Peroxynitrite-modified sphingomyelin liposomes and their use in combating mrsa infection

Abstract

The application discloses a p-hydroxycinnamaldehyde modified sphingomyelin liposome and application thereof in anti-MRSA infection. The p-hydroxycinnamaldehyde is bonded with the sphingomyelin through a two-step esterification reaction to obtain a bonding product sphingomyelin-p-hydroxycinnamaldehyde, and the p-hydroxycinnamaldehyde modified sphingomyelin liposome is prepared through a film hydration method. The liposome provided by the application is spherical, and the particle size distribution is uniform. The sphingomyelin component in the liposome can effectively neutralize the exotoxin of MRSA, and relieve the damage of the exotoxin of MRSA to normal cells of the body. The p-hydroxycinnamaldehyde in the liposome can induce the M0 type and M2 type macrophages in the body to polarize into M1 type macrophages with stronger phagocytosis and killing ability, relieve the immunosuppression caused by MRSA infection, and enhance the clearance ability of the macrophages to MRSA. The liposome provided by the application has application prospects in the preparation of drugs or preparations for treating diseases caused by MRSA infection.

Description

Technical Field

[0001] This invention relates to a liposome for antibacterial infection, and more particularly to a liposome that specifically neutralizes methicillin-resistant Staphylococcus aureus. Staphylococcus aureus Liposomes containing MRSA exotoxins and promoting macrophage cell transformation, and their application in the preparation of drugs or formulations for treating diseases caused by methicillin-resistant Staphylococcus aureus (MRSA) infection, belong to the field of liposomes and applications for anti-MRSA infection. Background Technology

[0002] In the global public health field, bacterial resistance has become a major challenge threatening human health. Among these, the prevalence and spread of methicillin-resistant Staphylococcus aureus (MRSA) is particularly severe, having become one of the leading pathogens causing clinical infectious diseases. According to the World Health Organization, more than 700,000 people die globally each year from drug-resistant bacterial infections, with MRSA infections accounting for 15%-20% of these deaths. Furthermore, with the increasing problem of antibiotic overuse, the resistance spectrum of MRSA continues to expand. It is not only completely resistant to β-lactam antibiotics (such as penicillin and cephalosporins), but its resistance rate to commonly used antibiotics such as macrolides and fluoroquinolones is also rising year by year, leading to a "no effective treatment" dilemma in clinical practice. Therefore, developing novel strategies to combat MRSA infections is of great significance.

[0003] MRSA can secrete various exotoxins, such as α-hemolysin, leukocytoxin, enterotoxin, and toxic shock syndrome toxin-1. Clinical studies have shown that MRSA-secreted exotoxins are key to host tissue damage and disease progression. Among them, α-hemolysin, as one of the most important virulence factors of MRSA, can bind to the ADAM10 receptor on the surface of host cells, forming a pore structure, leading to intracellular osmotic imbalance, calcium ion influx, and ultimately causing cell swelling, apoptosis, or necrosis, especially with a strong killing effect on erythrocytes, epithelial cells, and immune cells. Therefore, neutralizing MRSA exotoxins has become an important target for anti-MRSA infection therapy.

[0004] MRSA can regulate the host immune system, creating an immunosuppressive microenvironment conducive to its own survival, which is a key reason why it evades host clearance and leads to chronic infection. Macrophages, as core cells of the host's innate immunity, play crucial roles in bacterial infection, including phagocytosis, antigen presentation, and immune regulation. Based on their functional state, macrophages can be divided into M1 and M2 types: M1 macrophages highly express pro-inflammatory factors such as iNOS, TNF-α, and IL-6, possessing strong phagocytic and bactericidal capabilities; M2 macrophages highly express markers such as Arg-1, IL-10, and CD206, mainly participating in anti-inflammatory, tissue repair, and immunosuppression processes. Studies have found that MRSA induces macrophage polarization towards the M2 type by secreting virulence factors (such as capsular polysaccharides and protein A) or activating host immunosuppressive pathways (such as the TGF-β / Smad pathway), leading to an imbalance between M1 and M2 macrophages. This abnormal polarization significantly weakens macrophages' ability to phagocytose and kill MRSA, allowing it to colonize macrophages long-term. This not only evades clearance by antibiotics and the host immune system but also spreads throughout the body via macrophage migration, causing persistent infection or recurrence. Therefore, restoring macrophage polarization balance and its bactericidal function is crucial for MRSA clearance.

[0005] p-Hydroxycinnamaldehyde (pHCA), a phenylpropanoid compound extracted from natural plants such as cinnamon, has become a research hotspot in the fields of infection, inflammation, and tumors in recent years due to its function in regulating macrophage polarization. Studies have shown that pHCA can promote the polarization of M0 and M2 macrophages towards M1 type by inhibiting inflammatory pathways such as NF-κB and MAPK, thereby enhancing the phagocytic and bactericidal capacity of macrophages. In MRSA infection models, pHCA pretreatment can significantly promote the clearance of intracellular MRSA. However, pHCA itself has disadvantages such as poor water solubility, low bioavailability, and rapid in vivo metabolism, which limits its clinical application.

[0006] Sphingomyelin (SM), a natural sphingolipid compound, is an important component of cell membranes, particularly abundant in the myelin sheath and lipid raft structures of nerves. Recent studies have revealed that sphingomyelin possesses unique biological activities. Its hydrophobic chains and polar heads can specifically bind to specific domains of MRSA exotoxins (such as α-hemolysin), forming stable complexes that block the interaction between toxins and host cell receptors, thus alleviating toxin-mediated cell damage. Furthermore, sphingomyelin is an excellent material for liposome preparation, exhibiting good biocompatibility and biodegradability, which can effectively improve drug stability and targeting.

[0007] Based on these properties of sphingomyelin, if it is combined with p-hydroxycinnamaldehyde to construct a liposome delivery system that combines exotoxin neutralization and immunomodulation functions, it is expected to achieve a synergistic therapeutic effect of "detoxification-immune activation", thereby effectively treating various diseases caused by MRSA infection. Summary of the Invention

[0008] One objective of this invention is to provide SP liposomes that specifically neutralize MRSA exotoxins and promote macrophage cell transformation, as well as a method for their preparation. A second objective of this invention is to apply the liposomes described herein to the preparation of drugs or formulations for treating diseases caused by MRSA infection.

[0009] The above-mentioned objectives of the present invention are mainly achieved through the following technical solutions: One aspect of the present invention is to provide SP liposomes that specifically neutralize MRSA exotoxins and promote macrophage cell transformation, wherein the SP liposomes are prepared by the following method: (1) Sphingomyelin was cyclically opened with succinic anhydride to synthesize sphingomyelin with a carboxyl terminus (SM-COOH); The sphingomyelin with a carboxyl terminus was esterified with p-hydroxycinnamaldehyde to obtain the SM-p-hydroxycinnamaldehyde complex. (2) SP liposomes were prepared by thin-film hydration of SM-p-hydroxycinnamaldehyde complex, sphingomyelin and cholesterol.

[0010] In a preferred embodiment of the present invention, in step (1), sphingomyelin is synthesized by ring-opening with succinic anhydride under anhydrous and oxygen-free conditions in the presence of 4-pyrrolidinylpyridine and anhydrous chloroform, thereby synthesizing sphingomyelin with a carboxyl terminus.

[0011] In a preferred embodiment of the present invention, in step (1), SM-COOH, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in N,N-dimethylformamide and activated under anhydrous and oxygen-free conditions; p-hydroxycinnamaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are added to the activated reaction product and reacted under anhydrous and oxygen-free conditions to obtain the SM-p-hydroxycinnamaldehyde complex.

[0012] In a preferred embodiment of the present invention, the method for preparing liposomes by thin-film hydration in step (2) includes: dissolving SP bond compound, SM and cholesterol in an appropriate amount of methanol and then evaporating the solution, resuspending the solution in an appropriate amount of 5% glucose aqueous solution and hydrating it in a water bath, ultrasonically breaking it up and then filtering it through a microporous membrane to obtain the liposomes; more preferably, the water bath temperature is preferably 50°C, the hydration time is preferably 30 minutes, the ultrasonic parameters are preferably 60W, 3 / 2s ultrasonic breaking for 12 minutes, and the microporous membrane is preferably filtered sequentially through 0.45 and 0.22 μm microporous membranes.

[0013] The SP liposomes provided by this invention are spherical; the average particle size of SP NPs is about 97.34 ± 2.3 nm, PDI=0.241±0.14, and the particle size distribution is relatively uniform.

[0014] Another aspect of the present invention is to apply the provided SP liposomes to the preparation of drugs or formulations for treating diseases caused by MRSA infection.

[0015] Another aspect of the present invention provides a pharmaceutical composition for inhibiting MRSA infection, said pharmaceutical composition comprising a therapeutically effective amount of SP liposomes as described herein and a pharmaceutically acceptable carrier or excipient. The carrier or excipient may be any one or more of a binder, disintegrant, lubricant, solubilizer, diluent, wetting agent, flavoring agent, or preservative. Those skilled in the art can prepare said pharmaceutical composition into clinically suitable pharmaceutical formulations, such as tablets, granules, capsules, or oral liquids, according to conventional pharmaceutical formulation methods in the art.

[0016] The neutralization performance test results of liposome SP NPs showed that the sphingomyelin component in the liposome SP NPs provided by this invention can effectively neutralize MRSA exotoxins, thereby alleviating the damage of MRSA exotoxins to normal cells. The characterization test results of the macrophage M1 polarization performance induced by the liposome SP NPs showed that the p-hydroxycinnamaldehyde in the liposomes can induce M0 and M2 macrophages in the body to polarize into M1 macrophages, which have a stronger ability to phagocytose and kill bacteria, thereby effectively relieving immunosuppression caused by MRSA infection and enhancing the macrophage's ability to clear MRSA. Therefore, the SP liposomes provided by this invention have important application prospects as active ingredients or carriers in the preparation of drugs or formulations for treating diseases caused by MRSA infection. Attached Figure Description

[0017] Figure 1 This is the SP synthesis roadmap.

[0018] Figure 2 The infrared spectra of SM, SM-COOH, and SP are shown.

[0019] Figure 3 This is the proton NMR spectrum of SP.

[0020] Figure 4 The results of determining the particle size distribution of SP NPs using dynamic light scattering method.

[0021] Figure 5 Transmission electron microscopy images of SP NPs were obtained for TEM.

[0022] Figure 6 Different liposomes can alleviate hemolysis of blood cells caused by MRSA exotoxin.

[0023] Figure 7 To alleviate L929 cell damage induced by MRSA exotoxin using different liposomes.

[0024] Figure 8 To alleviate RAW264.7 cell damage induced by MRSA exotoxin using different liposomes.

[0025] Figure 9 The transformation status of macrophages in different treatment groups. Detailed Implementation

[0026] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, these experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0027] Example 1: Preparation and characterization of liposomes (SP NPs) 1. Experimental Methods 1.1 Synthesis of SP-bonded compounds Accurately weigh 42 mg SM, 7.2 mg succinic anhydride, and 1.6 mg 4-pyrrolidinylpyridine into a round-bottom flask. Add 30 mL of anhydrous chloroform to completely dissolve them. React under anhydrous and oxygen-free conditions for 12 h. After evaporating the solvent, redissolve the SM-COOH in dimethyl sulfoxide and transfer it to a dialysis bag with a molecular weight cutoff of 500 M. Dialyze the solution using 30% ethanol-water for three days to obtain a sphingomyelin with a carboxyl terminus (denoted as SM-COOH). Accurately weigh 36 mg SM-COOH, 7.6 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1.2 mg 4-dimethylaminopyridine into a round-bottom flask. Dissolve the SM-COOH in an appropriate amount of N,N-dimethylformamide and activate it under anhydrous and oxygen-free conditions for 1 h. Then add 6.67 mg p-hydroxycinnamaldehyde, 7.6 mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 1.2 mg 4-dimethylaminopyridine. 4-Dimethylaminopyridine was reacted under anhydrous and oxygen-free conditions for 24 hours. The mixture was then dialyzed with distilled water for three days to obtain the SP bonded compound.

[0028] 1.2 Characterization of SP SP-bonded compounds were characterized by infrared spectroscopy and proton nuclear magnetic resonance spectroscopy.

[0029] 1.3 Synthesis of SP NPs SP NPs were prepared by thin-film hydration: 16.74 mg of SP bond compound, 1.4 mg of SM, and 1.91 mg of cholesterol were accurately weighed into a round-bottom flask, dissolved in an appropriate amount of methanol, evaporated to dryness, resuspended in an appropriate amount of 5% glucose aqueous solution, hydrated in a 50°C water bath for 30 minutes, ultrasonically broken up at 60 W for 3 / 2 s for 12 minutes, and then filtered through 0.45 μm and 0.22 μm microporous membranes respectively.

[0030] 1.4 Characterization of SP NPs The particle size and particle size distribution of SP NPs were determined using a Malvern particle size analyzer (DLS). The morphology of SP NPs was observed using a transmission electron microscope (TEM).

[0031] 2. Experimental Results 2.1 Synthesis and Characterization of SP-bonded Compounds SM-COOH was synthesized by ring-opening of sphingomyelin and succinic anhydride, and then SM-COOH was esterified with p-hydroxycinnamaldehyde to obtain the SM-p-hydroxycinnamaldehyde complex (SP). Figure 1 Compared to SM, the infrared spectrum of SM-COOH is at 1730 cm⁻¹. -1 The characteristic absorption peak of the carbonyl group of succinic anhydride appears at 1730 cm⁻¹, and the infrared spectrum of SP shows a peak at 1730 cm⁻¹. -1 and 1690cm -1The simultaneous appearance of characteristic absorption peaks at the carbonyl group of succinic anhydride and the benzene ring in the structure of p-hydroxycinnamaldehyde proves the successful bonding between sphingomyelin and p-hydroxycinnamaldehyde. Figure 2 The NMR spectrum showed that, compared with SM, SP exhibited a characteristic resonance peak at 5.7 ppm for the hydrogen atom on the benzene ring of p-hydroxycinnamaldehyde. Figure 3 The grafting rate of p-hydroxycinnamaldehyde was calculated to be 19%.

[0032] 2.2 Synthesis and Characterization of SP NPs The particle size and polydispersity index (PDI) of nanoparticles are core characterization indicators of nanomaterials, directly determining their macroscopic properties and application value. SP NPs were prepared via a thin-film hydration method, and the particle size, particle size distribution, and morphological characteristics were investigated using DLS and TEM, respectively. The average particle size of the SP NPs was approximately 97.34 ± 2.3 nm. Figure 4 The PDI was 0.241 ± 0.14, indicating relatively uniform dispersion. TEM images show that the SP NPs are quasi-spherical (…). Figure 5 ).

[0033] Experimental Example 1: Performance Test of Liposome SP NPs in Neutralizing MRSA Exotoxins 1. Experimental Methods 1.1 Isolation of MRSA exotoxin Mix the USA300 bacterial suspension in the plateau phase and tryptone soybean broth at a ratio of 1:50, shake at 37°C for 24 hours, then centrifuge at 5000g for 30 minutes using a low-temperature refrigerated centrifuge. Collect the supernatant and filter it through a 0.22μm microporous membrane. Concentration can be performed at 45°C using a concentrator as needed for the experiment.

[0034] 1.2 Characterization of the neutralizing properties of SP NPs against MRSA exotoxins Using sheep blood cells, mouse fibroblasts, and mouse mononuclear macrophage leukemia cells as test cells, the performance of SP NPs in neutralizing MRSA exotoxin and alleviating cell damage was investigated by hemolysis assay and lactate dehydrogenase (LDH) kit.

[0035] (1) SP NPs alleviate hemolysis of blood cells: Sheep blood cells were centrifuged three times with sterile physiological saline (1000 r / min, 10 min) to obtain a stock solution. Gradient concentrations of soybean lecithin liposomes, sphingomyelin liposomes, and SPNPs were added to the sheep blood cell stock solution, followed by the addition of MRSA exotoxin. The solution was incubated at 37 ℃ for 3 h. The supernatant was collected by centrifugation (1000 r / min, 10 min), and the absorbance at 570 nm was measured using a UV spectrophotometer. The hemolysis rate was calculated using the formula.

[0036] (2) SP NPs alleviate cell damage: LDH is a marker enzyme in cells and is normally only present in the cytoplasm of living cells. When cell membrane integrity is disrupted due to damage, apoptosis, or necrosis, LDH is released into the extracellular culture medium. Therefore, cell viability can be quantitatively characterized by LDH activity. Gradient concentrations of soybean lecithin liposomes, sphingomyelin liposomes, and SPNPs were added to the culture medium of mouse fibroblasts (L929) and mouse mononuclear macrophages (RAW264.7), followed by the addition of MRSA exotoxin. The cells were incubated at 37 °C for 24 h, and the amount of lactate dehydrogenase released in the supernatant of each group was measured using a lactate dehydrogenase kit.

[0037] 2. Experimental Results Using sheep blood cells as test cells, SM NPs and SP NPs reduced hemolysis induced by MRSA exotoxin, and the hemolysis rate decreased in a concentration-dependent manner. Figure 6 This indicates that SM NPs and SP NPs can alleviate the damage to Mianyang hematology cells caused by MRSA exotoxin. Furthermore, using L929 and RAW264.7 cells as test cells, SM NPs and SP NPs reduced the release of cellular LDH in a concentration-dependent manner. Figure 7 , Figure 8 This indicates that SM NPs and SP NPs can alleviate the damage caused by MRSA exotoxin to L929 and RAW264.7 cells.

[0038] Experiment Example 2: Characterization of macrophage M1 polarization performance induced by liposome SP NPs 1. Experimental Methods The effect of SPNPs prepared in Example 1 on macrophage phenotype was verified by flow cytometry. This experiment was conducted in parallel groups: one group used well-growing mouse monocytic macrophage leukemia cells, and the other group used 5 μg / 10... 5 M2 macrophages were induced by interleukin-4 treatment for 24 h. M1 macrophages and M2 macrophages were labeled with CD86 receptor and CD206 receptor, respectively. After incubation with soybean lecithin, sphingomyelin and SP liposomes at the same lipid concentration for 24 h, the cell phenotypes of each group were determined by flow cytometry.

[0039] 2. Experimental Results In the microenvironment of bacterial infection, macrophages tend to be in an M2 immunosuppressive state, which significantly weakens their anti-infection ability. While anti-inflammatory factors such as interleukin-10 (IL-10) secreted by M2 macrophages suppress inflammatory responses and prevent excessive immune damage, they also reduce their phagocytic and bactericidal efficiency. Simultaneously, their reduced antigen-presenting ability makes it difficult to activate adaptive immunity, leading to easier bacterial colonization and proliferation, and even infection spread. Conversely, M1 macrophages are a "key line of defense" against bacterial infection. M1 macrophages possess strong bacterial phagocytic and killing capabilities, and also secrete pro-inflammatory factors such as tumor necrosis factor-α and interleukin-12, recruiting immune cells such as neutrophils and efficiently presenting antigens to activate T cells. This synergistic effect of innate and adaptive immunity is crucial for rapidly controlling infection and clearing bacteria. We used flow cytometry, with untreated RAW264.7 cells as a negative control, RAW264.7 cells treated with 1 μg / mL lipopolysaccharide (LPS) as a positive control for M1 type, and RAW264.7 cells treated with 5 μg / mL interleukin-4 (IL-4) as a positive control for M2 type. Groups D, E, and F were directly treated with different liposomes, while groups G, H, and I were first treated with 5 μg / mL interleukin-4 (IL-4) to induce macrophage transformation to M2 type, simulating the bacterial infection microenvironment, and then treated with different liposomes. Subsequently, M1 type macrophages were labeled with CD86 antibody, and M2 type macrophages were labeled with CD206 antibody. Flow cytometry analysis showed that SL NPs did not affect the transformation of M0 and M2 type macrophages; SM NPs could induce macrophage transformation to M2 type; while SP NPs could induce M0 and M2 type macrophages to revert to M1 type. Figure 9 This indicates that SP NPs can play a role in the action of hydroxycinnamaldehyde, thereby remodeling macrophage immune activity.

Claims

1. A method for preparing sphingomyelin liposomes modified with hydroxycinnamaldehyde, characterized in that, include: (1) Sphingomyelin and succinic anhydride were ring-opened to synthesize sphingomyelin with a carboxyl terminus; Sphingomyelin with a carboxyl terminus was esterified with p-hydroxycinnamaldehyde to obtain SM-p-hydroxycinnamaldehyde complex. (2) SP liposomes were prepared by thin-film hydration of SM-p-hydroxycinnamaldehyde complex, sphingomyelin and cholesterol.

2. The preparation method according to claim 1, characterized in that, In step (1), sphingomyelin is synthesized by ring-opening with succinic anhydride under anhydrous and oxygen-free conditions in the presence of 4-pyrrolidinylpyridine and anhydrous chloroform, thereby synthesizing sphingomyelin with a carboxyl terminus.

3. The preparation method according to claim 1, characterized in that, In step (1), sphingomyelin with a carboxyl terminus, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in N,N-dimethylformamide and then activated under anhydrous and oxygen-free conditions.

4. The preparation method according to claim 3, characterized in that, p-hydroxycinnamaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were added to the product of the activation reaction and reacted under anhydrous and oxygen-free conditions to obtain the SM-p-hydroxycinnamaldehyde complex.

5. The preparation method according to claim 1, characterized in that, The method for preparing liposomes by membrane hydration in step (2) includes: dissolving SM-p-hydroxycinnamaldehyde complex, sphingomyelin and cholesterol in methanol and then evaporating the solution, resuspending the solution in 5% glucose aqueous solution and hydrating it in a water bath, ultrasonically breaking it up and filtering it through a microporous membrane to obtain the liposomes.

6. The preparation method according to claim 5, characterized in that, The water bath temperature is 50°C, and the hydration time is 30 minutes.

7. The preparation method according to claim 5, characterized in that, The ultrasonic parameters are: 60W, 3 / 2s ultrasonic disruption for 12 minutes; the microporous filter membrane is passed through 0.45 and 0.22 μm microporous filter membranes in sequence.

8. Liposomes prepared by the preparation method according to any one of claims 1-7.

9. Use of the liposomes of claim 8 in the preparation of a medicament or formulation for treating diseases caused by methicillin-resistant Staphylococcus aureus infection.

10. A pharmaceutical composition for treating methicillin-resistant Staphylococcus aureus (MRSA) infections, characterized in that, This includes therapeutically effective amounts of the liposomes of claim 8, as well as pharmaceutically acceptable carriers or excipients.

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