A fully active nanomedicine that synergistically and step by step fights bacterial biofilm infection, and its preparation method and application

Abstract

The present invention belongs to the technical field of non-antibiotic treatment of bacterial infection, and in particular, relates to a fully active nano drug that synergistically and step by step combats bacterial biofilm infection, and its preparation method and application. The fully active nano preparation includes berberine, tetraphenylporphyrin tetrasulfonic acid and lanthanum nitrate. The drug releases drugs specifically in the biofilm microenvironment, targets lanthanum ions to break the bacterial intracellular drug delivery barrier, TPPP-mediated sonodynamic therapy to strongly kill bacterial cells, and berberine efficiently interferes with bacterial quorum sensing, so as to synergize step by step and programmatically combat the biofilm of drug-resistant bacteria. The preparation process is simple and does not require the introduction of a carrier. The obtained preparation has clear ingredients, each component is used to its full potential, and the proportion of active drugs is as high as 100%. It can effectively overcome the limitations of traditional carrier drug delivery nano preparations and has a high potential for clinical transformation.

Description

Technical Field

[0001] The present invention belongs to the technical field of non-antibiotic treatment of bacterial infection, and in particular relates to a fully active nano drug for synergistically and step by step combating bacterial biofilm infection, and a preparation method and application thereof. Background Art

[0002] Bacterial pneumonia is a fatal disease worldwide, posing a serious threat to public health. Currently, antibiotic therapy is the main treatment for bacterial pneumonia, but problems such as poor efficacy, high recurrence rate, and easy to cause dysbiosis of normal flora are still unavoidable. Studies have found that most of the clinically difficult-to-treat bacterial pneumonia is closely related to the formation of biofilms (BF). BF refers to a large number of bacterial aggregates formed by bacteria adhering to contact surfaces, secreting polysaccharide matrices, fibrin, and lipid proteins, etc., and wrapping themselves in it. Compared with planktonic bacteria, bacteria that form complete biofilms can successfully resist clearance by the host immune system, and their resistance to antibiotics is increased by 10-1000 times, greatly reducing the effectiveness of traditional antibiotic therapy.

[0003] As an antibiotic-free therapy, sonodynamic antibacterial technology based on reactive oxygen species (ROS) is expected to solve the current dilemma. Sonodynamic therapy (SDT) mainly uses low-frequency ultrasound to induce cavitation to activate sound-sensitive factors (i.e., sonosensitizers) to convert surrounding oxygen, water, or hydrogen peroxide into ROS, thereby causing strong oxidative stress damage to bacterial membrane phospholipids, functional proteins, and nucleic acids, interfering with their normal life activities to inhibit and kill bacteria. Due to the non-specific mechanism of action and extremely low mutagenicity of ROS, sonodynamic antibacterial therapy has a broad antibacterial spectrum, is not easy to induce resistance, and is effective against a variety of drug-resistant bacteria. In addition, during SDT treatment, the external ultrasound only irradiates and activates the sonosensitizer at the disease site, avoiding the treatment load on the body's normal tissues; the low-frequency ultrasound used in the band has a tissue penetration depth of up to 10 cm, does not induce thermal effects, and has minimal side effects on the tissues around the lesions, which can achieve effective and non-invasive treatment of deep infections. However, SDT also has certain defects, such as poor water solubility of sonosensitizers, limited ROS production, and low bacterial targeting, which make it difficult to completely eradicate the infection in a timely manner using a single SDT, limiting its clinical application.

[0004] The use of multi-mechanism therapy to programmatically fight bacterial infections has significant advantages in the treatment of drug-resistant bacteria. Unlike sonodynamic therapy, which directly kills bacteria themselves, interfering with bacterial quorum sensing therapy targets bacterial cell-to-cell interactions. Quorum sensing (QS) is an intercellular communication system used by bacteria to regulate cooperative activities and physiological processes, which can help bacteria adapt to environmental changes and form biofilms. Studies have found that interfering with quorum sensing can inhibit the virulence and metabolic activity of bacteria. In addition, QS is related to bacterial stress responses, including heat stress, oxidative stress, and osmotic stress. Therefore, in addition to inhibiting the formation of biofilms, QS interference may also enhance the effect of sonodynamic therapy by destroying the stress response of biofilms. Studies have shown that a large number of effective active ingredients of traditional Chinese medicines (such as rhubarb, phellodendron, and coptis chinensis) have antibacterial effects. Berberine (BBR), also known as berberine, as the main active ingredient of the traditional Chinese medicine coptis chinensis, can effectively inhibit the formation of bacterial biofilms and does not induce drug resistance. It has unique advantages in the treatment of bacterial infections.

[0005] The special physiological structure of bacterial cells poses a great obstacle to the entry of drugs into the cells to exert their effects. How to effectively break the bacterial intracellular drug delivery barrier greatly affects the therapeutic effect of drugs. Since the 20th century, people have discovered that rare earth ions can inhibit bacterial growth by destroying the bacterial membrane phosphate, DNA and protein. Studies have found that lanthanum ions can occupy the calcium ion binding protein site of calcium binding protein in Escherichia coli, but do not exert physiological activity, thereby changing the structure of the cell membrane and enhancing the permeability of the cell membrane, which is conducive to the entry of antibacterial drugs into the bacteria to exert their effects. When lanthanum ions form complexes with other compounds, the positively charged part of the lanthanum ions is transferred to the ligand, reducing the polarity of the rare earth ions, enhancing the lipophilicity of the drug, and making it easier for the drug to enter the bacteria to exert its therapeutic effect.

[0006] In recent years, nanomedicines have made great progress in the medical field. At present, most nanomedicine research and development focuses on the use of nanocarriers for drug delivery. The introduction of carriers inevitably brings huge challenges to the biosafety and in vivo metabolism of the preparations, and there are problems such as cumbersome synthesis, low drug loading, poor repeatability, and large batch differences. Fully active nanomedicines refer to all chemical components in the preparation system as active ingredients, which not only play a role in treating diseases, but also assume the function of self-carriers. This type of nanomedicine formed by drug molecules through non-covalent bond forces such as hydrophobic effects, hydrogen bonds, van der Waals forces, π-π stacking and electrostatic effects can circumvent the defects of carrier-delivered nanoformulations and achieve spontaneous delivery of drug molecules in the body. Summary of the invention

[0007] The purpose of the present invention is to provide a fully active nanomedicine that is easy to prepare, green and safe, and integrates the sonosensitizer tetraphenylporphyrin tetrasulfonic acid, the QS inhibitor berberine hydrochloride and the biofilm disruptor lanthanum ion; to develop a therapeutic platform that can accurately release the biofilm microenvironment, target the breaking of the bacterial intracellular drug delivery barrier, strongly kill bacterial cells, and efficiently inhibit bacterial quorum sensing, so as to cooperate step by step and programmatically combat the biofilm of drug-resistant bacteria.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] A fully active nanomedicine that synergistically and stepwise combats bacterial biofilm infection is assembled by mixing a berberine methanol solution, a tetraphenylporphyrin tetrasulfonic acid aqueous solution, and a lanthanum nitrate aqueous solution in a molar ratio of 1~3:1:1~3, wherein the concentration of the berberine methanol solution is 20 mM, and the concentrations of the tetraphenylporphyrin tetrasulfonic acid aqueous solution and the lanthanum nitrate aqueous solution are both 10 mM.

[0010] Furthermore, the loading rates of La, berberine, and tetraphenylporphyrin tetrasulfonic acid in the nanomedicine were 3.85%, 32.69%, and 63.46%, respectively.

[0011] Furthermore, the bacteria is multidrug-resistant Escherichia coli.

[0012] Another object of the present invention is to provide a method for preparing a fully active nanomedicine that synergistically and step by step combats bacterial biofilm infection, comprising the following steps:

[0013] S1. Dissolve berberine hydrochloride powder in methanol, and perform ultrasonic treatment in a water bath at room temperature for 1-5 min until berberine is completely dispersed, and the final concentration is 1-20 mM to obtain a berberine hydrochloride solution; dissolve tetraphenylporphyrin tetrasulfonic acid powder and lanthanum nitrate powder in deionized water, respectively, and perform ultrasonic treatment in a water bath at room temperature for 1-5 min until the powder is completely dispersed, and the final concentration is 1-10 mM to obtain a tetraphenylporphyrin tetrasulfonic acid solution and a lanthanum nitrate solution;

[0014] S2. The berberine hydrochloride solution, the tetraphenylporphyrin tetrasulfonic acid solution and the lanthanum nitrate solution are mixed in a molar ratio of 1-3:1:1-3, and then added dropwise into deionized water, wherein the volume of the deionized water is 100 times that of the tetraphenylporphyrin tetrasulfonic acid solution, and stirred at room temperature for 12-72 h at a speed of 100-1500 r / min to allow the compounds to assemble to obtain a crude fully active nanoparticle product, and then washed with deionized water 2-3 times to obtain a pure fully active nanoparticle mother liquor;

[0015] S3. Centrifuge the fully active nanoparticle mother solution at 3000-15000 rpm at room temperature for 10-20 min, aspirate the supernatant, and the resulting precipitate is the fully active nanoparticle.

[0016] Furthermore, a fully active nanomedicine is used to prepare drugs for the treatment of pneumonia caused by bacterial biofilm infection. Under ultrasonic irradiation, it can precisely release drugs in the biofilm microenvironment, target the breaking of bacterial intracellular drug delivery barriers, kill bacterial cells, and efficiently inhibit bacterial quorum sensing, so as to combat drug-resistant biofilms in a programmed and step-by-step manner.

[0017] Furthermore, when used as a drug, it is prepared into a preparation for aerosol inhalation, providing an ultrasonically controllable, safe and harmless bacterial pneumonia treatment platform, ensuring maximum drug enrichment at the lesion site while avoiding systemic toxicity of the drug to the body.

[0018] Mechanism: The fully active nanomedicine of the present invention, which is synergistically programmed to fight bacterial biofilm, utilizes the charge adsorption and coordination effect between different drugs, first disperses different drugs in different solvents, fully dissolves them, and then uses a solution method to mix and assemble different drug solutions to form fully active nanomedicines, thereby achieving effective treatment of bacterial biofilm infection; wherein the sonosensitizer is tetraphenylporphyrin tetrasulfonic acid, the QS inhibitor is berberine hydrochloride, and the biofilm disruptor is lanthanum ion. The organic solvent used in the assembly process is at least one of methanol, ethanol, water, dimethyl sulfoxide, and tetrahydrofuran.

[0019] The advantages of the present invention are:

[0020] 1. The present invention realizes the orderly interaction of berberine, tetraphenylporphyrin tetrasulfonic acid and lanthanum ions in the aqueous phase, and the action process is green, safe, convenient, and does not require the addition of surfactants. The obtained fully active nano-drug ingredients are clear and have good controllability between batches. It effectively overcomes the characteristics of traditional nano-preparations such as complex components, cumbersome preparation, and high loss, and greatly enhances the biological application value and clinical transformation potential;

[0021] 2. Fully active nanomedicines can be administered by aerosol inhalation, have a good passive targeting effect on bacterial pneumonia lesions, reduce nonspecific distribution in normal tissues and organs, and reduce the occurrence of toxic side effects; combined with its excellent bacterial cell membrane destruction performance, sonodynamic antibacterial performance, and quorum sensing inhibition performance, this fully active nanomedicine provides an opportunity to achieve the treatment of pneumonia infected by drug-resistant bacterial biofilms;

[0022] 3. The fully active nanomedicine of the present invention has the functions of accurately releasing drugs in the biofilm microenvironment, targeted breaking of the bacterial intracellular drug delivery barrier, powerful killing of bacterial cells, and efficient inhibition of bacterial quorum sensing. It can work synergistically step by step to programmatically combat drug-resistant bacterial biofilm infection, and the preparation process is simple, no carrier needs to be introduced, the composition of the resulting preparation is clear, each component is fully utilized, and the proportion of active drugs is as high as 100%, which can effectively overcome the limitations of traditional carrier drug delivery nanoformulations and has high clinical transformation potential; in addition, the fully active nanomedicine can be used as a non-antibiotic treatment platform, through ultrasound regulation and infection-specific microenvironment, while achieving targeted treatment of bacterial biofilm infection, it effectively avoids damage to normal organs and tissues, and is not easy to induce bacterial resistance, and has high biological application value; finally, the fully active nanomedicine of the present invention can provide an efficient and specific treatment for bacterial pneumonia-related biofilm infection by combining the two technologies of nebulized inhalation drug administration and sonodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a flow chart of the method for preparing nanoparticles.

[0024] Figure 2 The microscopic morphology of the nanoparticles prepared in Example 2 of the present invention, wherein A is a TEM image of the nanoparticles, and images B and C are the particle size and potential images of the nanoparticles, respectively.

[0025] Figure 3 These are the UV images and fluorescence images of the nanoparticles and monomer compounds prepared in Example 2 of the present invention, wherein Figure A is the UV image and Figure B is the fluorescence image.

[0026] Figure 4 These are time-dependent and concentration-dependent ROS release graphs of the nanoparticles prepared in Example 2 of the present invention at the solution level, wherein Graph A is a horizontal time-dependent ROS release graph, and Graph B is a horizontal concentration-dependent ROS release graph.

[0027] Figure 5 This is a graph showing the in vivo anti-biofilm lung homogenate bacteria effect of the nanoparticle solution prepared in Example 2 of the present invention, wherein Figure A is a bacterial plating graph, Figure B is a bacterial count graph, and Figure C is a relative bacterial survival rate data graph.

[0028] Figure 6 This is a graph showing the blood routine data of mice with the anti-biofilm effect of the nanoparticle solution prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] The invention provides a fully active nano drug for synergistically and step by step combating bacterial biofilm infection. The nano drug is assembled by mixing a berberine methanol solution, a tetraphenylporphyrin tetrasulfonic acid aqueous solution and a lanthanum nitrate aqueous solution in a molar ratio of 1-3:1:1-3, wherein the concentration of the berberine methanol solution is 20 mM, the concentrations of the tetraphenylporphyrin tetrasulfonic acid aqueous solution and the lanthanum nitrate aqueous solution are both 10 mM; and the bacteria are multidrug-resistant Escherichia coli.

[0030] Figure 1 The preparation method of a fully active nanomedicine for synergistically combating bacterial biofilm infection in Examples 1 to 5 of the present invention is as follows: Figure 1 , the fully active nanomedicines in Examples 1 to 5 were prepared.

[0031] Example 1

[0032] a. Add berberine hydrochloride solution, tetraphenylporphyrin tetrasulfonic acid solution and lanthanum nitrate solution dropwise in a molar ratio of 3:1:1 into a large volume of deionized water with rapid stirring (speed: 1500 r / min), and stir at room temperature in the dark for 48 h to allow the compounds to fully interact with each other to obtain a crude fully active nanoparticle solution, wherein the volume ratio of deionized water to tetraphenylporphyrin tetrasulfonic acid solution is controlled at 100 times;

[0033] b. The obtained crude fully active nanoparticle solution was washed three times with deionized water to completely remove the organic solvent and unassembled compounds to obtain a pure fully active nanoparticle mother solution;

[0034] c. Centrifuge the fully active nanoparticle mother solution at 15,000 rpm at room temperature for 15 min, aspirate the supernatant, and the resulting precipitate is the fully active pure drug nanoparticle.

[0035] Example 2

[0036] a. Add berberine hydrochloride solution, tetraphenylporphyrin tetrasulfonic acid solution and lanthanum nitrate solution dropwise in a molar ratio of 2:1:1 into a large volume of deionized water with rapid stirring (speed: 1500 r / min), and stir at room temperature in the dark for 48 h to allow the compounds to fully interact with each other to obtain a crude fully active nanoparticle solution, wherein the volume ratio of deionized water to tetraphenylporphyrin tetrasulfonic acid solution is controlled at 100 times;

[0037] b. The obtained crude fully active nanoparticle solution was washed three times with deionized water to completely remove the organic solvent and unassembled compounds to obtain a pure fully active nanoparticle mother solution;

[0038] c. Centrifuge the fully active nanoparticle mother solution at 15,000 rpm at room temperature for 15 min, aspirate the supernatant, and the resulting precipitate is the fully active pure drug nanoparticle.

[0039] Example 3

[0040] a. Add berberine hydrochloride solution, tetraphenylporphyrin tetrasulfonic acid solution and lanthanum nitrate solution dropwise in a molar ratio of 1:1:1 into a large volume of deionized water with rapid stirring (speed: 1500 r / min), and stir at room temperature in the dark for 48 h to allow the compounds to fully interact with each other to obtain a crude fully active nanoparticle solution, wherein the volume ratio of deionized water to tetraphenylporphyrin tetrasulfonic acid solution is controlled at 100 times;

[0041] b. The obtained crude fully active nanoparticle solution was washed three times with deionized water to completely remove the organic solvent and unassembled compounds to obtain a pure fully active nanoparticle mother solution;

[0042] c. Centrifuge the fully active nanoparticle mother solution at 15,000 rpm at room temperature for 15 min, aspirate the supernatant, and the resulting precipitate is the fully active pure drug nanoparticle.

[0043] Example 4

[0044] a. Add berberine hydrochloride solution, tetraphenylporphyrin tetrasulfonic acid solution and lanthanum nitrate solution dropwise in a molar ratio of 1:1:2 into a large volume of deionized water with rapid stirring (speed: 1500 r / min), and stir at room temperature in the dark for 48 h to allow the compounds to fully interact with each other to obtain a crude fully active nanoparticle solution, wherein the volume ratio of deionized water to tetraphenylporphyrin tetrasulfonic acid solution is controlled at 100 times;

[0045] b. The obtained crude fully active nanoparticle solution was washed three times with deionized water to completely remove the organic solvent and unassembled compounds to obtain a pure fully active nanoparticle mother solution;

[0046] c. Centrifuge the fully active nanoparticle mother solution at 15,000 rpm at room temperature for 15 min, aspirate the supernatant, and the resulting precipitate is the fully active pure drug nanoparticle.

[0047] Example 5

[0048] a. Add berberine hydrochloride solution, tetraphenylporphyrin tetrasulfonic acid solution and lanthanum nitrate solution dropwise in a molar ratio of 1:1:3 into a large volume of deionized water with rapid stirring (speed: 1500 r / min), and stir at room temperature in the dark for 48 h to allow the compounds to fully interact with each other to obtain a crude fully active nanoparticle solution, wherein the volume ratio of deionized water to tetraphenylporphyrin tetrasulfonic acid solution is controlled at 100 times;

[0049] b. The obtained crude fully active nanoparticle solution was washed three times with deionized water to completely remove the organic solvent and unassembled compounds to obtain a pure fully active nanoparticle mother solution;

[0050] c. Centrifuge the fully active nanoparticle mother solution at 15,000 rpm at room temperature for 15 min, aspirate the supernatant, and the resulting precipitate is the fully active pure drug nanoparticle.

[0051] Performance comparison

[0052] The fully active nanomedicine obtained in Examples 1 to 5 that synergistically and stepwise fights bacterial biofilm infection was characterized by performance testing.

[0053] 1 Table 1 shows the loading rate and particle size potential data of a fully active nanomedicine for synergistically and stepwisely combating bacterial biofilm infection prepared in Examples 1 to 5 of the present invention.

[0054] Table 1 Loading rate and particle size potential data of fully active nanoparticles prepared in Examples 1 to 5

[0055]

[0056] As can be seen from Table 1, berberine hydrochloride solution, tetraphenylporphyrin tetrasulfonic acid solution and lanthanum nitrate solution can be co-assembled to form fully active nanomedicines at different molar ratios. However, under different feed ratios, the loading rate of berberine showed a downward trend, and the lanthanum ion showed a trend of first increasing and then decreasing. Since tetraphenylporphyrin tetrasulfonic acid plays a leading role in nanomedicine during sonodynamic therapy, berberine and lanthanum ions play an auxiliary role. Therefore, when berberine hydrochloride solution, tetraphenylporphyrin tetrasulfonic acid solution and lanthanum nitrate solution are fed at 2:1:1, the loading rates of tetraphenylporphyrin tetrasulfonic acid and berberine are high. In addition, at this feed ratio, the nanomedicine particle size is about 114.75 nm, the PDI is 0.102, and the potential is -14.5 mV, indicating that the drug particle size is small and evenly dispersed, which can better meet the treatment needs.

[0057] The fully active nanoparticles (La-TPPS-BBR) provided in the present invention have a particle size of about 120 nm, have ultra-high sonodynamic performance and good anti-biofilm performance, and have great potential for treating drug-resistant bacterial biofilm infections; an application of a fully active nanomedicine, which is used to prepare a drug for treating pneumonia infected with bacterial biofilms, and under ultrasonic irradiation, precisely releases the drug in the biofilm microenvironment, targets to break the bacterial intracellular drug delivery barrier, kills bacterial cells, and efficiently inhibits bacterial quorum sensing, so as to programmatically combat drug-resistant bacterial biofilms step by step; further, when used as a drug, it is prepared into a preparation for aerosol inhalation, providing an ultrasonically controllable, safe and harmless bacterial pneumonia treatment platform, ensuring that the drug is maximally enriched in the lesion site, while avoiding the drug's systemic toxicity to the body.

[0058] Performance test

[0059] In an embodiment disclosed in the present invention, a method for preparing fully active nanoparticles with ultra-high sonodynamic performance is provided. Figure 1 The present invention is a flow chart of a method for preparing nanoparticles according to an embodiment of the present invention. Figure 2 A in the figure is a TEM image of the nanoparticles prepared according to this example. As can be seen from the figure, the nanoparticles are square in shape, uniform in size, and non-sticky. Figure 2 Figures B and C are the particle size diagram and potential diagram of the nanoparticles prepared according to this example. The particle size of the nanoparticles is about 120 nm and the potential is about -14.5 mV. The particle size and potential provide guarantees for their in vivo application.

[0060] Based on the nanoparticles prepared in the above examples, basic performance tests were carried out. Figure 3 Figures A and B in Figure 3 are the UV absorption curves and fluorescence spectra of nanoparticles (La-TPPS-BBR) with small molecules of berberine (BBR) and small molecules of tetraphenylporphyrin tetrasulfonic acid (TPPS). Figure 3 As shown in A and B, after the interaction, the absorption curve of berberine shows a red shift and the absorption curve of TPPS shows a blue shift. There is obvious absorption in the near-infrared light region and fluorescence quenching occurs.

[0061] Figure 4 A and B are the time-dependent and concentration-dependent ROS release diagrams of nanoparticles (La-TPPS-BBR) at the solution level. It can be seen from the figure that under ultrasound (1 MHz, 1.0 W / cm2, 50% air occupancy), the ROS release of nanoparticles (La-TPPS-BBR) reaches the highest level when ultrasound is applied for 5 min, and as the concentration increases, the ROS release increases. It also has good ultrasonic controllability, laying the foundation for in vivo application.

[0062] Figure 5 A in the middle is a picture of the anti-biofilm lung homogenate bacteria coating of nanoparticles (La-TPPS-BBR) at the in vivo level. Figure 5Figures B and C show the bacterial count and relative bacterial survival rate of the lung homogenate of nanoparticles (La-TPPS-BBR) against biofilm in vivo. As can be seen from the figure, on the third day, the mouse lungs were still severely infected. Compared with other groups, the Nano group and Nano+US group showed a certain therapeutic effect. On the 7th day after treatment, the number of colonies in the Saline group and the US group was the largest and there was no significant difference, indicating that ultrasound alone did not kill bacteria and had good biosafety. Compared with the Saline group, the colonies in the BBR group and the TPPS group decreased to a certain extent. In addition, the colony count of the Nano group further decreased, indicating that the drug played a significant antibacterial effect after nano-ization. Furthermore, in the Nano+US group, almost no bacterial colonies were observed, indicating that under ultrasonic irradiation, the nanodrug was effectively activated. While playing the role of sonodynamic antibacterial, the bacterial intracellular drug delivery barrier was broken by targeting lanthanum ions, and berberine effectively interfered with bacterial quorum sensing, so as to cooperate step by step and effectively fight against pneumonia induced by drug-resistant bacteria.

[0063] Figure 6 Figures A, B, and C are respectively blood routine data of the nanoparticle solution prepared according to the embodiments of the present disclosure against biofilm in mice. The number of white blood cells, lymphocytes, and neutrophils in the blood are commonly used indicators of inflammation in the body, which can reflect the therapeutic effect of nanoparticles. Figure 6 Analysis of A, B, and C showed that since the Saline group and the US group had no antibacterial activity, the pneumonia in these two groups of mice was the most severe, and the total number of white blood cells, lymphocytes, and neutrophils was the highest. Compared with the Saline group, the total number of lymphocytes, white blood cells, and neutrophils in the blood of mice in the BBR group, TPPS+US group, and Nano group decreased, while the total number of lymphocytes, white blood cells, and neutrophils in the blood of the Nano+US group decreased significantly. This shows that fully active nanomedicines can effectively treat bacterial pneumonia in mice induced by MDR E. coli under ultrasound activation after aerosol inhalation administration.

Claims

1. A fully active nanomedicine that synergistically and step by step combats bacterial biofilm infection, characterized in that: The berberine methanol solution, tetraphenylporphyrin tetrasulfonic acid aqueous solution and lanthanum nitrate aqueous solution are mixed and assembled in a molar ratio of 1~3:1:1~3, wherein the concentration of the berberine methanol solution is 20 mM, and the concentrations of the tetraphenylporphyrin tetrasulfonic acid aqueous solution and the lanthanum nitrate aqueous solution are both 10 mM.

2. The fully active nanomedicine for synergistically combating bacterial biofilm infection as claimed in claim 1, characterized in that: Lanthanum ions La in nanomedicine 3+ The loading rate of is 3.85%, the loading rate of berberine is 32.69%, and the loading rate of tetraphenylporphyrin tetrasulfonic acid is 63.46%.

3. The fully active nanomedicine for synergistically combating bacterial biofilm infection as claimed in claim 1, characterized in that: The bacteria were multidrug-resistant Escherichia coli.

4. The method for preparing a fully active nano drug for synergistically combating bacterial biofilm infection step by step as claimed in any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Dissolve berberine hydrochloride powder in methanol, and perform ultrasonic treatment in a water bath at room temperature for 1-5 min until berberine is completely dispersed, and the final concentration is 20 mM to obtain a berberine methanol solution; dissolve tetraphenylporphyrin tetrasulfonic acid powder and lanthanum nitrate powder in deionized water, respectively, and perform ultrasonic treatment in a water bath at room temperature for 1-5 min until the powder is completely dispersed, and the final concentration is 10 mM to obtain a tetraphenylporphyrin tetrasulfonic acid aqueous solution and a lanthanum nitrate aqueous solution; S2. The berberine hydrochloride solution, the tetraphenylporphyrin tetrasulfonic acid solution and the lanthanum nitrate solution are mixed in a molar ratio of 1-3:1:1-3, and then added dropwise into deionized water, wherein the volume of the deionized water is 100 times that of the tetraphenylporphyrin tetrasulfonic acid solution, and stirred at room temperature for 12-72 h at a speed of 100-1500 r / min to allow the compounds to assemble to obtain a crude fully active nanoparticle product, and then washed with deionized water 2-3 times to obtain a pure fully active nanoparticle mother liquor; S3. Centrifuge the fully active nanoparticle mother solution at 3000-15000 rpm at room temperature for 10-20 min, aspirate the supernatant, and the resulting precipitate is the fully active nanoparticle.

5. The use of the fully active nano drug prepared by the preparation method according to claim 4, characterized in that: It is used to prepare drugs for the treatment of pneumonia caused by bacterial biofilm infection, and under ultrasonic irradiation, it can precisely release drugs in the biofilm microenvironment, target breaking of bacterial intracellular drug delivery barriers, kill bacterial cells, and efficiently inhibit bacterial quorum sensing, thereby combating drug-resistant biofilms in a programmed and step-by-step manner.

6. The use according to claim 5, characterized in that: When used as a medicine, the drug is prepared into a preparation for aerosol inhalation.

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