Polymyxin B prodrug nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to polymyxin B prodrug nanoparticles as well as a preparation method and application thereof. The preparation method of the polymyxin B prodrug nanoparticles comprises the following steps: dissolving 4-(hydroxymethyl) phenylboronic acid pinacol ester in dichloromethane, adding carbonyl diimidazole, reacting at room temperature to obtain activated 4-(hydroxymethyl) phenylboronic acid pinacol ester, dissolving the activated 4-(hydroxymethyl) phenylboronic acid pinacol ester, polymyxin B and 4-dimethylaminopyridine in dimethyl sulfoxide, and reacting at room temperature to obtain a prodrug PPMB; the preparation method comprises the following steps: adding a lecithin solution, distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000-maleimide and distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000 into water, then dropwise adding a PPMB methanol solution, and reacting at room temperature to obtain the polymyxin B prodrug nanoparticles. The invention also provides the polymyxin B prodrug nanoparticles prepared by the preparation method and application of the polymyxin B prodrug nanoparticles. According to the invention, the problems of high in-vivo toxicity and insufficient targeting property of drugs are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbiology, and in particular, to a polymyxin B prodrug nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Pneumonia is a widespread respiratory disease that poses a serious threat to human health. Its causes are complex and diverse, and bacterial infection is one of the main inducing factors. In recent years, with the widespread use of antibiotics, the problem of bacterial drug resistance has become increasingly prominent. In particular, pneumonia caused by drug-resistant bacteria has brought great challenges to clinical treatment. Traditional antibiotics often fail to play an effective role when facing drug-resistant strains, resulting in poor treatment effects and significantly increased treatment difficulties, bringing heavy disease burdens and economic pressures to patients.

[0003] Among the many drugs used to treat pneumonia, polymyxin B is an important option. Polymyxin B is mainly used to treat infections caused by Gram-negative bacilli (such as Pseudomonas aeruginosa, Klebsiella pneumoniae, etc.), which are common pathogenic bacteria causing pneumonia. In addition, polymyxin B also has a certain therapeutic effect on infections with multi-drug resistant bacteria (such as carbapenem-resistant bacteria), which makes it of important clinical value in the treatment of drug-resistant bacterial infections. However, although polymyxin B plays a certain role in the treatment of pneumonia, its direct use in pneumonia treatment has some disadvantages and limitations, restricting its widespread application.

[0004] First of all, polymyxin B has significant nephrotoxicity. During use, it may cause proteinuria, cylindruria, azotemia, and even renal function damage. For patients with renal insufficiency, the use of polymyxin B requires close monitoring of renal function to avoid further aggravating kidney damage. This nephrotoxicity restricts the widespread application of polymyxin B in clinical practice, especially in patients with impaired renal function. In addition, polymyxin B may also cause neurotoxicity. Its neurotoxicity is manifested as dizziness, ataxia, lethargy, and peripheral sensory abnormalities, and may even lead to respiratory depression in severe cases. These adverse reactions not only affect the quality of life of patients but also increase the risk of treatment, further restricting the clinical application of polymyxin B.

[0005] Secondly, the targeting of polymyxin B is insufficient. During treatment, the drug is difficult to be accurately delivered to the infection site, resulting in insufficient drug concentration at the infection site and excessive drug distribution in other parts of the body, thereby increasing the drug's toxic and side effects. This problem of insufficient targeting makes it difficult for polymyxin B to fully exert its antibacterial effect in the treatment of pneumonia, and also restricts its application prospects in the treatment of drug-resistant bacterial infections.

[0006] Furthermore, the therapeutic effect of polymyxin B has certain limitations. For some multi-drug resistant bacteria (such as carbapenem-resistant Acinetobacter baumannii), the antibacterial efficacy of polymyxin B may be insufficient, resulting in poor therapeutic effects. Its therapeutic effect highly depends on the sensitivity of the pathogenic bacteria. If the bacteria are resistant to polymyxin B, the treatment may be ineffective. This dependence makes it difficult for polymyxin B to play a stable therapeutic role in the face of an increasingly complex drug-resistant bacterial environment.

[0007] Therefore, developing a new drug delivery system or therapeutic strategy that can overcome the existing drawbacks and limitations of polymyxin B has important clinical significance and market demand for reducing the drug's toxic side effects, improving targeting, and the therapeutic effect of drug-resistant bacterial pneumonia. Summary of the Invention

[0008] The object of the present invention is to provide a polymyxin B prodrug nanoparticle, its preparation method and application, aiming to improve the in vivo distribution characteristics of polymyxin B through a nano-drug delivery system. This invention focuses on overcoming the systemic toxic side effects such as renal toxicity and neurotoxicity caused by the inability of polymyxin B to effectively accumulate at the lesion site in the prior art, and at the same time overcoming the bottleneck of the limited therapeutic effect of traditional antibiotics on multi-drug resistant Gram-negative bacteria. By constructing a targeted prodrug nano-delivery system, specific accumulation of the drug at the lesion can be achieved, while reducing the systemic exposure amount and increasing the local treatment concentration, thereby improving the antibacterial efficacy and reducing the toxic and side reactions.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] A preparation method of a polymyxin B prodrug nanoparticle, comprising the following steps:

[0011] S1. Under nitrogen protection, dissolve 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP) in anhydrous dichloromethane, then add CDI (carbonyldiimidazole), react at room temperature for the first time, wash, extract to obtain an organic phase, dry and concentrate the organic phase to obtain activated 4-(hydroxymethyl)phenylboronic acid pinacol ester, namely CDI-PBAP;

[0012] S2. Dissolve CDI-PBAP, PMB (polymyxin B) and DMAP (4-dimethylaminopyridine) in anhydrous DMSO (dimethyl sulfoxide), purge with nitrogen to remove oxygen, react at room temperature for the second time to obtain a precipitate, which is the prodrug PPMB;

[0013] S3. Add lecithin solution, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-maleimide, and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to water to obtain a mixture; dissolve the prodrug PPMB in methanol to obtain a PPMB methanol solution; drop the PPMB methanol solution into the mixture, stir and react at room temperature, remove the organic solvent and the excessive aqueous phase to obtain polymyxin B prodrug nanoparticles.

[0014] The prodrug PPMB is prepared by reacting 4-(hydroxymethyl)phenylboronic acid pinacol ester, PMB, and DMAP as raw materials at room temperature, avoiding the complexity and energy consumption brought by high temperature or low temperature. Meanwhile, the use of nitrogen protection also effectively prevents the occurrence of oxidation reactions, improves the selectivity of the reaction and the purity of the product, has good scalability and repeatability, and provides the possibility for the industrial production of prodrugs.

[0015] It has been experimentally proved that the polymyxin B prodrug nanoparticles prepared by the method of the present invention have better bioavailability and targeting. The prodrug PPMB prepared by this method is further processed into nanoparticles, which improves the solubility and stability of the drug. At the same time, the size and surface properties of the nanoparticles can be regulated to achieve targeted delivery and controlled release of the drug, thereby reducing the toxicity of the drug in the body and improving the bioavailability and therapeutic effect of the drug.

[0016] Preferably, the mass ratio of the PBAP (4-(hydroxymethyl)phenylboronic acid pinacol ester) to the CDI (carbonyldiimidazole) is 1.11:1.59.

[0017] Preferably, the mass ratio of the activated 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP), the PMB (polymyxin B), and the DMAP (4-dimethylaminopyridine) is 1.58:1.19:1.10.

[0018] Preferably, in the S1, the time of the first room temperature reaction is 20-40 min.

[0019] Preferably, the time of the first room temperature reaction is 30 min.

[0020] Preferably, in the S2, the time of the second room temperature reaction is 22-26 h.

[0021] Preferably, the time of the second room temperature reaction is 24 h.

[0022] Preferably, the preparation method of the polymyxin B prodrug nanoparticles includes the following steps:

[0023] S1. In a 50 mL round-bottom flask under nitrogen protection, dissolve 1.11 g (4.70 mmol) of 4-(hydroxymethyl)phenylboronic acid pinacol ester in 10 mL of anhydrous dichloromethane, then add 1.59 g (71.50 mmol) of CDI (carbonyldiimidazole), and react at room temperature for 30 minutes. After the reaction, wash with 10.00 mL of water, extract to obtain the organic phase, and then extract three times with 10.00 mL of saturated sodium chloride solution. Dry the organic phase with anhydrous sodium sulfate and concentrate by rotary evaporation to obtain the activated 4-(hydroxymethyl)phenylboronic acid pinacol ester, namely PBAP-CDI;

[0024] S2. Dissolve 1.58 g (6.03 mmol) of PBAP-CDI, 1.19 g (0.86 mmol) of PMB (polymyxin B), and 1.10 g (24.04 mmol) of DMAP (4-dimethylaminopyridine) in 20.00 mL of anhydrous DMSO (dimethyl sulfoxide), purge with nitrogen three times to remove oxygen, and react at room temperature for 24 hours. After the reaction, a precipitate is obtained. Mix the precipitate with deionized water, centrifuge and discard the supernatant to obtain a solid. Lyophilize the obtained solid to obtain the prodrug PPMB;

[0025] S3. Take 0.3 mL of lecithin solution and 0.3 mL of a mixture of DSPE-PEG2000-Mal (distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide) / DSPE-PEG2000 (wherein, in the mixture, the mass ratio of DSPE-PEG2000-Mal to DSPE-PEG2000 is 1:1, the organic solvent is ethanol, and the concentration is 30 mg / mL) and add them to 15 mL of deionized water. Stir at 65 °C for 30 minutes to obtain a mixed solution; ultrasonically dissolve 50 mg of the prodrug PPMB in 5 mL of methanol to obtain a PPMB methanol solution; under vigorous stirring, drop the PPMB methanol solution into the mixed solution and stir at room temperature for 2 hours to obtain a first mixed solution; remove the organic solvent and excess aqueous phase from the first mixed solution by vacuum distillation to obtain the activated maleimide (MAL)-PNP, namely the polymyxin B prodrug nanoparticles.

[0026] The present invention also provides a polymyxin B prodrug nanoparticle prepared by the above preparation method.

[0027] The present invention also provides an application of the polymyxin B prodrug nanoparticle prepared by the above preparation method in the preparation of a drug for treating pneumonia.

[0028] Preferably, the pneumonia is Klebsiella pneumoniae pneumonia with multidrug resistance.

[0029] Preferably, the polymyxin B prodrug nanoparticle and the targeted phage complex are used in the preparation of a drug for treating Klebsiella pneumoniae pneumonia with multidrug resistance.

[0030] Preferably, the preparation method of the polymyxin B prodrug nanoparticles and the targeted phage complex comprises the following steps:

[0031] Mix the activated maleimide (MAL)-PNP (i.e., polymyxin B prodrug nanoparticles) with the targeted phage and stir overnight to obtain the polymyxin B prodrug nanoparticles and the targeted phage complex, namely TPNP.

[0032] Among them, the phage can serve as a natural targeting molecule. By binding to specific receptors on the bacterial surface, it enhances the recognition and binding ability of the nanoparticles to bacteria. At the same time, the nanoparticles can protect the phage from degradation in the in vivo environment, extend its half-life, and increase its enrichment at the infection site. This synergistic effect can not only improve the therapeutic effect of the polymyxin B prodrug nanoparticles but also reduce the toxic and side effects on normal tissues, providing a new and effective means for the treatment of pneumonia. As a new type of drug delivery system, the phage can further effectively improve the stability and targeting of the polymyxin B prodrug nanoparticles, thus providing a new solution for the treatment of pneumonia caused by drug-resistant bacteria.

[0033] Preferably, the volume ratio of the lecithin solution, DSPE-PEG2000-Mal, and DSPE-PEG2000 is 2:1:1.

[0034] Preferably, after adding the lecithin solution, DSPE-PEG2000-Mal (distearoyl phosphatidylethanolamine-polyethylene glycol 2000-maleimide), and DSPE-PEG2000 (distearoyl phosphatidylethanolamine-polyethylene glycol 2000) to water, stir at 65 °C for 25 - 35 min to obtain the mixture.

[0035] Preferably, the time for stirring reaction at room temperature is 2 hours.

[0036] Preferably, the preparation method of the prodrug nanoparticles and the targeted phage complex comprises the following steps:

[0037] Mix the activated maleimide (MAL)-PNP with the phage and stir overnight under the condition of pH 7.4, so that maleimide specifically covalently binds to the thiol group at the end of the phage through a click reaction to form a stable disulfide bond, thereby obtaining the prodrug nanoparticles and the targeted phage complex TPNP.

[0038] Preferably, the targeted phage is Escherichia phage M13, preservation time: February 11, 2025, preservation address: China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, preservation number: CCTCC No: M 2025196.

[0039] The beneficial effects of the present invention are as follows:

[0040] The present invention provides a polymyxin B prodrug nanoparticle. By modifying polymyxin B (PMB) with a small molecule 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP), PMB and PBAP are covalently coupled, and 3 PBAP molecules are connected to each PMB molecule to synthesize a prodrug PPMB with better bioavailability and targeting. Then, the prodrug PPMB is encapsulated by lecithin and DSPE-PEG to form a stable polymyxin B prodrug nanoparticle, reducing the toxicity of the drug PMB in vivo. The problem of high toxicity and insufficient targeting of existing drugs in vivo is solved.

[0041] The present invention provides a polymyxin B prodrug nanoparticle and targeted phage complex (TPNP). By loading the polymyxin B prodrug nanoparticle with a targeted phage, the targeting property is further improved, and the drug can be efficiently delivered and its stability in vivo is significantly enhanced, prolonging its action time. TPNP can accurately target the pneumonia infection site, significantly improve the treatment effect, and reduce the damage to normal tissues at the same time. The polymyxin B prodrug nanoparticle and targeted phage complex not only enhance the killing effect of the drug on multi-drug resistant bacteria, solve the problem of limited treatment effect of existing drugs on multi-drug resistant bacteria, but also provide an innovative and efficient treatment strategy for the treatment of pneumonia caused by drug-resistant bacteria. And the preparation method has the advantages of mild conditions, simple operation and suitability for large-scale production, and has significant industrialization prospects. In the field of microbial technology, it has wide popularization value and practical application potential, providing a new solution for the treatment of drug-resistant bacterial infections. Description of the Drawings

[0042] Figure 1 is an infrared absorption spectrum diagram;

[0043] Figure 2 is a nuclear magnetic resonance spectrum;

[0044] Figure 3 is an LC-MS spectrum of the prodrug PPMB and its hydrolysis product;

[0045] Figure 4 is a TEM image of PNP;

[0046] Figure 5TEM image of polymyxin B prodrug nanoparticles and targeted phage complex TPNP;

[0047] Figure 6 Bacterial load changes of PNP and TPNP for pneumonia treatment;

[0048] Figure 7 Bacterial changes in Giemsa staining of histopathological sections;

[0049] Figure 8 HE inflammation index changes in histopathological sections;

[0050] Figure 9 DHE inflammation index changes in histopathological sections;

[0051] Figure 10 Fluorescence intensity results of the lungs. Detailed implementation manners

[0052] The following will illustrate the implementation manners of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention and not for limiting the protection scope of the present invention.

[0053] Example 1

[0054] A preparation method of polymyxin B prodrug nanoparticles, comprising the following steps:

[0055] S1. In a 50 mL round-bottom flask under nitrogen protection, dissolve 1.11 g (4.70 mmol) of 4-(hydroxymethyl)phenylboronic acid pinacol ester in 10 mL of anhydrous dichloromethane, then add 1.59 g (71.50 mmol) of CDI (carbonyldiimidazole), react at room temperature for 30 minutes. After the reaction, wash with 10.00 mL of water, extract to obtain the organic phase, then extract three times with 10.00 mL of saturated sodium chloride solution. The organic phase is dried with anhydrous sodium sulfate and concentrated by rotary evaporation to obtain activated 4-(hydroxymethyl)phenylboronic acid pinacol ester, namely PBAP-CDI;

[0056] S2. Dissolve 1.58 g (6.03 mmol) of activated 4-(hydroxymethyl)phenylboronic acid pinacol ester, 1.19 g (0.86 mmol) of PMB (polymyxin B), and 1.10 g (24.04 mmol) of DMAP (4-dimethylaminopyridine) in 20.00 mL of anhydrous DMSO (dimethyl sulfoxide). Deoxygenate by passing nitrogen three times and react at room temperature for 24 hours. After the reaction, a precipitate is obtained. Mix the precipitate with deionized water, centrifuge and discard the supernatant to obtain a solid. Lyophilize the obtained solid to obtain the prodrug PPMB;

[0057] The reaction equation for the preparation of the prodrug PPMB is:

[0058]

[0059] Among them, due to the influence of steric hindrance, the number of groups that may be attached at the two sites may be different, that is, the values of n1 and n2 will be different, but it will not affect the overall performance of the prodrug nanoparticles;

[0060] S3. Take 0.3 mL of lecithin solution and 0.3 mL of a mixture of DSPE-PEG2000-Mal (distearoylphosphatidylethanolamine-polyethylene glycol 2000-maleimide) / DSPE-PEG2000 (wherein, in the mixture, the mass ratio of DSPE-PEG2000-Mal to DSPE-PEG2000 is 1:1, the organic solvent is ethanol, and the concentration is 30 mg / mL) and add them to 15 mL of deionized water. Stir at 65 °C for 30 minutes to obtain a mixed solution; Ultrasonically dissolve 50 mg of the prodrug PPMB prepared in S1 in 5 mL of methanol to obtain a PPMB methanol solution; Under the condition of vigorous stirring, drop the PPMB methanol solution into the mixed solution and stir at room temperature for 2 hours to obtain a first mixed solution; Remove the organic solvent and excess aqueous phase from the first mixed solution by vacuum distillation to obtain activated maleimide (MAL)-PNP, that is, polymyxin B prodrug nanoparticles.

[0061] Example 2

[0062] A preparation method of a polymyxin B prodrug nanoparticle and a targeted phage complex, comprising the following steps:

[0063] S4. Mix the activated maleimide (MAL)-PNP (i.e., polymyxin B prodrug nanoparticles) prepared in Example 1 with the targeted phage (Escherichia phage M13), and stir overnight at pH 7.4, so that maleimide specifically covalently binds to the thiol group at the end of the phage through a click reaction to form a stable disulfide bond, thereby obtaining a polymyxin B prodrug nanoparticle and a targeted phage complex TPNP.

[0064] Among them, Escherichia phage M13, preservation time: February 11, 2025, preservation address: China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, preservation number: CCTCC No: M2025196.

[0065] The screening method of Escherichia phage M13 includes the following steps:

[0066] S1. Using the Ph.D.-7 phage display peptide library kit (New England Biolab, UK), take 1 mL of whole cells of multidrug-resistant Klebsiella pneumoniae (MDR-Kp) and mix it with 100 μL of phage library (about 2×10 9 clones) in PBS buffer, incubate at 37 °C for 1 h to obtain bound phages;

[0067] S2. Wash the bound phages 10 times with TBST (TBS + 0.1% [v / v] Tween-20) to remove unbound phages, centrifuge to obtain the washed bound phages, then add 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA) to the washed bound phages and act at room temperature for 10 minutes to disrupt the binding interaction between the centrifuged whole cells and the bound phages to obtain a mixture;

[0068] S3. Add 50 μL of 1 M Tris-HCl buffer (pH 9.1) to the mixture to neutralize the mixture to obtain eluted phages;

[0069] S4. According to the infection method of M13 phage, amplify the eluted phages in the host Escherichia coli ER2738 (OD 600≈0.5) to obtain amplified phages;

[0070] S5. Adopt a similar procedure for multidrug-resistant Pseudomonas aeruginosa (MDR-Pa) or multidrug-resistant Acinetobacter baumannii (MDR-Ab) respectively to obtain engineered phages, and add an equal volume of sterile glycerol to the obtained phages and store them at -20 °C.

[0071] S5. Take 1 mL of multi-drug resistant Pseudomonas aeruginosa (MDR-Pa) and mix it with the phages obtained from the first round of screening (i.e., the amplified phages obtained in S4) in PBS buffer. Incubate at 37 °C for 1 hour, wash 10 times with TBST (TBS + 0.1% [v / v] Tween-20) to remove unbound phages. Add 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA) to the washed bound phages, incubate at room temperature for 10 minutes, and then add 150 μL of 1 M Tris-HCl buffer (pH 9.1) to neutralize the mixture to obtain eluted phages.

[0072] Amplify the eluted phages in the host Escherichia coli ER2738 (OD600 ≈ 0.5). Concentrate and purify the amplified phages by 20% PEG / 2.5 M NaCl. Add an equal volume of sterile glycerol to the amplified phages and store at -20 °C;

[0073] S6. Take 1 mL of multi-drug resistant Acinetobacter baumannii (MDR-Ab) and mix it with the phages obtained from the second round of screening (i.e., the amplified phages obtained in S5) in PBS buffer. Incubate at 37 °C for 1 hour, wash 10 times with TBST (TBS + 0.1% [v / v] Tween-20) to remove unbound phages. Add 100 μL of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg / mL BSA) to the washed bound phages, incubate at room temperature for 10 minutes, and then add 150 μL of 1 M Tris-HCl buffer (pH 9.1) to neutralize the mixture to obtain eluted phages. Amplify the eluted phages in the host Escherichia coli ER2738 (OD600 ≈ 0.5). Concentrate and purify the amplified phages by 20% PEG / 2.5 M NaCl. Add an equal volume of sterile glycerol to the amplified phages and store at -20 °C.

[0074] Among them, in each round of biopanning, the selected phages are verified and sequenced by the Beijing Genomics Institute (China). Concentrate and purify the amplified phage particles by 20% PEG / 2.5 M NaCl. Add an equal volume of sterile glycerol to the finally obtained phages and store at -20 °C.

[0075] The obtained engineered phages are verified and sequenced by the Beijing Genomics Institute (China), and the binding peptide sequence of the obtained phages is Trp-Ser-Leu-Gly-Thr-Tyr-Gly (WSLGYTG).

[0076] Detection and analysis

[0077] 1) Infrared absorption spectroscopy analysis

[0078] The infrared absorption spectra of 4-(hydroxymethyl)phenylboronic acid pinacol ester, PMB, and the prepared prodrug PPMB used in Example 1 were analyzed, and the results are as Figure 1 shown.

[0079] From Figure 1 the comparative analysis, it can be seen that by comparing the Fourier transform infrared spectra (FT-IR) of the original drug PMB, the small molecule 4-(hydroxymethyl)phenylboronic acid pinacol ester, and the prodrug PPMB, obvious structural characteristic changes can be observed. In the spectrum of the prodrug PPMB, a significant characteristic peak of the C=O group appears at 1735 cm -1 −1, and at the same time, characteristic peaks of the benzene ring in the 4-(hydroxymethyl)phenylboronic acid pinacol ester molecule are detected in the range of 1680−1580 cm -1 −1, thus confirming that a chemical reaction has occurred between the amino group on PMB and 4-(hydroxymethyl)phenylboronic acid pinacol ester, resulting in a decrease in the number of amino groups in PPMB compared to PMB. These evidences together indicate that 4-(hydroxymethyl)phenylboronic acid pinacol ester has been successfully attached to the compound PPMB.

[0080] 2) Nuclear magnetic resonance analysis

[0081] The nuclear magnetic resonance analysis of PMB and the prepared prodrug PPMB used in Example 1 was carried out, and the results are as Figure 2 shown.

[0082] From Figure 2 the comparative analysis, it can be seen that the original drug PMB and the prodrug PPMB were subjected to 1 1H NMR characterization. By comparing the hydrogen spectra of the two, the attached 4-(hydroxymethyl)phenylboronic acid pinacol ester moiety in PPMB can be identified. In the NMR spectrum of PPMB, the peaks between 3.0 - 3.5 ppm correspond to the hydrogen atoms of the benzene ring (labeled green ②) on PMB, while the peaks between 5.0 - 5.5 ppm belong to the hydrogen atoms of the methylene group (labeled blue ①) on PBAP. The presence of these characteristic peaks further confirms that PMB has been successfully attached to 4-(hydroxymethyl)phenylboronic acid pinacol ester to form the prodrug PPMB.

[0083] 3) Responsiveness analysis

[0084] The specific operation steps are as follows:

[0085] To verify the responsive release characteristics of the prodrug, in vitro simulation of the ROS microenvironment of the lesion was used to study the drug release kinetics. PPMB was placed in H2O2, incubated at 37 °C with constant shaking, sampled, and detected by LC-MS. The experimental results show (as Figure 3 shown) that under the stimulation of H2O2, PPMB exhibits significant drug release responsiveness.

[0086] The results are as Figure 3 shown below.

[0087] Among them, Figure 3 the LC-MS spectra of PMB, PPMB, and PPMB hydrolysis (PPMB + H2O2) are successively shown in A-3C.

[0088] As can be seen from Figure 3 A, the commercial PMB shows a bimodal pattern in the LC-MS analysis of natural lipopeptide PMB because its main components are two: PMB1 and PMB2. The theoretical molecular weight of PMB1 is 1189.47, while the theoretical molecular weight of PMB2 is 1203.05. Figure 3 The different m / z values shown by the different charge numbers of PMB1 and PMB2 in A are PMB1: 1189.7 (z = 1), 595.9

[0089] (z = 2), 397.3 (z = 3), 298.2 (z = 4), and PMB2: 1203.8 (z = 1), 602.4 (z = 2), 401.9 (z = 3),

[0090] 301.7 (z = 4), which is consistent with the theoretical values.

[0091] As can be seen from Figure 3 B, the LC-MS results of PPMB show molecular ion peaks with molecular weights of 1004.5 and 1011.5, which correspond to the molecular weights after 3 PBAP units are attached to PMB1 and PMB2, respectively. This finding confirms that 3 PBAP units have been successfully introduced onto each PMB molecule, thereby enhancing the hydrophobicity of the original drug. By comparing the LC-MS spectra of PMB and PPMB hydrolysis ( Figure 3 .4A and 3.4C), it is found that the LC-MS results show that after treatment with H2O2, the released substances from PPMB have the same molecular weight as the unmodified PMB.

[0092] As can be seen from Figure 3 C, it is found that after PPMB is treated with H2O2, Figure 3 the bimodal peaks (labeled ① and ②) at 8 min in B disappear. The disappearance of this bimodal structure may imply that some chemical bonds or structures of PPMB have changed after treatment with H2O2. More importantly, this change does not lead to the degradation of PMB because the LC-MS spectrum of PPMB hydrolysis ( Figure 3 C) has the same characteristic peaks as the unmodified PMB ( Figure 3 A).

[0093] 4) Transmission electron microscopy analysis

[0094] The polymyxin B prodrug nanoparticles PNP prepared in Example 1 and the polymyxin B prodrug nanoparticles and targeted phage complex TPNP prepared in Example 2 were respectively subjected to transmission electron microscopy analysis, and the results are as Figure 4 and Figure 5 shown.

[0095] PPMB was transformed into solid spherical polymyxin B nanoparticles PNP by the nanoprecipitation method. These nanoparticles showed regular and consistent morphology under the electron microscope, with a smooth surface ( Figure 4 ). By the nanoprecipitation method, activated PNP containing maleimide was successfully prepared and conjugated with engineered phages through thiol-maleimide click reaction to form TPNP, namely the polymyxin B prodrug nanoparticles and targeted phage complex ( Figure 5 ).

[0096] 5) Experiment on the treatment of MDR Klebsiella pneumoniae pneumonia with TPNP

[0097] TPNP was administered to the pneumonia model animals by tail vein injection at a dose of 1 mg / kg, once every 12 h, for a total of two doses, and its therapeutic effect was observed. The therapeutic effect of TPNP on pneumonia was evaluated by detecting the bacterial load in the lungs of animals, inflammatory indicators, and histopathological changes. At the same time, control groups were set up, including administering the same dose and the same number of times of polymyxin B prodrug nanoparticles (PNP) and PBS, and the therapeutic advantages of TPNP were analyzed by comparison.

[0098] The specific operation is as follows:

[0099] After homogenizing the lung tissue, the bacterial load in the lungs of mice in each group was detected by the gradient dilution plate counting method.

[0100] Histopathological analysis:

[0101] Giemsa staining: Observe the distribution of bacteria in the lung tissue.

[0102] Hematoxylin-eosin (H&E) staining: Evaluate the inflammation and pathological changes of the lung tissue.

[0103] Oxidative stress assessment: The level of reactive oxygen species (ROS) in the lung tissue was detected using the DHE fluorescent probe.

[0104] The changes in the plate bacterial load are as Figure 6 shown, the bacteria in the Giemsa staining of the histopathological sections are as Figure 7 shown, and the changes in the HE inflammatory indicators of the histopathological sections are as Figure 8 shown. The DHE inflammation of the histopathological sections is as Figure 9 shown.

[0105] From Figure 6Analysis showed that the lung bacterial load in the TPNP treatment group was significantly lower than that in other groups. Specifically, after TPNP treatment, the lung bacterial load in mice decreased to approximately 2.93 Log10 CFU / lung. Compared with approximately 8.01 Log10 CFU / lung in the PBS control group, the number of bacteria decreased by five orders of magnitude. The bacterial load in the PNP group was approximately 4.14 Log10 CFU / lung, indicating that TPNP had a significant advantage in reducing the bacterial load.

[0106] From Figure 7 Analysis showed that the Giemsa staining results showed that the number of blue dots (representing bacteria) in the lung tissue of the TPNP group was significantly less than that in the PNP group, which was consistent with the results of bacterial load detection. This indicated that TPNP could more effectively clear lung bacteria, reduce infection foci, and thus significantly reduce the bacterial load.

[0107] From Figure 8 Analysis showed that the lung tissue of the PBS control group mice showed obvious pathological changes under H&E staining, including alveolar capillary congestion and dilation, alveolar structure destruction, inflammatory cell infiltration, alveolar wall thickening, pulmonary edema, etc. The pathological changes in the lung tissue of the TPNP treatment group were significantly reduced, the inflammatory cell infiltration decreased, the alveolar structure partially recovered, and the phenomena of pulmonary edema and hemorrhage were alleviated. In contrast, the improvement of pathological features in the PNP group was not as significant as that in the TPNP group, indicating that TPNP had an advantage in reducing lung inflammation and tissue damage.

[0108] From Figure 9 Analysis showed that the DHE fluorescence probe detection results showed that the red fluorescence signal (representing ROS level) in the lung tissue of the TPNP treatment group was significantly lower than that in the PNP group and the PBS group, indicating that TPNP could more effectively reduce the oxidative stress level in the lung tissue.

[0109] 5) Targeting test of polymyxin B prodrug nanoparticles and targeted phage complex

[0110] Experimental design: To verify the lesion targeting of polymyxin B prodrug nanoparticles and targeted phage complex in a mouse pneumonia infection model, polymyxin B prodrug nanoparticles and polymyxin B prodrug - targeted phage complex were fluorescently labeled to track and evaluate their distribution and targeting in the mouse model.

[0111] Experimental procedures:

[0112] The polymyxin B prodrug nanoparticles, the targeted phage complex, and the polymyxin B prodrug nanoparticles prepared in Example 2 and Example 1 were labeled with fluorescent dyes. Among them, the nanoparticles were labeled with Cy5 to ensure that both carried traceable fluorescent signals. A certain number of healthy mice were selected to establish a MDR Klebsiella pneumoniae pneumonia infection model.

[0113] The infected mice were randomly divided into two groups. One group received treatment with the polymyxin B prodrug nanoparticles and the targeted phage complex (TPNP) (treatment group), and the other group received treatment with the polymyxin B prodrug nanoparticles (control group). At 6 h after infection, the mice in the treatment group were given 1 mg / kg of the polymyxin B prodrug nanoparticles and the targeted phage complex by tail vein injection, once every 12 h for a total of two doses. The mice in the control group were given the same dose and the same number of doses of the polymyxin B prodrug nanoparticles. At the same time points after administration, in vivo imaging technology was used to perform fluorescence imaging on the mice to track the distribution of the fluorescent signal in the body, especially the enhancement of fluorescence in the lungs. The results are as Figure 10 shown.

[0114] From Figure 10 the analysis, it can be seen that the fluorescence intensity of the treatment group was significantly higher than that of the control group, indicating that the polymyxin B prodrug nanoparticles and the targeted phage complex successfully aggregated in the lungs, further proving that the polymyxin B prodrug nanoparticles and the targeted phage complex have precise targeting.

[0115] In summary, the present invention provides a polymyxin B prodrug nanoparticle. By using the small molecule 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP) to modify polymyxin B (PMB), PMB and PBAP are covalently coupled, and 3 PBAP molecules are connected to each PMB molecule to synthesize the prodrug PPMB with better bioavailability and targeting. Then, the prodrug PPMB is encapsulated by lecithin and DSPE-PEG to form stable polymyxin B prodrug nanoparticles, reducing the toxicity of the drug PMB in the body. It solves the problems of high toxicity and insufficient targeting of existing drugs in the body.

[0116] The present invention provides a polymyxin B prodrug nanoparticle and targeted phage complex (TPNP). By loading the targeted phage onto the polymyxin B prodrug nanoparticle, the targeting property is further enhanced, and the drug can be efficiently delivered, significantly improving its stability in vivo and prolonging its action time. TPNP can precisely target the pneumonia infection site, significantly improving the treatment effect while reducing damage to normal tissues. This polymyxin B prodrug nanoparticle and targeted phage complex not only enhances the bactericidal effect of the drug against multi-drug resistant bacteria, solving the problem of limited treatment effect of existing drugs on multi-drug resistant bacteria, but also provides an innovative and efficient treatment strategy for the treatment of pneumonia caused by drug-resistant bacteria. Moreover, the preparation method has the advantages of mild conditions, simple operation and suitability for large-scale production, with significant industrialization prospects. In the field of microbial technology, it has broad promotion value and practical application potential, providing a new solution for the treatment of drug-resistant bacterial infections.

[0117] The above embodiments are only the preferred embodiments of the present invention, and the protection scope is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the protection scope of the present invention.

Claims

1. A preparation method of polymyxin B prodrug nanoparticles, characterized in that, It includes the following steps: S1. Under nitrogen protection, dissolve 4-(hydroxymethyl)phenylboronic acid pinacol ester in anhydrous dichloromethane, then add carbonyldiimidazole, react at room temperature for the first time, wash, extract to obtain an organic phase, dry and concentrate the organic phase to obtain activated 4-(hydroxymethyl)phenylboronic acid pinacol ester; S2. Dissolve the activated 4-(hydroxymethyl)phenylboronic acid pinacol ester, polymyxin B and 4-dimethylaminopyridine in anhydrous dimethyl sulfoxide, purge with nitrogen to remove oxygen, react at room temperature for the second time to obtain a precipitate, which is the prodrug PPMB; S3. Add a lecithin solution, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-maleimide and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to water to obtain a mixture; dissolve the prodrug PPMB in methanol to obtain a PPMB methanol solution; drop the PPMB methanol solution into the mixture, stir and react at room temperature, remove the organic solvent and the excess aqueous phase to obtain polymyxin B prodrug nanoparticles.

2. The preparation method of the polymyxin B prodrug nanoparticles according to claim 1, characterized in that, The mass ratio of the 4-(hydroxymethyl)phenylboronic acid pinacol ester to the carbonyldiimidazole is 1.11:1.59; And / or, the mass ratio of the activated 4-(hydroxymethyl)phenylboronic acid pinacol ester, the polymyxin B and the 4-dimethylaminopyridine is 1.58:1.19:1.

10.

3. The preparation method of the polymyxin B prodrug nanoparticles according to claim 1, wherein, In S1, the reaction time at room temperature for the first time is 20-40 min; And / or, in S2, the reaction time at room temperature for the second time is 22-26 h.

4. The preparation method of the polymyxin B prodrug nanoparticles according to claim 1, wherein The volume ratio of the lecithin solution, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-maleimide and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is 2:1:1; And / or, after adding the lecithin solution, distearoyl phosphatidylethanolamine-polyethylene glycol 2000-maleimide and distearoyl phosphatidylethanolamine-polyethylene glycol 2000 to water, stir at 65°C for 25-35 min to obtain the mixture; And / or, the stirring reaction time at room temperature is 2 hours.

5. A polymyxin B prodrug nanoparticle, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the polymyxin B prodrug nanoparticles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of a drug for treating pneumonia.

7. The application according to claim 6, wherein The pneumonia is multi-drug resistant Klebsiella pneumoniae pneumonia.

8. The application according to claim 7, wherein The polymyxin B prodrug nanoparticles and the targeted phage complex are used in the preparation of a drug for treating multi-drug resistant Klebsiella pneumoniae pneumonia.

9. The application according to claim 8, characterized in that, The preparation method of the polymyxin B prodrug nanoparticles and the targeted phage complex includes the following steps: Mix the polymyxin B prodrug nanoparticles and the targeted phage, stir overnight to obtain TPNP.

10. The application according to claim 7, wherein The targeted phage is Escherichia phage M13, the preservation time: February 11, 2025, the preservation address: China Center for Type Culture Collection (CCTCC), Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, the preservation number: CCTCC No:M 2025196.

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