Cell sugar and lipid metabolism regulation type drug-loaded linoleic acid lipid hybrid vesicle as well as preparation method and application of cell sugar and lipid metabolism regulation type drug-loaded linoleic acid lipid hybrid vesicle

Abstract

The invention relates to a cell sugar and lipid metabolism regulation type drug-loaded linoleic acid lipid hybrid vesicle as well as a preparation method and application thereof, and the cell sugar and lipid metabolism regulation type drug-loaded linoleic acid lipid hybrid vesicle comprises a linoleic acid lipid hybrid vesicle, a sugar metabolism drug and a lipid metabolism drug, the glycometabolism drug and the lipid metabolism drug are loaded in the linoleic acid lipid hybrid vesicles; wherein the glycometabolism medicine is prepared from alkannin, tripterine, scutellarin or luteolin, and the lipid metabolism medicine is prepared from cinnamyl aldehyde, curcumin, myricetin, squalene or diclofenac sodium. The drug-loaded linoleic acid lipid hybrid vesicles prepared by the invention have uniform particle size and very stable structure and property, are naturally targeted to macrophages, can enhance the ability of host cells to remove intracellular bacteria, and have a remarkable effect on treatment of staphylococcus aureus and escherichia coli drug-resistant bacterial infection.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicle, and a preparation method and application thereof. Background Art

[0002] Bacterial infections are one of the greatest public health threats facing the world today. Intracellular bacteria are able to evade the body's immune system and grow and reproduce normally within host cells. They are more difficult to eliminate than extracellular bacteria and can easily cause more serious infections. Intracellular bacterial infections are characterized by latency, a long course, and difficulty in effectively treating with drugs. Traditional antibiotic treatments have limited efficacy, and long-term use can cause pathogens to develop drug resistance, increasing the risk of antibiotic resistance. Therefore, it is critical to seek alternatives to traditional antibiotics and optimize strategies for treating intracellular bacterial infections.

[0003] Metabolic therapy involves exploiting metabolic characteristics that differ from those of normal cells in diseased cells to identify therapeutic approaches that target metabolic pathways and ultimately control the disease. Metabolic therapy has been extensively studied in a variety of diseases, including cancer, cardiovascular disease, osteoarthritis, and autoimmune diseases, and related drugs have been approved for clinical use. Metabolic therapy offers advantages such as long-term efficacy, minimal side effects, and immune-boosting effects. Key to this approach is the positive regulation of metabolic differences between metabolically abnormal and healthy cells. Similarly, during bacterial infection, significant differences in lipid metabolism are observed between infected and normal cells. Following bacterial infection, lipid droplets increase in tissues and dissociate from mitochondria, increasing their contact with bacteria and promoting their antimicrobial activity. These reassembled lipid droplets in infected cells are termed "defense-type lipid droplets" (D-LDs). Compared to normal lipid droplets (F-LDs), D-LDs formed in response to pathogens such as bacteria exhibit significantly enhanced protein-mediated antimicrobial activity. Studies have shown that RNF213-driven LD protein ISGylation is directly involved in the conversion of F-LDs to D-LDs. On the other hand, cells infected with bacteria switch from oxidative phosphorylation to aerobic glycolysis. PKM2, a key gene regulating cellular energy conversion, catalyzes the final step of glycolysis. However, aerobic glycolysis is inefficient, leading to impaired mitochondrial function and ultimately cell death from starvation. Therefore, inhibiting PKM2 activity plays a key role in reversing abnormal glucose metabolism. Applying metabolic therapy to the treatment of bacterial infections can improve the poor efficacy of existing antibiotics and the serious problem of drug resistance, while also improving prognosis and enhancing immunity.

[0004] The effectiveness of traditional drug treatment is subject to many constraints, such as solubility, bioavailability, and cellular entry limitations, and effective delivery vehicles need to be constructed to improve this. Nano drug delivery systems can effectively improve the solubility and stability of drugs in the body, increase drug bioavailability, and achieve controllable and sustained drug delivery. Linoleic acid (LA) and phosphatidylserine (PS) can self-assemble to form linoleic acid lipid hybrid vesicles, which are highly biodegradable and biocompatible. PS guides the phagocytosis of macrophages through the "eat me" signal to achieve macrophage targeting. Linoleic acid lipid hybrid vesicles have beneficial physiological activity, self-assembly activity, and self-crosslinking activity, making them an excellent delivery vehicle. As an energy source, they provide fatty acids to the body and can be loaded with drugs to achieve synchronous regulation of glycolipid metabolism.

[0005] In summary, providing a new drug for treating intracellular bacterial infection has become one of the urgent problems to be solved in this field. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a cell sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicle and its preparation method and application, which adopts metabolic therapy for antibacterial treatment, providing an alternative to traditional antibiotic treatment, effectively avoiding the problems of drug resistance and poor antibiotic treatment effect for drug-resistant bacteria.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicle, wherein the cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicle comprises a linoleic acid lipid hybrid vesicle, a sugar metabolism drug, and a lipid metabolism drug, wherein the sugar metabolism drug and the lipid metabolism drug are loaded in the linoleic acid lipid hybrid vesicle;

[0009] Wherein, the sugar metabolism drug includes any one of shikonin, triptolide, scutellarin or luteolin, or a combination of at least two thereof;

[0010] The lipid metabolism drug includes any one of cinnamaldehyde, curcumin, myricetin, squalene or diclofenac sodium, or a combination of at least two of them.

[0011] The cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid-lipid hybrid vesicles involved in the present invention are a drug system that, through the synergistic action of various components, can treat intracellular bacterial infections, especially infections caused by intracellular parasites such as Staphylococcus aureus and Escherichia coli. Linoleic acid-lipid hybrid vesicles are used as carriers for the delivery of sugar and lipid metabolism-regulating drugs. They have a uniform particle size and a very stable structure and properties, and are highly biocompatible and degradable. The sugar metabolism drug provided by the present invention is a traditional Chinese medicine ingredient that has the function of inhibiting cellular aerobic glycolysis, and the lipid metabolism drug is a traditional Chinese medicine ingredient that has the function of increasing the number of cellular defensive lipid droplets, effectively eliminating intracellular bacteria while maintaining mitochondrial function.

[0012] Preferably, the mass ratio of the carbohydrate metabolism drug to the lipid metabolism drug is (1-2):1.

[0013] The specific values ​​of the above (1-2) may be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0014] The metabolic regulation drug ratio provided by the present invention is the optimal ratio obtained by optimizing drug dosage through plate counting method, integrating cytotoxicity, establishing the interaction and dose-effect relationship between the ratio of two drugs and overall drug efficacy.

[0015] Preferably, the linoleic acid lipid hybrid vesicles are lipid-like nanoparticles composed of membrane phospholipid molecules and polyunsaturated fatty acids.

[0016] Preferably, the membrane phospholipid molecules include phosphatidylserine.

[0017] Preferably, the polyunsaturated fatty acid comprises linoleic acid and its structural isomers, and the chemical formula is C 18 H 32 O2.

[0018] Preferably, the linoleic acid lipid hybrid vesicles contain phosphatidylserine and conjugated linoleic acid.

[0019] The present invention uses conjugated linoleic acid and phosphatidylserine as raw materials for preparing linoleic acid-lipid hybrid vesicles. Phosphatidylserine naturally targets macrophages that highly express phosphatidylserine receptors through the "eat me" signal. Drug loading within these vesicles effectively achieves targeted, controlled, and long-lasting release of the drug, reducing the frequency of dosing and promoting effective intracellular accumulation of the drug, reducing drug accumulation in non-target tissues, and reducing drug toxicity and side effects. Lipids also serve as cellular energy sources, providing energy for the cells.

[0020] Preferably, the mass ratio of the phosphatidylserine to the conjugated linoleic acid is 1:(8-20).

[0021] The specific values ​​of the above (8 to 20) can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.

[0022] The ratio of the feed mass of phosphatidylserine to the feed mass of conjugated linoleic acid provided by the present invention satisfies the above-mentioned specific numerical range, so that the structural performance of the prepared linoleic acid lipid hybrid vesicles is more stable, and at the same time, it can have a better advantageous ability to target phagocytic immune cells.

[0023] Preferably, the raw materials for preparing the cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicles include sugar metabolism drugs, lipid metabolism drugs, phosphatidylserine and conjugated linoleic acid.

[0024] Preferably, the mass ratio of the carbohydrate metabolism drug to the lipid metabolism drug is (1-2):1.

[0025] The specific values ​​of the above (1-2) may be 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2, etc.

[0026] Preferably, the mass ratio of the phosphatidylserine to the conjugated linoleic acid is 1:(8-20).

[0027] The specific values ​​of the above (8 to 20) can be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.

[0028] In a second aspect, the present invention provides a method for preparing the cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicles as described in the first aspect, the preparation method comprising the following steps:

[0029] (1) Mixing and dissolving the carbohydrate metabolism drug, lipid metabolism drug and the raw materials for preparing linoleic acid lipid hybrid vesicles in an organic solvent, and rotary evaporating to form a lipid film;

[0030] (2) adding sodium dodecyl sulfate dissolved in sodium hydroxide solution and sonicating to obtain a dual-drug loaded liposome solution;

[0031] (3) Shaking the dual-drug-loaded liposome solution to obtain the cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicles.

[0032] Preferably, the raw materials for preparing the linoleic acid lipid hybrid vesicles include phosphatidylserine and conjugated linoleic acid.

[0033] Preferably, the organic solvent includes any one of methanol, chloroform, ethanol or dimethyl sulfoxide, or a combination of at least two thereof.

[0034] Preferably, the concentration of the sodium hydroxide solution is 0.1 to 0.5 M (for example, 0.1 M, 0.2 M, 0.3 M, 0.4 M or 0.5 M, etc.).

[0035] Preferably, the mass ratio of the sodium lauryl sulfate to the conjugated linoleic acid is 1:(8-12).

[0036] The specific values ​​of the above (8 to 12) may be 8, 9, 10, 11 or 12, etc.

[0037] Preferably, the power of the ultrasound is 300-500 W (for example, 300 W, 320 W, 350 W, 380 W, 400 W, 420 W, 450 W, 480 W or 500 W, etc.), and the time is 10-30 min (for example, 10 min, 15 min, 20 min, 25 min or 30 min, etc.).

[0038] Preferably, the shaking speed is 200-600 rpm (for example, 200 rpm, 300 rpm, 400 rpm, 500 rpm or 600 rpm, etc.), and the shaking time is 12-18 h (for example, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h or 18 h, etc.).

[0039] In a third aspect, the present invention provides the use of the preparation method of the cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicles as described in the first aspect and / or the cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicles as described in the second aspect in the preparation of drugs for treating intracellular bacterial infections.

[0040] Preferably, the intracellular bacterial infection includes Staphylococcus aureus infection and / or Escherichia coli infection.

[0041] Other specific point values ​​within the above numerical ranges can be selected and will not be described in detail here.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention relates to a cell sugar and lipid metabolism-regulated drug-loaded linoleic acid lipid hybrid vesicle for treating intracellular bacterial infection, which has a uniform particle size, a very stable structure and properties, and naturally targets macrophages, and can enhance the ability of host cells to clear intracellular bacteria. The method of treating intracellular bacterial infection based on metabolic regulation provided by the present invention has a significant effect on the treatment of drug-resistant bacterial infections including Staphylococcus aureus and Escherichia coli, is not easy to induce the generation of drug resistance, has a high efficiency in clearing intracellular bacteria, and has a high drug loading capacity of the linoleic acid lipid hybrid vesicle, good biosafety, and has the characteristics of a wide source and low cost, etc., for treating intracellular drug-resistant bacterial infection. The use of metabolic therapy for antibacterial treatment provides an alternative to traditional antibiotic treatment, effectively avoids the problems of drug resistance generation and poor antibiotic treatment effect for drug-resistant bacteria, has high safety and low toxic side effects, can be used for intracellular bacterial infection diseases, is a very promising drug delivery platform for treating intracellular bacterial infection, and provides a comprehensive and efficient treatment strategy for clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a transmission electron microscopy characterization of lipid hybrid vesicles (PS / CLAV).

[0045] Figure 2 is the particle size distribution diagram of lipid hybrid vesicles (PS / CLAV).

[0046] Figure 3 This is the potential characterization diagram of lipid hybrid vesicles (PS / CLAV).

[0047] Figure 4 This is a transmission electron microscopy characterization of lipid vesicles (CLAV).

[0048] Figure 5 is the particle size distribution diagram of lipid vesicles (CLAV).

[0049] Figure 6 This is a potential characterization diagram of lipid vesicles (CLAV).

[0050] Figure 7 This is the particle size distribution diagram of dual-drug loaded lipid hybrid vesicles (PS / CLAV@CA / SHI).

[0051] Figure 8 This is the potential characterization diagram of dual-drug-loaded lipid hybrid vesicles (PS / CLAV@CA / SHI).

[0052] Figure 9 This is a statistical graph of the intracellular fluorescence intensity of Raw264.7 macrophages after treatment in each group of endocytosis experiments.

[0053] Figure 10 These are the fluorescence microscopy observation results of lipid droplet production in Raw264.7 macrophages after treatment in each group.

[0054] Figure 11 This is the result of fluorescence quantitative PCR test on the transcription level of RNF213, a lipid metabolism regulatory gene in macrophage Raw264.7 cells after treatment in each group.

[0055] Figure 12 This is the protein expression result of RNF213, a lipid metabolism regulatory gene in macrophage Raw264.7 cells after treatment in each group of WB experiment.

[0056] Figure 13 This is a graph showing the effect of dual-drug loaded lipid hybrid vesicles (PS / CLAV@CA / SHI) on the protein expression levels of lipid droplet proteins PLIN2 and PLIN5 in macrophage Raw264.7 cells.

[0057] Figure 14 This is the result of the bacterial killing experiment on Staphylococcus aureus by lipid droplets produced after treating macrophage Raw264.7 cells with dual-drug loaded lipid hybrid vesicles (PS / CLAV@CA / SHI).

[0058] Figure 15 This is the result of the bacterial killing experiment on Escherichia coli by lipid droplets produced after treating macrophage Raw264.7 cells with dual-drug loaded lipid hybrid vesicles (PS / CLAV@CA / SHI).

[0059] Figure 16 This is a graph showing the effects of treatments in each group of the Seahorse experiment on the glycolysis level of macrophage Raw264.7.

[0060] Figure 17 This is the plate coating result after each group treated the macrophage Raw264.7 cells in the Staphylococcus aureus intracellular bacteria clearance test.

[0061] Figure 18 These are the results of fluorescence microscopy observation after macrophage Raw264.7 cells were treated by each group in the Staphylococcus aureus intracellular bacteria clearance test.

[0062] Figure 19 This is the plate coating result after each group treated the macrophage Raw264.7 cells in the Escherichia coli intracellular bacteria clearance test.

[0063] Figure 20 These are the results of fluorescence microscopy observation after macrophage Raw264.7 cells were treated with each group in the Escherichia coli intracellular bacteria clearance test. DETAILED DESCRIPTION

[0064] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0065] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0066] The sources of the reagents used in the following examples are as follows:

[0067] Conjugated linoleic acid was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a purity of 95% and the model number was C922713; phosphatidylserine was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a purity of 50% and the model number was S832149; shikonin, with an analytical purity (AR), was purchased from Ruifen Biotechnology Co., Ltd.; cinnamaldehyde was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a purity of ≥95% (GC) and the model number was C80500.

[0068] Example 1

[0069] This embodiment provides a lipid hybrid vesicle (PS / CLAV), the preparation method of which comprises the following steps:

[0070] (1) Weigh 16 mg of conjugated linoleic acid and 2 mg of phosphatidylserine and dissolve them in 4 mL of a mixture of methanol and chloroform (1:1, v / v). Transfer the mixture to a 50 mL round-bottom flask and place it in a 37°C water bath at a speed of 100 r / min. Rotary evaporation was performed for 20 min to remove the solvent. The remaining components formed a thin film on the bottom wall of the flask.

[0071] (2) Weigh 1.6 mg of sodium dodecyl sulfate and dissolve it in 3 mL of 0.1 M NaOH.

[0072] (3) 3 mL of 0.1 M NaOH (containing SDS) was slowly added to the round-bottom flask after rotary evaporation, and ultrasonicated at room temperature for 10 min at a power of 300 W to disperse the lipid carrier.

[0073] The prepared PS / CLAV nanoparticles were characterized by transmission electron microscopy. Figure 1 As shown in Figure 2, the PS / CLAV nanoparticles have uniform particle size and bilayer vesicle structure, good dispersion and stable properties. The prepared PS / CLAV nanoparticles were characterized by particle size and potential, as shown in Figure 2. Figure 2 and Figure 3As shown, the PS / CLAV nanoparticles were 79.76 ± 4.87 nm and had a zeta potential of -30.1 ± 4.10 mV.

[0074] Example 2

[0075] This example provides a lipid vesicle (CLAV), which differs from Example 1 in that it does not contain PS. The preparation method thereof comprises the following steps:

[0076] (1) Weigh 16 mg of conjugated linoleic acid and 2 mg of phosphatidylserine and dissolve them in 4 mL of a mixture of methanol and chloroform (1:1, v / v). Transfer the mixture to a 50 mL round-bottom flask and place it in a 37°C water bath at a speed of 100 r / min. Rotary evaporation was performed for 20 min to remove the solvent. The remaining components formed a thin film on the bottom wall of the flask.

[0077] (2) Weigh 1.6 mg of sodium dodecyl sulfate and dissolve it in 3 mL of 0.1 M NaOH.

[0078] (3) 3 mL of 0.1 M NaOH (containing SDS) was slowly added to the round-bottom flask after rotary evaporation, and ultrasonicated at room temperature for 10 min at a power of 300 W to disperse the lipid carrier.

[0079] The prepared CLAV nanoparticles were characterized by transmission electron microscopy. Figure 4 As shown in Figure 2, CLAV nanoparticles have uniform particle size and vesicle structure, good dispersion and stable properties. The particle size and potential of the prepared CLAV nanoparticles were characterized, as shown in Figure 2. Figure 5 and Figure 6 As shown, the CLAV nanoparticles were 25.43 ± 9.186 nm and had a zeta potential of -48 ± 6.21 mV.

[0080] Example 3

[0081] This embodiment provides a drug-loaded lipid hybrid vesicle (PS / CLAV@CA / SHI) for regulating cellular sugar and lipid metabolism. The preparation method thereof comprises the following steps:

[0082] (1) Weigh 16 mg of conjugated linoleic acid, 2 mg of phosphatidylserine, 1 mg of cinnamaldehyde, and 2 mg of shikonin and dissolve them in 4 mL of a mixture of methanol and chloroform (1:1, v / v). Transfer the mixture to a 50 mL round-bottom flask and place it in a 37°C water bath at a speed of 100 r / min. Rotary evaporation was performed for 20 min to remove the solvent. The remaining components formed a thin film on the bottom wall of the flask.

[0083] (2) Weigh 1.6 mg of sodium dodecyl sulfate and dissolve it in 3 mL of 0.1 M NaOH.

[0084] (3) 3 mL of 0.1 M NaOH (containing SDS) was slowly added to the round-bottom flask after rotary evaporation, and ultrasonicated at room temperature for 10 min at a power of 300 W to disperse the lipid carrier.

[0085] (4) The dual-drug-loaded liposome solution was shaken at 200 rpm for 12 h to obtain the drug-loaded lipid hybrid vesicles.

[0086] The prepared PS / CLAV@CA / SHI nanoparticles were characterized by particle size and potential, as shown in Figure 2. Figure 7 and Figure 8 As shown, the average particle size of PS / CLAV@CA / SHI nanoparticles is 84.74±6.16nm and the zeta potential is -28.1±5.31mV.

[0087] Test Example 1

[0088] This test example detects the endocytosis effect of PS / CLAV@CA / SHI nanoparticles in macrophage RAW264.7 cells:

[0089] (1) Cell culture:

[0090] Macrophage RAW264.7 cells (within 20 generations) were cultured in DMEM complete medium containing 10% newborn calf serum (NCS), cultured at 37° C. with 5% CO 2 , and passaged at a ratio of 1:3 when the cell density reached 80% to 90%.

[0091] (2) Cell plating:

[0092] 6-well cell culture plate, the density of Raw264.7 cells is 2×10 6 After culturing for 1 day in the cell culture plate, the cell density reached 100%.

[0093] (3) Endocytosis experiment:

[0094] The cell endocytosis experiment was carried out using the method reported in the literature (Zhao Q, Liu J, Liu S, et al. Multipronged Micelles-Hydrogel for Targeted and Prolonged Drug Delivery in Chronic Wound Infections [J]. ACS Applied Materials & Interfaces, 2022, 14 (41): 46224-46238.). The RAW264.7 cell experimental cell line cultured as above was used, PBS without any drug was used as the control group (NC), fluorescein coumarin 6 (C6) was used as the small molecule drug group, and lipid hybrid vesicles loaded with coumarin 6 (PS / CLAV@C6) were used as the nanoparticle group (refer to the preparation method of Example 1). Finally, CLAV@C6 (refer to the preparation method of Example 2) was added as the nanoparticle control group without adding modified molecules. After incubating macrophages with different groups of drugs, the cells were washed with PBS to remove the fluorescent drugs that failed to be internalized into the cells. The mean fluorescence intensity of the cells was detected by flow cytometry to evaluate the endocytosis of macrophages.

[0095] The statistical results are as follows Figure 9 As shown, compared with the PBS group and the C6-containing small molecule drug group, the C6-loaded lipid hybrid vesicles (PS / CLAV@C6) and C6-loaded lipid vesicles (CLAV@C6) nanoparticles and their control group significantly increased the concentration of C6 in macrophages. Since small molecule drugs can only enter the cell through passive transport, the intracellular drug accumulation concentration is low, manifested as lower fluorescence intensity, demonstrating the advantage of nanoparticles as carriers. In addition, since CLAV does not have the ability to target macrophages, while PS / CLAV can actively target cells through PS receptors, PS / CLAV@C6 exhibits higher fluorescence intensity than CLAV@C6, indicating higher intracellular drug accumulation. This suggests that PS / CLAV can enhance the endocytosis of small molecule drugs by macrophages and has advantages over lipid carriers. This process improves drug bioavailability while reducing drug accumulation in non-target tissues, minimizing drug toxicity and side effects, and improving biosafety.

[0096] Test Example 2

[0097] This test case investigates the effect of PS / CLAV@CA / SHI nanoparticles on macrophage lipid metabolism.

[0098] (1) Cell culture:

[0099] Macrophage RAW264.7 cells (within 20 generations) were cultured in DMEM complete medium containing 10% newborn calf serum (NCS), cultured at 37° C. with 5% CO 2 , and passaged at a ratio of 1:3 when the cell density reached 80% to 90%.

[0100] (2) Cell plating:

[0101] The density of Raw264.7 cells in the confocal microplate was 1.2×10 6 After culturing for 1 day in the cell culture plate, the cell density reached 100%.

[0102] (3) Drug-treated cells:

[0103] PBS without any drug was used as the negative and positive control groups, respectively, while 500 ng / mL LPS / LTA was used as the drug treatment groups. The drug treatment groups included the cinnamaldehyde (CA) treatment group (using a concentration of 0.625 μg / mL), the shikonin (SHI) treatment group (using a concentration of 1.25 μg / mL), the cinnamaldehyde / shikonin (CA / SHI) dual-drug treatment group (using a concentration of cinnamaldehyde and shikonin of 0.625 μg / mL and 1.25 μg / mL, respectively), the CLAV prepared in Example 2, and the PS / CLAV@CA / SHI treatment group prepared in Example 3 (using a concentration of cinnamaldehyde and shikonin of 0.625 μg / mL and 1.25 μg / mL, respectively). Mouse macrophages were treated for 12 hours.

[0104] (4) Intracellular lipid droplet staining and observation:

[0105] The cells were washed twice with PBS, fixed with 4% paraformaldehyde (1 mL / well) for 20 min, washed twice with PBS, permeabilized with 1% Triton for 20 min, and washed twice with PBS. Nile red at a working concentration of 5-10 μg / mL was then added to stain intracellular lipid droplets, and cell nuclei were stained with DAPI. Finally, super-resolution confocal microscopy was used to study the effect of PS / CLAV@CA / SHI on the lipid droplet content in macrophages.

[0106] In addition, the transcription and protein levels of RNF213, a key gene regulating defensive lipid droplets, were determined by fluorescence quantitative PCR and Western blot to determine the regulatory effect of PS / CLAV@CA / SHI on the conversion of defensive lipid droplets.

[0107] like Figure 10As shown in the figure, PS / CLAV@CA / SHI can significantly increase the number of intracellular lipid droplets, which is manifested by a significant increase in red fluorescence. The effect of each treatment group on macrophage lipid droplet production is PS / CLAV@CA / SHI>CLAV>CA≈SHI≈CA+SHI≈LPS / LTA≈Control. This shows that lipid carriers can provide energy substances for cells to increase the number of lipid droplets. PS / CLAV@CA / SHI further promotes cellular lipid metabolism by encapsulating sugar and lipid metabolism regulating drugs. Figure 11 and Figure 12 As shown in the figure, fluorescence quantitative PCR and Western Blot showed that PS / CLAV@CA / SHI could significantly increase the transcription and protein expression levels of the RNF213 gene.

[0108] (5) Lipid droplet extraction and lipid droplet bacteria killing experiment:

[0109] References (Marta Bosch, Miguel Alba Fajardo, et al. Mammalian lipid droplets are innate immune hubs integrating cellmetabolism and host defense. [J]. Science, 2020, 370: eaay8085) reported in the lipid droplet bacterial killing experiment. PBS without any drug and 500 ng / mL of LPS / LTA were used as negative and positive control groups, respectively, and the PS / CLAV @ CA / SHI treatment group prepared in Example 3 (the concentrations of cinnamaldehyde and shikonin were 0.625 μg / mL and 1.25 μg / mL, respectively) were incubated with mouse macrophages Raw264.7 for 12 hours, and the cells were collected and the cell lipid droplets were extracted using a lipid droplet extraction kit. Western Blot was used to detect the defensive lipid droplet markers PLIN2 and PLIN5 to characterize the proportion of defensive lipid droplets in the cells. The isolated lipid droplets were incubated with Staphylococcus aureus T144 strain and Escherichia coli B2 strain for bacterial killing experiments. The specific steps are as follows: Staphylococcus aureus (T144) and Escherichia coli (B2) were cultured to the logarithmic culture phase, diluted 1:100 (about 1.5×10 5 CFU / mL). Mix 100 μL of the diluted bacterial solution with 25 μg of LD protein. Then add PBS to 200 mL. Incubate in a shaker at 37°C for the designated time. Serially dilute three times on LB agar plates, incubate overnight at 37°C, and observe colony growth.

[0110] The results are as follows Figure 13As shown in the figure, compared with other treatment groups, the lipid droplets produced by macrophages after PS / CLAV@CA / SHI treatment highly expressed PLIN5 and low expressed PLIN2, showing the typical characteristics of defensive lipid droplets. Figure 14 and Figure 15 As shown in the figure, compared with the lipid droplets of untreated cells and cells stimulated with LPS / LTA, the lipid droplets produced by macrophages after PS / CLAV@CA / SHI treatment had a more significant bactericidal effect on Staphylococcus aureus and Escherichia coli.

[0111] Test Example 3

[0112] This test investigates the effect of PS / CLAV@CA / SHI nanoparticles on macrophage glucose metabolism.

[0113] (1) Cell culture:

[0114] Macrophage RAW264.7 cells (within 20 generations) were cultured in DMEM complete medium containing 10% newborn calf serum (NCS), cultured at 37° C. with 5% CO 2 , and passaged at a ratio of 1:3 when the cell density reached 80% to 90%.

[0115] (2) Cell plating:

[0116] In Seahorse XF96 cell plates, the density of Raw264.7 cells was 1.0 × 10 4 After culturing for 1 day in the cell culture plate, the cell density reached 100%.

[0117] (3) Drug-treated cells:

[0118] PBS without any drug was used as the negative and positive control groups, respectively, while 500 ng / mL LPS / LTA was used as the drug treatment groups. The drug treatment groups included the cinnamaldehyde (CA) treatment group (using a concentration of 0.625 μg / mL), the shikonin (SHI) treatment group (using a concentration of 1.25 μg / mL), the cinnamaldehyde / shikonin (CA / SHI) dual-drug treatment group (using a concentration of cinnamaldehyde and shikonin of 0.625 μg / mL and 1.25 μg / mL, respectively), the CLAV prepared in Example 2, and the PS / CLAV@CA / SHI treatment group prepared in Example 3 (using a concentration of cinnamaldehyde and shikonin of 0.625 μg / mL and 1.25 μg / mL, respectively). Mouse macrophages were treated for 12 hours.

[0119] (4) Seahorse tests cellular glycolysis levels:

[0120] Cellular glycolysis levels were measured using the method reported in the reference (Li X, Shen H, Zhang M, et al. Glycolytic reprogramming in macrophages and MSCs during inflammation. [J]. Front Immunol, 2023, 14: 1199-751). The extracellular acidification rate (ECAR) reflects the level of cellular glycolysis. By calculating the curvature trend of each level of the ECAR hippocampal curve and related parameters, the stress changes in cellular energy metabolism under different treatments can be assessed. For the ECAR test of glycolysis, Seahorse basal medium containing 4mM glutamine was prepared. During the ECAR assay, the probe card was sequentially injected with a final concentration of 25mM glucose, 4μM oligomycin, and 50mM 2-DG at a volume of 25μL / well, and the changes in extracellular pH were recorded. The effects of PS / CLAV@CA / SHI on the glycolysis level of macrophages were analyzed.

[0121] like Figure 16 As shown in the figure, compared with other treatment groups, the glycolysis level of macrophages after PS / CLAV@CA / SHI treatment was significantly decreased, which was manifested by the lower extracellular acidification rate at each stage compared with other groups, indicating that glycolysis was not the main mode of macrophages after PS / CLAV@CA / SHI treatment, and PS / CLAV@CA / SHI achieved the regulation of cells towards inhibiting aerobic glycolysis.

[0122] Test Example 4

[0123] This test investigated the clearance effect of PS / CLAV@CA / SHI nanoparticles on intracellular Staphylococcus aureus and Escherichia coli in macrophages.

[0124] (1) Cell culture:

[0125] Macrophage RAW264.7 cells (within 20 generations) were cultured in DMEM complete medium containing 10% newborn calf serum (NCS), cultured at 37° C. with 5% CO 2 , and passaged at a ratio of 1:3 when the cell density reached 80% to 90%.

[0126] (2) Cell plating:

[0127] The density of Raw264.7 cells in the confocal microplate was 1.2×10 6 After culturing for 1 day in the cell culture plate, the cell density reached 100%.

[0128] (3) Staphylococcus aureus / Escherichia coli invade cells:

[0129] Staphylococcus aureus fluorescent strain GFP T144 and Escherichia coli fluorescent strain GFP B2 were cultured to the logarithmic growth phase, and the concentration was adjusted to McFarland turbidimetric value = 0.5 (1.5×10 8 CFU / mL). Bacteria at a dose of 10 times the cell count were added to the confocal microplate and incubated at 37°C in a 5% constant temperature incubator for 2 h. Subsequently, bacteria that did not invade the cells were washed away with DMEM containing 1% FBS, and 100 μg / mL of gentamicin was added for 30 min to eliminate extracellular bacteria.

[0130] (4) Effect of drug group treatment on clearing intracellular bacteria:

[0131] The cell endocytosis experiment was performed according to the method reported in the reference (Junhua H,yingxian C,et al.A Comparative Analysis of the Antibacterial Spectrum of Ultrasmall Manganese Ferrite Nanozymes with Varied Surface Modifications[J].ACS Applied Materials&Interfaces,2024,16(12):14385-14404.). Mouse macrophages invaded by the fluorescent Staphylococcus aureus strain GFP29213 and the fluorescent Escherichia coli strain GFP25922 were used as experimental cell lines. PBS without any drug was used as the control group. The drug treatment groups included the cinnamaldehyde (CA) treatment group (using a concentration of 0.625 μg / mL), the shikonin (SHI) treatment group (using a concentration of 1.25 μg / mL), the cinnamaldehyde / shikonin (CA / SHI) dual-drug treatment group (using concentrations of cinnamaldehyde and shikonin of 0.625 μg / mL and 1.25 μg / mL, respectively), and the PS / CLAV@CA / SHI treatment group prepared in Example 3 (using concentrations of cinnamaldehyde and shikonin of 0.625 μg / mL and 1.25 μg / mL, respectively). Mouse macrophages invaded by the fluorescent Staphylococcus aureus strain GFP T144 and the fluorescent Escherichia coli strain GFP B2 were treated for 4 hours. After the cells were broken, the bacteria were collected and plate counted to clarify the clearance effects of different treatment groups on intracellular parasitic Staphylococcus aureus and Escherichia coli.

[0132] Furthermore, the effectiveness of PS / CLAV@CA / SHI in eliminating intracellular bacteria was analyzed using fluorescence microscopy. The number of surviving fluorescent strains within the cells was observed and counted under an inverted fluorescence microscope.

[0133] The statistical results are as follows Figures 17-20As shown, the effectiveness of each treatment group in clearing intracellular bacteria, from strong to weak, is PS / CLAV@CA / SHI > CA / SHI > CA ≈ SHI > CLAV ≈ Control. Compared to the other treatment groups, after PS / CLAV@CA / SHI treatment, PS naturally targets macrophages that overexpress PS receptors, enabling efficient intracellular transport of CA and SHI, synergistically regulating cellular glycolipid metabolism, increasing the number of defensive lipid droplets while inhibiting aerobic glycolysis. PS / CLAV enhances drug bioavailability while providing energy to the body, effectively eliminating intracellular Staphylococcus aureus and Escherichia coli.

[0134] In summary, the present invention uses four specific components as raw materials for preparing linoleic acid lipid hybrid vesicles that regulate cell sugar and lipid metabolism. The prepared drug-loaded linoleic acid lipid hybrid vesicles have significant effects on the treatment of drug-resistant bacterial infections including Staphylococcus aureus and Escherichia coli, are not easy to induce the development of drug resistance, have high efficiency in clearing intracellular bacteria, have high drug loading capacity of linoleic acid lipid hybrid vesicles, good biosafety, and have the characteristics of wide sources and low cost.

[0135] The applicant declares that the above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the scope of protection and disclosure of the present invention.

[0136] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0137] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A drug-loaded linoleic acid-lipid hybrid vesicle that regulates cell sugar and lipid metabolism, characterized in that: The cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicles include linoleic acid lipid hybrid vesicles, sugar metabolism drugs and lipid metabolism drugs, and the sugar metabolism drugs and lipid metabolism drugs are loaded in the linoleic acid lipid hybrid vesicles; Wherein, the sugar metabolism drug includes any one of shikonin, triptolide, scutellarin or luteolin, or a combination of at least two thereof; The lipid metabolism drug includes any one of cinnamaldehyde, curcumin, myricetin, squalene or diclofenac sodium, or a combination of at least two of them.

2. The cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicle according to claim 1, characterized in that: The mass ratio of the sugar metabolism drug to the lipid metabolism drug is (1-2):

1.

3. The cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicle according to claim 1 or 2, characterized in that: The linoleic acid lipid hybrid vesicles are lipid-like nanoparticles composed of membrane phospholipid molecules and polyunsaturated fatty acids; Preferably, the membrane phospholipid molecules include phosphatidylserine; Preferably, the polyunsaturated fatty acid comprises linoleic acid and its structural isomers, and the chemical formula is C 18 H 32 O2; Preferably, the linoleic acid lipid hybrid vesicles contain phosphatidylserine and conjugated linoleic acid; Preferably, the mass ratio of the phosphatidylserine to the conjugated linoleic acid is 1:(8-20).

4. The cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicle according to any one of claims 1 to 3, characterized in that: The raw materials for preparing the cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicles include sugar metabolism drugs, lipid metabolism drugs, phosphatidylserine and conjugated linoleic acid.

5. The cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicle according to claim 4, characterized in that: The mass ratio of the sugar metabolism drug to the lipid metabolism drug is (1-2):1; Preferably, the mass ratio of the phosphatidylserine to the conjugated linoleic acid is 1:(8-20).

6. A method for preparing the cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicles according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) Mixing and dissolving the carbohydrate metabolism drug, lipid metabolism drug and the raw materials for preparing linoleic acid lipid hybrid vesicles in an organic solvent, and rotary evaporating to form a lipid film; (2) adding sodium dodecyl sulfate dissolved in sodium hydroxide solution and sonicating to obtain a dual-drug loaded liposome solution; (3) Shaking the dual-drug-loaded liposome solution to obtain the cell sugar and lipid metabolism regulating drug-loaded linoleic acid lipid hybrid vesicles.

7. The preparation method according to claim 6, characterized in that The raw materials for preparing the linoleic acid lipid hybrid vesicles include phosphatidylserine and conjugated linoleic acid.

8. The preparation method according to claim 6 or 7, characterized in that The organic solvent includes any one of methanol, chloroform, ethanol or dimethyl sulfoxide or a combination of at least two thereof; Preferably, the concentration of the sodium hydroxide solution is 0.1 to 0.5 M; Preferably, the mass ratio of the sodium lauryl sulfate to the conjugated linoleic acid is 1:(8-12); Preferably, the power of the ultrasound is 300-500W, and the time is 10-30min; Preferably, the shaking speed is 200-600 rpm and the shaking time is 12-18 hours.

9. Use of the preparation method of the cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicles according to any one of claims 1 to 5 and / or the cellular sugar and lipid metabolism-regulating drug-loaded linoleic acid lipid hybrid vesicles according to any one of claims 6 to 8 in the preparation of drugs for treating intracellular bacterial infections.

10. The use according to claim 9, characterized in that The intracellular bacterial infection includes Staphylococcus aureus infection and / or Escherichia coli infection.

Images