An inhalable bionic nanomaterial for treating pulmonary bacterial infection pneumonia and its preparation method and application

Abstract

The present invention provides an inhalable bionic nanomaterial for treating bacterial pneumonia of the lungs, and a preparation method and application thereof, belonging to the technical field of drug preparation. Mesoporous polydopamine nanoparticles A are prepared by a one-pot synthesis method using block polyether and 3,3',5,5'-tetramethylbenzidine as templates; levofloxacin is added to A, stirred and centrifuged, and freeze-dried to obtain nanoparticles B; RAW264.7 cells are resuspended after centrifugation, homogenized and centrifuged again to collect the supernatant, and the supernatant centrifugal precipitation is the prepared macrophage membrane C; the configured B solution and C are mixed, and a bionic nanomaterial D wrapped in a macrophage membrane coating is prepared by a direct extrusion method. The inhalable bionic nanomaterial prepared by the present invention can cross the complex lung barrier to target the inflammatory site to respond to the release of drugs, and can remove reactive oxygen to relieve lung inflammation, which is of great significance for the treatment of lung infection and has promotion and application value.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to an inhalable bionic nanomaterial for treating pulmonary bacterial infection pneumonia, and a preparation method and application thereof. Background Art

[0002] Lower respiratory tract infections (LRTIs) are one of the leading causes of death worldwide in recent years. Among them, acute lower respiratory tract infections have become the main cause of hospitalization and death in children, accounting for 15% of global child deaths. Bacteria such as Streptococcus pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus are important pathogens in pneumonia. For the treatment of bacterial pneumonia, inhalation administration can directly deliver antibiotics to the infected area, with high compliance and safety, and has therefore received increasing attention in recent years. However, due to the special physiological and pathological structure of the lower respiratory tract, there are still many problems that hinder the application of inhalation administration. For example, the mucus layer acts as a physical barrier to antibiotics, which reduces the efficacy of inhalation therapy; alveolar macrophages are prone to phagocytosis of foreign substances, limiting the treatment efficiency of pneumonia. Micro-nanoparticles (NPs) have been studied for loading antibiotics and delivering them into the lungs by inhalation, but their ability to overcome the complex lung barrier remains to be optimized. Therefore, a drug delivery system with biomimetic targeting is of great significance for the inhalation administration of drugs to treat lung infections.

[0003] In addition, persistent inflammatory responses produce excessive reactive oxygen species, which can directly damage nucleic acids, proteins, and lipids, causing irreversible damage to the body. If the optimal treatment time is missed, reactive oxygen species and inflammatory responses will initiate a mutually reinforcing cycle and spread throughout the body, leading to systemic inflammatory syndrome. With the occurrence of systemic inflammatory syndrome, excessive reactive oxygen species will selectively attack major organs, leading to organ dysfunction and even death. Therefore, how to improve the inflammatory environment of the lungs while ensuring effective clearance of Streptococcus pneumoniae is an important issue to be solved in clinical practice.

[0004] Currently, there are no reports on the preparation method of using inhalable bionic nanomaterials to overcome the complex lung barrier response to release drugs and effectively remove reactive oxygen to relieve lung inflammation. Summary of the invention

[0005] In view of the above-mentioned problems existing in the prior art, one of the purposes of the present invention is to provide a bionic drug delivery system that can overcome the complex lung barrier and effectively alleviate lung inflammation. The second purpose of the present invention is to provide a method for preparing a bionic drug delivery system that can overcome the complex lung barrier and effectively alleviate lung inflammation. The third purpose of the present invention is to achieve effective antibacterial and simultaneously remove ROS to cure local inflammation caused by bacteria through bionic targeted delivery to avoid the clearance of drugs by the lung barrier.

[0006] The present invention provides the following technical solutions:

[0007] A method for preparing an inhalable bionic nanomaterial for treating pulmonary bacterial infection pneumonia, characterized in that it comprises the following steps:

[0008] The block polyether and 3,3',5,5'-tetramethylbenzidine were dissolved in a mixture of water and ethanol, and 120 mg of dopamine hydrochloride was added after stirring. After stirring and reacting, the mixture was centrifuged and the precipitate was collected. The template of the product was removed by solvent extraction to obtain nanoparticles A. Nanoparticles A and levofloxacin were dissolved in PBS, and the precipitate was collected by centrifugation after stirring and reacting to prepare nanoparticles B containing levofloxacin. RAW264.7 cells were taken, and the precipitate after centrifugation was resuspended and homogenized, and then centrifuged again to collect the supernatant. Macrophage membrane C was obtained after the supernatant was centrifuged and precipitated; the macrophage membrane C was mixed with nanoparticles B and ultrasonically treated in an ice bath; a bionic nanomaterial containing a macrophage membrane coating was prepared by direct extrusion.

[0009] Furthermore, the mass ratio of the block polyether to 3,3',5,5'-tetramethylbenzidine is (1:1) to (1:3); and the mass ratio of the nanoparticles A to levofloxacin is (2:1) to (1:2).

[0010] The present invention provides a method for preparing an inhalable bionic nanomaterial for treating pulmonary bacterial infection pneumonia, comprising the following steps:

[0011] 1) Dissolve block polyether (F127, 0.6-0.8 g) and 3,3',5,5'-tetramethylbenzidine (TMB, 1.0-1.2 g) (mass ratio 1:1-1:3) in a mixture of water and ethanol and stir for 30 min; then, add 120 mg of dopamine hydrochloride, stir and react at room temperature for a certain time, centrifuge, collect the precipitate, wash the product with ethanol and acetone several times, and remove the template by solvent extraction to prepare nanoparticles A;

[0012] 2) dissolving nanoparticles A (30-50 mg) and levofloxacin (30-50 mg) (mass ratio 2:1 to 1:2) in PBS (pH 7.4) and stirring for 12 h; then, collecting the precipitate by centrifugation to prepare nanoparticles B containing levofloxacin;

[0013] 3) Take RAW264.7 cells (the cell line was provided by the Cell Bank of the Chinese Academy of Sciences), centrifuge at 800g for 5 min, resuspend the precipitate in a hypotonic buffer (composed of NaHCO3 and ethylenediaminetetraacetic acid) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and use a homogenizer to remove the nucleus 20 times. Centrifuge at 3200g at 4°C to collect the supernatant. The supernatant is centrifuged at 15000g to precipitate the prepared macrophage membrane C;

[0014] 4) The macrophage membrane C and the nanoparticles B are mixed in a mass ratio of 1:1, and ultrasonically treated in an ice bath for a certain period of time; then a bionic nanomaterial D containing a macrophage membrane coating is prepared by a direct extrusion method.

[0015] Preferably, the mass ratio of F127 to TMB is 1:1.5.

[0016] Preferably, the volume ratio of water to ethanol is 13:12.

[0017] Preferably, the time for ultrasonic treatment of the macrophage membrane C and the nanoparticles B in the ice bath is 1 to 3 minutes.

[0018] Preferably, the macrophage membrane C and the nanoparticles B are ultrasonically treated in an ice bath for 2 minutes.

[0019] Preferably, the mass ratio of the nanoparticles A to levofloxacin is 1:1.

[0020] Preferably, after the macrophage membrane C and the nanoparticles B are mixed, they are ultrasonically treated in an ice bath at a power of 80 to 120 W, ultrasonically treated for 1 to 3 seconds, and then stopped for 4 to 6 seconds.

[0021] Preferably, after the macrophage membrane C and the nanoparticles B are mixed, they are ultrasonically treated in an ice bath at a power of 100 W, ultrasonicated for 2 seconds, and then stopped for 5 seconds.

[0022] The invention also provides an inhalable bionic nano material prepared by the preparation method.

[0023] Preferably, the hydrated particle size of the inhalable biomimetic nanomaterial is 100-150 nm.

[0024] The present invention also provides application of the inhalable bionic nanomaterial prepared by the preparation method in drug delivery.

[0025] The present invention also provides the use of the above-mentioned inhalable bionic nanomaterial in the preparation of a drug for treating pulmonary bacterial infection pneumonia.

[0026] Preferably, the loaded drugs include procaine penicillin, piperacillin, benzathine penicillin, amoxicillin, mezlocillin, amoxicillin-clavulanate potassium, levofloxacin or cephalosporins.

[0027] Preferably, the strains of pulmonary bacterial pneumonia include Streptococcus pneumoniae, Staphylococcus aureus or Pseudomonas aeruginosa.

[0028] The present invention constructs a bionic inhalable nanomaterial (LE@MPDA-MM) with the ability to overcome the complex lung barrier, efficiently remove pneumonia pathogens and anti-inflammatory properties. The present invention takes the model of pneumococcal infection as an example. Pneumococcus is the main cause of pneumonia deaths worldwide. In 2016, the number of deaths caused by it exceeded the sum of the number of deaths caused by all other causes of pneumonia. Although antibacterial agents and antibiotics are widely used to treat pneumococcal infection, the phagocytosis of alveolar macrophages in the lungs seriously limits the treatment efficiency of pneumococcal pneumonia. Considering the inflammatory tropism of macrophages and their ability to cross barriers, the present invention uses a bionic strategy to utilize the glycosyl groups on the surface of the macrophage membrane and the natural inflammatory tropism to drive the carrier to effectively target and accumulate in the site of lung inflammation to efficiently remove pneumococcus; in addition, the excessive ROS caused by lung inflammation directly destroys nucleic acids, proteins and lipids, causing irreversible damage to the body, which is also a problem to be solved. Therefore, the present invention designs a bionic nano delivery carrier with pH responsiveness and ROS scavenging ability, which can effectively respond to the slightly acidic environment of the lung infection site to release drugs, and can also scavenge reactive oxygen, reduce lung infection, and relieve inflammatory response. Different from the current lung infection treatment methods, the present invention has a simple design and safe materials, can effectively deliver antibiotics across barriers to kill bacteria and avoid drug resistance and other problems, and can also reduce lung infection and relieve inflammatory response, which is of great significance for the treatment of lung infection.

[0029] The inhalable bionic nanomaterial prepared by the invention can be used for the effective delivery and responsive release of drugs at the site of lung inflammation, and the pneumonia infection strains include Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0030] Beneficial Effects

[0031] 1) The inhalable bionic nanomaterial of the present invention has abundant sources of synthetic materials, is easy to obtain, has low cost, is simple to synthesize, has a short time period, and is easy to clinically transform.

[0032] 2) The inhalable bionic nanomaterial provided by the present invention avoids being phagocytosed by alveolar macrophages in the lungs by virtue of the inherent properties of macrophages and the inflammation-directed chemotaxis ability, and effectively drives the accumulation of the carrier in the inflammatory tissue of the lungs.

[0033] 3) The inhalable biomimetic nanomaterial provided by the present invention contains mesoporous polydopamine, which has a high drug loading rate and can effectively encapsulate antibiotic drugs; on the other hand, the nanoparticles contain more amino groups, are pH sensitive, and promote the release of drugs under acidic conditions; in addition, the nanoparticles are rich in catechol functional groups, which can effectively eliminate reactive oxygen, inhibit inflammation, and reduce pathological damage to lung tissue, and have promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Potential and particle size analysis diagram of the inhalable biomimetic nanomaterial prepared in Example 1 (Figure A is the potential, Figure B is the particle size);

[0035] Figure 2 Transmission electron microscopy image of the inhalable biomimetic nanomaterial prepared in Example 1;

[0036] Figure 3 The drug loading efficiency diagram of the inhalable biomimetic nanomaterial prepared in Example 1;

[0037] Figure 4 pH responsive release kinetics of the inhalable biomimetic nanomaterial prepared in Example 1;

[0038] Figure 5 Figure 1 shows the ROS scavenging ability of the inhalable biomimetic nanomaterial prepared in Example 1;

[0039] Figure 6 Figure 1 shows the levels of inflammatory factors in Raw264.7 cells stimulated by LPS after being treated with the inhalable biomimetic nanomaterials prepared in Example 1 (Figure A is TNF-α, Figure B is IL-6);

[0040] Figure 7 Effect of the inhalable bionic nanomaterials prepared in Example 1 on bacterial activity (Figure A is the relationship between the OD600 value of the bacterial solution and the concentration of LE@MPDA-MM; Figure B is the relationship between the OD600 value of the bacterial solution and time);

[0041] Figure 8 The in vitro anti-phagocytic ability of the inhalable biomimetic nanomaterial prepared in Example 1;

[0042] Fig. 9 A diagram showing the ability of the inhalable biomimetic nanomaterial prepared in Example 1 to cross the mucus barrier;

[0043] Fig.10 Figure 1 shows the in vivo bactericidal effect of the inhalable biomimetic nanomaterial prepared in Example 1 (Figure A is a plate photo, and Figure B is the result of the total bacterial colony count);

[0044] Fig.11 Figure 1 shows the in vivo anti-inflammatory effect of the inhalable biomimetic nanomaterial prepared in Example 1 (Figure A is IL-6, Figure B is TNF-α); DETAILED DESCRIPTION

[0045] In order to make the technical solutions, advantages and purposes of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of this application.

[0046] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0047] Example 1

[0048] A method for preparing an inhalable biomimetic nanomaterial comprises the following steps:

[0049] 1) 0.72 g F127 and 1.08 g TMB were dissolved in a mixture of water (130 mL) and ethanol (120 mL) and stirred for 30 min; then, 120 mg dopamine hydrochloride was added, stirred at room temperature for 24 h, centrifuged, and the product was washed with ethanol and acetone, and ultrasonically treated three times (30 min each time) in a mixed solution of ethanol and acetone (2:1 v / v) to remove the template to prepare nanoparticles A;

[0050] 2) mixing the nanoparticle A solution (40 mg, 8 mL) with the levofloxacin hydrochloride solution (LE, 40 mg, 8 mL) and stirring for 12 h; then, collecting the precipitate by centrifugation to prepare nanoparticles B containing levofloxacin;

[0051] 3) Take RAW264.7 cells, centrifuge at 800g for 5 min, resuspend the precipitate in a hypotonic buffer (composed of NaHCO3 and ethylenediaminetetraacetic acid) containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and use a homogenizer to remove the nucleus 20 times. Centrifuge at 4°C and 3200g to collect the supernatant. The supernatant is centrifuged at 15000g to precipitate the prepared macrophage membrane C;

[0052] 4) Mixing the macrophage membrane C with the nanoparticles B, and ultrasonically treating them in an ice bath for 2 min (100 W, ultrasonic for 2 s, stop for 5 s); then preparing the bionic nanomaterial D coated with the macrophage membrane by direct extrusion method;

[0053] Example 2

[0054] The bionic spray prepared in Example 1 is used to treat pneumonia caused by Streptococcus pneumoniae.

[0055] Specifically, the colony of Streptococcus pneumoniae was transferred to 10 mL of THB medium and cultured at 37° C. overnight to obtain bacteria in the exponential growth phase.

[0056] Detection and analysis

[0057] 1) The inhalable biomimetic nanomaterial prepared in Example 1 was subjected to a fluid dynamics analysis of the potential level and particle size distribution. The results are as follows: Figure 1 shown.

[0058] from Figure 1 The analysis showed that the average size of polydopamine nanomaterials containing levofloxacin (LE@MPDA) was about 90 nm. After coating with macrophage membranes (MMs), the average size increased to about 138 nm. The potential of LE@MPDA was -32.2 mV, and the potential of MM was -23.4 mV. After coating MMs on the surface of LE@MPDA, the potential of LE@MPDA-MM was similar to that of macrophage membranes, which was -22.2 mV, indicating that MMs were successfully covered on the surface of LE@MPDA.

[0059] 2) The inhalable biomimetic nanomaterial prepared in Example 1 was analyzed by transmission electron microscopy, with a scale of 50 nm. The results are as follows: Figure 2 shown.

[0060] from Figure 2 From the observation and analysis, it can be seen that the bionic nanomaterial (LE@MPDA-MM) has a spherical structure with an average diameter of about 127nm.

[0061] 3) Investigation of drug loading efficiency of the inhalable biomimetic nanomaterial prepared in Example 1

[0062] The specific operation is as follows: 1 mg of the inhalable biomimetic nanomaterial prepared in Example 1 is taken, 5 mL of anhydrous methanol solution is added, and the particles are destroyed by high-speed vortexing for 10 min, followed by centrifugation and collection of the supernatant. The LE amount in the supernatant is calculated by the LE standard curve, and the drug loading rate (LE) is determined as follows:

[0063] LE(%)=WA / (WA+WB)×100

[0064] WA is the mass of LE, WB is the mass of total carrier, and WC is the mass of total LE.

[0065] Due to the multi-hole structure of polydopamine, the drug loading rate is as high as about 12%, which lays the foundation for the efficient removal of pathogens in the pneumonia area ( Figure 3 ).

[0066] 4) The inhalable biomimetic nanomaterial prepared in Example 1 was subjected to pH-responsive drug release analysis

[0067] The specific operation is as follows: To explore the drug release behavior of inhalable biomimetic nanomaterials, 1 mg of inhalable biomimetic nanomaterials (LE@MPDA-MM) was dispersed in 5 mL of different solvents, and a dialysis bag (MWCO: 8-14 kDa) containing 1 mL of LE@MPDA-MM solution was immersed in 10 mL of phosphate buffer (0.1 M, pH 7.4, containing 0.5% tween-80; or 0.1 M, pH 6.0, containing 0.5% tween-80). 2 mL of the solution was taken at different time intervals and replaced with an equal amount of fresh PBS with the same pH value. The cumulative release of LE was quantified by HPLC at 293 nm.

[0068] The results showed that the cumulative release efficiency of LE@MPDA-MM in pH 7.4 PBS medium was only about 22% in 24 hours, while the cumulative release in pH 6.0 PBS was about 73% in 24 hours, which was more than 3 times that of pH 7.4 PBS release medium. This is because under low pH conditions, the protonation of the amino group in the MPDA molecule is enhanced, and the π-π interaction between MPDA and LE is weakened, thereby increasing the release of the drug. The above results show that LE@MPDA-MM can quickly release drugs in the weakly acidic environment of inflammation, thereby helping to eliminate bacteria at the site of inflammation ( Figure 4 ).

[0069] 5) The ROS scavenging ability of the inhalable biomimetic nanomaterial prepared in Example 1 was investigated

[0070] The specific operation is: Mlg cells (2×10 5 ) were inoculated in a 6-well plate overnight, and the old medium was replaced with serum-free medium containing 200 μM hydrogen peroxide and incubated for 1.5 h. Then, MPDA NPs, LE@MPDA NPs and LE@MPDA-MM NPs were added to the six-well plate respectively and incubated for another 4 h. The group without preparation treatment was used as the positive control, and the cells without hydrogen peroxide treatment were used as the negative control. Subsequently, DCFH-DA was added to detect the free radical level and stained with Hoechst. Finally, the cells were observed under a fluorescence microscope.

[0071] like Figure 5 As shown, compared with the positive control, the fluorescence intensity of Mlg cells treated with MPDA was significantly reduced, confirming the ability of MPDA to scavenge free radicals.

[0072] 6) The anti-inflammatory properties of the inhalable bionic nanomaterial prepared in Example 1 were investigated

[0073] The specific operation is as follows: In order to detect the anti-inflammatory ability of bionic nanomaterials, RAW 264.7 cells were inoculated in 6-well plates overnight, and LPS (1 μg / mL, 24 h) was used to induce an inflammatory environment. Experimental group 1: blank culture medium (without LPS), set as a negative control; Experimental group 2: LPS, set as a positive control group; Experimental group 3: LPS+MPDA NPs; Experimental group 4: LPS+LE@MPDANPs; Experimental group 5: LPS+LE@MPDA-MM NPs, cells were treated separately according to the grouping and incubated for 4 h; then, IL-6 and TNF-α in the supernatant were detected using an Elisa kit, and the results are shown in Figure 2. Figure 6 shown.

[0074] The results showed that after treatment with the MPDA-containing preparation, the IL-6 and TNF-α secretion levels of RAW 264.7 cells stimulated by LPS were significantly reduced, indicating that bionic nanomaterials containing MPDA can effectively inhibit inflammation.

[0075] 7) The antibacterial ability of the inhalable biomimetic nanomaterial prepared in Example 1 was investigated

[0076] Investigation of minimum inhibitory concentration: To investigate the minimum inhibitory concentration (MIC), the pneumococcal colonies were transferred to 10 mL of THB medium and cultured in a 37°C incubator overnight to obtain bacteria in the exponential growth phase. The bacterial suspension was diluted to a concentration of 2 × 10 5 CFU / mL, a certain amount of LE@MPDA-MM suspension was mixed with an equal amount of bacterial suspension in a 96-well plate, and the final concentration of LE in the sample ranged from 1 to 16 μg / mL. All 96-well plates were gently incubated at 37°C for 12 h, and the MIC was determined by OD600 (Bio-Road Model 680, UK) using a microplate reader.

[0077] To investigate the in vitro antibacterial effect of bionic nanomaterials: the bacterial suspension was added to THB culture medium at a ratio of 1:100, the MIC of LE@MPDA-MM was used as the investigation concentration, and the culture was gently cultured at 37°C. The changes in OD600 at different time points were detected to evaluate the in vitro antibacterial effect of bionic nanomaterials.

[0078] The results showed that when the concentration of LE@MPDA-MM was only 8.5μg / mL, it showed a significant antibacterial effect. When the concentration was 34μg / ml, the OD600 value of the bacterial solution was almost close to the OD600 value of the blank group (pure culture medium). Therefore, the MIC value of Streptococcus pneumoniae in the LE@MPDA-MM group was 34μg / mL ( Figure 7 A). In addition, the in vitro antibacterial experiment also showed that LE@MPDA-MM has excellent antibacterial effect. After dozens of hours of culture, the OD600 value is always close to the blank group.

[0079] 8) Evaluation of the anti-phagocytic properties of biomimetic nanomaterials in vitro

[0080] In this study, RAW264.7 cells were used as a model to calculate the in vitro anti-phagocytic performance of the biomimetic nanosystem. RAW 264.7 cells were plated at 10 5 The cells were seeded at a density of 100 cells / well in a 12-well plate and cultured for 24 h. LE@MPDA was pre-coated with liposomes to form LE@MPDA-lip, and then LE@MPDA-lip and LE@MPDA-MM were stained with DIO dye. Finally, they were co-incubated with the above cells for 4 h, and the anti-phagocytic ability was evaluated by flow cytometry.

[0081] like Figure 8 It was shown that the fluorescence level of macrophages treated with LE@MPDA-MM was lower than that of the LE@MPDA group, which was mainly due to the fact that the MM coating could effectively block the nonspecific phagocytosis of macrophages.

[0082] 9) Investigate the performance of biomimetic nanomaterials across the mucus barrier

[0083] Preparation of artificial mucus: 500 mg of deoxyribonucleic acid, 250 mg of mucin, 0.295 mg of diethylenetriaminepentaacetic acid, 1 mL of RPMI 1640 amino acid solution, 250 μL of egg yolk emulsion, 250 mg of sodium chloride and 110 mg of potassium chloride were mixed and dissolved in 50 mL of DNase-free water and equilibrated at 25°C for 2 h.

[0084] Investigation of the performance of biomimetic nanomaterials across the mucus barrier: Experimental grouping, Experimental group 1: LE@MPDA group; Experimental group 2: LE@MPDA-MM. Artificial mucus was placed on the hardened gelatin gel; then, the sample was stained with Coomassie Brilliant Blue and deposited on the surface of the artificial mucus layer. The images of the sample were recorded at 0min, 10min, 30min, 60min and 120min respectively.

[0085] Fig. 9 The results showed that LE@MPDA-MM had a higher diffusion rate in mucus compared with LE@MPDA, indicating that the MM coating played an important role in overcoming the mucus barrier.

[0086] 10) Investigate the antibacterial and anti-inflammatory properties of biomimetic nanomaterials in vivo

[0087] Construction of mouse lung infection model: Female BALB / c mice (8 weeks old, 18-20 g) were selected and inoculated with Streptococcus pneumoniae intranasally by nasal drops under isoflurane anesthesia. 8CFU / mouse. The inoculated mice were kept upright for 5 min to establish a mouse pneumonia infection model. Experimental group 1: mice intranasally inoculated with normal saline served as the control group, i.e., the “healthy control” group; Experimental group 2: PBS group, as a positive control; Experimental group 3: LE; Experimental group 4: LE@MPDA; Experimental group 5: LE@MPDA-MM.

[0088] In vivo antibacterial activity: After bacterial inoculation, mice were killed, lungs were washed twice with 400 μL saline, lungs were collected, and lung tissues were homogenized. 4 The samples were cultured overnight at 37°C and the bacterial growth on the plates was observed.

[0089] Investigation of anti-inflammatory properties in vivo: After treating mice, the levels of inflammatory factors (TNF-α, IL-6 and IL-1β) in the bronchoalveolar lavage fluid (BAL) of mice were detected.

[0090] Fig.10 The results showed that in the lung homogenate plate, compared with the LE group, basically no obvious colonies were observed in the LE@MPDA-MM group, which was the same as the total bacterial colony count results. This indicates that inhalation administration of LE@MPDA-MM can effectively overcome the mucus penetration ability and responsive drug release characteristics, and efficiently eliminate Streptococcus pneumoniae.

[0091] Fig.11 The results showed that after inhalation treatment, the TNF-α and IL-6 in the lung lavage fluid of the LE@MPDA group and LE@MPDA-MM group were significantly lower than those of the other groups, indicating that the inflammatory symptoms of bacterial pneumonia in the MPDA-containing group were improved, indicating that the MPDA preparation-containing group can effectively reduce lung infection and relieve inflammatory response after inhalation treatment.

[0092] The present invention constructs a biomimetic inhalable nanomaterial (LE@MPDA-MM) with the ability to overcome the mucus barrier and anti-inflammatory properties. The present invention takes the model of pneumococcal infection as an example. Pneumococcal infection is the main cause of pneumonia deaths worldwide. In 2016, the number of deaths caused by it exceeded the sum of deaths caused by all other pneumonias. Although antibacterial agents and antibiotics are widely used to treat pneumococcal infection, the mucus in the respiratory tract and the phagocytosis of alveolar macrophages in the lungs seriously limit the treatment efficiency of pneumococcal pneumonia. Considering the inflammatory tropism and barrier-crossing ability of macrophages, the present invention uses the glycosyl groups on the surface of macrophage membranes and the natural inflammatory tropism to drive the carrier to effectively target and accumulate in the pulmonary inflammation site to efficiently remove pneumococcus; the excessive ROS caused by pulmonary inflammation directly destroys nucleic acids, proteins and lipids, causing irreversible damage to the body. Therefore, the present invention designs polydopamine nanoparticles loaded with antibiotics with pH responsiveness and ROS scavenging ability, which can effectively respond to the slightly acidic environment of the pulmonary infection site to release drugs, and can also remove reactive oxygen, reduce pulmonary infection, and relieve inflammatory response. Different from the current treatment methods for pulmonary infection, the present invention is simple in design and safe in materials. It can effectively deliver antibiotics across barriers to kill bacteria, while reducing pulmonary infection and relieving inflammatory response, which is of great significance for the treatment of pulmonary infection.

[0093] During the actual preparation and research process, it was also found that the size of the nanomaterial can be effectively adjusted by changing the synthesis feed ratio of MPDA, providing strong technical support for effective pulmonary drug delivery.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the protection scope of the present invention.

Claims

1. A method for preparing an inhalable bionic nanomaterial for treating pulmonary bacterial infection pneumonia, characterized in that: The following steps are involved: The block polyether and 3,3',5,5'-tetramethylbenzidine were dissolved in a mixture of water and ethanol, dopamine hydrochloride was added after stirring, the reaction was centrifuged and the precipitate was collected after stirring, and the product was subjected to solvent extraction to remove the template to obtain nanoparticles A; nanoparticles A and levofloxacin were dissolved in PBS, the reaction was stirred and the precipitate was collected by centrifugation to prepare nanoparticles B containing levofloxacin; RAW264.7 cells were taken, the precipitate after centrifugation was resuspended and homogenized, and the supernatant was collected by centrifugation again, and the supernatant was centrifuged and precipitated to obtain macrophage membrane C; the macrophage membrane C was mixed with nanoparticles B and ultrasonically treated in an ice bath; the ultrasonic treatment time was 1-3 min; the ultrasonic treatment power was 80-120 W, the ultrasonic treatment was 1-3 s, and the stop was 4-6 s; the bionic nanomaterial containing macrophage membrane coating was prepared by direct extrusion method; The mass ratio of the block polyether to 3,3',5,5'-tetramethylbenzidine is (1:1) to (1:3); the mass ratio of the nanoparticle A to levofloxacin is (2:1) to (1:2).

2. The method for preparing the inhalable bionic nanomaterial for treating pulmonary bacterial infection pneumonia according to claim 1, characterized in that: The volume ratio of water to ethanol is 13:

12.

3. An inhalable bionic nanomaterial, characterized in that: Prepared by the method of claim 1 or 2.

4. The inhalable biomimetic nanomaterial according to claim 3, characterized in that: The particle size of the inhalable bionic nanomaterial is 100-150 nm.

5. Use of the inhalable bionic nanomaterial according to claim 4 in the preparation of a drug for treating pulmonary bacterial infection pneumonia; the strains of pulmonary bacterial infection pneumonia include one or more of Streptococcus pneumoniae, Staphylococcus aureus or Pseudomonas aeruginosa.

Images