthesize MPDA-PEG-Mal;

S3. Loading nitric oxide donors into the mesoporous structure of mesoporous polydopamine through conjugation effect to obtain mesoporous polydopamine NO nanoparticles.

Further, S1 specifically includes the following steps:

S1.1 Dissolve maltotriose in anhydrous pyridine, add acetic anhydride, and react at room temperature for 24h-48h to obtain fully acetylated maltotriose;

S1.2 Dissolve the fully acetylated maltotriose obtained in S1.1 in anhydrous dichloromethane (DCM), add 2-hydroxybromoethane, stir under low temperature nitrogen for 30min-50min, add trifluoride Boroethyl ether continued to react for 30min-50min to obtain brominated maltotriose;

S1.3 Dissolve the brominated maltotriose obtained in S1.2 in anhydrous DMF, add sodium azide, heat to 60°C-80°C under nitrogen, and react for 24h-48h to obtain azide maltotriose sugar;

S1.4 Dissolve the azide maltotriose obtained in S1.3 in anhydrous methanol, add sodium methoxide powder, stir at room temperature for 6h-8h, adjust the pH of the solution to neutral with a strong acid type cation exchange resin, filtering and removing the strong acid cation exchange resin to obtain deprotected azide maltotriose;

S1.5 Dissolve the deprotected azide maltotriose obtained in S1.4 in water, add N ₃ -PEG-NH ₂ powder, copper sulfate solution and sodium ascorbate solution, react at high temperature for 3-4 days, and then dialyze 2-3 days to get PEG-maltotriose.

Further, S2 specifically includes the following steps:

Dissolve the PEG-maltotriose and MPDA obtained in S1 in deionized water, adjust the pH of the solution to 8-9 with Tris hydrochloride, and react in the dark for 24h-48h to obtain MPDA-PEG-Mal.

Further, S3 specifically includes the following steps:

Dissolve the MPDA-PEG-Mal obtained in S2 in deionized water, and dissolve BNN6 in absolute ethanol. After the two are mixed, they are reacted in the dark for 24h-48h. After the reaction, the mesoporous polydopamine NO nanoparticles BNN6@MPDA are obtained. -PEG-Mal.

Further, the room temperature is 25°C-30°C; the low temperature is -20°C-0°C; and the high temperature is 70°C-90°C.

Further, in step S1.1, the molar ratio of maltotriose to acetic anhydride is 1:7-10; the amount of anhydrous pyridine is calculated by adding 100 mg-200 mg of maltotriose per 10 mL of anhydrous pyridine;

In step S1.2, the molar ratio of fully acetylated maltotriose to 2-hydroxybromoethane is 1:1.5-3; the amount of anhydrous dichloromethane is added per 10 mL of anhydrous dichloromethane 100mg-200mg full acetylated maltotriose;

In step S1.3, the molar ratio of the brominated maltotriose to sodium azide is 1:2-4; the dosage of the anhydrous DMF is 50mg-100mg sugar meter;

In step S1.4, the molar ratio of the azide maltotriose to sodium methoxide is 1:0.5-3; sugar meter;

In step S1.5, the molar ratio of the azide-deprotected maltotriose, N ₃ -PEG-NH ₂ , copper sulfate, and sodium ascorbate is 1:1.5-2:1.2-1.5:1.2-1.5; The amount of deionized water is calculated by adding 100mg-150mg of azidated deprotected maltotriose per 10mL of deionized water.

Further, the mass ratio of MPDA to PEG-maltotriose is 1:2-4; the amount of deionized water is calculated by adding 10 mg-20 mg of MPDA to 10 mL of deionized water.

Further, the mass ratio of MPDA-PEG-Mal to BNN6 is 1:1-2; the amount of dehydrated ethanol is calculated by adding 10 mg-20 mg BNN6 to 10 mL of dehydrated ethanol.

Another object of the present invention is to provide a kind of mesoporous polydopamine NO nanoparticles prepared by the above method for preparing mesoporous polydopamine NO nanoparticles.

Another object of the present invention is to provide an application of mesoporous polydopamine NO nanoparticles in the preparation of medicines for diagnosing and treating bacterial infections.

The outstanding effects of the present invention are:

(1) The present invention uses maltotriose to specifically target bacteria, thereby realizing real-time monitoring of bacterial infection sites in the body, solving the problem of inability to locate deep-seated bacterial infections clinically, and simultaneously connecting the targeting molecules to the load The nano-particles of bacterial therapeutic drugs can precisely act on the site of bacterial infection, which can effectively kill bacteria and achieve the effect of treating bacterial infection.

(2) The present invention utilizes the photothermal performance of MPDA and the thermal triggering property of BNN6 to use photothermal conversion to promote NO to kill bacteria. It can also be combined with other nanocarriers to use photodynamic therapy and photoacoustic therapy, multi-method and multi-mode The treatment of bacterial infection in the body can be applied to a variety of internal infections such as osteomyelitis, muscle bacterial infection, and bacterial infection after implantation surgery.

(3) The present invention solves the problem of limited application range of photothermal and NO bactericidal nanoparticles, uses maltotriose as a targeting molecule, and widely applies this nanomaterial to the problem of deep-seated bacterial infection in the human body. It can be supplemented by photothermal means to enhance the bactericidal effect of NO, which greatly expands the application range of this type of material.

(4) The present invention combines the maltotriose molecule with bacterial targeting and mesoporous dopamine loaded with NO and has photothermal effect, endows mesoporous polydopamine with new functions, and achieves the diagnosis and treatment of bacterial infection in vivo . Maltotriose can precisely target the site of bacterial infection. When the bactericidal effect of NO is not particularly good, the photothermal effect is introduced for synergistic sterilization. On the one hand, it improves the multi-drug resistance of bacteria by dissipating biofilm, and on the other hand, the photothermal effect is enhanced. The effect of NO is achieved, so that the material can continue to sterilize, and has a high bactericidal efficiency, and can be widely used in clinical practice.

The specific implementation of the present invention will be described in further detail below in conjunction with the examples, so as to make the technical solution of the present invention easier to understand and grasp.

Description of drawings

Fig. 1 is the photothermal effect diagram of the material under different concentrations and powers in Example 3 of the present invention;

Fig. 2 is the change figure of NO with time under the irradiation of 808nm laser in Example 4 (1) of the present invention;

Fig. 3 is the change diagram of NO with time under the irradiation of 808nm laser in Example 4 (2) of the present invention;

Fig. 4 is the in vitro antibacterial effect figure of embodiment 5 of the present invention;

Fig. 5 is the micrograph of muscle infection recovery process in the embodiment of the present invention 6;

Fig. 6 is the in vivo imaging diagram of Example 7 of the present invention;

Fig. 7 is a synthetic route diagram of the mesoporous polydopamine NO nanoparticles BNN6@MPDA-PEG-Mal of the present invention.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention.

Anhydrous dichloromethane, anhydrous methanol, and absolute ethanol described in the embodiments of the present invention are all purchased from the market; deionized water is obtained by purifying water through instruments;

The preparation method of the anhydrous pyridine (Py) follows the following steps: adding calcium hydride to Py, stirring for 6h-24h, and then distilling under reduced pressure to obtain anhydrous Py. Add 5-10 grams to the meter.

The preparation method of the anhydrous N,N-dimethylformamide (DMF) follows the following steps: adding calcium hydride to DMF, stirring for 6h-24h, and then distilling under reduced pressure to obtain anhydrous DMF, the hydrogenation The amount of calcium added is 1-2 grams per 500 mL of DMF.

The nude mice are BALB/C rats (20 g), purchased from the Animal Experiment Center of Southern Medical University, Guangzhou.

The synthesis route of the synthesized mesoporous polydopamine NO nanoparticles BNN6@MPDA-PEG-Mal of the present invention is shown in Figure 7.

Example 1

Synthetic PEG-maltotriose

(1) Dissolve a certain amount of maltotriose in anhydrous pyridine, then add acetic anhydride dropwise under nitrogen atmosphere, react at room temperature for 24 hours after the addition, and detect the reaction degree by TLC. After the reaction is over, pour it into a separatory funnel, add ethyl acetate, and wash with 10% saturated sodium bicarbonate solution, deionized water, and saturated brine, respectively, until the pH of the aqueous phase is neutral, and the organic phase is washed with anhydrous Drying over sodium sulfate, spin-drying the solvent, separating the product by silica gel column chromatography, and finally obtaining peracetylated maltotriose after vacuum drying. Wherein, the molar ratio of the maltotriose and acetic anhydride is 1:7; the amount of the anhydrous pyridine is based on the addition of 100 mg of maltotriose per 10 mL of anhydrous pyridine; the number of washings is 3 times, each Each solution was 20mL.

(2) Dissolve a certain amount of fully acetylated maltotriose obtained in step (1) in anhydrous dichloromethane, then add 2-hydroxybromoethane, and stir for 30 minutes in an ice bath under nitrogen. Take a certain amount of boron trifluoride ether solution, slowly add it into the flask, continue to stir in the ice bath for 30 min, then remove the ice bath, raise the temperature to room temperature, and pour the reaction solution into ice water after 24 hours of reaction, add a certain Measure ethyl acetate, then wash the organic phase with saturated sodium bicarbonate, deionized water, and saturated brine for 3 times, dry the organic phase with anhydrous sodium sulfate, spin the solvent, and separate the product by silica gel column chromatography to obtain Brominated maltotriose. The molar ratio of the fully acetylated maltotriose to 2-hydroxybromoethane is 1:1.5; the amount of the anhydrous dichloromethane is calculated by adding 200mg of fully acetylated maltotriose per 10mL of anhydrous dichloromethane ; The amount of ethyl acetate is calculated by adding 50 mg of fully acetylated maltotriose in every 50 mL of ethyl acetate.

(3) Dissolve the brominated maltotriose obtained in step (2) in a certain amount of anhydrous DMF, add a certain amount of sodium azide, heat up to 80°C under nitrogen, stir for 24 hours, TLC detects the reaction, and the reaction ends After extraction with ethyl acetate, the organic phase was washed three times with deionized water and saturated brine, then dried with anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain azide maltotriose. The molar ratio of the brominated maltotriose and sodium azide is 1:1.5; the consumption of anhydrous DMF is based on adding 100mg of brominated maltotriose in every 10mL of anhydrous DMF; Add 50mg of brominated maltotriose to every 50mL of ethyl acetate;

(4) Dissolve a certain amount of maltotriose azidose obtained in step (3) in anhydrous methanol at room temperature of 35°C, stir and dissolve, add sodium methoxide powder, adjust the pH of the solution to be alkaline, stir for 6 hours, add 732 type polystyrene strong acid ion exchange resin, adjust the pH of the solvent to be neutral, filter the cation exchange resin with a funnel, spin the filtered solution to obtain an oily liquid, add water to dissolve, and then freeze-dry to obtain a light yellow powder. The mol ratio of described azidated maltotriose and sodium methylate is 1:3; The consumption of described anhydrous methanol is calculated by adding 200mg azidated maltotriose per 10mL anhydrous methanol;

(5) Under the condition of nitrogen protection, dissolve the azide-deprotected maltotriose obtained in step (4) in deionized water, then add N ₃ -PEG-NH ₂ , stir under nitrogen for 15 min-30 min, then Add a certain amount of CuSO4·5H2O and sodium ascorbate, raise the temperature to 70°C and react for 72 hours. After the reaction, dialyze in a dialysis bag for 3 days with deionized water, and freeze-dry to obtain the product PEG-maltotriose. The molar ratio of the azide-deprotected maltotriose to N ₃ -PEG-NH ₂ , CuSO4·5H2O, and sodium ascorbate is 1:1.5:1.2:1.2; the amount of deionized water per 10mL of deionized water Add 100 mg of azidated deprotected maltotriose meter.

Example 2

Synthesis of Mesoporous Polydopamine NO Nanoparticles BNN6@MPDA-PEG-Mal

(1) Dissolve dopamine hydrochloride and F127 in a mixed solution of equal volumes of ethanol and deionized water, ultrasonically oscillate to dissolve evenly, then add mesitylene to the flask, and ultrasonically oscillate for 5 minutes to make the solution milky white and turbid. Ammonia water was added dropwise under the condition of stirring, and the color of the reaction gradually deepened. After reacting for 2 hours, the product was collected by centrifugation, washed and purified with a mixture of ethanol and water, and the final product, mesoporous polydopamine MPDA, was suspended in deionized water. The molar ratio of the dopamine hydrochloride, F127, mesitylene and ammonia water is 1:0.2:0.5:0.1. The volume ratio of ethanol and water in the mixed solvent of ethanol and water is 1:1; the amount of ammonia water is calculated by adding 200 μL of ammonia water to every 10 mL of ethanol and water mixed solvent.

(2) Get the gained MPDA and dissolve it in deionized water, add the PEG-maltotriose synthesized in Example 1, add Tris hydrochloride to adjust the pH of the solution to be 8-9, react in a dark place for 24h, wash with deionized water for 3 Next, unreacted PEG-maltotriose was removed, and finally the obtained product MPDA grafted with PEG-maltotriose (MPDA-PEG-Mal) was suspended in deionized water. The mass ratio of MPDA to PEG-maltotriose is 1:2; the amount of deionized water is based on the addition of 10 mg MPDA to 10 mL of deionized water.

(3) MPDA-PEG-Mal was dissolved in deionized water, BNN6 was dissolved in absolute ethanol, and then the two were mixed, and stirred at room temperature for 24 hours in the dark, and after the reaction was completed, excess BNN6 was washed with deionized water, Then the product BNN6-loaded MPDA-PEG-Mal (BNN6@MPDA-PEG-Mal) was suspended in water. The mass ratio of the MPDA-PEG-Mal and BNN6 is 1:1; the consumption of the dehydrated alcohol is calculated by adding 10mg BNN6 to 10mL dehydrated alcohol.

Example 3

Photothermal Performance Evaluation Experiment

Take MPDA synthesized in Example 2, MPDA grafted with PEG-maltotriose (MPDA-PEG-Mal), and MPDA-PEG-Mal loaded with BNN6 (BNN6@MPDA-PEG-Mal) 5mg were dissolved in 5mL Take 200 μL of deionized water and add it to the 96 ^- well plate. At the same time, set the PBS group and the pure BNN6 group as the control group. Take 200 μL each and add it to the 96-well plate. Take the indication of the thermometer, irradiate for 5 minutes in total, observe the temperature change of each group, and draw the different values read into a curve to evaluate the photothermal performance of the material. For the photothermal performance of different concentrations of BNN6@MPDA-PEG-Mal, prepare solutions of 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, and 0 mg/mL respectively, and read the value every 3 s. The laser was irradiated together for 5 minutes, and the obtained temperature change was drawn as a curve, indicating the photothermal effect of different concentrations of materials. The results are shown in Figure 1. Compared with the PBS group, MPDA has a better photothermal effect, and can be raised to 60°C in 5 minutes. After modifying PEG-Mal and loading BNN6, it has less effect on the photothermal performance of the material. It is proved that the material has good photothermal performance.

Example 4

NO release ability experiment

(1) Prepare 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, and 0 mg/mL MPDA-PEG-Mal loaded with BNN6 (BNN6@MPDA-PEG-Mal), and take Place 200 μL in a 96-well plate, irradiate with a 1W/cm ² 808nm laser, absorb 50 μL of the material at 0 min, 1 min, 3 min, 5 min, 10 min, 15 min, and 20 min, and centrifuge to get the supernatant, add Griess reagent, and incubate for 15 min. Measure the absorbance at OD=540nm, and then convert the absorbance into NO release through the standard curve equation to evaluate the NO release capacity of the material. The results are shown in Figure 2. With the increase of time, the concentration of NO gradually increases, indicating that the material has a better light-controlled release effect.

(2) Prepare 1mg/mL BNN6@MPDA-PEG-Mal, take 200μL and place it in a 96-well plate, and irradiate it with 0W/cm2, 0.2W/cm2, 0.5W/cm2, 1W/cm2 lasers, respectively, at 0min , 1min, 3min, 5min, 10min, 15min, 20min, etc., take 50 μL of the solution, centrifuge to get the supernatant, incubate with Griess reagent for 15min, measure its absorbance at OD=540nm with a microplate reader, and convert it to NO release. The light-controlled NO release ability of the material was evaluated. The results are shown in Figure 3. Under the irradiation of different power lasers, the release rate of NO gradually increased. Among them, the irradiation power of 1W/cm2 released NO the fastest, and was selected as the most suitable irradiation power.

Example 5

Antibacterial test in vitro

Four kinds of materials of 1 mg/mL were prepared respectively, that is, the maltotriose-modified MPDA-PEG (MPDA-PEG-Mal) prepared in Example 2, the loaded BNN6 (BNN6@MPDA-PEG-Mal) and the simple BNN6 were added to In 1mL of methicillin-resistant Staphylococcus aureus at the same concentration and in the same logarithmic phase of growth (bacterial concentration: 1.0*10 ⁸ CFU/mL), irradiate with laser light for 10 minutes and continue to culture for 4 hours, then centrifuge to remove the material. Plate the remaining bacterial liquid and place it in an incubator for 12 hours to continue culturing, and calculate the number of bacteria on the agar plate. The results are shown in Figure 4. Compared with the NO-loaded material group (BNN6@MPDA-PEG -Mal), the antibacterial effect of N+BNN6@MPDA-PEG-Mal after laser irradiation was significantly enhanced, and there was no significant difference in other control groups, indicating that both pure photothermal and pure NO There is no better bactericidal effect, but after the two are coordinated, they have better bactericidal effect.

Example 6

In vivo antibacterial experiment

To construct a mouse leg muscle infection model, use the MPDA (NIR+BNN6@MPDA-PEG-Mal) obtained in Example 2 with photothermal and targeting capabilities to treat the rat wound for 12 days, and treat the mouse leg after 12 days Hematoxylin-eosin (H&E) staining and pathological analysis were performed on the muscle parts of the body to analyze the healing performance of the material on muscle bacterial infection; under the same conditions, the wound healing rate of rats treated with normal saline was used as a blank control. The experimental results are shown in Figure 5. From the slices, it can be clearly seen that yellow pus and tissue edema appeared in the muscles of the legs of the mice. Compared with the muscle infection of the blank control group, it was found that after 12 days of administration through tail vein injection, And the next day, 808nm laser was used to irradiate the legs, and the whole process of leg infection recovery was observed. From the slices, it can be seen that the wound has a large amount of granulation tissue tissue, the internal swelling has basically disappeared, and most of the muscle tissue has recovered. The experimental results It is proved that the NIR+BNN6@MPDA-PEG-Mal antibacterial material has efficient in vivo and in vitro antibacterial and significant effect of promoting muscle tissue healing, and is expected to become a new type of intelligent in vivo antibacterial drug.

Example 7

Bacteria-specific targeting assays

The intramuscular injection of MRSA in the left leg of the mouse represents a bacterial infection, while the intramuscular injection of bacterial endotoxin (LPS) in the right leg represents the inflammatory response caused by an aseptic infection, and the BNN6@ prepared in Example 2 is injected into the tail vein. MPDA-PEG-Mal nanoparticles were used to observe the fluorescence intensity of the left leg of the mouse at 0h, 3h, 6h, 12h, and 24h through an in vivo imaging instrument. The results are shown in Figure 6. As time goes by, the fluorescence of the bacterial infection site on the left leg becomes stronger and stronger, indicating that there is a large amount of enrichment at this site. The decreasing trend of fluorescence after 12h indicates that the first 12h has a better aggregation effect, and also indicates the specific targeting of maltotriose to bacteria.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, any person familiar with the technical field within the technical scope disclosed in the present invention, according to the technical solution of the present invention Any equivalent replacement or change of the inventive concepts thereof shall fall within the protection scope of the present invention.

Claims (6)

1. a preparation method of mesoporous polydopamine NO nanoparticles, is characterized in that comprising the following steps: S1. Linking maltotriose to the PEG chain through a click reaction to synthesize PEG-maltotriose; S2. By reacting with mesoporous polydopamine (MPDA), maltotriose is fixed on the surface of mesoporous polydopamine to synthesize MPDA-PEG-Mal; S3. Loading nitric oxide donors into the mesoporous structure of mesoporous polydopamine through conjugation effect to obtain mesoporous polydopamine NO nanoparticles; Wherein, S1 specifically includes the following steps: S1.1 Dissolve maltotriose in anhydrous pyridine, add acetic anhydride, and react at room temperature for 24h-48h to obtain fully acetylated maltotriose; S1.2 Dissolve the fully acetylated maltotriose obtained in S1.1 in anhydrous dichloromethane, add 2-hydroxybromoethane, stir under low temperature nitrogen for 30min-50min, add boron trifluoride ether to continue React for 30min-50min to obtain brominated maltotriose; S1.3 Dissolve the brominated maltotriose obtained in S1.2 in anhydrous DMF, add sodium azide, heat to 60°C-80°C under nitrogen, and react for 24h-48h to obtain azide maltotriose sugar; S1.4 Dissolve the azide maltotriose obtained in S1.3 in anhydrous methanol, add sodium methoxide powder, stir at room temperature for 6h-8h, adjust the pH of the solution to neutral with a strong acid cation exchange resin, filtering and removing the strong acid cation exchange resin to obtain deprotected azide maltotriose; S1.5 Dissolve the deprotected azide maltotriose obtained in S1.4 in deionized water, add N ₃ -PEG-NH ₂ powder, copper sulfate solution and sodium ascorbate solution, and react at high temperature for 3-4 days, After dialysis for 2-3 days, PEG-maltotriose was obtained; S2 specifically includes the following steps: Dissolve the PEG-maltotriose and MPDA obtained in S1 in deionized water, adjust the pH of the solution to 8-9 with Tris hydrochloride, and react in the dark for 24h-48h to obtain MPDA-PEG-Mal; S3 specifically includes the following steps: Dissolve the MPDA-PEG-Mal obtained in S2 in deionized water, and dissolve BNN6 in absolute ethanol. After the two are mixed, they are reacted in the dark for 24h-48h. After the reaction, the mesoporous polydopamine NO nanoparticles BNN6@MPDA are obtained. -PEG-Mal; The room temperature is 25°C-30°C; the low temperature is -20°C-0°C; and the high temperature is 70°C-90°C. 2. the preparation method of a kind of mesoporous polydopamine NO nanoparticles as claimed in claim 1, is characterized in that: in step S1.1, the mol ratio of described maltotriose and acetic anhydride is 1:7-10; The dosage of the anhydrous pyridine is calculated by adding 100 mg-200 mg maltotriose per 10 mL of anhydrous pyridine; In step S1.2, the molar ratio of fully acetylated maltotriose to 2-hydroxybromoethane is 1:1.5-3; the amount of anhydrous dichloromethane is added per 10 mL of anhydrous dichloromethane 100mg-200mg full acetylated maltotriose; In step S1.3, the molar ratio of the brominated maltotriose to sodium azide is 1:2-4; the dosage of the anhydrous DMF is 50mg-100mg sugar meter; In step S1.4, the molar ratio of the azide maltotriose to sodium methoxide is 1:0.5-3; sugar meter; In step S1.5, the molar ratio of the deprotected azide maltotriose, N ₃ -PEG-NH ₂ , copper sulfate, and sodium ascorbate is 1:1.5-2:1.2-1.5:1.2-1.5; The amount of deionized water mentioned above is calculated by adding 100 mg-150 mg of deprotected azide maltotriose per 10 mL of deionized water. 3. the preparation method of a kind of mesoporous polydopamine NO nanoparticle as claimed in claim 1, is characterized in that: described MPDA and PEG-maltotriose mass ratio are 1:2-4; The dosage is based on adding 10mg-20mg MPDA to 10mL deionized water. 4. the preparation method of a kind of mesoporous polydopamine NO nanoparticle as claimed in claim 1, is characterized in that: the mass ratio of described MPDA-PEG-Mal and BNN6 is 1:1-2; Described dehydrated alcohol The dosage is based on adding 10mg-20mg BNN6 to 10mL absolute ethanol. 5. A kind of mesoporous polydopamine NO nanoparticle prepared according to the preparation method of a kind of mesoporous polydopamine NO nanoparticle according to any one of claims 1-4. 6. The application of a kind of mesoporous polydopamine NO nanoparticles according to claim 5 in the preparation of medicines for diagnosing and treating bacterial infections.

Images