Fluorine tag modified semeglutide nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to a fluorine tag modified semeglutide nano-particle as well as a preparation method and application of the fluorine tag modified semeglutide nano-particle. According to the semeglutide nano-particles, the side chain of the 26th amino acid lysine of the semeglutide is subjected to fluorination modification, and the side chain of the 26th amino acid lysine of the semeglutide is subjected to fluorination modification; the particle size of the semeglutide nano-particles is 120 nm to 130 nm. The fluorination modification refers to modification of a perfluorocarbon chain. According to the prepared fluorine tag modified semeglutide nanoparticles, the bioavailability of oral semeglutide is improved; the adverse reaction of the gastrointestinal tract caused by oral administration of the semeglutide is overcome; and the economic cost of oral taking of the semeglutide is also reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide drugs, and particularly to a fluorine-tag modified semaglutide nanoparticle and its preparation method and application. Background Art

[0002] GLP-1 (glucagon-like peptide-1) is a hormone secreted by the intestine, which can regulate blood glucose levels, promote insulin secretion, and inhibit gastric emptying, thus generating a sense of satiety. GLP-1 receptor agonists help control blood glucose and body weight by mimicking the action of this natural hormone. The earliest GLP-1 receptor agonist was liraglutide, but its side effects include the trouble of daily injection, resulting in low patient compliance.

[0003] Semaglutide is a drug used to treat type 2 diabetes and obesity and belongs to the category of GLP-1 receptor agonists. Its development has undergone years of research and has been approved globally. Semaglutide was developed by the Danish pharmaceutical company Novo Nordisk. It is a modified form of GLP-1 receptor agonists with a longer half-life (about 1 week), so it can be injected once a week. This makes semaglutide have higher compliance compared to other GLP-1 receptor agonists in the treatment of diabetes and obesity. Therefore, semaglutide can improve the therapeutic effect by prolonging its action time in the body while reducing the injection frequency of patients.

[0004] As a GLP-1 receptor agonist, semaglutide mainly exerts its effects through the following mechanisms: (1) Enhancing insulin secretion: When blood glucose rises, semaglutide can stimulate the pancreas to secrete insulin, which helps lower blood glucose. (2) Inhibiting glucagon secretion: Semaglutide can also inhibit the pancreas from secreting glucagon, a hormone that promotes blood glucose elevation, thus further helping to control blood glucose.

[0005] Delaying gastric emptying: By slowing down the gastric emptying rate, semaglutide increases the sense of satiety and helps reduce body weight. (3) Promoting weight loss: Due to suppressing appetite and increasing satiety, semaglutide helps patients reduce food intake, thus promoting weight loss.

[0006] Semaglutide was first approved by the US FDA in 2017 for the treatment of type 2 diabetes. Subsequently, with the accumulation of more clinical data, semaglutide has also been approved for the treatment of obesity and is widely used globally. In 2017, semaglutide was approved as an injection for the treatment of patients with type 2 diabetes under the brand name Ozempic. In 2021, the oral formulation of semaglutide (brand name Wegovy) was approved by the FDA for the treatment of obesity. Its effect is similar to the injection formulation, but the oral form greatly improves patient compliance.

[0007] Novo Nordisk developed an oral formulation of semaglutide, using SNAC (Sodium N-[8-(2-hydroxybenzoyl)amino]caprylate) as a drug delivery excipient to facilitate its absorption through the gastrointestinal tract. This oral formulation can help patients take the drug more conveniently and avoid the trouble of injection. However, currently, the bioavailability of the SNAC-modified semaglutide oral formulation is relatively low, about 0.4 - 1%; the SNAC-modified semaglutide oral formulation is prone to cause gastrointestinal adverse reactions; and the economic cost of the SNAC-modified semaglutide oral formulation is relatively high. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides a fluorine-labeled semaglutide nanoparticle and a preparation method thereof. The fluorine-labeled semaglutide nanoparticle prepared by the present invention improves the bioavailability of oral semaglutide; overcomes the gastrointestinal adverse reactions of oral semaglutide; and also reduces the economic cost of oral semaglutide.

[0009] The present invention is achieved through the following technical solutions:

[0010] The first object of the present invention is to provide a fluorine-labeled semaglutide nanoparticle, wherein the side chain of the 26th amino acid lysine of the semaglutide nanoparticle is fluorinated; the particle size of the semaglutide nanoparticle is 120nm - 130nm.

[0011] In an embodiment of the present invention, the fluorination modification is to modify a perfluorocarbon chain.

[0012] In an embodiment of the present invention, the perfluorocarbon chain is derived from one or more of a pentafluoromonomer, a nonafluoromonomer, a tridecafluoromonomer, and a heptadecafluoromonomer; preferably a tridecafluoromonomer.

[0013] In an embodiment of the present invention, the pentafluoromonomer is selected from one or more of pentafluoropropionic acid, pentafluorobutyric acid, pentafluorovaleric acid, pentafluorohexanoic acid, pentafluoroheptanoic acid, pentafluorooctanoic acid, pentafluorononanoic acid, and pentafluorodecanoic acid;

[0014] And / or, the nonafluoromonomer is selected from one or more of nonafluoropentanoic acid, nonafluorohexanoic acid, nonafluoroheptanoic acid, nonafluorooctanoic acid, nonafluorononanoic acid, and nonafluorodecanoic acid;

[0015] And / or, the tridecafluoromonomer is selected from one or more of perfluoroheptanoic acid, tridecafluorooctanoic acid, tridecafluorononanoic acid, and tridecafluorodecanoic acid;

[0016] And / or, the heptadecafluoromonomer is selected from heptadecafluorononanoic acid and / or heptadecafluorodecanoic acid.

[0017] The second object of the present invention is to provide a method for preparing the fluorine-tagged semaglutide nanoparticles, comprising the following steps:

[0018] S1. Mix and stir a perfluoro monomer, 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) in an organic solvent to obtain a mixed solution;

[0019] S2. Add a semaglutide solution to the mixed solution obtained in step S1 and stir;

[0020] S3. Dialyze the solution obtained in step S2 to obtain the fluorine-tagged semaglutide nanoparticles.

[0021] In one embodiment of the present invention, in step S1, the molar ratio of the perfluoro monomer, EDC, and NHS is 1:1.5 - 2:1.2 - 2, preferably 1:1.5:1.2 ；

[0022] In one embodiment of the present invention, in step S1, the organic solvent is selected from one or more of dimethyl sulfoxide (DMSO), dichloromethane, and acetonitrile, preferably dimethyl sulfoxide.

[0023] In one embodiment of the present invention, in step S2, the concentration of the semaglutide solution is 1 mg / mL - 20 mg / mL, specifically 1 mg / mL, 2 mg / mL, 3 mg / mL, 5 mg / mL, 15 mg / mL, 20 mg / mL, preferably 15 mg / mL.

[0024] The third object of the present invention is to provide the use of the fluorine-tagged semaglutide nanoparticles in the preparation of a medicament for treating type 2 diabetes.

[0025] The fourth object of the present invention is to provide the use of the fluorine-tagged semaglutide nanoparticles in the preparation of a medicament for treating obesity.

[0026] Semaglutide is modified with different fluorine atoms after NHS esterification reaction. Among them, semaglutide modified with 13F (perfluoroheptanoic acid) has the best mucus penetration ability and cell uptake ability;

[0027] Semaglutide modified with fluorine tags self-assembles into nanoparticles through fluorine-fluorine interactions, and the particle size and PDI are 120nm - 130nm and 0.2 respectively, showing good transcellular transport ability and uniform dispersion;

[0028] Semaglutide modified with fluorine tags has higher hypoglycemic ability, reduces economic costs, and improves patient compliance.

[0029] The above technical solutions of the present invention have the following advantages compared with the prior art:

[0030] 1. The present invention provides a semaglutide nanoparticle modified with fluorine tags and its preparation method. Different degrees of fluorination modification are carried out on the side chain of the 26th amino acid lysine in the drug backbone. According to the fluorine effect, it can self-assemble into nanoparticles in PBS solution to maintain the stability of the drug when passing through the gastrointestinal tract.

[0031] 2. The nanoparticles have a smaller particle size (about 120nm) than conventional drugs and are easier to penetrate the mucin network structure in the mucus layer. The perfluorocarbon chain on its surface can also reduce the non-specific adsorption between the carrier and mucin, greatly enhancing the ability of the carrier to penetrate the mucus layer with the advantages of small particle size and non-adsorption.

[0032] 3. The surface of the nanoparticles has a certain density of positive charge and is modified with perfluorocarbon chains that can help penetrate the cell membrane, so it has good endocytosis ability and effectively improves the transport effect of the drug on the small intestinal epithelium; the particles are transported through the process of transcytosis, without damaging the tight junctions between intestinal epithelial cells, reducing gastrointestinal adverse reactions.

[0033] 4. The overall preparation process is simple, efficient, has good drug efficacy, and has broad application prospects. Description of the Drawings

[0034] In order to make the content of the present invention easier to be clearly understood, the following further detailed description of the present invention is made according to the specific embodiments of the present invention in combination with the drawings, where

[0035] Figure 1 shows the particle sizes of the nanoparticles in Examples 1 - 4 and Comparative Example 1 described in Test Example 1 of the present invention;

[0036] Figure 2 shows the potential diagrams of the nanoparticles in Examples 1 - 4 and Comparative Example 1 in Test Example 2 of the present invention;

[0037] Figure 3 It is the cell viability graph of the nanoparticles in Examples 1 to 4 and Comparative Example 1 in Test Example 3 of the present invention;

[0038] Figure 4 It is the cell uptake level graph of the nanoparticles in Caco-2 cells in Test Example 4 of the present invention;

[0039] Figure 5 It is the Transwell experiment result graph of the nanoparticles on Caco-2 cells in Test Example 5 of the present invention;

[0040] Figure 6 It is the mutual adsorption strength result graph of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention with mucin of mass fractions 0.1%, 0.3% and 0.5% in Test Example 6;

[0041] Figure 7 It is the experimental graph of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention simulating epithelial cell endocytosis in vitro in Test Example 7;

[0042] Figure 8 It is the experimental graph of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention simulating epithelial cell exocytosis in vitro in Test Example 7;

[0043] Figure 9 It is the experimental graph of the insulin level of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention in a rat diabetes model in Test Example 8;

[0044] Figure 10 It is the experimental graph of the blood glucose level of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention in a rat diabetes model in Test Example 8;

[0045] Figure 11 It is the experimental graph of the biodistribution of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention at different time points in the small intestine of rats in Test Example 9;

[0046] Figure 12 It is the experimental graph of the biodistribution of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention at different time points in the kidneys and livers of rats after treatment in Test Example 9;

[0047] Figure 13 It is the experimental graph of the body weight change of the rats after treatment with the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention in Test Example 10;

[0048] Figure 14 It is the experimental graph of the change in intestinal permeability of rats after administration of the fluorine-labeled semaglutide nanoparticles obtained in Example 3 of the present invention in Test Example 11. Detailed implementation mode

[0049] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited do not limit the present invention.

[0050] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0051] Semaglutide was purchased from Nanjing Peptide Biotechnology Co., Ltd., product number: NJP27130;

[0052] Rats were purchased from Changzhou Cavens Experimental Animal Co., Ltd., product number: C000121;

[0053] The pentafluorinated monomer pentafluoropropionic acid was purchased from Shanghai Macklin Biochemical Co., Ltd., product number: C15749106;

[0054] The nonafluorinated monomer nonafluoropentanoic acid was purchased from Anhui Zesheng Technology Co., Ltd., product number: A01005548;

[0055] The tridecafluorinated monomer perfluoroheptanoic acid was purchased from Anhui Zesheng Technology Co., Ltd., product number: W810317;

[0056] The heptadecafluorinated monomer heptadecafluorononanoic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number: G2208164;

[0057] EDC was purchased from Anhui Zesheng Technology Co., Ltd., product number: A010938;

[0058] NHS was purchased from Sigma-Aldrich (Shanghai) Co., Ltd., product number: B010017.

[0059] Example 1

[0060] This example provides a method for preparing fluorine-labeled semaglutide nanoparticles, which is specifically as follows:

[0061] Dissolve 15 mg of semaglutide in 1 mL of dimethyl sulfoxide solution to prepare a 15 mg / mL semaglutide solution. After adding 4.4 mg of 5F monomer pentafluoropropionic acid to the reaction flask, add 1 mL of dimethyl sulfoxide solution, stir at 37 °C, add 100 μL of the prepared EDC dimethyl sulfoxide solution (32 mg / mL), and then continue to add 100 μL of the prepared NHS dimethyl sulfoxide solution (15 mg / mL), and activate with magnetic stirring for 12 h. After adding 1 mL of the prepared 15 mg / mL semaglutide solution, continue magnetic stirring for 12 h. Pipette out the fluorine-labeled semaglutide solution after stirring for 12 h, precipitate it with anhydrous ether pre-cooled at 4 °C, and then centrifuge at 4000 rpm for 30 min; after centrifugation, discard the ether solution, and repeat the precipitation of fluorine-labeled semaglutide with anhydrous ether 3 times; remove the unreacted monomers to obtain fluorine-labeled semaglutide nanoparticles.

[0062] Example 2

[0063] This example provides a method for preparing fluorine-labeled semaglutide nanoparticles, which is as follows:

[0064] Dissolve 15 mg of semaglutide in 1 mL of dimethyl sulfoxide solution to prepare a 15 mg / mL semaglutide solution. After adding 4.2 mg of 9F monomer nonafluoropentanoic acid to the reaction flask, add 1 mL of dimethyl sulfoxide solution, stir at 37 °C, add 100 μL of the prepared EDC dimethyl sulfoxide solution (32 mg / mL), and then continue to add 100 μL of the prepared NHS dimethyl sulfoxide solution (15 mg / mL), and activate with magnetic stirring for 12 h. After adding 1 mL of the prepared 15 mg / mL semaglutide solution, continue magnetic stirring for 12 h. Pipette out the fluorine-labeled semaglutide solution after stirring for 12 h, precipitate it with anhydrous ether pre-cooled at 4 °C, and then centrifuge at 4000 rpm for 30 min; after centrifugation, discard the ether solution, and repeat the precipitation of fluorine-labeled semaglutide with anhydrous ether 3 times; remove the unreacted monomers to obtain fluorine-labeled semaglutide nanoparticles.

[0065] Example 3

[0066] This example provides a method for preparing fluorine-labeled semaglutide nanoparticles, which is as follows:

[0067] Dissolve 15 mg of semaglutide in 1 mL of dimethyl sulfoxide solution to prepare a 15 mg / mL semaglutide solution. Add 4.0 mg of 13F monomer perfluorooctanoic acid to the reaction flask, then add 1 mL of dimethyl sulfoxide solution, stir at 37 °C, add 100 μL of the prepared EDC dimethyl sulfoxide solution (32 mg / mL), and continue to add 100 μL of the prepared NHS dimethyl sulfoxide solution (15 mg / mL). Activate and stir magnetically for 12 h. After adding 1 mL of the prepared 15 mg / mL semaglutide solution, continue to stir magnetically for 12 h. Pipette out the semaglutide solution with fluorinated label after stirring for 12 h, precipitate it with anhydrous ether pre-cooled at 4 °C, and centrifuge at 4000 rpm for 30 min; after centrifugation, discard the ether solution, and repeat the precipitation of fluorinated semaglutide with anhydrous ether 3 times; remove the unreacted monomers to obtain fluorinated label-modified semaglutide nanoparticles.

[0068] Example 4

[0069] This example provides a method for preparing fluorinated label-modified semaglutide nanoparticles, which is as follows:

[0070] Dissolve 15 mg of semaglutide in 1 mL of dimethyl sulfoxide solution to prepare a 15 mg / mL semaglutide solution. Add 3.8 mg of 17F monomer perfluorononanoic acid to the reaction flask, then add 1 mL of dimethyl sulfoxide solution, stir at 37 °C, add 100 μL of the prepared EDC dimethyl sulfoxide solution (32 mg / mL), and continue to add 100 μL of the prepared NHS dimethyl sulfoxide solution (15 mg / mL). Activate and stir magnetically for 12 h. After adding 1 mL of the prepared 15 mg / mL semaglutide solution, continue to stir magnetically for 12 h. Pipette out the semaglutide solution with fluorinated label after stirring for 12 h, precipitate it with anhydrous ether pre-cooled at 4 °C, and centrifuge at 4000 rpm for 30 min; after centrifugation, discard the ether solution, and repeat the precipitation of fluorinated semaglutide with anhydrous ether 3 times; remove the unreacted monomers to obtain fluorinated label-modified semaglutide nanoparticles.

[0071] Comparative Example 1

[0072] This comparative example provides a method for preparing stearic acid-modified semaglutide nanoparticles, which is as follows:

[0073] Dissolve 15 mg of semaglutide in 1 mL of dimethyl sulfoxide solution to prepare a 15 mg / mL semaglutide solution. After adding 2 mg of stearic acid (2.35 μL) to the reaction flask, add 1 mL of dimethyl sulfoxide solution, stir at 37 °C, add 100 μL of the prepared EDC dimethyl sulfoxide solution (32 mg / mL), and then continue to add 100 μL of the prepared NHS dimethyl sulfoxide solution (15 mg / mL), and activate magnetic stirring for 12 h. After adding 1 mL of the prepared 15 mg / mL semaglutide solution, continue magnetic stirring for 12 h. Pipette out the semaglutide solution containing stearic acid after stirring for 12 h, precipitate it with anhydrous ether pre-cooled at 4 °C, and then centrifuge at 4000 rpm for 30 min; after centrifugation, discard the ether solution, and repeat the precipitation of semaglutide containing stearic acid with anhydrous ether 3 times; remove the unreacted monomers to obtain semaglutide nanoparticles without fluorine modification.

[0074] Comparative Example 2

[0075] This comparative example provides a commercially available semaglutide modified with SNAC, purchased from Nanjing Peptide Biotechnology Co., Ltd., product number: NJP27222.

[0076] Test Example 1

[0077] The prepared Comparative Example 1 and Examples 1-7 were respectively subjected to particle size and dispersity coefficient (PDI) detection, and the results are as Figure 1 shown. The particle size of the semaglutide nanoparticles in Examples 1-7 was 120 nm - 130 nm, the particle size of Example 3 was about 120 nm, and the PDI was 0.2, having good nanoparticle characteristics.

[0078] Test Example 2

[0079] The prepared Comparative Example 1 and Examples 1-4 were respectively subjected to zeta potential detection, and the results are as Figure 2 shown. The zeta potential of Examples 1-4 was greater than that of Comparative Example 1, having good positive zeta potential.

[0080] Test Example 3

[0081] Inoculate Caco-2 cells at 2×10 4 cells / well into a 96-well plate and culture for 24 h. Add Comparative Example 1 and Examples 1-4 at a dose of 1 mg / mL, incubate with the cells for 24 h, and then use MTT assay to evaluate the viability. The results are expressed as the percentage of the viability of control cells not treated with NC. The results are as Figure 3 shown. The effects of Comparative Example 1 and Examples 1-4 on cell viability were all small.

[0082] Test Example 4

[0083] The Caco-2 cells were seeded at a density of 2×10 4 cells per well in a 6-well plate and cultured in MEM medium (manufacturer: Shanghai Yuanpei, product number: L570KJ) containing 10% FBS for 24 h. The FITC-labeled nanoparticles of Examples 1-4 and Comparative Examples 1-2 were added to the wells at a dose of 10 μL per well and incubated for 4 h. Subsequently, they were washed once with cold PBS solution, 500 μL of trypsin (manufacturer: Shanghai Beyotime, product number: C203-100 mL) was added to each well for digestion for 1 min. After aspirating the trypsin, 500 μL of serum-free MEM medium was used to blow the cells off the bottom of the dish. After collecting the cell suspension, it was centrifuged at 1000 rpm for 5 min, and the bottom precipitated cells were retained. After adding 100 μL of PBS solution, flow cytometry was performed using BD's Accuri C6 Plus flow cytometer. The results are shown in Figure 4 , and the cellular uptake levels of the nanoparticles of Examples 1-4 were all superior to those of Comparative Example 1 and Comparative Example 2, and Example 3 was selected as the group with better endocytic ability.

[0084] Test Example 5

[0085] A model simulating the transport of small intestinal epithelial cells was established in vitro. First, Caco-2 cells were seeded at a density of 5×10 4 cells / well on the apical side of Transwell, and then cultured for 17-21 days to form a monolayer of cells. The media on the apical side and the basolateral side were changed daily. When the transepithelial electrical resistance (TEER) value of the monolayer reached 450-550 Ω / cm 2 , it indicated that the model was established. Before the formal experiment started, the media on the apical side and the basolateral side of Transwell were first changed to Hanks balanced salt buffer (formula: 8 g / L NaCl, 0.4 g / L KCl, 1 g / L glucose, 60 mg / L KH2PO4, 47.5 mg / L Na2HPO4, adjusted to pH 7.2). After the system was equilibrated for 0.5 h, the FITC-labeled nanoparticles of Examples 1-4 and Comparative Example 1 were added to the apical side at an addition amount of 2 μL of nanoparticle solution per well and incubated for 4 h. The above operation steps were repeated, and 1% mucin (manufacturer: Sigma-Aldrich, product number: M1778) by mass fraction was added to the Hanks balanced salt buffer on the apical side of Transwell, and then the FITC-labeled nanoparticles of Examples 1-4 and Comparative Example 1 were added to the apical side and incubated for 4 h. Finally, 50 μL of the buffer solution on the basolateral side was taken, and its fluorescence intensity was detected by an enzyme-linked immunosorbent assay (ELISA) reader, and its apparent permeability coefficient (P app ) was calculated. The formula used was P app = Q / Act. Where Q is the total amount (ng) of the permeated example, A is the diffusion area (cm 2), c is the initial concentration of the top side example (ng / cm 3 ), t is the penetration time. Figure 5 In the absence of mucin, the nanoparticles obtained in Examples 1 to 4 and Comparative Example 1 were detected on the Transwell app The values are higher than those of Comparative Example 2. However, in the presence of mucin, the P values of the nanoparticles of Examples 1 to 4 and Comparative Example 1 are app The value is significantly decreased, and the nanoparticles in Example 3 are more preferred. In summary, the addition of tridecafluoromonomer perfluoroheptanoic acid can enhance the system's cell transport capacity in the presence of a mucus layer.

[0086] Test Example 6

[0087] The FITC-labeled nanoparticles of Examples 1 to 4 and Comparative Example 1 were dispersed in mucin solutions with mass fractions of 0.1%, 0.3%, and 0.5%, respectively. After vortexing, they were placed in an oscillator and incubated at room temperature. After 30 minutes, the mixture was centrifuged at 1500 rpm for 2 minutes, and the precipitate was washed twice with PBS. Then 200 μL NaOH (5 mol / L) was added to the precipitate to treat it; finally, the fluorescence intensity was measured using a fluorescence spectrometer. Under the condition of maintaining the same mass fraction of mucin, the adsorption intensity of Examples 1 to 4 on mucin was less than that of Comparative Example 1 and Comparative Example 2, and more preferably, Examples 3 and 4 were the groups with the best anti-mucin adsorption. Among them, the results of Example 3 and Comparative Example 1 are shown in FIG. Figure 6 It is further shown that the addition of an appropriate proportion of fluorinated monomers helps to enhance the system's ability to resist mucin adsorption.

[0088] Test Example 7

[0089] Endocytosis and exocytosis are two important processes in epithelial cell transcytosis. Therefore, the present invention explored the transport mechanism across the Caco-2 cell monolayer in Example 3. First, Caco-2 cells were plated at 5×10 4Cell density inoculation per well. The endocytic pathway of Example 3 in Caco-2 cells was explored by conducting cell uptake studies at 4 °C or in the presence of various endocytosis inhibitors, including wortmannin (WTM, a macropinocytosis inhibitor), chlorpromazine (CPZ, a clathrin-mediated endocytosis inhibitor), genistein (GNT, a caveolin-mediated endocytosis inhibitor), and methyl-β-cyclodextrin (mβCD, a lipid raft inhibitor), which were added to Caco-2 cells in 6-well plates respectively. The uptake level of Example 3 was significantly reduced at 4 °C or by GNT and CPZ, indicating the caveolin- and clathrin-mediated endocytic pathways. The exocytosis of Example 3 was further investigated using various exocytosis inhibitors, monensin (a Golgi-to-plasma membrane pathway inhibitor, Golgi / PM) and brefeldin A (an endoplasmic reticulum-to-Golgi pathway inhibitor, ER / Golgi). Flow cytometry experiments were performed on cells after co-incubating Example 3 with Caco-2 cells for 4 h. The results showed that monensin and brefeldin A increased the intracellular content of Example 3 to approximately 148% and 152% respectively, confirming that both of these pathways were involved in the exocytosis of Example 3. In addition, GNT, CPZ, monensin, and brefeldin A decreased the transport efficiency of Example 3 across the Caco-2 cell monolayer, indicating that the caveolin, clathrin, ER / Golgi, and Golgi / PM pathways were all involved in the transcytosis of Example 3, as specifically shown in Figures 7 - 8 shown.

[0090] Test Example 8

[0091] A rat diabetes model was induced by a single intraperitoneal injection of streptozotocin (STZ) (manufacturer: Shanghai Macklin, product number: S817944) at a dose of 65 mg / kg. The model was successfully established after 10 days. Female SD rats were orally gavaged with the nanoparticles described in Example 3 and Comparative Example 1 at a dose of 150 μg / kg of semaglutide (6 rats per group). Rats not administered the drug (normal saline) were used as Control Group 1, and rats without modeling and without drug administration were used as the blank group. At the time points of 0.08 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after administration, a SanNuo blood glucose meter (manufacturer: SanNuo Biochemical Sensing, product number: 5G01B240312) was used to detect the blood glucose of the rats, and 50 μL of blood was collected from the tail vein of each rat. The levels of GLP-1 and insulin in the serum were determined by ELISA. It can be Figures 9 - 10 seen that orally administered fluorine-containing nanoparticles were more effective than fluorine-free nanoparticles in inhibiting the increase in blood glucose levels in the diabetic model, and at the same time promoting the levels of insulin and GLP-1 in the blood of rats, resulting in better blood glucose lowering efficacy for the diabetic model.

[0092] Test Example 9

[0093] Rats in Example 3, Comparative Example 1 and the blank group (without modeling and without drug administration) were orally gavaged with Cy5-modified drugs. After 0.5 h, 4 h, 8 h, 12 h and 24 h, small animal imaging of the intestines (duodenum, jejunum, ileum and colon) and organs of rats in each group was performed. It was found that as time passed, the fluorescence in the intestines of rats in the blank group disappeared at 0.5 h, while the fluorescence in the other two groups remained, and that in Example 3 was higher than that in Comparative Example 1. At 4 h, the fluorescence in the intestines of rats in the Comparative Example 1 group disappeared, while that in Example 3 remained. At 8 h, the fluorescence in the intestines of rats in the Example 3 group remained and disappeared until 24 h. Moreover, the fluorescence in the intestines of rats in the Example 3 group was higher at 4 h than at other time points. At the corresponding time points, small animal imaging of the liver and kidneys of rats was performed and fluorescence quantification was carried out. It could be seen that the fluorescence intensities in the liver and kidneys of rats in the Example 3 group were higher than those in other groups, proving that Example 3 entered the systemic circulation metabolism after gastrointestinal metabolism. Specifically, as Figure 11 and Figure 12 shown.

[0094] Test Example 10

[0095] Rats were fed a 60% high-fat diet for 24 weeks to induce an obesity model. The average body weight of the rats reached 600 g and exceeded 20% of the body weight of non-modeled rats, indicating successful modeling. The nanoparticles described in Example 3 were orally gavaged to female SD rats at a dose of 300 μg / kg of semaglutide (6 rats per group), and rats without drug administration (normal saline) were used as the control group. After administration, the body weight of the rats was monitored using an electronic scale at a monitoring time point of every three days. It could Figure 13 be seen that oral administration of fluorine-containing nanoparticles inhibited the increase in body weight levels in the rat obesity model compared with fluorine-free nanoparticles, and had a better body weight control effect.

[0096] Test Example 11

[0097] The intestinal permeability was evaluated using FITC-dextran. Briefly, rats were deprived of food and water for 4 h, and then Example 3 (300 μg / kg) or PBS and FITC-dextran (4 kDa, 400 mg / kg) were administered by gavage. After 8 h, rat blood was collected, and the content of FITC-dextran in the serum was measured by fluorescence spectrophotometry (λ ex = 488 nm, λ em = 520 nm). Specifically, as Figure 14 shown; it could be seen that the fluorine-labeled semaglutide nanoparticles obtained by oral administration of Example 3 had no damage to the gastrointestinal tract, did not change the intestinal permeability, and did not cause intestinal leakage.

[0098] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A fluorine-labeled semaglutide nanoparticle, characterized in that, The side chain of the 26th amino acid of the fluorine-labeled semaglutide nanoparticles is fluorinated; the particle size of the semaglutide nanoparticles is 120 nm - 130 nm.

2. The semaglutide nanoparticles according to claim 1, wherein The fluorination modification is to modify a perfluorocarbon chain.

3. The semaglutide nanoparticles according to claim 2, wherein The perfluorocarbon chain is selected from one or more of a pentafluorinated monomer, a nonafluorinated monomer, a tridecafluorinated monomer, and a heptadecafluorinated monomer.

4. The semaglutide nanoparticles according to claim 3, characterized in that, The pentafluorinated monomer is selected from one or more of pentafluoropropionic acid, pentafluorobutyric acid, pentafluorovaleric acid, pentafluorohexanoic acid, pentafluoroheptanoic acid, pentafluorooctanoic acid, pentafluorononanoic acid, and pentafluorodecanoic acid; and / or, the nonafluorinated monomer is selected from one or more of nonafluorovaleric acid, nonafluorohexanoic acid, nonafluoroheptanoic acid, nonafluorooctanoic acid, nonafluorononanoic acid, and nonafluorodecanoic acid; and / or, the tridecafluorinated monomer is selected from one or more of perfluoroheptanoic acid, tridecafluorooctanoic acid, tridecafluorononanoic acid, and tridecafluorodecanoic acid; and / or, the heptadecafluorinated monomer is selected from heptadecafluorononanoic acid and / or heptadecafluorodecanoic acid.

5. The preparation method of the fluorine-labeled semaglutide nanoparticles according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Mix and stir a perfluorinated monomer, 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in an organic solvent to obtain a mixed solution; S2. Add a semaglutide solution to the mixed solution obtained in step S1 and stir; S3. Dialyze the solution obtained in step S2 to obtain fluorine-labeled semaglutide nanoparticles.

6. The preparation method according to claim 5, wherein, In step S1, the molar ratio of the perfluorinated monomer, 1-(3-dimethylpropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.5 - 2:1.2 - 2.

7. The preparation method according to claim 5, characterized in that, In step S1, the organic solvent is selected from one or more of dimethyl sulfoxide, dichloromethane, and acetonitrile.

8. The preparation method according to claim 5, characterized in that, In step S2, the concentration of the semaglutide solution is 1 mg / mL - 20 mg / mL.

9. Use of the fluorine-labeled semaglutide nanoparticles according to any one of claims 1 - 4 in the preparation of a medicament for treating type 2 diabetes.

10. Use of the fluorine-labeled semaglutide nanoparticles according to any one of claims 1 - 4 in the preparation of a medicament for treating obesity.

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