Beta-1, 3 / alpha-1, 3-glucan modified liposome preparation as well as preparation method and application thereof

Abstract

The invention provides a beta-1, 3 / alpha-1, 3-glucan modified liposome preparation preparation method, which comprises the following steps: S1, glucan activation treatment; s2, constructing a lipid phase; s3, forming a co-assembled compound; s4, carrying out aqueous-phase film formation on the liposome; s5, carrying out ultrasonic emulsification and purification to obtain a beta-glucan liposome particle suspension with the particle size of about 120 nm; according to the invention, soluble beta-1, 3 / alpha-1, 3-glucan is innovatively modified on the surface of liposome through a lipid-assisted molecule chimeric technology to construct a nano-composite with targeting delivery and immunoregulation functions, and the beta-glucan triple helix structure exposed on the surface of the nano-composite directly targets an immune cell receptor, so that a local immunoregulation pathway is activated to the maximum extent.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of polysaccharide liposome preparation, in particular to a kind of β-1,3 / α-1,3-glucan modified liposome preparation and its preparation method and application. BACKGROUND

[0002] As a kind of high molecular weight soluble polysaccharide with glucose as basic unit connected by glycosidic bond, glucan has great application potential in many fields due to its unique chemical structure and biological activity, and has biological activities such as lowering blood lipids, lowering blood sugar, anti-inflammatory, antioxidant and anti-tumor.

[0003] β-1,3 / α-1,3-glucan is made of sucrose as main raw material, through Rhizobium pusense fermentation, alcohol precipitation, filtration, separation, drying, crushing and other processes. β-1,3 / α-1,3-glucan is a natural polysaccharide with a new structure, which is a chain structure of repeated units connected by 7 β-1,3-glycosidic bonds and 2 α-1,3-glycosidic bonds, without side chain. This unique bonding mode not only enhances the molecular stability, but also endows it with special physicochemical properties and biological activities. It has been officially approved by the National Health Commission as a new food raw material on April 25, 2025.

[0004] β-1,3 / α-1,3-glucan can activate innate immunity through pattern recognition receptors such as Dectin-1 on the surface of macrophages and dendritic cells, and bidirectionally regulate immune response: on the one hand, it moderately enhances phagocytic activity and releases pro-inflammatory factors (such as TNF-α) to eliminate pathogens; more importantly, it can inhibit Th2 overactivation by inducing the secretion of anti-inflammatory factors such as IL-10 and TGF-β, promoting the polarization of M2 macrophages and the differentiation of Treg cells, and relieving inflammatory response. However, the immune efficacy of β-glucan is highly dependent on its physical form and delivery efficiency, and free molecules are difficult to be efficiently taken up by immune cells.

[0005] Therefore, the present application aims to modify the surface of β-1,3 / α-1,3-glucan liposomes by lipid-assisted molecular chimeric technology to construct a nanocomposite with targeted delivery and immune regulation functions. SUMMARY

[0006] In view of the above-mentioned shortcomings of the prior art, the present application aims to provide a kind of β-1,3 / α-1,3-glucan modified liposome preparation and its preparation method and application, β-1,3 / α-1,3-glucan is modified on the surface of liposome by lipid-assisted molecular chimeric technology, and a nanocomposite with targeted delivery and immune regulation functions is constructed.

[0007] To achieve the above object and other related objects, in a first aspect, the present application provides the following technical solution: A preparation method of a β-1,3 / α-1,3-glucan modified liposome preparation, the preparation steps comprising: S1: glucan activation treatment, mixing β-1,3 / α-1,3-glucan with a purity of > 95% with dimethyl sulfoxide (DMSO) at a weight-volume ratio of 1:15-1:20 (w / v: kg / L) and completely dissolving to obtain a transparent viscous liquid; S2: Lipid phase construction, mixing polyethylene glycol (PEG, MW=2000) with dichloromethane at a weight-volume ratio (w / v: kg / L) of 1:4-1:6 and vortexing to completely dissolve to form a uniform lipid organic phase; S3: Formation of co-assembled complex, the transparent viscous liquid obtained in step S1 is added dropwise into the uniform lipid organic phase obtained in step S2 at a volume ratio (v / v) of 1:2-1:3, and is uniformly stirred at 800 rpm to form a glucan-PEG chimeric precursor; S4: Liposome water phase film formation, the glucan-PEG chimeric precursor obtained in step S3 is injected into the bottom layer of pre-cooled physiological saline along the pipe wall at a flow rate of 15-20% of the volume of dichloromethane in step S2; S5: Ultrasonic emulsification and purification, the mixture obtained in step S4 is subjected to ultrasonic treatment until a milky white homogeneous emulsion is formed, and the obtained milky white homogeneous emulsion is filtered in sequence through 0.4 μm and 0.22 μm filter membranes to obtain a β-glucan liposome particle suspension with a particle size of about 120 nm.

[0008] As a preferred solution of step S1, the dissolving method in step S1 is oscillation in a 50°C water bath.

[0009] As a preferred solution of step S4, the temperature of the pre-cooled physiological saline in step S4 is 4°C.

[0010] As a preferred solution of step S5, the ultrasonic treatment in step S5 is performed using a water bath ultrasonic instrument with a power of 150 W and a frequency of 40 kHz.

[0011] In a second aspect, the present application provides a β-1,3 / α-1,3-glucan modified liposome preparation prepared by any one of the above preparation methods.

[0012] In a third aspect, the present application provides the use of a β-1,3 / α-1,3-glucan modified liposome preparation in a nasal inflammation treatment spray.

[0013] As described above, the present application provides a kind of β-1,3 / α-1,3-glucan modified liposome preparation and its preparation method and application, with the following beneficial effects: 1. In the glucan activation treatment step, the unique properties of dimethyl sulfoxide (DMSO) are used as a strong polar aprotic solvent. The additional thermodynamic energy provided by heating at 50°C makes it easier for DMSO molecules to "pry open" the tight hydrogen bond network, greatly accelerating the dissolution process. Water bath oscillation provides external force to ensure uniform heat and concentration, preventing local overheating or clumping, allowing the solvent to make more complete contact with the undissolved glucan.

[0014] 2. In the lipid phase construction step, PEG serves as a spatial stabilizer for the lipid bilayer, with its long hydrophilic end providing a binding site for subsequent glucan anchoring. 3. In the liposome water phase film formation step, under the induction of water-organic phase interfacial tension, the lipid molecules automatically align with the hydrophilic head facing the water and the hydrophobic tail facing away from the water. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 TEM imaging diagram.

[0016] Figure 2 DLS analysis diagram.

[0017] Figure 3 Zeta potential determination results diagram.

[0018] Figure 4 Anti-inflammatory experiment 1 results diagram.

[0019] Figure 5 Anti-inflammatory experiment 2 results diagram. DETAILED DESCRIPTION

[0020] Now a variety of exemplary embodiments of the present application will be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0021] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All documents mentioned in this specification are incorporated by reference to disclose and describe in connection with the methods and / or materials described herein. In the event of conflict between any incorporated document and the content of this specification, the content of this specification will control.

[0022] Many modifications and variations of the specific embodiments of the application can be practiced in accordance with the principles of the application, and such variations are within the scope of the application as disclosed herein. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative of the application.

[0023] The instruments, reagents, materials and the like involved in the following examples are conventional instruments, reagents, materials and the like that are already available in the prior art, and can be obtained through regular commercial channels, unless otherwise specified. The experimental methods, detection methods and the like involved in the following examples are conventional experimental methods, detection methods and the like that are already available in the prior art, unless otherwise specified.

[0024] A preparation method of a β-1,3 / α-1,3-glucan modified liposome preparation, the preparation steps comprising: S1: glucan activation treatment, mixing β-1,3 / α-1,3-glucan with purity > 95% with dimethyl sulfoxide (DMSO) according to weight volume ratio 1:15-1:20 (w / v:kg / L) and completely dissolving to obtain transparent viscous liquid; wherein, the dissolving mode is oscillation in 50℃ water bath; this step utilizes the unique properties of dimethyl sulfoxide (DMSO) strong polar aprotic solvent, combined with 50℃ oscillation external force to destroy the hydrogen bond network of glucan, improve the reaction activity; S2: Lipid phase construction, mixing polyethylene glycol (PEG, MW=2000) with dichloromethane according to weight volume ratio (w / v:kg / L) 1:4-1:6 and vortex oscillation until completely dissolved to form a uniform lipid organic phase; this step PEG as a space stabilizer of lipid bilayer, its long chain hydrophilic end provides a binding site for subsequent glucan anchoring; S3: Co-assembly complex formation, the transparent viscous liquid obtained in step S1 is added dropwise into the uniform lipid organic phase obtained in step S2 with volume ratio (v / v) of 1:2-1:3 using a pipette, and is uniformly stirred at 800 rpm to form a glucan-PEG chimeric precursor; S4: Liposome water phase film formation, the glucan-PEG chimeric precursor obtained in step S3 is injected along the pipe wall into the bottom layer of pre-cooled physiological saline at a temperature of 4℃ at a flow rate of 15-20% of the volume of dichloromethane in step S2; the tension of the water-organic phase interface is used to induce the automatic alignment of the lipid molecules with the hydrophilic head facing the water and the hydrophobic tail away from the water; S5: ultrasonic emulsification and purification, the mixed solution obtained in step S4 was subjected to ultrasonic treatment in a water bath ultrasonic instrument with a power of 150 W and a frequency of 40 kHz until a milky white homogeneous emulsion was formed, and the obtained milky white homogeneous emulsion was filtered through 0.4 μm and 0.22 μm filter membranes in turn to obtain a β-glucan modified liposome (β-Glu@LP) particle suspension with a particle size of about 120 nm.

[0025] A β-1,3 / α-1,3-glucan modified liposome preparation was prepared by the above steps. Transmission electron microscopy (TEM) imaging (see Figure 1 ) showed that the particle size of the β-glucan modified liposome (β-Glu@LP) was about 120 nm; dynamic light scattering (DLS) analysis showed that the polydispersity index (PDI) of the nano-assembly was less than 0.25 (see Figure 2 ), indicating that it had good size uniformity and dispersion state; and the Zeta potential determination results (see Figure 3 ) confirmed that the particle surface had a net negative charge.

[0026] To verify the application effect of the β-1,3 / α-1,3-glucan modified liposome preparation prepared by the above steps in the treatment of rhinitis spray, the following experiments were carried out: Anti-inflammatory experiment 1: In the RAW264.7 murine macrophage model cultured in vitro, lipopolysaccharide (LPS) was used to induce macrophages to polarize to pro-inflammatory M1. After 12 hours of β-Glu@LP treatment in the experimental group, the secretion level of pro-inflammatory factor TNF-α in the cell culture supernatant was detected. The results showed that the secretion level of TNF-α under non-inflammatory state was 11.29 ± 0.41 pg / mL, while that in the LPS-induced inflammation group was significantly increased to 90.78 ± 1.03 pg / mL; after β-Glu@LP treatment, the TNF-α content in the inflammation group was significantly reduced to 66.40 ± 1.23 pg / mL (see Figure 4 ).

[0027] Anti-inflammatory experiment 2: Bone marrow-derived macrophages (BMDM) were isolated from the femur and tibia of 4-week-old C57BL / 6 mice, and after in vitro induction and differentiation into M1 macrophages, they were treated with PBS or β-Glu@LP for 48 hours. The fluorescence intensity of CD86 in the β-Glu@LP treatment group was significantly weakened, and the fluorescence intensity of CD206 was significantly enhanced, indicating that β-Glu@LP could effectively reverse the pro-inflammatory CD86 + M1 phenotype and promote its transformation to anti-inflammatory CD206 + M2 phenotype (see Figure 5Wherein DAPI marks the nucleus, CD86 and CD206 are the target proteins of immunofluorescence staining, and MERGE is the fluorescence superimposed graph.

[0028] Although the existing mainstream therapies such as nasal glucocorticoids, antihistamines and the like can relieve symptoms, they have significant limitations: the drugs target symptoms rather than the immune root, long-term use of hormones can cause side effects such as mucosal atrophy, antihistamines have limited effect on nasal congestion, decongestants have rebound risk, and immunotherapy is long and costly. The present application innovatively modifies soluble β-1,3 / α-1,3-glucan on the surface of liposomes through lipid-assisted molecular intercalation technology, constructs a nanocomposite with targeted delivery and immunomodulatory functions, and the exposed β-glucan triple helix structure on the surface directly targets immune cell receptors, maximizes the activation of local immune regulation pathways; at the same time, the lipid bilayer core can load other active ingredients to achieve synergistic treatment. Dispersed in saline to form a spray type, it has the dual advantages of moisturizing the nasal cavity, diluting secretions and precise immune intervention. At present, there is no related report on the use of liposome carriers to load soluble β-1,3 / α-1,3-glucan for nasal immune regulation, and this technology is expected to fill this gap and provide a revolutionary solution for rhinitis treatment.

[0029] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for preparing a β-1,3 / α-1,3-glucan modified liposome formulation, characterized by, The preparation steps include: S1: glucan activation treatment, β-1, 3 / α-1, 3-glucan with purity > 95% is mixed with dimethyl sulfoxide (DMSO) at a weight volume ratio of 1:15-1:20 (w / v: kg / L) and completely dissolved to obtain a transparent viscous liquid; S2: lipid phase construction, polyethylene glycol (PEG, MW=2000) is mixed with dichloromethane at a weight volume ratio (w / v: kg / L) of 1:4-1:6 and vortexed to completely dissolve to form a uniform lipid organic phase; S3: co-assembly complex formation, the transparent viscous liquid obtained in step S1 is added dropwise into the uniform lipid organic phase obtained in step S2 at a volume ratio (v / v) of 1:2-1:3, and uniformly stirred at 800 rpm to form a glucan-PEG chimeric precursor; S4: liposome water phase film formation, the glucan-PEG chimeric precursor obtained in step S3 is injected along the pipe wall into the bottom layer of pre-cooled physiological saline at a flow rate of 15-20% of the dichloromethane in step S2; S5: ultrasonic emulsification and purification, the mixture obtained in step S4 is subjected to ultrasonic treatment until a milky white homogeneous emulsion is formed, and the obtained milky white homogeneous emulsion is filtered through 0.4 μm and 0.22 μm filter membranes in turn to obtain a β-glucan liposome particle suspension with a particle size of about 120 nm.

2. A process for the preparation of a β-1,3 / α-1,3-glucan modified liposome formulation according to claim 1, characterized in that, The dissolution method in step S1 is oscillation in a 50°C water bath.

3. A process for the preparation of a β-1,3 / α-1,3-glucan modified liposome formulation according to claim 1, characterized in that, The temperature of the pre-cooled physiological saline in step S4 is 4°C.

4. A process for the preparation of a β-1,3 / α-1,3-glucan modified liposome formulation according to claim 1, characterized in that, The ultrasonic treatment in step S5 is performed using a water bath ultrasonic instrument with a power of 150W and a frequency of 40kHz.

5. A beta-1,3 / alpha-1,3-glucan modified liposome formulation characterized in that, Prepared by the preparation method of any one of claims 1-4.

6. The use of a β-1, 3 / α-1, 3-glucan modified liposome preparation in claim 5 in a nasal inflammation treatment spray.

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