Preparation method of novel antibacterial liposome for enhancing phagocytosis of neutrophil

Abstract

The invention relates to a preparation method of a novel antibacterial liposome for enhancing phagocytosis of neutrophil. Belongs to the technical field of biological medicine, and comprises the following steps: weighing phosphatidylcholine and other components according to a molar ratio of POPC: DOPE: DOTAP: Cholesterol of 2: 1: 3: 4, dissolving the components in chloroform, carrying out rotary evaporation and gradual pressure reduction at 25 DEG C to form a lipid membrane, carrying out PBS hydration, vortex and ultrasonic treatment, extruding through a nuclear track membrane and a support membrane, and finally determining related parameters by using a dynamic light scattering technology. The novel antibacterial lipidosome is unique in formula and good in biocompatibility, can be combined with neutrophil with high selectivity, enhances the phagocytic ability of the neutrophil, and improves the anti-infection level of an organism. In addition, bacteria can be rapidly combined, multiplication of the bacteria is delayed, growth of the bacteria is efficiently inhibited, a new way is provided for clinical antibacterial treatment, and the method has great application potential in coping with bacterial infection diseases.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and particularly discloses a preparation method of a novel antibacterial liposome capable of enhancing neutrophil phagocytosis. Background Art

[0002] Neutrophils are an important component of the human immune system, clearing pathogens from the body through phagocytosis. However, in certain conditions, neutrophil phagocytosis can be inhibited, resulting in reduced bacterial clearance efficiency. Therefore, enhancing neutrophil phagocytosis is key to improving the body's ability to resist infection.

[0003] Liposomes have been extensively studied as drug delivery systems due to their biocompatibility and targeting properties. The development of novel liposome technologies, particularly those that rapidly bind to bacteria and delay their doubling time, and those that selectively bind to neutrophils and enhance their phagocytosis, offers new possibilities for antimicrobial therapy.

[0004] Therefore, it is necessary to develop a method for preparing a new antibacterial liposome that enhances the phagocytic effect of neutrophils to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a novel antibacterial liposome capable of enhancing the phagocytic function of neutrophils.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: A method for preparing a novel antibacterial liposome for enhancing neutrophil phagocytosis comprises the following steps: S1. Accurately weigh the required components of phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyldimethylalanine (DOTAP), and cholesterol (Cholesterol) according to the molar ratio of POPC:DOPE:DOTAP:Cholesterol = 2:1:3:4; S2. Dissolve the lipid prepared above in chloroform, evaporate the chloroform using a rotary evaporator at 25°C to form a lipid film, gradually reduce the vacuum pressure during evaporation to 275, 240, 100, and 10 mbar, and maintain each pressure for 10 minutes until the chloroform is completely evaporated to form a uniform lipid film; evaporate the chloroform using a rotary evaporator at 25°C to form a lipid film, gradually reduce the vacuum pressure during evaporation to 275, 240, 100, and 10 mbar, and maintain each pressure for 10 minutes until the chloroform is completely evaporated to form a uniform lipid film; S3. Hydrate the lipid film with PBS pH 7.4 and vortex for 1 min to ensure that the lipids are fully dispersed. S4, sonication for 5 minutes to further disperse the liposomes and reduce the size of the liposomes; S5, extruding the liposome solution through 100 nm and 50 nm nuclear pore membranes and two 10 mm PE support membranes to obtain liposomes of uniform size; S6. Use dynamic light scattering (DLS) technology to determine the size, zeta potential, and particle concentration of liposomes.

[0007] The beneficial effects of the present invention are: The novel antibacterial liposomes prepared by this invention exhibit significant beneficial effects. Their unique formulation features a specific molar ratio of lipid components (POPC:DOPE:DOTAP:Cholesterol = 2:1:3:4). These liposomes exhibit excellent biocompatibility and highly selectively bind to neutrophils, effectively enhancing their phagocytic capacity, thereby improving the body's pathogen clearance and boosting infection resistance. Furthermore, they rapidly bind to bacteria, slowing their doubling time and contributing to more efficient inhibition of bacterial growth and reproduction during antibacterial therapy. This provides a powerful new approach for clinical antibacterial therapy and holds great potential for addressing bacterial infection-related diseases.

[0008] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Schematic diagram of the method for measuring the size of liposomes using dynamic light scattering technology according to the present invention; Figure 2 Schematic diagram of measuring the zeta potential of liposomes using dynamic light scattering technology according to the present invention; Figure 3 Schematic diagram of the method for measuring the particle concentration of liposomes using dynamic light scattering technology according to the present invention; Figure 4 Schematic diagram of the combination of liposomes and Escherichia coli shown in the present invention; Figure 5 Schematic diagram of the liposomes of the present invention delaying the doubling time of Escherichia coli and reducing the number of bacterial proliferation; Figure 6 Schematic diagram of the specific binding of liposomes to neutrophils according to the present invention; Figure 7 Schematic diagram of the liposome-enhanced neutrophil phagocytosis of Escherichia coli shown in the present invention; Figure 8 Schematic diagram of the co-localization of liposomes, Escherichia coli and neutrophils shown in the present invention. DETAILED DESCRIPTION

[0010] The following embodiments of the present invention are described in further detail with reference to the accompanying drawings and examples. The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0011] Unless otherwise specified, the technical solutions described in the present invention are all conventional solutions in the field; the reagents or materials described are all from commercial channels unless otherwise specified.

[0012] The present application provides a method for preparing a novel antibacterial liposome for enhancing neutrophil phagocytosis, comprising the following steps: S1. Accurately weigh the required components of phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyldimethylalanine (DOTAP), and cholesterol (Cholesterol) according to the molar ratio of POPC:DOPE:DOTAP:Cholesterol = 2:1:3:4; S2. Dissolve the lipid prepared above in chloroform, evaporate the chloroform using a rotary evaporator at 25°C to form a lipid film, gradually reduce the vacuum pressure during evaporation to 275, 240, 100, and 10 mbar, and maintain each pressure for 10 minutes until the chloroform is completely evaporated to form a uniform lipid film; evaporate the chloroform using a rotary evaporator at 25°C to form a lipid film, gradually reduce the vacuum pressure during evaporation to 275, 240, 100, and 10 mbar, and maintain each pressure for 10 minutes until the chloroform is completely evaporated to form a uniform lipid film; S3. Hydrate the lipid film with PBS pH 7.4 and vortex for 1 min to ensure that the lipids are fully dispersed. S4, sonication for 5 minutes to further disperse the liposomes and reduce the size of the liposomes; S5, extruding the liposome solution through 100 nm and 50 nm nuclear pore membranes and two 10 mm PE support membranes to obtain liposomes of uniform size; S6, see Figures 1 to 3 The size, zeta potential, and particle concentration of liposomes were determined using dynamic light scattering (DLS) technology. Example

[0013] Observing the interaction between liposomes and bacteria 1. Material preparation: The liposome solution was prepared according to the molar ratio of POPC:DOPE:DOTAP:Cholesterol = 2:1:3:4; The E. coli strain was pre-cultured to the logarithmic growth phase; Agarose plate culture medium; Fluorescent dye Cy5 used to label liposomes; the nucleic acid-binding dye DAPI used to label E. coli; confocal laser scanning microscopy; Microplate reader.

[0014] 2. Observe the binding of liposomes to bacteria: (1) Mix the fluorescent dye Cy5 with the liposome solution to label the liposomes; (2) Cultivate the bacteria to the logarithmic growth phase, collect the bacterial cells by centrifugation, wash with PBS and resuspend to a concentration of OD600nm = 0.1; (3) Cy5-labeled liposomes and DAPI-labeled Escherichia coli were mixed in a 1:1 volume ratio and placed on the sample stage of a confocal microscope; (4) Use confocal laser scanning microscopy to observe the interaction between liposomes and bacteria in real time; (5) Adjust the microscope parameters to obtain the best imaging effect of liposomes (red fluorescence) and bacteria (blue fluorescence); (6) See Figure 4 , liposomes can bind to E. coli within 10 seconds.

[0015] 3. Bacterial growth curve: (1) Cultivate the bacteria to the logarithmic growth phase, collect the bacterial cells by centrifugation, wash with PBS and resuspend to a concentration of OD600nm = 0.1; (2) In a 96-well plate, inoculate the bacteria into the culture medium and set up an experimental group (with liposomes added) and a control group (without liposomes added); (3) Add an equal amount of liposome solution to the bacterial solution into the wells of the experimental group, and add an equal amount of PBS to the control group; (4) Place the 96-well plate in a microplate reader, set the temperature to 37°C, and set the OD600nm reading interval (read every 5 minutes); (5) Start the microplate reader to monitor bacterial growth in real time for 16 hours and record the changes in OD600nm; (6) Record the OD600nm value of each well and calculate the bacterial growth curve; (7) See Figure 5 , liposomes delay the doubling time of Escherichia coli and reduce the number of bacterial proliferation. Example

[0016] Observation of the interaction between liposomes and neutrophils 1. Material preparation: Liposome solution prepared according to the molar ratio of POPC:DOPE:DOTAP:Cholesterol=2:1:3:4, human peripheral blood cells, red blood cell lysate, Phrodo E. Coli particles, a fluorescently labeled E. coli particle for phagocytosis experiment, fluorescent dye Cy5 for labeling liposomes, flow cytometric antibodies for labeling different cell subpopulations, flow cytometer, confocal laser scanning microscope.

[0017] 2. Observe the specificity of liposome binding to peripheral blood cells: (1) Collect peripheral blood and add appropriate amount of red blood cell lysis solution to lyse red blood cells; (2) Wash and resuspend the lysed peripheral blood leukocytes; (3) Add flow cytometry antibodies according to the ratio specified in the antibody instructions to label different cell subpopulations; (4) Mix the antibody-labeled peripheral blood cells with the liposomes and immediately detect them using a flow cytometer; (5) See Figure 6 , liposomes can specifically bind to neutrophils.

[0018] 3. Observe the effect of liposomes on neutrophil phagocytosis: (1) Collect peripheral blood and add cell lysis buffer at a ratio of 1:5 to lyse red blood cells; (2) Wash and resuspend the lysed peripheral blood leukocytes; (3) Incubate leukocytes with Phrodo E. coli particles and Cy5-labeled liposomes at 37°C for 1 hour; (4) Analyze the fluorescence signal of Phrodo E. coli particles phagocytosed by neutrophils using flow cytometry; (5) Observe the sample after neutrophils were co-incubated with Phrodo E. coli particles and liposomes under a laser confocal microscope; (6) See Figure 7-Figure 8 , liposomes significantly enhanced the phagocytosis of Escherichia coli particles by neutrophils.

[0019] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0020] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for preparing a novel antibacterial liposome for enhancing neutrophil phagocytosis, characterized in that: The following steps are involved: S1. Accurately weigh the required components of phosphatidylcholine (POPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyldimethylalanine (DOTAP), and cholesterol (Cholesterol) according to the molar ratio of POPC:DOPE:DOTAP:Cholesterol = 2:1:3:4; S2. Dissolve the lipids prepared above in chloroform and evaporate the chloroform using a rotary evaporator at 25°C to form a lipid film. During the evaporation process, gradually reduce the vacuum pressure to 275, 240, 100, and 10 mbar, and maintain each pressure for 10 minutes until the chloroform is completely evaporated and a uniform lipid film is formed. S3. Hydrate the lipid film with PBS pH 7.4 and vortex for 1 min to ensure that the lipids are fully dispersed. S4, sonication for 5 minutes to further disperse the liposomes and reduce the size of the liposomes; S5, extruding the liposome solution through 100 nm and 50 nm nuclear pore membranes and two 10 mm PE support membranes to obtain liposomes of uniform size; S6. Use dynamic light scattering technology to determine the size, zeta potential, and particle concentration of liposomes.

Images