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

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a nanoparticle TK-PSBs / CBD-Met and a preparation method and application thereof. The nanoparticle TK-PSBs / CBD (at) Met provided by the invention is a nanoparticle formed by wrapping a thioketone modified pulmonary surface activity bionic liposome with cannabidiol and metformin, and in vitro experiments prove that the nanoparticle TK-PSBs / CBD (at) Met can effectively inhibit a BEAS-2B cell fibrosis process induced by silicon dioxide. Animal experiments show that the nanoparticles TK-PSBs / CBD (at) Met can significantly improve lung tissue structure damage and abnormal collagen deposition caused by SiO2 exposure, and relieve the pathological progress of silicosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to nanoparticles TK-PSBs / CBD@Met and a preparation method and application thereof. Background Art

[0002] Silicosis is the most common, fastest-progressing, and most devastating type of occupational pneumoconiosis. It is a systemic disease characterized by diffuse pulmonary fibrosis, primarily caused by long-term and repeated inhalation of free silica crystals. Its pathological hallmark is interstitial pulmonary fibrosis. During the pathogenesis of pulmonary fibrosis, changes in the local lung microenvironment determine the tissue's response to various insults. The nature and intensity of these responses determine the development, progression, and outcome of tissue inflammation, wound healing, and fibrosis. The primary effector cells in the development of silicotic fibrosis include macrophages, fibroblasts, epithelial cells, endothelial cells, and lymphocytes. When macrophages become dysfunctional, their repair and homeostatic functions are disrupted, leading to the secretion of large amounts of inflammatory cytokines and platelet-derived growth factor, which indirectly activate and regulate the activation and migration of endothelial cells, epithelial cells, and fibroblasts in the lung tissue, inducing extracellular matrix deposition and influencing the progression of fibrosis. Epithelial cells and fibroblasts regulate each other in structure and function. In the pathological process, epithelial cells mainly show signs of aging, regulating immunity and fluid balance, while fibroblasts are activated and differentiated into myofibroblasts and secrete collagen. The interaction between the two leads to silicosis fibrosis.

[0003] Therefore, regulating and inhibiting epithelial cell senescence is a very promising approach to treating silicotic fibrosis. Although cannabidiol and metformin can regulate the senescence of BEAS-2B epithelial cells and thus exert an anti-silicotic fibrosis effect, systemic administration of cannabidiol and metformin alone exhibits nonspecific distribution in the body, low bioavailability, short retention time, and a large effective dose, which limits the therapeutic effect. How to improve the bioavailability and targeted distribution of cannabidiol and metformin is a key issue in the treatment of silicotic fibrosis with cannabidiol and metformin. Summary of the Invention

[0004] The purpose of the present invention is to provide nanoparticles TK-PSBs / CBD@Met and a preparation method and application thereof.

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

[0006] The nanoparticles TK-PSBs / CBD@Met provided by this invention are formed by encapsulating cannabidiol and metformin in thioketone-modified lung surfactant biomimetic liposomes. Animal experiments have shown that the nanoparticles TK-PSBs / CBD@Met can trigger liposome disassembly in the environment of pulmonary fibrosis, targeting the lesions. Compared with the administration of cannabidiol and metformin alone, they significantly alleviate the structural damage and abnormal collagen deposition of lung tissue caused by SiO2 exposure, thereby improving the bioavailability of the drugs.

[0007] The present invention also provides a method for preparing nanoparticles TK-PSBs / CBD@Met, and the preparation method is as follows; S1, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, thioketone, cholesterol and chloroform were mixed and then rotary evaporated to form a uniform biomimetic membrane TK-PSBs, and a mixed solution containing resveratrol and wolfberry polysaccharides was added to hydrate the biomimetic membrane TK-PSBs to form bilayer liposomes; S2. The bilayer liposomes were sonicated at 85-95W for 9-11 minutes and then repeatedly frozen and thawed 3-5 times. The liposomes were centrifuged at 10,000-12,000 rpm for 55-65 minutes and the supernatant was discarded to obtain nanoparticles TK-PSBs / CBD@Met.

[0008] Furthermore, the volume ratio of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, thioketone, cholesterol, chloroform and mixed solution is 9-11:1-3:1-3:0.5-1.5:0.5-1.5:1-3.

[0009] Furthermore, the mass ratio of cannabidiol to metformin in the mixed solution is 0.5~1.5:1~3.

[0010] The present invention also provides the use of the nanoparticles TK-PSBs / CBD@Met in preparing a drug for treating silicosis fibrosis.

[0011] Furthermore, the nanoparticles TK-PSBs / CBD@Met in the drug serve as the only active ingredient.

[0012] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0013] Furthermore, the auxiliary materials include any one or more of fillers, stabilizers, diluents, and adjuvants.

[0014] Furthermore, when the dosage form of the drug is a solution dosage form, the solution dosage form includes an oral liquid preparation, a gavage preparation, and an injection dosage form.

[0015] Furthermore, the solution dosage form 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.

[0016] The present invention has the following beneficial effects: The nanoparticles TK-PSBs / CBD@Met provided by the present invention can effectively overcome the influence of the pulmonary surfactant barrier on drug delivery. Through bionic design, cannabidiol and metformin are encapsulated in nanoliposomes to form a dual drug delivery system. This structure can trigger the disintegration of liposomes in the environment of pulmonary fibrosis to achieve precise release of drugs. This is to increase the retention time of the drug in the alveolar area and enhance its targeted delivery efficiency to damaged BEAS-2B cells. This nanoparticle reduces the degradation and inactivation of the drug in the blood circulation, further improves the bioavailability of the drug, and reduces the nonspecific distribution of the drug in the body, thereby reducing the toxicity to normal cells and having better biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Characterization diagram of TK-PSBs / CBD@Met, where A is the transmission electron microscope image, B is the particle size image, C is the Fourier transform infrared spectrum image, and D is the Fourier transform infrared spectrum wavenumber segment magnification image.

[0018] Figure 2 These are immunofluorescence images of fibronectin in cells, where A is the immunofluorescence image of fibronectin and B is the statistical graph of the relative area of ​​fibronectin.

[0019] Figure 3 These are immunofluorescence images of α-smooth muscle actin in cells, where A is the immunofluorescence image of α-smooth muscle actin and B is the relative area statistical graph of α-smooth muscle actin.

[0020] Figure 4 These are protein immunofluorescence images, where A is the immunofluorescence image of p21, p16, vimentin, and E-cadherin, B is the relative area statistical graph of p21, C is the relative area statistical graph of p16, D is the relative area statistical graph of vimentin, and E is the relative area statistical graph of E-cadherin.

[0021] Figure 5 Figures are protein immunoblotting images, where A is the immunoblotting image of type I collagen, and B is the statistical graph of the relative expression of type I collagen. *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05.

[0022] Figure 6 This is a chemically stained section of mouse lung.

[0023] Figure 7 Immunofluorescence images of p21, p16, and α-smooth muscle actin in mouse lung.

[0024] Figure 8 HE staining images of mouse heart, liver, spleen, lung and kidney. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to specific examples, but these examples should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0026] Example 1: Development of nanoparticles TK-PSBs / CBD@Met.

[0027] 1. Preparation method of nanoparticles TK-PSBs / CBD@Met.

[0028] 1. Preparation Method: 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol, thioketone, cholesterol, and chloroform were mixed at a volume ratio of 20:4:4:2:2 and rotary evaporated to form a uniform lipid film TK-PSBs on the container wall. 4 mL of a mixed solution of cannabidiol (CBD) and metformin (Met) (the mass ratio of CBD, Met, and solvent in the mixture was 1:2:500) was added to hydrate the TK-PSBs lipid film to form bilayer liposomes. The bilayer liposomes were sonicated at 90W for 10 minutes, and then freeze-thawed four times at -80°C and 32°C. The mixture was centrifuged at 11,000 rpm for 60 minutes, and the supernatant was discarded to obtain TK-PSBs / CBD@Met nanoparticles.

[0029] 2. Detection Method: The prepared nanoparticle TK-PSBs / CBD@Met sample was observed under a transmission electron microscope, and the sample morphology was recorded and analyzed. Dynamic light scattering (DLS, Nano ZS90 Zetasizer, Malvern) was used to detect the particle size of the nanoparticles, and Fourier transform infrared spectroscopy was used to characterize its maximum absorbance and the chemical bonds contained, proving the synthesis of the nanoparticles.

[0030] 3. Test results: Figure 1As shown, transmission electron microscopy and dynamic light scattering characterization revealed that the nanoparticles TK-PSBs / CBD@Met exhibited uniformly dispersed elliptical nanostructures 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 nanoparticles TK-PSBs / CBD@Met, indicating that the two drugs were successfully co-loaded into the liposomal nanocarrier.

[0031] 2. In vitro efficacy testing of nanoparticles TK-PSBs / CBD@Met.

[0032] 1. Cell Culture: Thaw cells (MRC-5 and BEAS-2B) stored in liquid nitrogen immediately in a 37°C water bath. Disinfect the centrifuge tubes with alcohol and transfer to a biosafety cabinet. Dilute the cryoprotectant to 4 mL with DMEM basal medium. Centrifuge at 1000 rpm for 5 minutes, then remove the supernatant. Resuspend the cells in complete medium containing dual-antibody (penicillin 100 U / mL + streptomycin 100 μg / mL) and 10% heat-inactivated fetal bovine serum. Plate the cells into culture flasks and place them in a 37°C CO2 incubator.

[0033] 2. Experimental methods: This experiment set up four groups, namely the Control group, SiO2 group, Met+CBD group, and TPCM group. Among them, the SiO2 group, Met+CBD group, and TPCM group all induced BEAS-2B cell senescence by adding 50μg / mL SiO2 suspension and continuously intervening for 4 days; the Met+CBD group gave the aged BEAS-2B cells 500μg / mL Met and 3μg / mL CBD combined intervention for 24 hours, and the TPCM group gave the aged BEAS-2B cells 10μg / mL TK-PSBs / CBD@Met intervention for 24 hours. The Control group was normal BEAS-2B cells supplemented with PBS.

[0034] The culture fluid from each group was collected and centrifuged to obtain the supernatant. The supernatant was then incubated with MRC-5 cells for 24 hours. Finally, immunofluorescence staining and western blotting were used to assess the effect of TK-PSBs / CBD@Met on the alleviation of silicotic fibrosis.

[0035] 0. Experimental results: Figure 2 and 3As shown, immunofluorescence staining of the fibrosis markers fibronectin and α-smooth muscle actin showed a significant fibrotic phenotype in the SiO2 group compared to the control group, as evidenced by strong positive expression of fibronectin and α-smooth muscle actin. The Met+CBD group showed a moderate fibrosis alleviation effect, while the TPCM group exhibited a more significant anti-fibrotic effect, with significantly reduced expression of both markers. Figure 4 and Figure 5 The mechanism of TK-PSBs / CBD@Met in alleviating silicotic fibrosis was explored. During this process, the aging markers p21 and p16 were upregulated, and the expression of type I collagen increased. TK-PSBs / CBD@Met could effectively alleviate the upregulation of p21 and p16 and reduce the expression of type I collagen. Therefore, the following conclusion was drawn: TK-PSBs / CBD@Met can regulate the senescence of BEAS-2B epithelial cells and thus exert an anti-silicotic fibrosis effect.

[0036] 3. Application of nanoparticles TK-PSBs / CBD@Met for improving silicosis fibrosis in vivo.

[0037] 1. Experimental Animals: SPF-grade C57BL / 6J mice were purchased from Beijing Weishanglide Biotechnology Co., Ltd. and housed on the third floor of the Experimental Animal Center of Ningxia Medical University. The husbandry and experimental procedures complied with the Ethical Guidelines for the Use of Laboratory Animals.

[0038] 1. Experimental Methods: The experimental group consisted of 8 C57BL / 6J mice in each group, including the control group, the SiO2 group, the Met+CBD group, and the TPCM group. After isoflurane anesthesia, the mice were intratracheally instilled with 0.1 mL of a 200 mg / mL SiO2 suspension to establish a silicosis model. The control group received a non-invasive tracheal instillation of 0.1 mL of normal saline, the SiO2 group received a non-invasive tracheal instillation of 0.1 mL of normal saline, the Met+CBD group received a non-invasive tracheal instillation of 0.1 mL of a mixture of Met and CBD (the concentration ratio of Met to CBD in the mixture was 500 μg / mL:3 μg / mL), and the TPCM group received a non-invasive tracheal instillation of 0.1 mL of a 5 μg / mL nanoparticle TK-PSBs / CBD@Met solution. Each dosing interval for the four groups was 72 hours, resulting in a 28-day experimental period. On day 28, lung tissue was collected for HE, Masson's, Sirius Red staining, and immunofluorescence analysis to assess pulmonary fibrosis. Heart, liver, spleen, and kidney tissue were also collected for HE staining to assess the biosafety of the different treatment groups.

[0039] 2. Experimental results: Figure 6 and 7As shown, the lung tissues of mice in the SiO2 group displayed typical fibrotic features, including the formation of silicic nodules, alveolar structural destruction, and abnormal collagen deposition. Furthermore, the expression levels of α-smooth muscle actin and the senescence markers p21 and p16d were significantly upregulated. Following drug intervention, both the Met+CBD and TPCM groups significantly inhibited the progression of fibrosis. The nanoparticle TK-PSBs / CBD@Met group demonstrated a superior intervention effect, partially alleviating the pathological phenotype.

[0040] The biosafety evaluation of nanomedicines is a key prerequisite for their clinical transformation or experimental research. Figure 8 As shown in the results, HE staining observation revealed that the heart, liver, spleen and kidney of the experimental animals in the TPCM group all showed intact tissue structures, and no characteristic pathological damage was found. The nanoparticles TK-PSBs / CBD@Met have ideal biosafety characteristics.

[0041] In summary, the present invention has designed a highly safe and biodegradable nanoparticle, TK-PSBs / CBD@Met. The nanoparticles are physically encapsulated with cannabidiol and metformin in TK-PSBs, with a biomimetic membrane forming the outermost layer of the nanocomposite material, which is loaded with cannabidiol and metformin. This nanoparticle utilizes the anti-silicotic fibrosis effects of cannabidiol and metformin to achieve targeted treatment for silicotic fibrosis.

[0042] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes preferred embodiments.

[0043] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0044] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Nanoparticles TK-PSBs / CBD@Met, characterized by: The nanoparticles TK-PSBs / CBD@Met are formed by encapsulating cannabidiol and metformin in a thioketone-modified lipid membrane. The particle size of the nanoparticles TK-PSBs / CBD@Met is 90-180 nm.

2. The method for preparing the nanoparticles TK-PSBs / CBD@Met according to claim 1, characterized in that: The preparation method is as follows: S1. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, thioketone, cholesterol and chloroform were mixed and then rotary evaporated to form a uniform biomimetic membrane TK-PSBs. A mixed solution containing cannabidiol and metformin was added to hydrate the biomimetic membrane TK-PSBs to form bilayer liposomes; S2. The bilayer liposomes were sonicated at 85-95W for 9-11 minutes and then repeatedly frozen and thawed 3-5 times. The liposomes were centrifuged at 10,000-12,000 rpm for 55-65 minutes and the supernatant was discarded to obtain nanoparticles TK-PSBs / CBD@Met.

3. The method for preparing nanoparticles TK-PSBs / CBD@Met according to claim 2, characterized in that: The volume ratio of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, thioketone, cholesterol, chloroform and mixed solution is 9-11:1-3:1-3:0.5-1.5:0.5-1.5:1-3.

4. The method for preparing nanoparticles TK-PSBs / CBD@Met according to claim 2, characterized in that: The mass ratio of cannabidiol to metformin in the mixed solution is 0.5-1.5:1-3.

5. Use of the nanoparticles TK-PSBs / CBD@Met according to claim 1 in the preparation of a drug for treating silicosis fibrosis.

6. The use of the nanoparticles TK-PSBs / CBD@Met in the preparation of a drug for treating silicosis fibrosis according to claim 5, characterized in that: The nanoparticles TK-PSBs / CBD@Met in the drug serve as the only active ingredient.

7. The use of the nanoparticles TK-PSBs / CBD@Met in the preparation of a drug for treating silicosis fibrosis according to claim 5, characterized in that: The drug also includes pharmaceutically acceptable excipients.

8. The use of the nanoparticles TK-PSBs / CBD@Met in the preparation of a drug for treating silicosis fibrosis according to claim 7, characterized in that: The auxiliary materials include any one or more of fillers, stabilizers, diluents, and adjuvants.

9. The drug according to claim 5, characterized in that When the dosage form of the drug is a solution dosage form, the solution dosage form includes an oral liquid preparation, a gavage preparation, and an injection dosage form.

10. The drug according to claim 9, characterized in that The solution dosage form is a solution consisting of water and the nanoparticles TK-PSBs / CBD@Met, or a solution consisting of physiological saline and the nanoparticles TK-PSBs / CBD@Met.

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