Nanoparticles for targeted elimination of intracellular bacteria and preparation method thereof

Abstract

The present invention provides nanoparticles for targeted elimination of intracellular bacteria and a preparation method thereof. The nanoparticles are composed of a biodegradable nanoparticle matrix, a potassium ion-responsive linear polymer grafted onto the surface of the nanoparticle matrix, and a drug loaded into the pore structure of the nanoparticle matrix. The nanoparticle matrix has a pore structure, the drug is a drug for treating bacterial infection, and the potassium ion-responsive linear polymer can achieve conformational transition and charge reversal after recognizing and responding to potassium ions. The nanoparticles are taken into the cells through the endocytosis of macrophages. In the high potassium ion concentration environment within the cells, the potassium ion-responsive linear polymer on the surface of the nanoparticles complexes with potassium ions, causing the nanoparticles, which are negatively charged in the extracellular fluid, to undergo charge reversal within the cells, thereby inducing the nanoparticles to target and bind to the intracellular bacteria, while simultaneously undergoing conformational transition and releasing the loaded drug.

Description

Technical Field

[0001] The invention belongs to the field of materials for resisting intracellular bacterial infection, and relates to nanoparticles for targeted elimination of intracellular bacteria and a preparation method thereof. Background Art

[0002] When bacteria invade the body, immune cells rapidly engulf, digest, and kill the bacteria. However, some bacteria can inhibit the fusion of lysosomes and phagosomes, allowing them to escape the phagosome and enter the cytoplasm, where they can survive within the host cell and form intracellular bacteria. Intracellular bacteria are commonly found in phagocytes such as macrophages, neutrophils, and dendritic cells. Common intracellular bacteria include bacteria, Escherichia coli, and Mycobacterium tuberculosis. Intracellular bacteria can evade the immune system and the bactericidal effects of most drugs, leading to recurrence of infectious diseases and the development of chronic infections. However, due to the protection of the host cell outer membrane and the complex intracellular environment, drugs exhibit disadvantages such as poor penetration, low intracellular accumulation, easy inactivation, and different distribution from intracellular pathogens. As a result, most drugs have poor efficacy against intracellular bacterial infections. Clinically, to treat infectious diseases caused by intracellular bacteria, long-term and high-dose medications are often required, which can lead to toxic side effects and the development of drug resistance. Therefore, the development of drug delivery systems that can efficiently deliver drugs to the cell interior and target intracellular pathogens for drug release is urgent.

[0003] Nanoparticle drug carriers, such as micelles, liposomes, and inorganic nanoparticles, can enter macrophages via endocytosis. Combined with controlled-release switches that respond to the intracellular microenvironment, these drugs can be effectively delivered into the cell, promoting intracellular accumulation and thereby improving drug bioavailability and reducing toxic side effects. However, the distribution of drugs within the cell differs from that of pathogens, preventing some drugs from reaching the subcellular structures where pathogens reside, leading to ineffective intracellular killing of pathogens. In response to this, researchers have recently developed a variety of nanoparticle drug carriers that target intracellular bacteria, aiming to increase drug concentrations in the surrounding environment and thereby enhance their anti-intracellular activity. Among these, bacterial targeting strategies based on electrostatic interactions have attracted considerable attention due to their simple principles and ease of implementation. Under physiological conditions, the teichoic acid in the bacterial cell wall contains numerous highly acidic phosphate groups, resulting in a more negatively charged bacterial surface than that of mammalian cells. Strong electrostatic interactions between positively charged nanoparticles and bacteria induce the accumulation of positively charged nanoparticles on the bacterial surface, achieving targeted action. However, positively charged nanoparticles also have strong interactions with proteins in the blood, which can affect the stability of the nanoparticles in the body. Currently, some researchers have developed nanodrug carriers with charge reversal capabilities to solve the above problems. However, to achieve controlled drug release and charge reversal at the same time, two functional units are usually required or the nanocarrier is modified in two or more steps, which is a cumbersome operation process. Therefore, the development of nanodrug carriers for the treatment of intracellular bacterial infections based on electrostatic interactions remains challenging. Summary of the Invention

[0004] To address the problem that nanocarriers for treating intracellular bacterial infections find it difficult to simultaneously achieve controlled intracellular drug release and charge reversal, the present invention provides a nanoparticle for targeted elimination of intracellular bacteria and a method for preparing the nanoparticle, thereby improving the bioavailability of the drug while enhancing the therapeutic effect on intracellular bacterial infections and simplifying the preparation process.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is as follows:

[0006] Nanoparticles for targeted elimination of intracellular bacteria, the nanoparticles comprising a biodegradable nanoparticle matrix, a potassium ion-responsive linear polymer grafted onto the surface of the nanoparticle matrix, and a drug loaded into the pore structure of the nanoparticle matrix; the nanoparticle matrix having a pore structure; the drug being a drug for treating bacterial infection; and the potassium ion-responsive linear polymer being able to undergo conformational transition and charge reversal after recognizing and responding to potassium ions;

[0007] The nanoparticles are taken into the cells through the endocytosis of macrophages. In the high potassium ion concentration environment inside the cells, the potassium ion-responsive linear polymer complex potassium ions located on the surface of the nanoparticles cause the negatively charged nanoparticles in the extracellular fluid to undergo charge reversal inside the cells, inducing the nanoparticles to target and bind to the bacteria inside the cells, while undergoing conformational change and releasing the loaded drugs.

[0008] In the technical solution for the nanoparticles for targeted removal of intracellular bacteria, the nanoparticle matrix should have the ability to load drugs, good biocompatibility, biodegradability, and modifiability to achieve drug loading and controllable modification of potassium ion-responsive linear polymers on the nanoparticle matrix. The modifiability means that the surface of the nanoparticle matrix should have the ability to graft potassium ion-responsive linear polymers. For example, when the surface of the nanoparticle matrix has a large number of hydroxyl groups, the surface of the nanoparticle matrix can have the ability to graft potassium ion-responsive linear polymers. On the basis of meeting these requirements, the nanoparticle matrix can be selected from nanomaterials with a negative surface charge and capable of grafting potassium ion-responsive linear polymers. Furthermore, the nanoparticle matrix can be selected from nanomaterials with a negative surface charge, hydroxyl groups on the surface, and a porous structure. For example, the nanoparticle matrix can be selected from any one of mesoporous silica nanoparticles, mesoporous titanium dioxide nanoparticles, carbon nanotubes, and composite gold nanoparticles. The specific selection can be made according to the actual application requirements.

[0009] In the above-mentioned technical solution for nanoparticles for targeted removal of intracellular bacteria, the potassium ion-responsive linear polymer is obtained by free radical polymerization of N-isopropylacrylamide monomer, benzo-18-crown-6-acrylamide monomer and coupling agent monomer according to a molar ratio of 1:(0.05~0.30):(0.03~0.10).

[0010] In the above-mentioned technical solution for nanoparticles for targeted removal of intracellular bacteria, a feasible method for preparing potassium ion-responsive linear polymers is as follows:

[0011] N-isopropylacrylamide monomer, benzo-18-crown-6-acrylamide monomer, coupling agent monomer and thermal initiator are added to solvent A and stirred thoroughly until the components are dissolved. Nitrogen gas is passed through the resulting mixture under stirring to remove oxygen from the mixture. The mixture is then transferred to a sealed container and reacted at 65-80°C for 16-36 hours. The potassium ion-responsive linear polymer is separated and purified from the resulting reaction solution by a precipitation method.

[0012] Furthermore, the potassium ion response characteristics of the potassium ion responsive linear polymer are affected by the ratio between the N-isopropylacrylamide monomer, the benzo-18-crown-6-acrylamide monomer and the coupling agent monomer, the reaction temperature and the reaction time, etc. In practical applications, the above factors can be flexibly adjusted according to the potassium ion response characteristics of the potassium ion responsive linear polymer. Preferably, when preparing the potassium ion responsive linear polymer, the molar ratio of the N-isopropylacrylamide monomer to the initiator is controlled to be 100: (0.03-0.5), the molar ratio of the benzo-18-crown-6-acrylamide monomer to the N-isopropylacrylamide monomer is 100: (5-30), and the molar ratio of the coupling agent monomer to the N-isopropylacrylamide monomer is (3-10): 100.

[0013] Furthermore, when preparing a potassium ion-responsive linear polymer, N-isopropylacrylamide monomer, benzo-18-crown-6-acrylamide monomer, coupling agent monomer and thermal initiator are added to solvent A and thoroughly stirred and mixed until the components are dissolved in a mixed solution, and the concentration of N-isopropylacrylamide monomer is 0.1 to 0.5 mol / L.

[0014] When preparing a potassium ion-responsive linear polymer, the potassium ion-responsive linear polymer is separated and purified from the resulting reaction solution by a precipitation method. The operation can be as follows: the resulting reaction solution is concentrated until insoluble matter is just produced, solvent A is added to the concentrated reaction solution to just dissolve the insoluble matter, the resulting solution is dropwise added to a stirred solvent B for precipitation, filtered to obtain a filter cake, and then the aforementioned dissolution with solvent A, precipitation with solvent B, and filtration are repeated 2 to 4 times. The resulting filter cake is dried to obtain the potassium ion-responsive linear polymer.

[0015] When preparing potassium ion-responsive linear polymers, the solvent A is tetrahydrofuran, N',N-dimethylformamide (DMF) or 1,4-dioxane, and the solvent B is anhydrous ether, methanol, petroleum ether or n-hexane.

[0016] When preparing potassium ion responsive linear polymer, the initiator is azobisisobutyronitrile, azobisisoheptanenitrile or dimethyl azobisisobutyrate.

[0017] In the aforementioned technical solution for nanoparticles for targeted removal of intracellular bacteria, the coupling agent monomer contains a double bond that can provide the necessary free radical polymerization and a coupling group that can be grafted onto the surface of the nanoparticle substrate. The specific coupling agent monomer selected is related to the choice of nanoparticle substrate. Silane coupling agents are particularly effective at forming a covalent bonding layer between inorganic and organic interfaces, particularly when the interface contains a large number of hydroxyl groups. Therefore, silane coupling agents containing carbon-carbon double bonds are a viable coupling agent monomer.

[0018] When the nanoparticle matrix includes mesoporous silica nanoparticles, mesoporous titanium dioxide nanoparticles, carbon nanotubes, or composite gold nanoparticles, the surface of these nanoparticle matrices inherently contains a large number of hydroxyl groups or easily generates a large number of hydroxyl groups after simple treatment. Therefore, for these nanoparticle matrices, silane coupling agents containing carbon-carbon double bonds are all viable coupling agent monomers. For example, 3-(triethoxysilyl)propyl)methacrylamide is a viable silane coupling agent monomer. When the coupling agent monomer used is 3-(triethoxysilyl)propyl)methacrylamide, the structure of the resulting potassium ion-responsive linear polymer is shown in Formula (I):

[0019]

[0020] In formula (I), x / y / z = (0.05 to 0.30) / 1 / (0.03 to 0.10).

[0021] In the aforementioned technical solution for nanoparticles for targeted elimination of intracellular bacteria, the mass ratio of the potassium-responsive linear polymer grafted onto the surface of the nanoparticle matrix to the nanoparticle matrix can be determined based on the desired potassium ion responsiveness of the nanoparticles in practical applications. Typically, during preparation, the potassium ion responsiveness of the nanoparticles can be adjusted by controlling the mass ratio of the potassium-responsive linear polymer to the drug-loaded nanoparticle matrix to a ratio of (0.5-4):1.

[0022] In the aforementioned technical solution for nanoparticles for targeted elimination of intracellular bacteria, the mass ratio of the drug loaded into the nanoparticle matrix's pore structure to the nanoparticle matrix itself is determined based on the dosage requirements for actual application, with the principle of achieving the desired anti-intracellular bacterial effect while minimizing cumulative toxicity. Typically, during preparation, the amount of drug loaded into the nanoparticle matrix's pore structure can be adjusted by controlling the drug-to-nanoparticle matrix mass ratio to a range of 0.05 to 8:1.

[0023] In the above-mentioned technical solution of nanoparticles for targeted elimination of intracellular bacteria, the drug is a drug used to treat bacterial infection. The specific type of drug can be selected according to actual application requirements. For example, common drugs used to treat bacterial infections include vancomycin, teicoplanin, tigecycline, rifampicin, gentamicin, levofloxacin, erythromycin and penicillin G.

[0024] The present invention also provides a method for preparing the above-mentioned nanoparticles for targeted elimination of intracellular bacteria, comprising the following steps:

[0025] (1) dispersing the nanoparticle matrix in solvent C, adding the drug, and mixing uniformly to obtain a mixed solution A, which is fully shaken or stirred to load the drug into the pore structure of the nanoparticle matrix, and then washed and dried to obtain the drug-loaded nanoparticle matrix;

[0026] (2) The drug-loaded nanoparticle matrix is ​​dispersed in solvent D, and a potassium ion-responsive linear polymer is added and mixed evenly to obtain a mixed solution B. The mixture is fully shaken or stirred to allow the potassium ion-responsive linear polymer to be grafted onto the surface of the drug-loaded nanoparticle matrix. The nanoparticle surface is then washed and dried to obtain nanoparticles for targeted removal of intracellular bacteria.

[0027] In the technical solution of the above-mentioned method for preparing nanoparticles for targeted removal of intracellular bacteria, step (2) preferably controls the addition mass ratio of the potassium ion-responsive linear polymer to the drug-loaded nanoparticle matrix to (0.5-4):1.

[0028] In the technical solution of the above-mentioned method for preparing nanoparticles for targeted removal of intracellular bacteria, in step (1), the mass ratio of the drug to the nanoparticle matrix is ​​preferably controlled to be (0.05-8):1.

[0029] In the technical solution of the above-mentioned method for preparing nanoparticles for targeted removal of intracellular bacteria, in step (1), the concentration of the drug in the mixed solution A is preferably controlled to be 1 to 20 mg / mL, and in step (2), the concentration of the potassium ion-responsive linear polymer in the mixed solution B is preferably controlled to be 5 to 40 mg / mL.

[0030] In the technical solution of the above-mentioned method for preparing nanoparticles for targeted elimination of intracellular bacteria, the solvent C can be deionized water, ethanol or a phosphate balanced salt solution, and the solvent D is determined according to the drug solubility and the reaction characteristics of the coupling group in the potassium ion-responsive linear polymer. For example, the solvent D can be deionized water, ethanol, methanol, acetone or tetrahydrofuran.

[0031] In the technical solution of the above-mentioned method for preparing nanoparticles for targeted removal of intracellular bacteria, step (1) is preferably carried out under oscillation conditions of 20 to 40°C and a rotation speed of 50 to 200 rpm, and step (2) is preferably carried out under oscillation conditions of 20 to 40°C and a rotation speed of 50 to 200 rpm.

[0032] The principle of the nanoparticles for targeted elimination of intracellular bacteria provided by the present invention to exert their anti-intracellular bacteria effect is as follows: the nanoparticles provided by the present invention are used by injection. After injection, they circulate throughout the body through the blood. The nanoparticles can be taken up into the cells by phagocytes containing bacteria through endocytosis. After the nanoparticles respond to the high concentration of potassium ions inside the cells, the potassium ion-responsive linear polymers located on the surface of the nanoparticles undergo conformational transition and charge reversal, so that the nanoparticles with negative charge in the extracellular fluid become positively charged inside the cells. On the one hand, the positively charged nanoparticles can be attracted by the bacteria with negative surface charge and target the bacteria inside the cells and bind to the bacteria inside the cells. On the other hand, the conformational transition and charge reversal of the potassium ion-responsive linear polymers can realize the release of the drugs loaded in the nanoparticles. The nanoparticles target bacteria and release drugs, which can effectively increase the drug concentration around the bacteria, thereby achieving a better therapeutic effect against intracellular bacterial infection. That is, the nanoparticles provided by the present invention can efficiently deliver drugs to the inside of cells, then target the bacteria inside the cells and release drugs, increasing the drug concentration in the environment surrounding the bacteria inside the cells, thereby better resisting intracellular bacterial infection. The potassium ion-responsive linear polymer described in the present invention achieves conformational transition and charge change based on the principle that the poly(N-isopropylacrylamide) linear polymer chain possesses both hydrophilic amide groups and hydrophobic isopropyl groups. At low temperatures, the amide groups form stable hydrogen bonds with water molecules, extending the polymer chain, resulting in a clear solution and high solution transmittance. At high temperatures, the hydrogen bonding weakens, destroying the hydration layer of the poly(N-isopropylacrylamide) linear polymer chain. The hydrophobic effect of the isopropyl groups becomes dominant, and the polymer chain undergoes a conformational transition from an extended state to a curled state due to hydrophobic association, leading to phase separation and a decrease in solution transmittance. When benzo-18-crown-6-acrylamide monomers are introduced into the linear polymer chain of poly(N-isopropylacrylamide), due to the large ring cavity structure of benzo-18-crown-6-acrylamide monomers, positively charged host-guest complexes with certain stability can be formed through the ion-dipole supramolecular interaction between the lone pair electrons of the oxygen-containing groups in the cavity and the ions, as well as the size effect, thereby affecting the conformational transition behavior and charge density of the linear polymer.

[0033] Compared with the prior art, the technical solution provided by the present invention produces the following beneficial technical effects:

[0034] 1. The present invention provides a nanoparticle for targeted elimination of intracellular bacteria. The nanoparticle is composed of a biodegradable nanoparticle matrix, a potassium ion-responsive linear polymer grafted onto the surface of the nanoparticle matrix, and a drug loaded into the pore structure of the nanoparticle matrix. The nanoparticle matrix has a pore structure, the drug is a drug for treating bacterial infection, and the potassium ion-responsive linear polymer can undergo conformational transition and charge reversal after recognizing and responding to potassium ions. The nanoparticle is taken up into the cell through endocytosis by macrophages. In the high potassium ion concentration environment inside the cell, the potassium ion-responsive linear polymer on the surface of the nanoparticle complexes with potassium ions, causing the nanoparticle, which is negatively charged in the extracellular fluid, to undergo charge reversal inside the cell, inducing the nanoparticle to target and bind to the intracellular bacteria, undergoing conformational transition and releasing the loaded drug. The nanoparticle provided by the present invention can efficiently deliver the drug into the cell, then target the intracellular bacteria and release the drug, thereby increasing the drug concentration in the environment surrounding the intracellular bacteria, thereby improving the bioavailability of the drug and enhancing the therapeutic effect of intracellular bacterial infection.

[0035] 2. The present invention has confirmed through antibacterial experiments that the nanoparticles provided by the present invention for targeted elimination of intracellular bacteria have excellent anti-intracellular bacterial ability, which can effectively solve the problems existing in the current intracellular bacteria treatment methods such as low intracellular drug accumulation and different distribution of drugs and intracellular pathogens, thereby achieving improved drug bioavailability and enhanced therapeutic effects of intracellular bacterial infections.

[0036] 3. The present invention also provides a method for preparing the aforementioned nanoparticles for targeted clearance of intracellular bacteria. This method utilizes the specific effect of crown ethers on potassium ions to prepare potassium-responsive linear polymers that can respond to potassium ions and achieve conformational transitions and charge reversals at physiological temperatures. This potassium-responsive linear polymer is grafted onto the surface of a drug-loaded nanoparticle matrix, endowing the drug-loaded nanoparticle matrix with the ability to target bacteria within cells and controlled drug release. Furthermore, the nanoparticles are negatively charged in the extracellular fluid, making them less likely to generate strong electrostatic interactions with proteins in the blood, which helps increase their stability in the blood. While prior art methods for constructing drug-loaded nanomaterials that are negatively charged in the extracellular fluid and have charge reversal capabilities often require two functional units or two or more steps of modification of the nanocarrier, resulting in a cumbersome process. Compared to these prior art methods, the present invention achieves controlled drug release and charge reversal within and outside the cell through a single modification step and a single functional monomer. This method is simple, requires mild conditions, and is highly effective, facilitating widespread application of the preparation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1This is the synthesis route of the potassium ion responsive linear polymer in Examples 1 to 6.

[0038] Figure 2 yes Figure 2 These are infrared spectra of the linear polymers, 3-MAAPTS monomers, and B18C6Am monomers prepared in Examples 1 to 6 and Comparative Examples 1 to 2.

[0039] Figure 3 These are the hydrogen nuclear magnetic spectra of the linear polymers prepared in Examples 1 to 6 and Comparative Examples 1 to 2.

[0040] Figure 4 These are the curves showing changes in transmittance of the linear polymers prepared in Examples 1 to 6 and Comparative Examples 1 to 2 as a function of temperature under different environments.

[0041] Figure 5 These are the ΔLCST calculation results of the linear polymers prepared in Examples 1 to 6 and Comparative Examples 1 to 2.

[0042] Figure 6 Schematic diagram of the preparation process of nanoparticles in Example 9.

[0043] Figure 7 Graph showing the particle size distribution of the nanoparticles prepared in Example 9 and Comparative Examples 3 and 5.

[0044] Figure 8 Graph showing the pore size distribution of the nanoparticles prepared in Example 9 and Comparative Examples 3 and 5.

[0045] Figure 9 1 and 2 are the drug release curves of the nanoparticles prepared in Example 9 and Comparative Example 5 in simulated intracellular fluid (s-ICF) and simulated extracellular fluid (s-ECF).

[0046] Figure 10 These are the Zeta potential test results of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5 in s-ICF and s-ECF.

[0047] Figure 11 These are the test results of the ability of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5, as well as Van, to inhibit bacterial growth in s-ICF and s-ECF.

[0048] Figure 12 3 are scanning electron micrographs of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5 targeting bacteria in s-ICF and s-ECF.

[0049] Figure 13 These are the test results of the anti-intracellular bacterial ability of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5. DETAILED DESCRIPTION

[0050] The following examples further illustrate the nanoparticles for targeted elimination of intracellular bacteria provided by the present invention and their preparation methods. It should be noted that the following examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by persons skilled in the art based on the above disclosure and implemented in accordance with the present invention remain within the scope of protection of the present invention.

[0051] In the following examples and comparative examples, mesoporous silica nanoparticles (MSN) were synthesized according to the method described in Nano Lett 2014, 14, 923-32. Benzo-18-crown-6-acrylamide monomer was synthesized according to the method described in Journal of Hazardous Materials 2021, 404, 124-157. N-isopropylacrylamide (NIPAM) was recrystallized from n-hexane / acetone before use. The coupling agent 3-(triethoxysilyl)propyl)methacrylamide (3-MAAPTS) monomer and other experimental materials were purchased.

[0052] Example 1

[0053] In this embodiment, according to Figure 1 The synthetic route shown is used to prepare a potassium ion-responsive linear polymer, namely poly(N-isopropylacrylamide-co-benzo-18-crown-6-acrylamide-co-N-(3-(triethoxysilyl)propyl)methacrylamide) (P(NIPAM-co-B18C6Am-co-3-MAAPTS), PNBM), the structure of which is shown in formula (I):

[0054]

[0055] In formula (I), x / y / z = 0.05 / 1 / 0.05. The specific synthesis steps are:

[0056] (1) N-isopropylacrylamide (NIPAM) monomer, benzo-18-crown-6-acrylamide (B18C6Am) monomer, coupling agent N-(3-(triethoxysilyl)propyl)methacrylamide (3-MAAPTS) monomer and initiator azobisisobutyronitrile (AIBN) were added to tetrahydrofuran (THF) solvent and stirred thoroughly until the components were fully dissolved; then, nitrogen was passed through the obtained mixture for 30 minutes under stirring to remove oxygen in the mixture; then, the mixture was transferred to a sealed container and reacted at 70°C for 24 hours to obtain a reaction solution.

[0057] In this step, the molar ratio of NIPAM monomer to AIBN was controlled to be 100:0.5, the molar ratio of B18C6Am monomer to NIPAM monomer was controlled to be 5:100, the molar ratio of 3-MAAPTS monomer to NIPAM monomer was controlled to be 5:100, and the concentration of NIPAM monomer in the mixed solution was controlled to be 0.2 mol / L.

[0058] (2) The reaction solution obtained in step (1) was concentrated until insoluble matter was just produced. THF was added to the concentrated reaction solution to just dissolve the insoluble matter. The resulting solution was added dropwise to anhydrous ether in a stirring state for precipitation. The solution was filtered to obtain a filter cake. The above-mentioned dissolution with THF, precipitation with anhydrous ether, and filtration were repeated three times. The filter cake was dried to obtain P(NIPAM-co-B18C6Am-co-3-MAAPTS), which was recorded as PNBM-5%.

[0059] Example 2

[0060] In this example, a potassium ion-responsive linear polymer was prepared using the same procedure as in Example 1, except that the molar ratio of the B18C6Am monomer to the NIPAM monomer added in step (1) was 10:100. The resulting potassium ion-responsive linear polymer was designated PNBM-10%.

[0061] Example 3

[0062] In this example, a potassium ion-responsive linear polymer was prepared using the same procedure as in Example 1, except that the molar ratio of the B18C6Am monomer to the NIPAM monomer added in step (1) was 15:100. The resulting potassium ion-responsive linear polymer was designated PNBM-15%.

[0063] Example 4

[0064] In this example, a potassium ion-responsive linear polymer was prepared using the same procedure as in Example 1, except that the molar ratio of the B18C6Am monomer to the NIPAM monomer added in step (1) was 20:100. The resulting potassium ion-responsive linear polymer was designated PNBM-20%.

[0065] Example 5

[0066] In this example, a potassium ion-responsive linear polymer was prepared using the same procedure as in Example 1, except that the molar ratio of the B18C6Am monomer to the NIPAM monomer added in step (1) was 25:100. The resulting potassium ion-responsive linear polymer was designated PNBM-25%.

[0067] Example 6

[0068] In this example, a potassium ion-responsive linear polymer was prepared using the same procedure as in Example 1, except that the molar ratio of the B18C6Am monomer to the NIPAM monomer added in step (1) was 30:100. The resulting potassium ion-responsive linear polymer was designated PNBM-30%.

[0069] Comparative Example 1

[0070] In this comparative example, the P(NIPAM) linear polymer was prepared by the same operation as in Example 1, except that the B18C6Am monomer and the 3-MAAPTS monomer were not added in step (1). The obtained P(NIPAM) linear polymer was referred to as PNM.

[0071] Comparative Example 2

[0072] In this comparative example, the linear polymer P(NIPAM-co-B18C6Am) was prepared by the same operation as in Example 1, except that no 3-MAAPTS monomer was added in step (1). The obtained linear polymer P(NIPAM-co-B18C6Am) was designated as PNBM-0%.

[0073] Example 7

[0074] In this example, the chemical compositions of the linear polymers prepared in Examples 1 to 6 and Comparative Examples 1 to 2 were examined.

[0075] Figure 2 IR spectra of the linear polymers, 3-MAAPTS monomers, and B18C6Am monomers prepared in Examples 1 to 6 and Comparative Examples 1 to 2; Figure 3 These are the hydrogen nuclear magnetic spectra of the linear polymers prepared in Examples 1 to 6 and Comparative Examples 1 to 2.

[0076] The infrared spectrum of PNM prepared in Comparative Example 1 is as follows Figure 2 As shown in the curve PNM, at 1383.70 and 1364.90 cm -1 A double peak of bending vibration of -CH3 in the isopropyl group of the NIPAM monomer appeared at , and the above characteristic peaks appeared in the infrared spectrum of PNM, indicating that PNM was prepared in Comparative Example 1.

[0077] The infrared spectra of PNBM-0%, B18C6Am monomer and 3-MAAPTS monomer prepared in Comparative Example 2 are as follows: Figure 2 As shown, at 1383.70, 1364.90cm -1 The bending vibration doublet of -CH3 in the isopropyl group of NIPAM monomer appeared at 782.49 cm -1The peak at belongs to the characteristic peak of the symmetrical stretching vibration peak of Si-O in the 3-MAAPTS structure. The above characteristic peak appears in the infrared spectrum of PNBM-0%, indicating that PNBM-0% was prepared in Comparative Example 2.

[0078] The infrared spectra of PNBM-5%, PNBM-10%, PNBM-15%, PNBM-20%, PNBM-25% and PNBM-30% prepared in Examples 1 to 6 are as follows: Figure 2 As shown, at 1383.70, 1364.90cm -1 The bending vibration doublet of -CH3 in the isopropyl group of NIPAM monomer appeared at 782.49 cm -1 The peaks at 1510.01, 1219.29, and 1132.51 cm-1 belong to the characteristic peaks of the symmetrical stretching vibration of Si-O in the 3-MAAPTS structure. -1 The characteristic peaks of B18C6Am appeared at the bottom, which are the stretching vibration peak of the C=C skeleton of the benzene ring in the B18C6Am structure, the asymmetric stretching vibration peak of CO in Ar-CO, and the stretching vibration peak of CN in -CONH-. The intensity of the characteristic absorption peak of B18C6Am in the PNBM linear polymer spectrum increases with the increase of the addition amount of B18C6Am. In addition, from Figure 3 The H-NMR spectra of the linear polymers prepared in Examples 1-6 reveal a characteristic peak for NIPAM at a chemical shift of 1.05 ppm, a characteristic peak for B18C6Am at a chemical shift of 6.70-7.70 ppm, and a characteristic peak for 3-MAAPTS at a chemical shift of 0.55 ppm. This further demonstrates that B18C6Am, 3-MAAPTS, and NIPAM were successfully copolymerized via thermally initiated free radical polymerization, resulting in a series of PNBM linear polymers with varying B18C6Am contents prepared in Examples 1-6.

[0079] Example 8

[0080] In this example, the thermal response phase transition behaviors of the linear polymers prepared in Examples 1 to 6 and Comparative Examples 1 to 2 in different solutions were investigated.

[0081] PNM, PNBM-0%, PNBM-5%, PNBM-15%, PNBM-20%, PNBM-25% and PNBM-30% were dissolved in deionized water, simulated extracellular fluid (s-ECF) and simulated intracellular fluid (s-ICF) respectively to prepare solutions with a concentration of 10 mg / mL. The curves of the permeability of each group of solutions as a function of temperature were measured. The results are shown in Figure 2. Figure 4The lower critical solution temperature (LCST) was calculated from the curve of transmittance versus temperature. In order to evaluate the degree of LCST migration, △LCST was defined as the difference between the LCST value of the same linear polymer in s-ICF and the LCST value in s-ECF at the same concentration. The results are shown in the figure. Figure 5 shown.

[0082] Depend on Figures 4-5 As shown, the transmittance of both PNM and PNBM linear polymers decreases significantly with increasing temperature in deionized water, s-ECF, and s-ICF. This is attributed to the temperature-responsiveness of the PNM backbone. When the PNM linear polymer is in s-ECF or s-ICF, its LCST shifts toward lower temperatures due to the salt effect. When only 3-MAAPTS is introduced into the PNIPAM linear polymer chain, the LCST shifts toward higher temperatures in low ion concentration solutions, while in s-ECF or s-ICF, the LCST shifts toward lower temperatures. Simultaneous introduction of B18C6Am and 3-MAAPTS monomers into the PNBM polymer chain in PNBM-5%, PNBM-15%, PNBM-20%, PNBM-25%, and PNBM-30% polymers reveals that the pendant macrocyclic cavities complex with metal ions, altering the polymer's charge density and other physicochemical properties, which in turn influence the extent of the LCST shift of the PNBM linear polymer. In s-ECF and s-ICF, K + The difference in concentration also leads to differences in the electrostatic repulsion between polymer chains in s-ECF and s-ICF. As the molar ratio of B18C6Am increases, the difference in electrostatic repulsion gradually increases. In general, the PNBM linear polymers prepared by the method of the present invention have good temperature responsiveness and K + The phase change behavior of the catalyst can be flexibly controlled by adjusting the amount of B18C6Am added.

[0083] Example 9

[0084] In this example, mesoporous silica nanoparticles loaded with antibiotics and grafted with PNBM linear polymers on the surface were prepared, denoted as Van@MSN-PNBM. The nanoparticles are nanoparticles used for targeted removal of intracellular bacteria. The preparation process is as follows: Figure 6 As shown, the specific preparation steps are:

[0085] (1) Mesoporous silica nanoparticles (MSNs) were dispersed in 0.2 mol / L phosphate buffer, and an appropriate amount of vancomycin (Van) was added and mixed evenly. The concentration of Van in the mixture was controlled to be 5 mg / mL, and the mass ratio of Van to MSN was 1. The mixture was shaken at 150 rpm at 25°C to load Van into the MSNs. The mixture was then centrifuged, washed, and dried to obtain Van-loaded MSNs.

[0086] (2) Van-loaded MSNs were dispersed in ethanol, and PNBM-15% prepared in Example 3 was added and mixed uniformly. The concentration of PNBM-15% in the resulting mixture was controlled to be 20 mg / mL, and the mass ratio of PNBM-15% to Van-loaded MSNs was 2. The mixture was shaken at 150 rpm at 25°C to graft PNBM-15% onto the surface of Van-loaded MSNs. The mixture was then centrifuged, washed, and dried to obtain Van@MSN-PNBM.

[0087] Comparative Example 3

[0088] In this comparative example, MSNs were prepared according to the method described in Nano Lett 2014, 14, 923-32.

[0089] Comparative Example 4

[0090] In this comparative example, the surface-grafted PNBM-15% linear polymer MSN prepared in Example 3 was prepared. The operation was basically the same as step (2) of Example 9, with the only difference being that the MSN prepared in Comparative Example 3 was used to replace the Van-loaded MSN in step (2) of Example 9, and the prepared nanoparticles were recorded as MSN-PNBM.

[0091] Comparative Example 5

[0092] In this comparative example, Van-loaded MSNs were prepared by the same operation as step (1) of Example 9, and the prepared nanoparticles were denoted as Van@MSN.

[0093] Example 10

[0094] In this example, the particle size distribution and pore size distribution of the nanoparticles Van@MSN-PNBM, MSN-PNBM and Van@MSN prepared in Example 9 and Comparative Examples 3 and 5 were investigated.

[0095] Figure 7 、 8 The particle size distribution and pore size distribution of the nanoparticles prepared in Example 9 and Comparative Examples 3 and 5 are shown respectively. Figure 7It can be seen that the particle sizes of the nanoparticles prepared in Example 9 and Comparative Examples 3 and 5 are between 60 and 90 nm. The nanoparticles prepared in Example 9 have the largest particle size due to the inclusion of Van and linear polymers. Figure 8 It can be seen that only the nanoparticles prepared in Comparative Example 3 have an obvious pore size distribution curve, with a pore size of about 7.6 nm. The pore size distribution of the nanoparticles prepared in Example 9 and Comparative Example 5 is not obvious, indicating that the nanoparticles are successfully loaded with Van.

[0096] Example 11

[0097] In this example, the potassium ion-responsive drug release behavior of the nanoparticles prepared in Example 9 and Comparative Example 5 in s-ICF and s-ECF was investigated.

[0098] The drug release behavior of the nanoparticles was investigated by dynamic dialysis. The specific operation was as follows: 6 mg of the nanoparticles prepared in Example 9 and Comparative Example 5 were accurately weighed and added to 1 mL of release medium (s-ICF or s-ECF), respectively. The resulting solutions were transferred into dialysis bags, respectively placed in 20 mL of release medium, and placed in an air bath shaking incubator at a temperature of 37°C and an oscillation speed of 100 rpm. At a specific time point, an appropriate amount of dialysate was removed and fresh release medium was added to maintain its total volume unchanged. The Van content in the dialysate was detected by ultraviolet spectrophotometer, and the drug release curve was drawn.

[0099] The drug release curves of the nanoparticles prepared in Example 9 and Comparative Example 5 in simulated intracellular fluid and simulated extracellular fluid are as follows: Figure 9 As shown. The drug release curves of Van@MSN prepared in Comparative Example 5 and Van@MSN-PNBM prepared in Example 9 in s-ICF and s-ECF showed the same trend. The drug release rate gradually decreased with the increase of time, and the drug release basically reached equilibrium after 36 hours. The cumulative release of Van@MSN in s-ICF and s-ECF after 48 hours were 32.82% and 30.92%, respectively, indicating that there was no significant difference in the drug release behavior of Van@MSN in s-ICF and s-ECF. Van@MSN-PNBM has good potassium ion response characteristics, and the cumulative release in s-ICF and s-ECF after 48 hours were 55.13% and 23.94%, respectively.

[0100] After PNBM grafting, the cumulative release of Van in s-ECF decreased by 6.98%. This is due to the hydrophobic nature of the PNBM polymer layer at 37°C, which hinders the diffusion of solutes and water molecules between the nanoparticle pores and the release medium, thereby reducing the release rate and cumulative release of Van. This suggests that the PNBM polymer layer protects Van within the nanoparticles during blood circulation, reducing premature drug leakage to a certain extent. However, after PNBM grafting, the cumulative release in s-ICF fluid increased by 22.31%. This is due to the hydrophilic nature of the PNBM polymer layer after complexing potassium ions in simulated intracellular fluid, allowing Van release. Furthermore, the electrostatic repulsion between the positively charged complex and Van cations accelerates Van dissolution and diffusion, resulting in an increase in both the release rate and cumulative release. This potassium-responsive drug release behavior ensures low drug leakage from nanoparticles and rapid intracellular drug release under human physiological conditions, thereby reducing drug toxicity and enhancing therapeutic efficacy.

[0101] Example 12

[0102] In this example, the ability and mechanism of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5 to inhibit bacterial growth in s-ICF and s-ECF were investigated.

[0103] Nanoparticle size and Zeta potential analyzers were used to determine the surface charge of the nanoparticles in s-ICF and s-ECF, respectively. The scattering angle of the test was 90° and the test temperature was 25°C. The growth curve method was used to investigate the ability of the nanoparticles to inhibit bacterial growth in s-ICF and s-ECF. The group in which the bacterial growth medium was the original culture medium without any drugs and nanoparticles was set as the control group (Control); the same mass of nanoparticles prepared in Comparative Example 3 or Comparative Example 4, or s-ECF or s-ICF solutions containing free Van with the same Van content and nanoparticles prepared in Example 9 and Comparative Example 5 were added to the experimental group. The interaction between bacteria and nanoparticles was observed after 2 hours of co-culture of bacteria and nanoparticles using the climbing slide method.

[0104] Figure 10 These are the Zeta potential test results of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5 in s-ICF and s-ECF. Figure 11 These are the test results of the ability of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5, as well as Van, to inhibit bacterial growth in s-ICF and s-ECF. Figure 12 3 are scanning electron micrographs of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5 targeting bacteria in s-ICF and s-ECF.

[0105] from Figure 10It can be seen that the Zeta potentials of MSN, MSN-PNBM, Van@MSN and Van@MSN-PNBM in s-ECF are -8.0, -4.6, -6.3 and -5.1 mV, respectively, and the Zeta potentials in s-ICF are -7.9, 0.5, -6.1 and 3.3 mV, respectively. In s-ICF, the linear polymers on the surface of the nanoparticles of MSN-PNBM and Van@MSN-PNBM form positively charged complexes with potassium ions, thus making the surface of the nanoparticles positively charged. Figure 11 It can be seen that MSN and MSN-PNBM have no significant effect on bacterial growth, indicating that they have no antibacterial activity. Free Van is completely unable to inhibit bacterial growth in s-ICF and s-ECF, while Van@MSN-PNBM exhibits the ability to inhibit the growth of some bacteria in s-ICF, while Van@MSN-PNBM exhibits better antibacterial activity. Figure 12 It can be seen that only MSN-PNBM and Van@MSN-PNBM with positive surface charge can bind to the bacterial cell wall and accumulate on the bacterial surface, thereby increasing the local Van concentration and achieving the purpose of sterilization.

[0106] Example 13

[0107] In this example, the ability of the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5 to resist intracellular bacteria was examined.

[0108] Macrophages are immune cells whose primary function is to phagocytose and digest cellular debris and pathogens in either fixed or free form, and to activate lymphocytes or other immune cells to respond to pathogens. When bacteria infect a host, they are generally first engulfed by phagocytes. However, if engulfment fails to kill the bacteria, it may actually contribute to their spread. Only by generating specific cellular immunity against the bacteria can intracellular parasites be gradually eliminated. Free drug molecules have a limited ability to enter infected cells, resulting in low bioavailability when used to treat intracellular bacterial infections. Van@MSN-PNBM, however, can enter phagocytes through endocytosis, deliver Van into the cell, and release Van in response to high intracellular potassium ion concentrations.

[0109] In order to verify the anti-intracellular bacterial activity of Van@MSN-PNBM, an infection model of mouse mononuclear macrophage leukemia cells (Raw264.7) infected with methicillin-resistant Staphylococcus aureus (MRSA) was established, and the bacterial-infected cells were incubated with the nanoparticles prepared in Example 9 and Comparative Examples 3 to 5, as well as free Van, for 24 hours. The Van contained in the nanoparticles in Example 9 and Comparative Example 5 was the same as the dose of free Van, and a control group (Control) was set as a drug-free culture medium. The survival of intracellular MRSA was then examined by the dilution plate method. The results are shown in Figure 2. Figure 13 As shown in Figure 3 . Counting results after co-culture with nanoparticles showed that MSN and MSN-PNBM had no significant antibacterial activity against intracellular MRSA, while Van@MSN and Van@MSN-PNBM exhibited good antibacterial activity against intracellular MRSA. Compared with the same dose of free Van, Van@MSN-PNBM exhibited significantly better antibacterial activity, demonstrating the superiority of MSN-PNBM as a drug carrier for the treatment of intracellular bacteria.

Claims

1. Nanoparticles for targeted elimination of intracellular bacteria, characterized in that: The nanoparticles are composed of a biodegradable nanoparticle matrix, a potassium ion-responsive linear polymer grafted onto the surface of the nanoparticle matrix, and a drug loaded into the pore structure of the nanoparticle matrix. The nanoparticle matrix has a pore structure, the drug is a drug for treating bacterial infections, and the potassium ion-responsive linear polymer can achieve conformational change and charge reversal after recognizing and responding to potassium ions. The nanoparticle matrix is ​​a nanomaterial with a negative surface charge, hydroxyl groups on the surface, and a porous structure. The nanoparticle matrix is ​​a mesoporous silica nanoparticle. The potassium ion responsive linear polymer is composed of N -isopropyl acrylamide monomer, benzo-18-crown-6-acrylamide monomer and coupling agent monomer are obtained by free radical polymerization according to a molar ratio of 1: (0.05-0.30): (0.03-0.10); the coupling agent monomer is 3-(triethoxysilyl)propyl)methacrylamide; The nanoparticles are taken into the cells through the endocytosis of macrophages. In the high potassium ion concentration environment inside the cells, the potassium ion-responsive linear polymer complex potassium ions located on the surface of the nanoparticles cause the negatively charged nanoparticles in the extracellular fluid to undergo charge reversal inside the cells, inducing the nanoparticles to target and bind to the bacteria inside the cells, while undergoing conformational change and releasing the loaded drugs.

2. The nanoparticles for targeted elimination of intracellular bacteria according to claim 1, characterized in that: The preparation method of the potassium ion responsive linear polymer is as follows: Will N -Isopropyl acrylamide monomer, benzo-18-crown-6-acrylamide monomer, coupling agent monomer, and thermal initiator were added to solvent A and thoroughly stirred until the components dissolved. Nitrogen was passed through the resulting mixture under stirring to remove oxygen from the mixture. The mixture was then transferred to a sealed container and reacted at 65-80°C for 16-36 hours. The potassium ion-responsive linear polymer was separated and purified from the resulting reaction solution by precipitation.

3. The method for preparing nanoparticles for targeted elimination of intracellular bacteria according to claim 1 or 2, characterized in that: The following steps are involved: (1) Dispersing the nanoparticle matrix in solvent C, adding the drug, and mixing uniformly to obtain a mixed solution A. Fully oscillating or stirring to load the drug into the pore structure of the nanoparticle matrix, and then washing and drying to obtain the drug-loaded nanoparticle matrix; (2) The drug-loaded nanoparticle matrix is ​​dispersed in solvent D, and potassium ion-responsive linear polymer is added and mixed evenly to obtain a mixed solution B. The mixture is fully shaken or stirred to allow the potassium ion-responsive linear polymer to be grafted onto the surface of the drug-loaded nanoparticle matrix. The nanoparticle surface is then washed and dried to obtain nanoparticles for targeted removal of intracellular bacteria.

4. The method for preparing nanoparticles for targeted elimination of intracellular bacteria according to claim 3, characterized in that: In step (2), the mass ratio of the potassium ion responsive linear polymer to the drug-loaded nanoparticle matrix is ​​controlled to be (0.5~4):

1.

5. The method for preparing nanoparticles for targeted elimination of intracellular bacteria according to claim 3, characterized in that: In step (1), the mass ratio of the drug to the nanoparticle matrix is ​​controlled to be (0.05~8):

1.

6. The method for preparing nanoparticles for targeted elimination of intracellular bacteria according to any one of claims 3 to 5, characterized in that: In step (1), the concentration of the drug in the mixed solution A is controlled to be 1 to 20 mg / mL; in step (2), the concentration of the potassium ion-responsive linear polymer in the mixed solution B is controlled to be 5 to 40 mg / mL.

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