Nanoparticle TK-PSBs / CBD@Met, preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and more particularly relates to nanoparticles TK-PSBs / CBD@Met, a preparation method therefor and application thereof. The nanoparticles TK-PSBs / CBD@Met are nanoparticles formed by wrapping cannabidiol and metformin with thione-modified lung surfactant biomimetic liposomes. In vitro experiments have confirmed that the nanoparticles TK-PSBs / CBD@Met can effectively inhibit the fibrosis process of BEAS-2B cells induced by silicon dioxide. Animal experiments have shown that the nanoparticles TK-PSBs / CBD@Met can significantly improve the damage to the lung tissue structure and abnormal deposition of collagen caused by SiO2 exposure, and alleviate the pathological progression of silicosis fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to nanoparticles TK-PSBs / CBD@Met, their preparation methods, and applications. Background Technology

[0002] Silicosis is the most common, fastest-progressing, and most serious type of occupational pneumoconiosis. It refers to a systemic disease characterized by diffuse pulmonary fibrosis, primarily caused by long-term, repeated inhalation of free silica crystal particles, with interstitial pulmonary fibrosis as its pathological feature. During the pathogenesis of pulmonary fibrosis, changes in the local microenvironment of lung tissue determine the tissue's response to various injuries. The nature and intensity of these responses determine the tissue's inflammatory response, wound healing, and the occurrence, development, and outcome of fibrosis. The main effector cells in the development of silicosis fibrosis include macrophages, fibroblasts, epithelial cells, endothelial cells, and lymphocytes. When macrophage function is disordered, their repair and homeostasis functions are disrupted, leading to the secretion of large amounts of inflammatory cytokines and platelet-derived growth factor, indirectly activating and regulating the activation and migration of endothelial cells, epithelial cells, and fibroblasts in lung tissue, and inducing extracellular matrix deposition, thus affecting the progression of fibrosis. Epithelial cells and fibroblasts regulate each other in structure and function. In the pathological process, epithelial cells mainly undergo aging, regulate immunity and humoral balance, while fibroblasts are activated and differentiate into myofibroblasts and secrete collagen. The interaction between the two leads to silicosis fibrosis.

[0003] Therefore, regulating and inhibiting epithelial cell senescence is a promising approach for treating silicosis fibrosis. While cannabidiol (CBD) and metformin can regulate BEAS-2B epithelial cell senescence and thus exert anti-silicosis fibrosis effects, systemic administration of CBD and metformin alone suffers from drawbacks such as non-specific distribution, low bioavailability, short residence time, and high effective doses, thus limiting therapeutic efficacy. Improving the bioavailability and targeted distribution of CBD and metformin is a key issue in the treatment of silicosis fibrosis using these two methods. Summary of the Invention

[0004] The purpose of this invention is to provide nanoparticles TK-PSBs / CBD@Met, their preparation methods, and applications.

[0005] The present invention provides nanoparticles TK-PSBs / CBD@Met, wherein the nanoparticles TK-PSBs / CBD@Met are formed by encapsulating cannabidiol and metformin in a lipid membrane modified with thioketone, and the particle size of the nanoparticles TK-PSBs / CBD@Met is 90~180 nm.

[0006] The TK-PSBs / CBD@Met nanoparticles provided by this invention are formed by encapsulating cannabidiol and metformin in thioketone-modified lung surfactant biomimetic liposomes. Animal experiments show that the TK-PSBs / CBD@Met nanoparticles can trigger liposome disintegration in a pulmonary fibrosis environment, targeting the lesion site. Compared with administration of cannabidiol and metformin alone, they significantly improve lung tissue structural damage and abnormal collagen deposition caused by SiO2 exposure, and improve drug bioavailability.

[0007] This invention also provides a method for preparing TK-PSBs / CBD@Met nanoparticles, the preparation method being as follows;

[0008] S1. 1,2-Dipalmitoyl-sn-glycerol-3-phosphorylcholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphorylglycerol, thioketone, cholesterol and chloroform are mixed and then rotary evaporated to form a uniform biomimetic membrane TK-PSBs. A mixed solution containing resveratrol and wolfberry polysaccharide is added to hydrate the biomimetic membrane TK-PSBs to form a bilayer liposome.

[0009] S2. After sonicating the bilayer liposomes at 85-95W for 9-11 min, the mixture was repeatedly frozen and thawed 3-5 times. After centrifuging at 10000-12000 rpm for 55-65 min, the supernatant was discarded to obtain nanoparticles TK-PSBs / CBD@Met.

[0010] Furthermore, the volume ratio of the 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol, thioketone, cholesterol, chloroform, and the mixed solution is 9~11:1~3:1~3:0.5~1.5:0.5~1.5:1~3.

[0011] Furthermore, the mass ratio of cannabidiol to metformin in the mixed solution is 0.5~1.5:1~3.

[0012] The present invention also provides the application of the nanoparticles TK-PSBs / CBD@Met in the preparation of drugs for treating silicosis fibrosis.

[0013] Furthermore, the drug contains TK-PSBs / CBD@Met nanoparticles as its sole active ingredient.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] Furthermore, the excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.

[0016] Furthermore, when the dosage form of the drug is a solution dosage form, the solution dosage form includes oral liquid preparations, gastric enemas, and injectable dosage forms.

[0017] Furthermore, the solution formulation is a solution composed of water and the nanoparticles TK-PSBs / CBD@Met, or a solution composed of physiological saline and the nanoparticles TK-PSBs / CBD@Met.

[0018] The present invention has the following beneficial effects:

[0019] The TK-PSBs / CBD@Met nanoparticles provided by this invention can effectively overcome the impact of the pulmonary surfactant barrier on drug delivery. Through biomimetic design, cannabidiol and metformin are encapsulated within nanoliposomes to form a dual-drug delivery system. This structure can trigger liposome disintegration in the pulmonary fibrosis environment, achieving precise drug release. This improves drug retention time in the alveolar region and enhances its targeted delivery efficiency to damaged BEAS-2B cells. These nanoparticles reduce drug degradation and inactivation in the bloodstream, further improving drug bioavailability and reducing non-specific drug distribution in vivo, thereby reducing toxicity to normal cells and exhibiting better biocompatibility. Attached Figure Description

[0020] Figure 1 The image shows the characterization of TK-PSBs / CBD@Met, where A is a transmission electron microscope image, B is a particle size distribution, C is a Fourier transform infrared spectrum, and D is a magnified view of the wavenumber band of the Fourier transform infrared spectrum.

[0021] Figure 2 The images show immunofluorescence patterns of fibronectin in cells, where A is the immunofluorescence pattern of fibronectin and B is a statistical chart of the relative area of ​​fibronectin.

[0022] Figure 3 The images show immunofluorescence patterns of α-smooth muscle actin in cells, where A is the immunofluorescence pattern of α-smooth muscle actin and B is a statistical graph of the relative area of ​​α-smooth muscle actin.

[0023] Figure 4 The images show immunofluorescence patterns of proteins. In this image, A shows the immunofluorescence patterns of p21, p16, vimentin, and E-cadherin; B shows the relative area statistics of p21; C shows the relative area statistics of p16; D shows the relative area statistics of vimentin; and E shows the relative area statistics of E-cadherin.

[0024] Figure 5 The graphs are Western blot images of proteins. In the graphs, A is the immunoblot image of type I collagen, and B is the statistical graph of the relative expression level of type I collagen. *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05.

[0025] Figure 6 Image of a chemically stained section of mouse lung.

[0026] Figure 7 Immunofluorescence images of p21, p16, and α smooth muscle actin in mouse lungs.

[0027] Figure 8 HE staining images of mouse heart, liver, spleen, lung, and kidney. Detailed Implementation

[0028] The present invention will now be described in detail with reference to specific embodiments, but these should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments can be obtained commercially unless otherwise specified.

[0029] Example 1: Development of nanoparticles TK-PSBs / CBD@Met.

[0030] I. Preparation method of TK-PSBs / CBD@Met nanoparticles.

[0031] 1. Preparation Method: 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol, thioketone, cholesterol, and chloroform were mixed in a volume ratio of 20:4:4:2:2 and then rotary evaporated onto the container wall to form a uniform lipid membrane TK-PSBs. 4 ml of a mixed solution of cannabidiol (CBD) and metformin (Met) (the mass ratio of CBD, metformin, and solvent in the mixed solution was 1:2:500) was added to hydrate the lipid membrane TK-PSBs, forming a bilayer liposome. The bilayer liposomes were sonicated at 90 W for 10 min, then subjected to four freeze-thaw cycles at -80 °C and 32 °C, centrifuged at 11000 rpm for 60 min, and the supernatant was discarded to obtain nanoparticles TK-PSBs / CBD@Met.

[0032] 2. Detection method: The prepared TK-PSBs / CBD@Met nanoparticle samples were observed under a transmission electron microscope, and the morphology of the samples was recorded and analyzed. The particle size of the nanoparticles was detected by dynamic light scattering (DLS, Nano ZS90 Zetasizer, Malvern), and the maximum absorbance and chemical bonds contained therein were characterized by Fourier transform infrared spectroscopy to prove the synthesis of nanoparticles.

[0033] 3. Test results: such as Figure 1As shown, transmission electron microscopy and dynamic light scattering characterization revealed that the TK-PSBs / CBD@Met nanoparticles exhibited a uniformly dispersed elliptical nanostructure with an average particle size of 163 nm. Fourier transform infrared spectroscopy confirmed the dual-drug loading characteristics: characteristic peaks of cannabidiol and metformin were observed in the TK-PSBs / CBD@Met nanoparticles, indicating that the two drugs were successfully co-loaded in the liposome nanocarrier.

[0034] II. In vitro efficacy testing of TK-PSBs / CBD@Met nanoparticles.

[0035] 1. Cell Culture: Cells (MRC-5, BEAS-2B) stored in liquid nitrogen were rapidly thawed in a 37°C water bath. Centrifuge tubes were sterilized with alcohol and transferred to a biosafety cabinet. The cryopreservation solution was diluted to 4 mL with DMEM basal medium, centrifuged at 1000 rpm for 5 min, and the supernatant was removed. The cells were resuspended in complete medium containing penicillin 100 U / mL + streptomycin 100 μg / mL and 10% heat-inactivated fetal bovine serum. The cells were then seeded into culture flasks and incubated in a 37°C CO2 incubator.

[0036] 2. Experimental Methods: This experiment consisted of four groups: Control group, SiO2 group, Met+CBD group, and TPCM group. The SiO2 group, Met+CBD group, and TPCM group were induced to senescence in BEAS-2B cells by adding 50 μg / mL SiO2 suspension for 4 consecutive days. The Met+CBD group was treated with 500 μg / mL Met and 3 μg / mL CBD for 24 hours to induce senescent BEAS-2B cells. The TPCM group was treated with 10 μg / mL TK-PSBs / CBD@Met for 24 hours to induce senescent BEAS-2B cells. The Control group consisted of normal BEAS-2B cells supplemented with PBS.

[0037] The culture media from each group were collected and centrifuged to obtain the supernatant. The collected supernatant was then co-incubated with MRC-5 cells for 24 hours. Finally, the efficacy of TK-PSBs / CBD@Met in alleviating silicosis fibrosis was assessed by immunofluorescence staining and Western blotting.

[0038] 0. Experimental Results: such as Figure 2 and 3As shown, the expression levels of fibrosis markers fibrosis protein and α-smooth muscle actin were detected by immunofluorescence staining. The SiO2 group showed a significantly more pronounced fibrosis phenotype than the Control group, with strong positive expression of both fibrosis protein and α-smooth muscle actin. The Met+CBD group showed a certain degree of fibrosis relief, while the TPCM group showed a more significant anti-fibrotic effect, with significantly reduced expression levels of both markers. Figure 4 and Figure 5 This study explored the mechanism by which TK-PSBs / CBD@Met alleviates silicosis fibrosis. During this process, aging markers p21 and p16 were upregulated, and type I collagen expression increased. TK-PSBs / CBD@Met effectively alleviated the upregulation of p21 and p16 and reduced type I collagen expression. Therefore, the following conclusion was drawn: TK-PSBs / CBD@Met can regulate the aging of BEAS-2B epithelial cells, thereby exerting an anti-silicosis fibrosis effect.

[0039] III. Application of TK-PSBs / CBD@Met nanoparticles in improving in vivo silicosis fibrosis.

[0040] 1. Laboratory animals: SPF-grade C57BL / 6J mice were used in the experiment. They were purchased from Beijing Weishang Lide Biotechnology Co., Ltd. and housed on the third floor of the Laboratory Animal Center of Ningxia Medical University. The feeding and experimental procedures followed the ethical guidelines for the use of laboratory animals.

[0041] 1. Experimental Methods: The experiment was divided into Control group, SiO2 group, Met+CBD group, and TPCM group, with 8 C57BL / 6J mice in each group. After isoflurane anesthesia, mice were used to establish a silicosis model by intratracheal instillation of 0.1 mL of a 200 mg / mL SiO2 suspension. The Control group consisted of normal mice receiving a non-invasive intratracheal instillation of 0.1 mL of physiological saline. The SiO2 group consisted of silicosis model mice receiving a non-invasive intratracheal instillation of 0.1 mL of physiological saline. The Met+CBD group consisted of silicosis model mice receiving a non-invasive intratracheal instillation of 0.1 mL of a mixture of Met and CBD (Met to CBD concentration ratio 500 μg / mL:3 μg / mL). The TPCM group consisted of silicosis model mice receiving a non-invasive intratracheal instillation of 0.1 mL of a 5 μg / mL TK-PSBs / CBD@Met nanoparticle solution. The four groups were administered drugs at 72-hour intervals, with an experimental period of 28 days. On day 28, lung tissue was harvested for HE, Masson's red, and Sirius red staining, and immunofluorescence was used to observe pulmonary fibrosis. Heart, liver, spleen, and kidney were harvested for HE staining to assess the biocompatibility of different treatment groups.

[0042] 2. Experimental results: such as Figure 6 and 7As shown, the lung tissues of mice in the SiO2 group exhibited typical fibrotic characteristics such as silicotic nodule formation, alveolar structure destruction, and abnormal collagen deposition. Furthermore, the expression levels of α-smooth muscle actin and aging markers p21 and p16d were significantly upregulated. After drug intervention, both the Met+CBD and TPCM groups significantly inhibited the fibrotic process, with the TK-PSBs / CBD@Met nanoparticles showing superior intervention effects and partially alleviating the pathological phenotype.

[0043] The evaluation of the biosafety of nanomedicines is a crucial prerequisite for their clinical translation or experimental research. For example... Figure 8 As shown, HE staining revealed that the heart, liver, spleen, and kidneys of the experimental animals in the TPCM group all exhibited intact tissue structures, with no characteristic pathological damage. The nanoparticles TK-PSBs / CBD@Met possess ideal biocompatibility characteristics.

[0044] In summary, this invention designs a highly safe and biodegradable nanoparticle, TK-PSBs / CBD@Met. The TK-PSBs / CBD@Met nanoparticles of this invention physically encapsulate cannabidiol and metformin with TK-PSBs, and a biomimetic membrane is placed on the outermost layer of the nanocomposite material, loading cannabidiol and metformin. Utilizing the anti-silicosis fibrosis effect of cannabidiol and metformin, targeted therapy for silicosis fibrosis can be achieved.

[0045] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0046] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0047] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Nanoparticles TK-PSBs / CBD@Met, characterized in that, The preparation method of the nanoparticles TK-PSBs / CBD@Met is as follows; S1. 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol, thioketone, cholesterol and chloroform are mixed and then rotary evaporated to form a uniform biomimetic membrane TK-PSBs. A mixed solution containing cannabidiol and metformin is added to hydrate the biomimetic membrane TK-PSBs to form a bilayer liposome. S2. After sonicating the bilayer liposomes at 85-95W for 9-11 min, the mixture was repeatedly freeze-thawed 3-5 times, centrifuged at 10000-12000 rpm for 55-65 min, and the supernatant was discarded to obtain nanoparticles TK-PSBs / CBD@Met. The particle size of the TK-PSBs / CBD@Met nanoparticles was 90-180 nm. The volume ratio of 1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphorylglycerol, thioketone, cholesterol, chloroform, and the mixed solution was 10:2:2:1:1:

2. The mass ratio of cannabidiol to metformin in the mixed solution was 1:

2.

2. The use of the nanoparticles TK-PSBs / CBD@Met according to claim 1 in the preparation of drugs for treating silicosis fibrosis.

3. The application of the nanoparticles TK-PSBs / CBD@Met according to claim 2 in the preparation of drugs for treating silicosis fibrosis, characterized in that, The drug contains TK-PSBs / CBD@Met nanoparticles as its sole active ingredient.

4. The application of the nanoparticles TK-PSBs / CBD@Met according to claim 3 in the preparation of drugs for treating silicosis fibrosis, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The application of the nanoparticles TK-PSBs / CBD@Met according to claim 4 in the preparation of drugs for treating silicosis fibrosis, characterized in that, The excipients include any one or more of stabilizers, diluents, and adjuvants.

6. The application according to claim 2, characterized in that, When the dosage form of the drug is a solution, the solution dosage form includes oral liquid preparations, oral boluses, and injectable dosage forms.

7. The application according to claim 6, characterized in that, The solution formulation is a solution composed of water and the nanoparticles TK-PSBs / CBD@Met, or a solution composed of physiological saline and the nanoparticles TK-PSBs / CBD@Met.

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