Multifunctional polymyxin B nanoparticles as well as preparation and application thereof

Abstract

The invention relates to the technical field of drugs, in particular to multifunctional polymyxin B nanoparticles and preparation and application thereof.The multifunctional nanospheres (HPK) of polymyxin B (PMB) are synthesized by connecting hyaluronic acid and polymyxin B through Schiff base bonds and combining KAFAK, and the multifunctional nanospheres (HPK) serve as candidate drugs for refractory infectious diseases and can release PMB in response to the infected microenvironment, so that the effect of treating the refractory infectious diseases is achieved. The targeted infection site removes bacteria and various inflammatory mediators to reduce cytokine storm, including removal of LPS and active oxygen, and inhibition of macrophage activation. Hyaluronic acid (HA) serves as a carrier of PMB, the HA and the PMB are tightly connected through a pH sensitive bond Schiff base bond, the hyaluronic acid can change the biological distribution condition of the PMB, and a targeted CD44 receptor, hyaluronidase and the Schiff base bond can achieve PMB acidic microenvironment targeted delivery and meanwhile cooperate with PMB to resist bacteria and KAFAK to resist inflammation. The design and preparation of HPK effectively achieve the effects of reducing toxicity and enhancing efficacy on PMB.

Description

Technical Field

[0001] The present application relates to the technical field of pharmaceuticals, and specifically relates to a multifunctional polymyxin B nanoparticle and its preparation and application. Background Art

[0002] Due to various reasons, infectious diseases for which conventional treatment methods are difficult to achieve good results, refractory infectious diseases remain one of the key factors leading to population death globally. Among them, mixed infections (such as sepsis) and drug-resistant bacterial infections (such as severe pneumonia caused by drug-resistant bacteria) are particularly prominent, and have already posed an extremely severe threat to human life and health. The host of infectious diseases is invaded by a large number of pathogens, and pathogen-associated molecules such as lipopolysaccharide (LPS) are introduced into the host body. After the host immune system recognizes LPS, it will promote the generation and release of pro-inflammatory cytokines, chemokines, and reactive oxygen species, thereby triggering an inflammatory response against the pathogen. In the context of refractory infectious diseases, the inflammatory response continues and is difficult to subside, causing the host to fall into a vicious cycle of excessive inflammation and compensatory immunosuppression. This not only leads to a high risk of additional infections, but also often results in fatal consequences such as disseminated intravascular coagulation (DIC) and multiple organ dysfunction. In addition, different from extracellular bacteria, intracellular bacteria have the unique ability to invade and proliferate within host cells. Increasing experimental evidence has strongly shown that intracellular bacterial pathogens have greatly increased the treatment difficulty and complexity of refractory infectious diseases.

[0003] At present, for refractory infectious diseases, broad-spectrum antibiotics, surgery, and supportive treatment are mainly adopted. The core purpose is to control the early infection status and maintain the normal functioning of end organs. However, the high mortality rate and the lack of recent achievements in drug R & D fully demonstrate that there is an extremely urgent and unmet great demand for effective medical intervention measures in the treatment of refractory infectious diseases. LPS and reactive oxygen species can induce the release of inflammatory cytokines. Inflammatory mediators circulating in the body will cause systemic inflammation to remain active continuously, thereby triggering cell damage, organ failure, and even potential death risks. If these inflammatory mediators circulating in the circulatory system cannot be cleared in time, they will continuously induce systemic inflammatory responses in patients, thereby causing irreversible damage to endothelial cells, epithelial cells, and immune cells, and ultimately leading to acute organ failure. In addition, after bacteria invade cells, they will hide in a dormant form, becoming the source of repeated infections. Due to their unique survival strategies inside cells, significantly reduced metabolic rates, limited intracellular drug accumulation, impaired antibiotic activity, and slow growth of persistent cells, etc., the therapeutic efficacy of conventional antibiotics is greatly weakened. Based on this, we infer that reducing the level of reactive oxygen species, clearing various inflammatory mediators, and eliminating intracellular bacteria are the key points in the treatment of refractory infectious diseases.

[0004] In addition, in refractory infectious diseases, antibiotic-resistant bacteria can form bacterial biofilms, constructing physical-chemical double barriers and effectively mutating to escape the attack of the host immune system, which makes it extremely difficult to completely clear biofilm infections. Even after experiencing the conventional antibiotic treatment cycle, patients usually still present recurrence symptoms. The bacterial biofilm constructs physical-chemical double barriers, which not only causes its resistance to conventional antibiotics, but also weakens the local innate immunity by interfering with the inflammatory response and creating an immunosuppressive microenvironment. A great deal of effort has been devoted to the treatment of refractory biofilm infections, trying to start from aspects such as preventing bacterial adhesion and killing pathogens. However, since traditional antibacterial strategies are difficult to destroy the complex biofilm structure and completely eradicate mature biofilm infections, the treatment of established biofilm infections still faces numerous difficulties and the progress is rather difficult.

[0005] In the search for new treatment methods for refractory infectious diseases, we noticed the existing drug, polymyxin B (PMB), which is widely known as a powerful weapon against Gram-negative bacterial infections. PMB belongs to cationic antimicrobial peptides and is an extremely effective LPS adsorbent. By virtue of its property of having an adverse effect on the bacterial membrane, PMB is effective against the vast majority of Gram-negative bacteria. Moreover, PMB can bind to LPS with high affinity, thereby inhibiting the acute inflammatory response. Existing studies have confirmed that PMB can effectively alleviate LPS-induced sepsis in mice and is regarded as a potential treatment approach for Gram-negative bacterial infectious diseases. Given that Gram-negative bacteria account for nearly 70% of severe refractory infectious diseases, PMB is expected to become a key weapon in the treatment of refractory infectious diseases. However, the nephrotoxicity and neurotoxicity of PMB greatly limit its clinical application at effective doses in the treatment of refractory infectious diseases. Although several attempts have been made to develop PMB derivatives with lower toxicity, they all inevitably lose their antibacterial activity.

[0006] Therefore, finding a safe and effective polymyxin B pharmaceutical preparation is the current key research direction. Summary of the Invention

[0007] In order to solve the above-mentioned technical problems existing in the prior art, the present application provides a multifunctional polymyxin B nanoparticle and its preparation and application.

[0008] A multifunctional polymyxin B nanoparticle is synthesized by linking hyaluronic acid and polymyxin B through Schiff base bonds and combining with an anti-inflammatory peptide.

[0009] Further, the anti-inflammatory peptide is KAFAKLAARLYRKALARQLGVAA, KAFAK.

[0010] The preparation method of the above-mentioned multifunctional polymyxin B nanoparticle specifically includes the following steps:

[0011] (1) Dissolve hyaluronic acid (HA) in deionized water and add NaIO4;

[0012] (2) Stir under dark conditions, and then add ethylene glycol and continue stirring to stop the reaction;

[0013] (3) Dialyze with deionized water to remove NaIO4, and then perform freeze-drying to obtain a white spongy product OHA;

[0014] (4) Add OHA and PMB to degassed deionized water by ultrasonic treatment and incubate under nitrogen protection in the dark;

[0015] (5) Dialyze the reaction product again to remove unreacted PMB, and obtain a light yellow sponge-like product HA-PMB (HP) by freeze-drying method;

[0016] (6) Construct multifunctional polymyxin B nanoparticles (i.e., HPK) by electrostatic self-assembly under the induction of anti-inflammatory peptides.

[0017] Further, in the step (1), specifically, 0.2 - 0.6 parts of hyaluronic acid are dissolved in 8 - 12 parts of deionized water, and 0.05 - 0.06 parts of NaIO4 are added. The above parts are by weight.

[0018] Further, in the step (2), specifically, stir for 10 - 15 hours under light-shielded conditions, and then add ethylene glycol and continue to stir for 0.2 - 1 hour to stop the reaction.

[0019] Further, the dialysis in the step (3) is carried out for 36 - 72 hours using deionized water with a molecular weight of 500 - 3000 Da.

[0020] Further, in the step (4), the molar ratio of OHA to PMB is (15 - 20):1.

[0021] Further, in the step (4), incubate for 48 hours.

[0022] Further, in the step (5), use a microspectrophotometer to measure the grafting rate of PMB.

[0023] Further, in the step (6), specifically, it is carried out under the induction of 20 - 30 μg / mL anti-inflammatory peptide (i.e., KAFAKLAARLYRKALARQLGVAA, KAFAK).

[0024] The above-mentioned multifunctional polymyxin B nanoparticles are used in the preparation of drugs for refractory infectious diseases, especially in the preparation of drugs for pneumonia and sepsis caused by drug-resistant bacteria.

[0025] Compared with the prior art, the technical effects of the present application are reflected in:

[0026] The present application provides a multifunctional polymyxin B nanosphere, and relevant research has confirmed that this multifunctional polymyxin B nanosphere has good biosafety. It can effectively remove various inflammatory mediators and is thus applied to the treatment of severe pneumonia and sepsis caused by carbapenem-resistant Pseudomonas aeruginosa (CRPA) infection. In addition to exhibiting excellent antibacterial activity, HPK can also remove a variety of inflammatory mediators (such as lipopolysaccharide (LPS) and reactive oxygen species, etc.) and inhibit the activation of macrophages. In the treatment experiments conducted on severe pneumonia caused by CRPA infection and sepsis induced by the cecal ligation and puncture (CLP) model, the results clearly show that the multifunctional polymyxin B nanosphere has good biocompatibility in vivo, and it can increase the survival rates of mice in these two diseases to 100% and 86% respectively. Moreover, through pathological evaluation, it can be found that the systemic inflammatory response of the treated mice has been significantly alleviated, and the condition of organ failure has also been relieved. This good regulatory effect of HPK on inflammation reveals its potential in the treatment of other critically ill infectious diseases with a high risk of cytokine storm, and also provides an effective method that can target multiple inflammatory mediators for the treatment of refractory infectious diseases. In addition, its simple synthesis method also indicates that it is expected to be widely promoted and applied clinically in the future. Brief Description of the Drawings

[0027] Figure 2 is the construction and characterization of HPK. Among them: (A) Process flow chart for the preparation of HPK nanoparticles. (B) 1 1H NMR spectrum of the successful synthesis of Schiff base between HA and PMB. (C) and (D) DLS determination of the particle size of HPK and statistical zeta potential distribution of hemolysis rate. (E) TEM, elemental mapping and HAADF images of HPK, scale bar = 100 nm. (F) and (G) Images and hemolysis rates of PBS, HA, PMB, HP, HPK, with normal saline and ddH2O as negative control and positive control. (H) Cumulative release curves of PMB in HPK or physical mixture of PMB and HA (PMB+HA) under the conditions of pH 5.5 and pH 7.4, and in the presence of hyaluronidase.

[0028] Figure 3It is the observation of in vitro bactericidal effect. Among them: (A) Bacterial growth curves of CRPA and Escherichia coli under co-incubation with HA, PMB, HP, and HPK at different concentrations; (B) SYTO9 / PI staining laser confocal images of CRPA and E. coli after different treatments; (C) PI staining results of CRPA and Escherichia coli detected by flow cytometry after treatment with PBS, HA, PMB, HP, and HPK; (D) Representative SEM images of CRPA and E. coli after incubation with PBS, HA, PMB, HP, and HPK for 4 hours (green and yellow represent CRPA and E. coli respectively, and red fluorescent dots represent HPK), scale bar = 1 μm; (E) Schematic diagram of intracellular sterilization; (F) and (G) Quantitative analysis of bacteria forming colonies on LB agar plates and HUVEC (pre-infected with CRPA and E. coli) after treatment with PBS, HA, free PMB, HP, and HPK. (H) and (I) Quantitative analysis of the colonies and cell content of the lysate of RAW264.7 cells pre-infected with CRPA and E. coli formed on LB agar plates after treatment with PBS, HA, free PMB, HP, and HPK.

[0029] Figure 4 It is the mechanism of bacterial biofilm formation and inhibition. Among them: (A) Schematic diagram of the inhibition or disruption of bacterial biofilm formation by HPK. (B) Crystal violet staining showed the inhibitory or disruptive effect on biofilms after treatment with PBS, HA, PMB, HP, and HPK. (C) and (D) Quantitative determination of crystal violet absorption values by UV-vis showed the disruptive or inhibitory effect of different formulations on CRPA biofilms. (E) and (F) The inhibitory or disruptive situation of CRPA biofilms under different treatment conditions was observed using SYTO9 / PI staining technology, and laser confocal images and 3D cross-sectional schematic diagrams (the first to third columns are top views; the fourth column is a side view).

[0030] Figure 5 It is the efficacy of HPK in the treatment of severe pneumonia. Among them (A) Schematic diagram of the treatment process of severe pneumonia. (B) Survival rates of mice with severe pneumonia infected with CRPA after treatment with HA, free PMB, HP, and HPK for 7 days. In addition, the negative control group was treated with PBS (n = 7). (C) and (D) Changes in the body weight and body temperature of mice (n = 7). (E) Representative pictures of CRPA bacterial colonies on LB agar in lung homogenates of mice receiving different treatment regimens. (F) Number of bacterial colonies in lung tissue homogenates of mice in different groups after treatment.

[0031] Figure 6These are the results of observing the efficacy of HPK in the treatment of sepsis. Among them: (A) Schematic diagram of the sepsis treatment process. (B) Changes in the survival time of mice in each group after treatment with HA, free PMB, HP, and HPK for 7 days. (C) and (D) show the changes in the body weight and body temperature of septic mice (n = 7). (E) and (F) Representative pictures of colonies on LB agar of plasma and peritoneal lavage fluid (PLF) samples from pneumonia mice treated with different therapies. (G) and (H) Quantification of colonies on agar in the plasma and peritoneal lavage fluid of mice at different concentrations.

[0032] Figure 1 This is the research mechanism diagram of this application. Detailed implementation manners

[0033] The technical solution of this application will be further limited below in combination with specific implementation manners, but the scope of protection required is not limited only to the description made.

[0034] Research experiment on highly efficient elimination of multiple inflammatory mediators and biofilms by multifunctional polymyxin B nanoparticles for the treatment of refractory infectious diseases

[0035] Materials

[0036] Strains: Strains such as Escherichia coli (E. coli) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) were provided by the Microbiology Laboratory of the Affiliated Hospital of Guizhou Medical University.

[0037] Animals: Male ICR mice were from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China). All surgical operations and in vivo experiments were carried out according to the protocols detailed in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States.

[0038] The first part: Preparation and characterization of HPK

[0039] Methods

[0040] 1.1 Preparation method of multifunctional polymyxin B nanoparticles (HPK)

[0041] Dissolve 0.4 g of hyaluronic acid (HA) in 10 mL of deionized water, and add 0.053 g of NaIO4. Subsequently, stir for 12 hours under dark conditions, then add ethylene glycol and continue stirring for 0.5 hour to stop the reaction. Next, perform dialysis for 48 hours using deionized water with a molecular weight of 1000 Da to remove NaIO4. Then, perform freeze-drying to obtain a white sponge-like product OHA. Then, add OHA and PMB to 30 mL of ultrasonically degassed deionized water at a molar ratio of 17:1, and incubate for 48 hours under nitrogen protection in the dark. Finally, dialyze the reaction product again to remove unreacted PMB, and obtain a pale yellow sponge-like product HA-PMB (HP) by freeze-drying. The grafting rate of PMB was determined using a microspectrophotometer. Finally, HPK was prepared by electrostatically adsorbing 25 μg / mL KAFAK.

[0042] 1.2 Multifunctional Polymyxin B Characterization

[0043] The morphology of the nanoparticles was observed using transmission electron microscopy (TEM) and high-angle annular dark-field imaging scanning transmission electron microscopy (HAADF-STEM). In addition, through 1 1H NMR confirmed the successful synthesis between the Schiff base and PMB. The particle size and zeta potential of HPK nanoparticles were measured using a DLS nanoparticle size analyzer. The PMB content was detected using a UV-visible spectrophotometer (λ = 215 nm) at room temperature.

[0044] 1.3 Hemolysis Examination

[0045] Fresh whole blood and red blood cells collected from mice were diluted, and their concentration was adjusted to approximately 2% (volume / volume) using physiological saline. Then, the red blood cell suspension was mixed with gradient dilution series solutions of HA (10 mg / mL), PMB, HP, and HPK (equivalent to 2 mg / mL PMB) respectively, and incubated at 37 °C for 2 hours. Finally, non-hemolyzed red blood cells were separated by centrifugation at 1500 rpm for 10 min, and an equal volume of supernatant (100 μL) was placed in a 96-well plate for UV absorbance measurement at 545 nm. In addition, red blood cell suspensions treated with physiological saline or 2% Triton-X were used as negative control and positive control. The hemolysis rate was calculated according to the following formula: Hemolysis rate (%) = [(OD545nm of treated sample - OD545nm of negative control) / (OD545nm of positive control - OD545nm of negative control)] × 100%.

[0046] 1.4 PMB Release Examination

[0047] The dialysis method was used to study the release of polymyxin B (PMB). Briefly, physical mixtures of PMB and HAc (PMB+HA), HP and HPK (1 mg / mL, 1 mL) were introduced into a dialysis bag with a cut-off molecular weight of 3.5 kDa, and then added to PBS buffer containing 0.4 mg / mL hyaluronidase. Continuous oscillation was carried out at 37 °C and 60 rpm for 100 rpm under the conditions of pH 5.5 and 7.4. 0.1 mL of the release medium was collected regularly and replaced with fresh PBS buffer. After collecting the samples, they were centrifuged at 20,000 rpm for 30 minutes, and the supernatant was taken for further analysis. Subsequently, the concentration of PMB was determined using a UV-visible spectrophotometer.

[0048] Results of the preparation and characterization of the first part of HPK

[0049] Before preparing the HPK nanoparticles, the authors first modified PMB with hyaluronic acid (HA) using Schiff base (abbreviated as HP) to enhance the targeted delivery to the infected lesion ( Figure 2 A). The 1 1H NMR analysis results showed that the amino proton peak disappeared at 7.85 ppm, and a new resonance hemiacetal proton peak appeared at 6.3 - 6.5 ppm, indicating the successful formation of the Schiff base structure between HA and PMB ( Figure 2 B). Subsequently, HP was assembled with HA to obtain uniform HPK nanoparticles with an average size of 180.2 ± 16.5 nm and a surface charge of -23.6 ± 7.3 mV ( Figure 2 C and 2D). In addition, the experimental data of transmission electron microscopy (TEM) and high-angle annular dark-field image scanning transmission electron microscopy (HAADF-STEM) further confirmed the spherical structure composed of carbon, oxygen, and nitrogen ( Figure 2 E). In addition, it was found that no hemolysis occurred in each group during the hemolysis test. In summary, the results showed that HPK was safe and had no hemolytic toxicity when administered in vivo. ( Figure 2 F and 2G)

[0050] In addition, the pH-sensitive modification of PMB can promote the release of drugs from HPK on demand in the mildly acidic environment (about 6.0) of the infected lesion. Therefore, the pH-sensitive release of PMB was detected at pH 5.5 and pH 7.4 respectively. As Figure 2As shown in Figure H, at pH 5.5 and pH 7.5, the cumulative release rate of PMB in the physical mixture of HA and PMB (HA + PMB) was significantly faster than that of HP and HPK. When the pH was 5.5, almost 100% of PMB in the HP and HPK groups was released after 60 hours, while when the pH was 7.4, the cumulative release rates of the HP and HPK groups were both less than 80%. Finally, when compared with the HPK group, the addition of hyaluronidase significantly increased the release rate of PMB in HPK, especially when the pH was 5.5. In summary, these results indicate that the Schiff base chemical linkage method achieved the hyaluronidase sensitivity and pH-sensitive release effects of HA and PMB, and this is crucial for enhancing targeted therapy and antibacterial effects against infected lesions.

[0051] The second part: Study on in vitro antibacterial effect

[0052] Methods

[0053] 2.1 Determination of MIC and bacterial growth curve

[0054] By measuring the minimum inhibitory concentration (MIC) and observing the growth curve, the antibacterial effects of HPK against CRPA and E. coli were investigated. We used the microplate broth dilution method to determine the MIC values of each preparation (PBS, HA, PMB, KAFAK, HP, HPK). In the growth curve analysis, after incubating CRPA and E. coli (1×10 5 CFU / mL) with PBS, HA, PMB, HP, and HPK for different times, the bacterial content of each group was measured by UV-vis detection of OD600nm to plot the bacterial growth curve. To ensure the consistency and reliability of the results, each experiment was repeated three times.

[0055] 2.2 SYTO9 / PI bacterial live / dead staining

[0056] To visually evaluate the bactericidal effects of HPK against different strains (including E. coli and CRPA), we used the SYTO9 / PI staining method. First, each bacterium (1×10 8 CFU / mL) was inoculated into a 24-well plate containing glass slides and treated with PBS, HA (10 μg / mL), PMB, HP, and HPK for 12 hours respectively. Then, SYTO9 / PI was mixed at a volume ratio of 1:1 to obtain a dye mixture, which was reacted for 15 minutes under dark conditions at room temperature. Next, 5 μL of SYTO9 / PI was added to each well and incubated for 15 minutes to complete bacterial staining. Finally, each bacterial sample was imaged using a confocal laser scanning microscope, and the experiment was repeated at least three times.

[0057] 2.3 Flow cytometry was used to detect bacterial viability.

[0058] In addition, quantitative determination of the bactericidal rate plays an important role in evaluating the anti-infective effects of each preparation against CRPA and E. coli. Briefly, CRPA and E. coli at 1×10 8 CFU / mL were exposed to PBS, HA, PMB, HP, and HPK and incubated at 37 °C for 12 h. Subsequently, all these bacteria were collected and incubated with PI (2 mM) for 15 min in the dark. The bacterial viability was detected by quantitatively detecting the bacteria stained positively with PI by flow cytometry.

[0059] 2.4 Observation of bacterial morphology by scanning electron microscopy (SEM)

[0060] Using scanning electron microscopy (SEM), CRPA and E. coli at a concentration of 1×10 8 CFU / mL were treated with PBS, HA, PMB, HP, and HPK at 37 °C respectively, and after being treated with PMB at an equal concentration of 2 μg / mL for 4 h, the bacterial specimens were fixed with 2.5% glutaraldehyde. Subsequently, the changes in bacterial morphology under different treatments were observed.

[0061] 2.5 Bactericidal effect of HPK intracellularly

[0062] RAW264.7 and HUVEC cells were cultured in 24-well plates at a concentration of 1×10 5 cells / well and incubated overnight at 37 °C. Subsequently, they were co-incubated with CRPA and E. coli for 2 h, and then the infected cells were treated with PBS, HA, PMB, HP, and HPK for 4 h. After that, they were washed 3 times with PBS, and the cells were lysed with 0.1% Triton X-100. Finally, the cell lysate cultures of each group were evenly spread on agar plates and monitored for 24 h.

[0063] Results

[0064] To evaluate the inhibitory effect of HPK on bacteria, we determined the bacterial survival curves of CRPA and Escherichia coli under different formulations. After incubation with PBS, HA, PMB, HP, and HPK containing 0.5 μg / mL, 1 μg / mL, and 2 μg / mL PMB for 24 h, the results were obtained by monitoring the OD620nm absorbance of the bacterial solution. The specific data are shown in Figure 3 A. The results showed that when the PMB concentration was 2 μg / mL, the preparations containing PMB could completely inhibit the visible growth of the three bacteria, especially for E. coli and CRPA; when the PMB concentration was 1 μg / mL, the bacterial growth was completely inhibited. Subsequently, we further studied the antibacterial ability of HPK using the SYTO9 / PI live / dead staining kit. The laser confocal microscopy scanning images showed (Figure 3 B), when compared with the free PMB group, the number of red fluorescent dots in CRPA, HP, and HPK of E. coli increased significantly, indicating a significant improvement in the bactericidal effect of the HPK group. Subsequently, flow cytometry was used to quantitatively analyze the inactivation rate of HPK against bacteria, and the data showed that the PI fluorescence intensity increased sharply in the groups containing PMB when compared with the PBS or HA group (see Figure 3 C), especially in the HPK group, where the optimal bactericidal effect was obtained.

[0065] Scanning electron microscopy (SEM) further provided us with direct evidence of the antibacterial effect of nanoparticles against CRPA and E. coli. As Figure 3 shown in D, the bacteria in the PBS and HA groups presented a typical complete and smooth rod-like appearance; while after treatment with the PMB-related groups (including free PMB, HP, and HPK groups), significant morphological changes were clearly observed in CRPA and E. coli, that is, the bacterial membrane shrank sharply and even ruptured. In addition, obvious attachment of HPK could be clearly seen on the surface of the damaged bacteria, indicating a close interaction between HPK and the bacterial membrane. According to our previous research results, this interaction may be due to the binding of the polycationic ring of PMB to the negatively charged LPS on the surface of the bacterial membrane, and further disrupted the cell membrane of the bacteria. In summary, for Gram-negative bacteria CRPA and E. coli, HPK has excellent antibacterial effects.

[0066] In addition, different from extracellular bacteria, intracellular bacteria have unique abilities to invade and proliferate within host cells. An increasing number of experimental evidences emphasize that intracellular bacterial pathogens significantly increase the complexity and challenges of treating refractory infectious diseases. Therefore, for a complete cure of the infection, the intracellular bactericidal effect plays a crucial role. In this work, we explored the intracellular bactericidal ability of HPK in HUVEC and RAW264.7 cells ( Figure 3E to 3H). By observing the colony images, it was found that after incubating CRPA for 2 hours, the number of colonies in the cell lysates of HUVEC and RAW264.7 cells in the PBS group was significantly higher than that in the PMB group (including the free PMB group, HP group, and HPK group). In particular, there were significant differences between the PMB group and the HPK group in HUVEC and RAW264.7. These results indicate that HPK can effectively enhance its inhibitory ability against microorganisms in the internal parts of host cells, and this effect is related to the fact that HA-modified nanoparticles greatly promote the absorption of PMB by the host. In addition, similar differences were also found between free PMB and HPK in RAW264.7 in the Escherichia coli infection model, and this was caused by the effective uptake of HPK by RAW264.7 cells. Summarizing the above results, it can be seen that HPK can efficiently control and eliminate microorganisms in the internal parts of host cells and further promote the post-infection repair process.

[0067] Part III In vitro inhibition and disruption of biofilms

[0068] Methods

[0069] 3.1 Inhibition of biofilm formation

[0070] In the biofilm inhibition assay, 1×10 8 CFU / mL of CRPA and E. coli bacteria were inoculated into 24-well plates, treated with PBS, HA, PMB, HP, or HPK (equivalent to 100 μg / mL PMB) for 24 h, the inhibitory activity of the biofilm was measured, and crystal violet staining was used for imaging. In addition, the SYTO9 / PI staining method was also used to investigate the formation of bacterial biofilms.

[0071] 3.2 Disruption of biofilms

[0072] CRPA and E. coli (1×10 8 CFU / mL) were inoculated into 24-well plates and cultured at 37 °C for 24 h to form mature biofilms. Subsequently, the mature biofilms were incubated with PBS, HA, PMB, HP, HPK (equivalent to 100 μg / mL PMB) for 12 h. Then, the supernatant was discarded, and the biofilms were gently washed three times with PBS. Finally, crystal violet or SYTO9 / PI staining was used, and imaging was performed using a camera or a laser confocal microscope, respectively.

[0073] Results

[0074] Biofilms are composed of various nutrients, such as extracellular DNA, proteins, polysaccharides, and bacterial complex communities. Its main function is to provide a safe haven (usually with a pH value of about 5.5), promote bacterial growth, assist bacteria in resisting antibiotic invasion, and promote bacterial recurrent infections. Therefore, in the treatment of bacterial infections, disrupting or inhibiting the formation of biofilms plays a key role (Figure 4 A). In this study, the crystal violet staining method was first used to evaluate the ability of HPK in these two aspects. The results showed that in the PMB, HP, and HPK treatment groups, compared with the PBS and HA groups, the purple staining was significantly reduced ( Figure 4 B). Especially in the HP and HPK treatment groups, the inhibitory effect on CRPA biofilm formation was significant. Similar results were obtained by dissolving the crystal violet in each treatment group and measuring the absorbance using a 595 nm ultraviolet-visible spectrum ( Figure 4 C and 4D). In addition, we also evaluated the ability of Escherichia coli to form and disrupt biofilms with HPK, and verified it by SYTO9 / PI staining and laser confocal microscopy. The results showed that when moving from the PBS to the HPK treatment group, the red fluorescent dots gradually increased in intensity while the green fluorescence intensity showed the opposite trend; at the same time, the biofilm thickness decreased from an average of 40 μm to 5 μm ( Figure 4 E). In addition, significant changes in fluorescence intensity similar to those described above were also observed in preventing biofilm formation ( Figure 4 F). The above results indicate that HPK has the effect of effectively eradicating and preventing biofilm formation.

[0075] In addition, through further observation by SYTO9 / PI staining and laser confocal scanning, the above results were confirmed. Figure 4 E shows the results of biofilm disruption. From the PBS to the HPK group, the red fluorescent dots gradually increased in intensity, while the green fluorescence intensity showed the opposite trend. At the same time, the biofilm thickness decreased from an average of 40 μm to 5 μm. In addition, in Figure 4 F, the inhibition of biofilm formation results also showed significant changes in fluorescence intensity similar to those in Figure 4 E. These results clearly indicate that HPK has a significant inhibitory effect on biofilm formation and an eradication effect.

[0076] Part 4: In Vivo Treatment Study of Severe Pneumonia

[0077] Methods

[0078] 4.1 Evaluation of Severe Pneumonia

[0079] Male ICR mice at 4 weeks of age and weighing approximately 25 g were randomly divided into 5 groups (n = 7) and were respectively given LPS (intraperitoneal injection, 2.5 mg / kg) and infected with CRPA (intratracheal intubation, 1×10 10Induce pneumonia by injecting lipopolysaccharide (LPS, 50 μg / mL, 50 μL) into the abdominal cavity. Eight hours after modeling, various preparations such as PBS, HA (25 mg / mL), PMB (5 mg / kg), HP, and HPK (equivalent to 5 mg / kg PMB) were injected intravenously. Subsequently, the mortality, body temperature, and body weight of mice in each group were continuously monitored for 7 days to preliminarily evaluate the anti-infection effect of HPK in vivo. To comprehensively evaluate the anti-infection effect of lung tissue homogenate, the lung tissue homogenate was serially diluted and cultured on LB agar plates for colony quantification. In addition, after the mice were sacrificed, organs including the heart, liver, spleen, lungs, and kidneys were collected for H&E staining and further examination under a pathological slide scanner. In addition, immunostaining was performed using myeloperoxidase (MPO) antibody and CD86 / CD206, and images were obtained through a pathological slide scanner to evaluate neutrophil inflammatory infiltration in the lung tissues of different groups.

[0080] At the same time, blood biochemical analysis was performed to evaluate the in vivo safety of each group. After modeling, mice were injected intravenously with PBS, HA, PMB, HP, and HPK (n = 5). Twenty-four hours later, serum samples from each group were taken for blood tests to observe changes in the liver and kidney functions of the mice.

[0081] Results

[0082] In this study, a mouse model of CRPA severe pneumonia was established by combining lipopolysaccharide with CRPA infection. Figure 5 A shows the flowchart of modeling and treatment. Briefly, lung infection was induced by intraperitoneal injection of LPS and intratracheal culture of clinically identified CRPA bacterial specimens. Subsequently, mice with severe pneumonia caused by CRPA infection were treated with PBS, HA, PMB, HP, and HPK (at a concentration of 5 mg / kg of PMB), and changes in body temperature, body weight, and colony plating in the bronchoalveolar lavage fluid of the mice were observed within 7 days. At the same time, to investigate the effect of HPK in reducing the toxicity of PMB, the concentration of PMB was increased by 10-fold, and the safety of HPK was investigated through an acute toxicity experiment.

[0083] As Figure 5 shown in B, the survival rate of mice in PBS was only 20%, that in the PMB group reached 40%, while in the HPK group it reached 100%. In addition, when the concentration of PMB was increased to 10 times the original, all mice were observed to die immediately after injection. However, the mortality rate of mice in the HPK (10-fold) group was only 30%, indicating that the toxicity of PMB was significantly reduced. Additionally, in terms of the curves of body weight and body temperature changes ( Figure 5 C and Figure 5D), it can be clearly seen that the mice in the HPK group quickly recovered from severe weight loss and hypothermia; in contrast, the body temperature and weight of the surviving mice in the PBS group continued to decline. Finally, to observe the survival of bacteria in the lungs, the surviving mice in different treatment groups were euthanized, and lung and other organ samples were collected for bacterial colony analysis. As Figure 5 shown in E, there were almost no visible bacterial colonies in the HPK group, while there were a large number of densely distributed ones in the PBS group. In addition, in Figure 5 F, the quantitative data showed a significant difference between the PBS and PMB groups, and the number of CRPA bacteria in the PMB group was much more than that in the HPK group. The above results indicate that HPK treated by nano-modification can effectively resist CRPA infection.

[0084] Part V Study on HPK in the Treatment of Sepsis

[0085] Methods

[0086] Observation on the Treatment of Sepsis

[0087] A mouse sepsis model was established by cecal ligation and puncture (CLP). Briefly, the mice were anesthetized with isoflurane before the CLP operation, and then a longitudinal incision of about 1 cm was made on the midline with scissors. Subsequently, the cecum was carefully removed and ligated tightly with 4-0 silk at 0.5 cm from the end of the cecum. In addition, the ligated end was punctured with a syringe to drain the cecal contents. Finally, after repositioning the cecum after treatment, the wound of the mouse was sutured. After modeling, the ICR sepsis mice were divided into 5 groups and given PBS, HA (25 mg / mL), PMB (5 mg / kg), HP, and HPK (equivalent to 5 mg / kg of PMB) via the tail vein. The body temperature, weight, and survival rate were monitored for 24 hours per week. After euthanasia, the abdominal cavity was rinsed with PBS, 10 μL of peritoneal lavage fluid (PLF) was collected for culture, and the colony-forming units (CFU) were counted after incubation. The heart, liver, spleen, lung, and kidney tissues of the mice were collected after death, stained with H&E, and pictures were taken using a pathological section scanner.

[0088] Results

[0089] Sepsis is the main cause of death in critically ill patients and usually occurs when the body has an overwhelming immune response to infection. However, there is still a lack of specific treatment methods for sepsis, and its treatment always depends on the control of infection and inflammation. Therefore, we further evaluated the bactericidal effect of HPK on sepsis in mice by cecal ligation and puncture (CLP). As Figure 6 shown in A, HPK was intravenously injected 5 hours after CLP, and the mice were observed and recorded for 7 days. Finally, the surviving mice were euthanized for bacterial clone culture and immunohistochemical analysis. Figure 6The survival curves of mice are shown in Figure 6 B. All the mice in the PBS group died within 5 days, while the mortality rates of the free PMB and HPK groups were only 50% and 15% respectively. These results indicate that PMB has a good therapeutic effect on sepsis and that this effect can be significantly enhanced by using HPK. In addition, the following was observed on the curves of temperature and body weight changes (

[0090] C and 6D): The mice in the PBS group showed a rapid decrease in body weight and body temperature and even died within the first 96 h. In contrast, the mice in the HPK group recovered normal body weight loss and body temperature decrease. These results are closely related to the bactericidal and PMB toxicity-reducing effects of HPK. Figure 6 E and 6F and Figure 6 G and 6H show that, compared with other treatments, the colony counts of septic mice in the HPK group and the HP group were significantly reduced, and the inhibition rate differed by 100-fold. In particular, dense colonies were observed on the colony plates of the abdominal lavage fluid in the PBS and HA groups, while almost none were visible in the HPK group. These results indicate that HPK has excellent bactericidal effects and thus treats sepsis.

[0091] Conclusion

[0092] Refractory infectious diseases are often accompanied by high mortality and poor prognosis, which has led to increasing attention to the research and development of new drugs in this field. In this context, we have successfully developed a multifunctional polymyxin B nanosphere. In the process of new drug development, it is crucial that the research strategy can minimize potential toxic effects, and we have paid special attention to this aspect. Through relevant studies, we have confirmed that this multifunctional polymyxin B nanosphere has good biosafety. It can be used to treat severe pneumonia and sepsis caused by CRPA infection by effectively removing various inflammatory mediators. In addition to showing excellent antibacterial activity, HPK can also remove a variety of inflammatory mediators and inhibit the activation of macrophages. In the treatment experiments for severe pneumonia and sepsis caused by CRPA infection, the results clearly showed that the multifunctional polymyxin B nanosphere has good biocompatibility in vivo, and it can increase the survival rate of mice in these two diseases to 100% and 86%, respectively. Moreover, after pathological evaluation, it can be found that the systemic inflammatory response of the treated mice was significantly reduced, and the organ failure was also alleviated. This HPK's good regulation of inflammation shows its potential in treating other critical infectious diseases with a high risk of cytokine storms, and also provides an effective method for targeting multiple inflammatory mediators in the treatment of refractory infectious diseases. In addition, its simple synthesis method also indicates that it is expected to be widely promoted and applied in the clinic in the future.

[0093] Finally, it should be pointed out that the above embodiments are only representative examples of the present application. Obviously, the technical solution of the present application is not limited to the above embodiments, and there may be many variations. All variations that can be directly derived or associated with the contents disclosed in the present application by a person of ordinary skill in the art should be considered as the protection scope of the present application.

Claims

1. A multifunctional polymyxin B nanoparticle, characterized in that: It is synthesized by linking hyaluronic acid and polymyxin B through Schiff base bonds and combining them with anti-inflammatory peptides.

2. The multifunctional polymyxin B nanoparticles according to claim 1, characterized in that: The anti-inflammatory peptide is KAFAKLAARLYRKALARQLGVAA, KAFAK.

3. The method for preparing the multifunctional polymyxin B nanoparticles according to claim 1, characterized in that: The specific steps include: (1) Dissolve hyaluronic acid (HA) in deionized water and add NaIO4; (2) stirring under light-proof conditions, then adding ethylene glycol and continuing stirring to stop the reaction; (3) dialyzing with deionized water to remove NaIO4, followed by freeze drying to obtain a white sponge-like product, OHA; (4) OHA and PMB were added to ultrasonically degassed deionized water and incubated in dark conditions under nitrogen protection; (5) dialyzing the reaction product again to remove unreacted PMB, and obtaining a light yellow sponge product HA-PMB (HP) by lyophilization; (6) Multifunctional polymyxin B nanoparticles (i.e., HPK) were constructed by electrostatic self-assembly under the induction of anti-inflammatory peptides.

4. The method for preparing multifunctional polymyxin B nanoparticles according to claim 3, characterized in that: The step (1) specifically comprises dissolving 0.2-0.6 parts of hyaluronic acid in 8-12 parts of deionized water, and adding 0.05-0.06 parts of NaIO4.

5. The method for preparing multifunctional polymyxin B nanoparticles according to claim 1, characterized in that: The step (2) is specifically to stir the mixture for 10-15 hours in a light-proof condition, and then add ethylene glycol and continue stirring for 0.2-1 hour to stop the reaction.

6. The method for preparing multifunctional polymyxin B nanoparticles according to claim 1, characterized in that: The dialysis in step (3) is performed using deionized water with a molecular weight of 500-3000Da for 36-72 hours.

7. The method for preparing multifunctional polymyxin B nanoparticles according to claim 4, characterized in that: In the step (4), the molar ratio of OHA to PMB is (15-20):

1.

8. The method for preparing multifunctional polymyxin B nanoparticles according to claim 1, characterized in that: The step (6) is specifically carried out under the induction of 20-30 μg / mL of an anti-inflammatory peptide (ie, KAFAKLAARLYRKALARQLGVAA, KAFAK).

9. Use of the multifunctional polymyxin B nanoparticles according to claim 1 in the preparation of drugs for refractory infectious diseases.

10. The use according to claim 9, characterized in that: In the aspect of preparing drugs for refractory infectious diseases, it is the aspect of preparing drugs for pneumonia and sepsis caused by drug-resistant bacteria.

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