Nanoparticle TK-PSBs (at) Met as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a nanoparticle TK-PSBs (at) Met and a preparation method and application thereof. According to the nanoparticle TK-PSBs (at) Met provided by the invention, liposome nanoparticles are synthesized in a customized manner and are coated with metformin, so that the targeting delivery capability of a drug to a lung is enhanced, the drug accumulation of a diseased region is improved, and the treatment accuracy and effectiveness are further improved, thereby improving the treatment effect.

Description

Technical Field

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

[0002] Nanodrug delivery systems typically refer to nanoscale particles or carriers used to encapsulate, protect, transport, and control the release of drugs. Nanodrug delivery systems can improve drug bioavailability, increase drug uptake by target cells, prolong drug residence time in the body, and improve pharmacokinetics, thereby significantly enhancing the therapeutic effect of drugs and reducing side effects. Specifically, tailored liposomes, which vary in size, charge, lipid composition, and ratio, can be used to treat diseases of specific organs, thereby enhancing drug circulation, accumulation in lesions, intracellular delivery, and organ targeting, improving the therapeutic efficacy of specific diseases.

[0003] Since its first synthesis in 1922, metformin (abbreviated as Met) has been the most commonly used hypoglycemic agent and is used as a first-line drug for the treatment of type 2 diabetes in many clinical guidelines. In the past few years, researchers have paid more attention to the immunomodulatory, anti-aging, and anti-inflammatory properties of metformin, using it as an adjuvant treatment for cancer, age-related diseases, obesity, inflammatory diseases, and COVID-19. However, despite its significant pharmacological effects, metformin's limited oral availability and pharmacokinetic properties, where its elimination rate is faster than its absorption rate, greatly limit its efficacy.

[0004] Silicosis refers to a systemic disease characterized by diffuse fibrosis of the lung tissue caused by long-term inhalation of free silica dust during occupational activities and its retention in the lungs. Impairment of lung function increases with the progression of the disease, even after the patient is no longer exposed. The main pathological changes of silicosis are inflammatory cell infiltration of the lungs, formation of silicotic nodules, and diffuse fibrosis of the lung tissue. The pathogenesis is complex, involving a complex interaction between epithelial cells, fibroblasts, immune cells, and endothelial cells. There is increasing evidence that fibrosis is driven by repeated damage to the alveolar epithelium. Damaged alveolar epithelial cells can release excessive reactive oxygen species, thereby promoting the secretion of key pro-fibrotic mediators, leading to dysregulated repair and pathogenic activation of fibroblasts, which in turn limits the repair of damaged alveoli and accelerates fibrosis.

[0005] Currently, there is no definitive cure for silicosis. Clinical treatment primarily relies on supportive interventions, including oxygen supplementation for hypoxic patients, prompt treatment of infections, and pulmonary rehabilitation. Lung transplantation is the only viable cure for end-stage silicosis, but lung transplantation is costly, resource-limited, and the median survival after transplantation is suboptimal. Nintedanib and phenanthroline are widely used clinically for anti-fibrotic treatments, but they can only slow the decline in lung function; they cannot cure or reverse established fibrosis, and both have varying degrees of side effects. Summary of the Invention

[0006] The purpose of the present invention is to provide nanoparticles TK-PSBs@Met and a preparation method and application thereof.

[0007] The present invention provides nanoparticles TK-PSBs@Met. The nanoparticles TK-PSBs@Met are formed by encapsulating metformin in a thioketone-modified liposome membrane. The particle size of the nanoparticles TK-PSBs@Met is 190-210 nm.

[0008] The nanoparticles TK-PSBs@Met provided by the present invention are formed by encapsulating metformin in a customized synthetic liposome membrane. This structure enhances the targeted delivery of the drug to the lungs and increases drug accumulation in the lesion site. In vivo experimental results show that the use of nanoparticles TK-PSBs@Met can significantly reduce inflammatory infiltration and shrink the area of ​​fibrosis, indicating that nanoparticles TK-PSBs@Met can effectively reverse silicosis fibrosis.

[0009] The present invention also provides a preparation method of nanoparticles TK-PSBs@Met, and the preparation method is as follows;

[0010] 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 metformin solution with a concentration of 1 g / mL was added to hydrate the biomimetic membrane TK-PSBs to form bilayer liposomes;

[0011] 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@Met.

[0012] Furthermore, the volume ratio of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, thioketone, cholesterol, chloroform and 1 g / mL metformin solution is 19-21:3-5:3-5:1-3:2-4:3-5.

[0013] The present invention also provides the use of nanoparticles TK-PSBs@Met in preparing a medicine for treating silicosis fibrosis.

[0014] Furthermore, the nanoparticles TK-PSBs@Met in the drug serve as the only active ingredient.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the auxiliary materials include any one or more of fillers, stabilizers, diluents, and adjuvants.

[0017] Furthermore, the content of the nanoparticles TK-PSBs@Met in the drug is 0.1 wt% to 99 wt%.

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

[0019] The present invention uses nanotechnology to develop a new type of nanoparticle TK-PSBs@Met for the treatment of silicosis fibrosis. The nanoparticle can improve the bioavailability of drugs and increase the uptake of drugs by target cells. It can not only slow down the decline of lung function, but also reverse the established fibrotic silicosis model. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Characterization diagram of nanoparticles TK-PSBs@Met, where A is the electron microscope image, B is the Zeta potential image, C is the particle size detection image, D is the stability detection statistical image, E is the Fourier transform infrared spectroscopy detection image, and F is the Fourier transform infrared spectroscopy detection image with amplified wavenumber.

[0021] Figure 2 This is a stained image of a lung section.

[0022] Figure 3 This is a staining image of mouse organ sections. DETAILED DESCRIPTION

[0023] 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.

[0024] Example 1: Development of nanoparticles TK-PSBs@Met.

[0025] 1. Preparation method of nanoparticles TK-PSBs@Met.

[0026] 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 20:4:4:2:3 and rotary evaporated to form a uniform liposome membrane TK-PSBs on the container wall. 4 ml of 1 mg / mL metformin solution was added to hydrate the biomimetic TK-PSB membrane to form bilayer liposomes. The mixture was then sonicated at 90W for 10 minutes, freeze-thawed four times at -80°C and 32°C, and centrifuged at 11,000 rpm for 60 minutes. The supernatant was discarded to obtain nanoparticles TK-PSBs@Met.

[0027] After adding 4 ml of distilled water to the uniform liposome membrane TK-PSBs to form a bilayer liposome, the mixture was ultrasonicated at 90W for 10 min, frozen and thawed four times at -80°C and 32°C, and centrifuged at 11000 rpm for 60 min. The supernatant was discarded to obtain drug-free nanoparticles TK-PSBs.

[0028] 2. Detection method: The prepared nanoparticle TK-PSBs@Met sample was placed under a transmission electron microscope for observation, and the morphology of the sample was recorded and analyzed. Dynamic light scattering (DLS, Nano ZS90 Zetasizer, Malvern) was used to detect the Zeta potential and particle size of the nanoparticles. Fourier transform infrared spectroscopy was used to characterize its maximum absorbance and the chemical bonds contained. The prepared nanoparticles TK-PSBs@Met were redissolved in a 10% volume fraction of fetal bovine serum solution and PBS every day for one week, and the particle size of the sample was measured to evaluate its stability in different media.

[0029] 3. Test results: Figure 1 As shown in Figure A, the nanoparticles TK-PSBs@Met were analyzed by transmission electron microscopy. The biomimetic membrane TK-PSBs was wrapped in the outer layer of metformin, showing an obvious "core-shell" structure. Figure 1 As shown in Figures B and C, the dynamic light particle size scattering system shows that the average diameter of the nanoparticles TK-PSBs@Met is 200nm and the Zeta potential is about -5.00mV. Figure 1 As shown in D, the nanoparticles TK-PSBs@Met showed good stability in both PBS and 10% fetal bovine serum solution. Figure 1 As shown in Figures E and F, infrared spectroscopy showed that the presence of C=N, NH, and CN indicated that TK-PSBs were successfully loaded with metformin.

[0030] 2. Application of nanoparticles TK-PSBs@Met in silicosis fibrosis.

[0031] 1. Experimental methods: The experimental group was divided into control group, model group, cell therapy group, and cell + drug therapy group. There were 8 C57BL / 6J mice in each group. After isoflurane anesthesia, 0.5 mL of 200 mg / mL SiO2 suspension was instilled into the trachea to establish the silicosis model mice. The control group mice were instilled with the same volume of normal saline without silicosis treatment. 48 hours after surgery, the model group mice were instilled with 0.5 mL of normal saline through the trachea. 48 hours after surgery, the cell therapy group mice were instilled with 0.5 ml of 5×10 8 Cells were treated with AECⅡ cell suspension at a concentration of 10 cells / mL. At 48 hours after surgery, mice in the cell + drug treatment group were injected with 0.5 ml of 30 mg / mL TK-PSBs@Met and 0.5 ml of 5×10 8 AECⅡ cell suspension was prepared at a concentration of 10 cells / mL. Each dosing interval was 48 hours. At the end of the 28th day, lung tissue sections were obtained and stained with HE, Masson's, and Sirius Red to observe pulmonary fibrosis. Heart, liver, spleen, and kidney sections were also stained with HE to assess the biosafety of the different treatment groups.

[0032] 2. Experimental results: Figure 2 As shown, TK-PSBs@Met can effectively inhibit epithelial cell fibrosis. Compared with the control group, the cell + drug treatment group was able to reduce SiO2 damage to lung tissue to form silicotic nodules, maintain relatively intact alveolar structure, reduce inflammatory infiltration, and shrink the area of ​​fibrosis, indicating that nanoparticles TK-PSBs@Met can effectively alleviate and reverse silicotic fibrosis.

[0033] Biosafety results such as Figure 3 As shown, no obvious organic lesions were found in the organs of mice in different treatment groups, indicating that the nanoformulation constructed by the present invention has good biosafety.

[0034] In summary, the present invention has designed a highly safe nanomedicine, TK-PSBs@Met, that effectively alleviates silicosis fibrosis. The nanomedicine comprises TK-PSBs that physically encapsulate metformin. During the pathogenesis of silicosis fibrosis, epithelial cells undergo epithelial-to-mesenchymal transition, accompanied by the production of inflammatory factors. TK-PSBs@Met responds to this inflammation, enabling target cells to effectively utilize the nanocomposite material, enhancing its effectiveness in preventing and treating silicosis fibrosis.

[0035] 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.

[0036] 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.

[0037] 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@Met, characterized by: The nanoparticles TK-PSBs@Met are formed by encapsulating metformin in a thioketone-modified liposome membrane. The particle size of the nanoparticles TK-PSBs@Met is 190-210 nm.

2. The method for preparing the nanoparticle TK-PSBs@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 metformin solution with a concentration of 1 g / mL 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@Met.

3. The method for preparing nanoparticles TK-PSBs@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 1 g / mL metformin solution is 19-21:3-5:3-5:1-3:2-4:3-5.

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

5. The use of the nanoparticles TK-PSBs@Met according to claim 4 in the preparation of a drug for treating silicosis fibrosis, characterized in that: The nanoparticles TK-PSBs@Met are the only active ingredient in the drug.

6. The use of the nanoparticles TK-PSBs@Met in the preparation of a drug for treating silicosis fibrosis according to claim 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. Use of the nanoparticles TK-PSBs@Met according to claim 6 in preparing a drug for treating silicosis fibrosis, characterized in that: The auxiliary materials include any one or more of fillers, stabilizers, diluents, and adjuvants.

8. The use of the nanoparticles TK-PSBs@Met in the preparation of a drug for treating silicosis fibrosis according to claim 4, characterized in that: The content of the nanoparticles TK-PSBs@Met in the medicine is 0.1 wt% to 99 wt%.

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