A nasally administrable semaglutide formulation, and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a nasal administration semaglutide preparation, a preparation method and application thereof, and comprises the following steps: N-acetylglucosamine is coupled with semaglutide lysine residues through an activated ester method to obtain glycosylated semaglutide; the glycosylated semaglutide is crosslinked with wheat agglutinin to form uniform nanoparticles with a diameter of 70-90 nm through self-assembly. The glycosylated semaglutide nanopreparation prepared by the application has high drug loading and high targeting, can maintain good colloidal stability and delivery performance under physiological conditions; after nasal administration, the nanopreparation has excellent adhesion and penetration capacity on the nasal mucosa, and improves the concentration of semaglutide in brain tissue; the nanopreparation has a weight loss effect, and provides a new solution for non-invasive long-term administration of semaglutide.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a nasal administration of smegglutide preparation, its preparation method and application. Background Technology

[0002] Semaglutide is a synthetically produced long-acting GLP-1 receptor agonist. Its molecular design includes three key modifications: first, the 4th lysine is replaced by arginine, which enhances its binding ability to plasma albumin and prolongs its circulation time in vivo; second, the 8th α-aminobutyric acid is replaced by alanine, which can resist the degradation of dipeptidyl peptidase-4 (DDP-4); third, the 26th lysine is linked to the C18 fatty acid diacid sidewall, forming a steric barrier through hydrophobic interaction and prolonging its half-life. This structure makes semaglutide as homologous to human GLP-1 amino acid sequence as high as 94%. The mechanism by which smegglutinin exerts its weight-loss effect primarily involves the peripheral system, where it promotes insulin secretion from glucose-dependent pancreatic β-cells, inhibits glucagon release from pancreatic α-cells, and delays gastric emptying, thereby lowering blood sugar. In the central nervous system, it acts on GLP-1 receptors in the diffuse hypothalamus and medullary chemoreceptor area, activating POMC / CART neurons in the arcuate nucleus (ARC) of the hypothalamus, releasing α-melanocyte-stimulating hormone (α-MSH), inhibiting NPY / AgRP neurons, and simultaneously regulating the nucleus tractus solitarius (NTS) pathway through the vagus nerve, thereby effectively suppressing appetite, enhancing satiety, and promoting energy expenditure. Existing semaglutide formulations mainly fall into two categories: once-weekly subcutaneous injections (such as Ozempic® and Wegovy®) and once-daily oral formulations (Rybelsus®). Currently, although semaglutide can effectively improve patients' blood sugar and weight, its clinical application is still limited by the shortcomings of its formulations. For example, because it requires subcutaneous injection, it is often accompanied by discomfort such as redness, swelling, and itching at the injection site, and it must be refrigerated for storage, which is costly and affects long-term patient compliance. Oral formulations, on the other hand, require higher doses due to gastrointestinal degradation and low bioavailability.

[0003] The nasal-to-brain drug delivery route has emerged as a promising strategy. Firstly, the unique anatomical structure of the nasal cavity provides a distinctive pathway for drug delivery; the total surface area of ​​the nasal cavity is approximately 150 cm². 2The highly vascularized epithelial cells in the respiratory and olfactory regions allow for rapid drug absorption and avoid the first-pass effect. Nasal administration allows semaglutide to directly cross the nasal mucosa, reaching the central nervous system via the olfactory and trigeminal nerves, and then being delivered to the brainstem and hypothalamus, bypassing the blood-brain barrier. It also rapidly reaches the target area through axonal transport or intercellular diffusion, improving bioavailability. Furthermore, studies have shown that nasal administration allows the drug to reach peak concentration within 20-40 minutes, significantly shorter than subcutaneous injection or oral administration. This route offers advantages such as non-invasiveness and ease of administration, while also reducing adverse reactions and improving patient compliance. Therefore, modifying the existing semaglutide structure and designing nasally delivered semaglutide formulations will be crucial for improving its clinical application and meeting the growing therapeutic needs of patients. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a nasal-administered smegglutide formulation, thereby addressing the problems mentioned in the background section.

[0005] The present invention is implemented as follows: a method for preparing a nasal-administered semaglutide formulation includes the following steps:

[0006] N-acetylglucosamine was coupled with lysine residues of smegglutinin via an activated ester method to obtain glycosylated smegglutinin.

[0007] Glycosylated smegglutinin was cross-linked with wheat lectin, and through self-assembly, uniform nanoparticles with a diameter of 70-90 nm were formed.

[0008] Another objective of this invention is to provide a nasal administration formulation of smegglutide, which is prepared using the above-described preparation method.

[0009] Another objective of this invention is to provide the application of a nasally administered smegglutide formulation in the preparation of a weight control drug.

[0010] This invention modifies the structure of existing semaglutide to enable nasal delivery to the brain, directly bypassing digestive degradation and the first-pass effect via the olfactory and trigeminal nerve pathways, thereby improving drug bioavailability and inducing weight loss; through the Lys... 26 and Lys 34 N-acetylglucosamine (GlcNAc) was introduced at two sites for modification. GlcNAc improved the overall water solubility and biocompatibility of the peptide chain, and also enabled the glycosyl surface to specifically bind to nasal mucosal receptors. Simultaneously, GlcNAc provided a hydrophilic structure for smegglutinin, and its own Lys... 26A C18 fatty acid chain is attached to the nanoparticle, providing it with a hydrophobic structure, driving hydrophobic interactions between molecules, and inducing spontaneous self-assembly of the nanoparticles. The hydrophobic ester chains aggregate to form a core, while the hydrophilic glycosyl segments face the aqueous phase, forming a core-shell structure. To further enhance the stability of the nanoparticles and their retention time in the nasal cavity, this invention introduces wheat lectin (WGA) to achieve multivalent cross-linking. WGA can firmly couple nanoparticles or the nasal mucosa surface through recognition with multiple sugar receptors, which not only improves the mechanical stability of the nanoparticles but also enhances the efficiency of receptor-mediated transepithelial transport. The WGA recognition sites on the particle surface can specifically bind to glycoprotein receptors rich in GlcNAc / sialic acid on the nasal mucosa, improving receptor-mediated cellular uptake efficiency. The self-assembly strategy of glycosylation modification and WGA crosslinking eliminates the need for additional lipid or polymer carriers, simplifying formulation composition and reducing toxicity risks. The semaglutide formulation prepared in the embodiments of the present invention has both hydrophilic and hydrophobic properties, self-assembled nanostructures, and receptor-targeted crosslinking, which significantly improves the stability of the nanostructure, retention in the nasal cavity, and receptor-mediated transport efficiency, thereby significantly improving its delivery capability from the nose to the brain.

[0011] In summary, the embodiments of the present invention have the following significant effects: the glycosylated semaglutide nanoformulation designed and prepared has high drug loading capacity and high targeting, and can maintain good colloidal stability and delivery performance under physiological conditions; after nasal administration, the nanoformulation has excellent adhesion and penetration ability on the nasal mucosa, increasing the concentration of semaglutide in brain tissue; the nanoformulation has the effect of reducing body weight, providing a new solution for non-invasive long-term administration of semaglutide. Attached Figure Description

[0012] Figure 1 This is a transmission electron microscope image of GlcNAc-Semaglutide-WGA nanoparticles from Example 1 of the present invention.

[0013] Figure 2 Transmission electron microscope image of GlcNAc-modified liraglutide nanoparticles crosslinked with WGA, provided in Example 3 of this invention.

[0014] Figure 3 MTT cell activity of nanoparticles in three cell lines provided in Example 4 of this invention;

[0015] Figure 4 The average adhesion rate of different particle types in the nasal mucosa model provided in Example 5 of the present invention is shown. * indicates a statistically significant difference between the two groups (**p<0.01, ***p<0.001, ****p<0.0001).

[0016] Figure 5The image shows the confocal microscopy cell uptake results provided in Example 6 of this invention, where the left image shows the nanoparticles of the fully modified group and the right image shows the nanoparticles of the control group.

[0017] Figure 6 The MFI-time curves of the three groups of nanoparticles provided in Example 6 of the present invention are shown. * indicates the significance level of the single modification group and the fully modified group compared with the control group (**p<0.01, ****p<0.0001), and # indicates the significance level of the fully modified group compared with the single modification group (#<0.05, ##<0.01).

[0018] Figure 7 The three groups of nanoparticles provided in Example 7 of this invention are used to evaluate their ability to cross the nasal epithelial barrier. * indicates the significance level of the single modification group and the fully modified group compared with the control group (*p<0.05, **p<0.01, ***p<0.001), and # indicates the significance level of the fully modified group compared with the single modification group (##p<0.01, ###p<0.001).

[0019] Figure 8 The cumulative release curves of the three groups of nanoparticles provided in Example 8 of the present invention in simulated nasal fluid are shown. * indicates the significance level of the single modification group and the fully modified group compared with the control group (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001), and # indicates the significance level of the fully modified group compared with the single modification group (#<0.05, ##p<0.01, ###p<0.001).

[0020] Figure 9 The following is a trend of mouse body weight change in each group provided in Example 10 of the present invention. * indicates the significance level of the smegglutide group, the single modification group and the nano-formulation group compared with the control group (*p<0.05, **p<0.01, ***p<0.001). Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0022] A nasal administration formulation of semaglutide, the preparation method of which includes the following steps:

[0023] Step 1: Dissolve smegglutinin powder in sodium bicarbonate buffer, dissolve GlcNAc-NHS in DMF, and gently sonicate or vibrate to dissolve.

[0024] Step 2: Under ice bath conditions, slowly add GlcNAc-NHS to the solution containing smegglutinin and stir gently to allow the reaction to proceed.

[0025] Step 3: After the reaction is complete, adjust the pH to neutral with acetic acid to stop the reaction. Dialyze with a dialysis bag to remove unreacted GlcNAc and impurities. Freeze dry to obtain glycosylated modified smegglutinin.

[0026] Step 4: Dissolve WGA in PBS to a concentration of 5 mg / mL, add EDC·HCl and NHS, mix well at room temperature to generate WGA-NHS intermediate;

[0027] Step 5: Dissolve the glycosylated smegglutinin in PBS and mix it with the activated WGA-NHS intermediate, wherein the mass ratio of glycosylated smegglutinin to WGA is 1:1. Stir slowly to promote self-assembly and formation of nanoparticles.

[0028] Step 6: After the reaction, allow the precipitate to stand at 4°C, centrifuge, collect the precipitate, wash twice with PBS, and resuspend in PBS for later use.

[0029] The raw material information used in the embodiments of the present invention is as follows:

[0030] Smegglutinin: S871921, purchased from Shanghai Maclean Biochemical Technology Co., Ltd., its amino acid sequence is as follows:

[0031] H-His 7 -Aib 8 -Glu 9 -Gly 10 -Thr 11 -Phe 12 -Thr 13 -Ser 14 -Asp 15 -Val 16 -Ser 17 -Ser 18 -Tyr 19 -Leu 20 -Glu 21 -Gly 22 -Gln 23 -Ala 24 -Ala 25 -Lys 26 -Glu 27 -Phe 28 -Ile 29 -Ala 30 -Trp 31 -Leu 32 -Val 33 -Lys34 -Gly 35 -Arg 36 -Gly 37 -OH (as shown in SEQ ID NO.1);

[0032] Liraglutide: L860407, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.;

[0033] Wheat lectin (WGA): T937863, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.;

[0034] Human nasal mucosal squamous epithelial cells (RPMI 2650): purchased from Wuhan Pronosei Life Science Technology Co., Ltd.;

[0035] Human neuroblastoma cells (SH-SY5Y): purchased from Wuhan Pronosai Life Science Technology Co., Ltd.;

[0036] Mouse microglia (BV2): purchased from Wuhan Pronosei Life Science Technology Co., Ltd.;

[0037] C57 / BL6 mice: 8 weeks old, male, purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0038] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0039] Example 1: A nasal administration formulation of semaglutide (GlcNAc-Semaglutide-WGA nanoparticles), the preparation method of which includes the following steps:

[0040] (1) Synthesis of GlcNAc-modified semaglutide: Weigh 50 mg of semaglutide and place it in 25 mL of 0.1 M sodium bicarbonate buffer (pH 8.5). Use ultrasonic dispersion to promote complete dissolution of the peptide chain. Separately, take 30 mg of N-acetyl-D-glucosamine-NHS ester (GlcNAc-NHS) and dissolve it in 3 mL of anhydrous dimethylformamide (DMF). Ensure that it is fully dissolved by gentle sonication or oscillation. In this embodiment of the invention, the activated lipid method is used. The semaglutide solution is placed in a low-temperature ice bath. The GlcNAc-NHS solution is slowly added to the reaction solution and gently stirred to avoid vigorous stirring that may cause peptide chain conformational damage or side reactions. The reaction is carried out at low temperature to reduce the risk of decomposition reaction and non-specific NHS chain breakage. Ensure that the reaction is complete and that GlcNAc-NHS is in Lys 26 and Lys 34The sites were fully coupled; after the reaction, the pH was adjusted to neutral by slowly adding acetate buffer to terminate the activity of NHS ester and reduce the generation of impurities from the hydrolysis of excess GlcNAc-NHS; then the product was placed in a dialysis bag and dialyzed at low temperature to completely remove unreacted GlcNAc-NHS, DMF and salt ions. After dialysis, it was freeze-dried to obtain a white powder, namely GlcNAc-modified semaglutide.

[0041] (2) WGA cross-linking promotes the assembly of nano-formulations: Weigh 10 mg of wheat lectin (WGA) and dissolve it in 2 mL of 0.1 M solution. PBS (pH 7.4) was added and gently vortexed at room temperature until completely homogeneous. Then, equimolar amounts of EDC·HCl and NHS were added, and the mixture was gently stirred at room temperature to activate the carboxyl groups on the WGA molecules, generating the WGA-NHS intermediate. This intermediate can form amide bonds with the free amino groups of the peptide chain in subsequent reactions, achieving multiple cross-linking on the nanoparticle surface. The prepared GlcNAc-Semaglutide was then diluted with PBS and filtered to remove any possible particles or undissolved impurities. GlcNAc-Semaglutide was then slowly added to the WGA-NHS solution with gentle stirring to promote the formation of self-assembled nanoparticles, avoiding non-specific aggregation or peptide chain conformation disruption caused by localized high concentrations, ensuring a complete reaction and promoting amide bond formation between WGA and the peptide chain. After the reaction, the mixture was allowed to stand at 4°C to promote initial precipitation. The precipitate was collected by high-speed centrifugation, and the precipitate consisted of WGA-crosslinked GlcNAc-Semaglutide nanoparticle clusters. These clusters were washed twice with PBS to remove free WGA and free peptides, and then resuspended in 5 mL of PBS. PBS was used as a backup to obtain GlcNAc-Semaglutide-WGA nanoparticles;

[0042] The prepared nanoparticles were analyzed using transmission electron microscopy, and the TEM images are shown below. Figure 1 As shown.

[0043] Example 2: Characterization and size optimization of GlcNAc-Semaglutide-WGA nanoparticles:

[0044] In this embodiment of the invention, by optimizing the ratio of GlcNAc-Semaglutide to WGA, the process of synthesizing nanoparticles according to Example 1 is followed. By adjusting the degree of modification and the crosslinking ratio, the particle size of the nanoparticles is controlled at 75-90 nm and the PDI is less than 0.25.

[0045] First, a modification gradient was designed: Lysyl ester of semaglutide was modified with GlcNAc-NHS ester. 26 and Lys 34Different molar ratios were designed for the site: Group A was GlcNAc: smegglutinin = 1:1 (theoretically modifying 1 lysine), Group B was 2:1 (theoretically modifying 2 lysines), and Group C was 4:1 (over-modification).

[0046] WGA crosslinking gradient design: Each group of modified products was mixed with WGA at the following mass ratios: 1:0.5, 1:1, 1:2, 1:4 (i.e., GlcNAc-Semaglutide:WGA); the concentration of each modified product was 1 mg / mL, dissolved in PBS buffer (pH 7.4); after mixing, the mixture was incubated and self-assembled to form nanoparticles. The average particle size, PDI and zeta potential of each sample were measured by dynamic light scattering (DLS), and the stability data were recorded.

[0047] The results are as follows:

[0048] The effects of GlcNAc modification degree on nanoparticle size and PDI are shown in Table 1:

[0049] Table 1

[0050]

[0051] Group A has an average particle size >180nm and a relatively high PDI (>0.3), indicating that insufficient modification leads to low crosslinking efficiency and incomplete particle assembly. Group B has a particle size of 85-110nm, a PDI <0.2, stable morphology, and uniform distribution. Group C has a relatively small particle size (40-60nm), but aggregation was observed in some groups, which is speculated to be due to excessive GlcNAc on the surface, resulting in an imbalance between charge and hydrophilicity that affects stability.

[0052] The effects of different WGA crosslinking ratios on nanoparticle size, PDI, and zeta potential are shown in Table 2.

[0053] Table 2

[0054]

[0055] When the modification ratio was 2:1 (Group B), the nanoparticles in the group with a modified peptide to WGA mass ratio of 1:1 exhibited the best properties: particle size of 83 ± 6.22 nm, PDI of 0.181 ± 0.12, and zeta charge of +10.6 mV. TEM observation showed that they were spherical and uniformly distributed.

[0056] Example 3: Preparation of GlcNAc-modified liraglutide and WGA cross-linked nanoparticles:

[0057] In this embodiment of the invention, following the method of Example 1, the structurally similar GLP-1 analog liraglutide was modified with GlcNAc at non-critical functional sites and self-assembled into nanoparticles via wheat lectin (WGA) crosslinking, verifying that the method provided in this embodiment of the invention is also applicable to this drug molecule.

[0058] First, weigh 50 mg of liraglutide into 25 mL of sodium bicarbonate buffer (approximately 2 mg / mL), and dissolve it by magnetic stirring at room temperature for 10 min. Dissolve 30 mg of GlcNAc-NHS in 5 mL of acetonitrile, add it to the peptide solution, and gently stir at 4 °C for 4 h in the dark. After the reaction, dialyze the product in a dialysis bag for 24 h (changing the water every 4 h). Concentrate the dialyzed product to 2 mg / mL, slowly add WGA (peptide:WGA mass ratio of 1:1), allow it to stand at room temperature for cross-linking and self-assembly, centrifuge, collect the precipitate, wash twice with PBS, and resuspend for later use.

[0059] Particle size, PDI, and Zeta potential were determined by dynamic light scattering (DLS), and morphology was observed by TEM. The average particle size of the nanoparticles was 80.6 ± 5.4 nm, the PDI was 0.143 ± 0.012, and the Zeta potential was +18.9 ± 2.1 mV. TEM images are shown below. Figure 2 As shown;

[0060] The results in summary indicate that liraglutide can also be successfully introduced into GlcNAc at non-critical sites, and nanoparticles with a diameter of approximately 70-90 nm and uniform distribution can be obtained through WGA crosslinking.

[0061] Example 4: Evaluation of the cytotoxicity and biocompatibility of GlcNAc-Semaglutide-WGA nanoparticles:

[0062] This invention provides a safety basis for subsequent nasal-brain delivery and animal experiments by detecting the cell biocompatibility and cytotoxicity of the prepared GlcNAc-Semaglutide-WGA nanoparticles.

[0063] The nanoparticle sample was obtained from the optimal ratio in Example 2 (GlcNAc to smegglutinin molar ratio 2:1, modified peptide to WGA mass ratio 1:1), with a particle size of approximately 85 nm and a PDI < 0.2. RPMI 2650 (human nasal mucosal squamous epithelial cells), SH-SY5Y (human neuroblastoma cells), and BV2 (mouse microglia) were seeded into 96-well plates, 1 × 10⁶ cells per well. 4 Cells were cultured for 24 hours and then incubated with nanoparticle solutions of different concentrations (5, 10, 25, 50, 100, 200 μg / mL) for another 24 hours.

[0064] Five replicates were set up for each group, including a blank control group (culture medium only) and a positive toxicity control group (0.1% Triton X-100 added). After incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and the mixture was incubated at 37°C for 4 hours. Then, 150 μL of DMSO was added to dissolve the crystals, and the absorbance at 570 nm was measured using a microplate reader.

[0065] The results are as follows Figure 3 As shown, the cell viability of the three cell lines remained above 90% after treatment at concentrations of 50 μg / mL and below, with no obvious toxic reactions. At a concentration of 200 μg / mL, cell viability decreased slightly, but remained within an acceptable range. High concentrations may cause slight irritation. In addition, microscopic observation revealed that the cells in the low-dose treatment group were morphologically intact and adhered well to the cell wall, while some cells in the high-dose group showed slight detachment or morphological changes. In summary, the nanoparticles prepared in the embodiments of this invention have good biocompatibility with nasal mucosa and central nervous system-related cells within the effective concentration range.

[0066] Example 5: Evaluation of the adhesion of GlcNAc-Semaglutide-WGA nanoparticles to the nasal mucosa:

[0067] To simulate the adhesion of nanoparticles to the nasal mucus layer, quantitative determination was performed using the Transwell system and quantitative fluorescence method. First, the nanoparticle samples were divided into three groups: a fully modified group (GlcNAc-Semaglutide-WGA nanoparticles, with a molar ratio of GlcNAc to semaglutide of 2:1 and a mass ratio of modified peptide to WGA of 1:1), a single modified group (GlcNAc-Semaglutide nanoparticles, with a molar ratio of GlcNAc to semaglutide of 2:1), and a control group (unmodified semaglutide). Next, nanoparticle suspensions from each group were taken, and the nanoparticles were labeled with fluorescein isothiocyanate (FITC). The FITC-labeled nanoparticles were then diluted with simulated nasal fluid. Then, the simulated mucus layer was constructed by removing the well plate from the upper chamber of the Transwell system, uniformly coating the surface of the well membrane with a mucin solution, and incubating at room temperature to allow the mucin to bind to the membrane surface and form a simulated mucus layer. For each group, FITC-labeled nanoparticle suspension was added to the upper chamber, and HBSS was added to the lower chamber. The cells were incubated at 37°C for 0.5, 1, 2, and 4 hours. After incubation, the Transwell cells were carefully removed, the upper chamber liquid was discarded, and the cells were washed with PBS to remove non-specific adsorption. Triton X-100 lysis buffer was then added to the upper chamber to disrupt the upper chamber cell layer. The lysis buffer was collected, centrifuged, and the supernatant was used to measure the FITC intensity using a fluorescence microplate reader. The adhesion rate was calculated as: (upper chamber capture amount) / (initial addition amount) × 100%.

[0068] The results are as follows Figure 4 As shown, compared with the control group and the single-modification group, the fully modified group exhibited a significantly higher adhesion rate. The results indicate that GlcNAc modification and WGA crosslinking significantly improved the adhesion of nanoparticles to the nasal mucosa. By performing these modifications on smegglutinin, its affinity on the nasal mucosa can be enhanced, which may contribute to better drug delivery.

[0069] Example 6: Evaluation of the cell's ability to take up nanoparticles:

[0070] The nanoparticles were grouped the same as in Example 5, and RPMI 2650 cells were cultured at 5 × 10⁻⁶. 4 1 cell / well and 1×10 5 Cells were seeded at a density of 100 cells / well in 24-well plates for flow cytometry and confocal microscopy observation. Cells were treated with nanoparticle suspension and quantified using flow cytometry. At each time point, cells were gently washed with PBS to remove unbound particles, digested with trypsin, collected by centrifugation, and resuspended in 300 μL PBS. FITC fluorescence intensity was detected on the flow cytometer, and the mean fluorescence intensity (MFI) was recorded. MFI-time curves were plotted, and images of cell surface and intracellular distribution were acquired under a confocal microscope.

[0071] Flow cytometry MFI-time curve as shown Figure 6 As shown, cell uptake in the fully modified group increased continuously over time, reaching 2.1 times that of the single-modification group; confocal images are shown below. Figure 5 As shown, the fully modified nanoparticles were largely internalized into the cytoplasm, while the fluorescence signal in the control group was mostly distributed in the cell membrane. In summary, the results indicate that the binding of WGA to the cell surface GlcNAc / sialic acid receptor significantly enhances endocytosis efficiency, prolongs intracellular retention time, and facilitates subsequent transepithelial transport.

[0072] Example 7: Evaluation of the ability of nanoparticles to cross the nasal epithelial barrier:

[0073] Human nasal squamous cell line RPMI 2650 was used and cultured in EMEM supplemented with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 1% penicillin / streptomycin, and 10% fetal bovine serum. The cells were grouped as in Example 5 and FITC-labeled nanoparticles were used. RPMI 2650 cells were cultured at a rate of 1 × 10⁻⁶ cells / year. 5 Cells / cm² were seeded in the upper chamber of Transwell and cultured in liquid-liquid culture until the cells were confluent and formed a continuous monolayer. The culture medium was then replaced. Subsequently, the culture medium in the upper chamber was removed and the culture was changed to gas-liquid culture conditions. When the cells formed a tightly polarized monolayer, it could be used for experiments to evaluate the ability of cells to cross the epithelial barrier.

[0074] FITC-labeled nanoparticles were resuspended in HBSS and filtered. The nanoparticle suspension was added to the upper chamber, and HBSS was added to the lower chamber. The Transwell was incubated at 37°C with gentle shaking to simulate nasal microcirculation. Samples from the lower chamber were collected at 0.5, 1, 2, and 4 hours, and the fluorescence intensity was measured using a fluorescence spectrophotometer. The transmittance was calculated by referring to the standard curve, where: transmittance = (cumulative transmittance in the lower chamber) / (initial amount added in the upper chamber) × 100%.

[0075] The results are as follows Figure 7 As shown, the cumulative permeability of the fully modified group within 4 h was significantly higher than that of the single-modified group and the unmodified control group. This indicates that WGA crosslinking significantly enhances the specific binding of nanoparticles to nasal mucosal receptors and promotes receptor-mediated transcytosis.

[0076] Example 8: Evaluation of drug release characteristics of GlcNAc-Semaglutide-WGA nanoparticles in a simulated nasal environment:

[0077] This invention evaluates the drug release behavior of GlcNAc-modified and WGA-crosslinked nanoparticles in simulated nasal liquids, comparing their sustained-release or controlled-release potential.

[0078] First, the nanoparticles were grouped as in Example 5, and nasal discharge was simulated using PBS buffer (pH 6.5) and kept at 37°C.

[0079] In vitro release experiments were conducted using the dialysis bag method: First, each group of samples (2 mL) was placed into a dialysis bag to ensure that drug molecules could pass through the dialysis membrane. Then, the dialysis bag was placed in 50 mL of simulated nasal fluid, kept at a constant temperature of 37°C, and shaken at a constant speed (50 rpm). At time points of 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, 1 mL of the released solution was taken out, and an equal volume of simulated nasal fluid was added. Each time, 1 mL of sample was taken, and the concentration of released semaglutide was quantitatively analyzed.

[0080] The results are as follows Figure 8 As shown, the control group exhibited rapid release characteristics, while the single-modification group and the fully modified group exhibited sustained-release effects, with WGA cross-linking showing continuous drug release.

[0081] Example 9: Animal experiments to evaluate pharmacokinetics and brain distribution:

[0082] This invention systematically evaluates the distribution kinetics of drugs in peripheral blood and brain tissue after intranasal administration of different formulations, and verifies whether the constructed GlcNAc-modified and WGA-crosslinked semaglutide nanoformulation can improve the drug delivery efficiency in the brain.

[0083] Eight-week-old male C57 / BL6 mice were randomly divided into four groups (n=6): control group (administered with an equal volume of physiological saline), semaglutide group (unmodified semaglutide solution), single modification group (GlcNAc-Semaglutide nanoparticles only), and nanoformulation group (GlcNAc-Semaglutide-WGA nanoparticles). Mice were fixed in a supine position, and the formulation was slowly dripped into the nasal cavity in multiple doses using a micropipette. Peripheral blood and brain tissue samples were collected at 0.25h, 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h after the first administration.

[0084] Table 3 shows the comparison of pharmacokinetic parameters of mouse brain tissue and plasma after intranasal administration of each formulation:

[0085] Table 3

[0086]

[0087] Experimental results showed that the concentration of the nano-formulation group in brain tissue was significantly higher than that in other groups, while the concentration in plasma was relatively low, indicating good brain-targeting distribution ability. Compared with unmodified smegglutinin, GlcNAc modification can improve the initial exposure level of the molecule in brain tissue, and after further assembly into nanoparticles by WGA crosslinking, it significantly improves the retention time and accumulation of the drug in brain tissue.

[0088] Example 10: Evaluation of the effect of nano-formulation on weight intervention in high-fat diet-induced obese mice:

[0089] This invention evaluates the effect of GlcNAc-Semaglutide-WGA nanoparticles on weight control. Similar to Example 9, 8-week-old male C57BL / 6 mice were used. All animals were acclimatized for 7 days and then uniformly fed a high-fat diet for 4 weeks to induce an obesity model. As in Example 9, the mice were divided into 4 groups (n=6): control group (administered with an equal volume of saline), semaglutide group (unmodified semaglutide solution), single-modification group (GlcNAc-Semaglutide nanoparticles only, GlcNAc to semaglutide molar ratio 2:1), and nanoformulation group (GlcNAc-Semaglutide-WGA nanoparticles, GlcNAc to semaglutide molar ratio 2:1, modified peptide to WGA mass ratio 1:1). All treatments and measurements were performed at 19:00. The mice were administered 10 µL of the formulation via nasal drip daily for 6 consecutive weeks. During the experiment, the mice were weighed at a fixed time each week, and the weight change curve was recorded. Each mouse was housed individually, and a fixed amount of food was given each day. The amount of food remaining was recorded the following morning. The daily food intake and energy intake of the mice were calculated, as shown in Table 4.

[0090] Table 4

[0091]

[0092] The trends of weight change in each group of mice are as follows: Figure 9 As shown, compared with the control group, the weight gain of the nano-formulation group slowed down significantly after the second week and the weight showed a downward trend after the fourth week; while the single modification group and the smegglutinin group both showed that the weight was maintained or slightly increased. The average daily energy intake of the nano-formulation group was also significantly lower than that of other groups. It can be seen that the delivery system provided by the embodiments of the present invention not only improves the efficiency of drug efficacy, but may also suppress appetite through the central mechanism, thereby achieving a dual intervention effect.

[0093] In summary, the GlcNAc-Semaglutide-WGA nanoparticles prepared in the embodiments of the present invention have a moderate particle size and are positively charged. They not only exhibit significant adhesion ability in a simulated nasal mucosa environment, but also have efficient receptor-mediated endocytosis and transepithelial transport characteristics. Nasal administration can significantly improve the brain delivery efficiency of peptides, thereby more effectively reducing body weight, and providing a novel drug delivery strategy for the treatment of obesity and metabolic diseases.

[0094] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a nasal-administered smegglutide formulation, characterized in that, Includes the following steps: N-acetylglucosamine was coupled with lysine residues of smegglutinin via an activated ester method to obtain glycosylated smegglutinin. Glycosylated smegglutinin was cross-linked with wheat lectin and formed uniform nanoparticles with a diameter of 70-90 nm through self-assembly. The step of coupling the N-acetylglucosamine with the lysine residues of smegglutinin via an activated ester method to obtain glycosylated smegglutinin specifically includes: Smegglutinin powder was dissolved in sodium bicarbonate buffer, and N-acetyl-D-glucosamine-NHS ester GlcNAc-NHS was dissolved in DMF; Under ice bath conditions, GlcNAc-NHS was added to a solution containing smegglutinin, and the reaction was carried out with gentle stirring at pH 7.8-8.6 and 0-10 °C. After the reaction was complete, the pH was adjusted to neutral to stop the reaction. The product was then dialyzed and freeze-dried to obtain glycosylated semaglutide with the N-acetylglucosamine modification site being the Lys group of semaglutide. 26 and Lys 34 site; The step of cross-linking glycosylated smegglutinin with wheat lectin to form uniform nanoparticles with a diameter of 70-90 nm through self-assembly specifically includes: Wheat lectin (WGA) was dissolved in PBS, and EDC·HCl and NHS were added. The mixture was stirred at room temperature to obtain the WGA-NHS intermediate. Glycosylated smegglutinin was dissolved in PBS, mixed with activated WGA-NHS intermediate, and stirred to promote self-assembly, forming uniform nanoparticles with a diameter of 70-90 nm.

2. The method for preparing the nasal-administered smegglutide formulation according to claim 1, characterized in that, The nanoparticles were collected by centrifugation at 4°C at a speed of 10,000-20,000 g for 10-30 min, and then washed 2-3 times with PBS.

3. The method for preparing the nasal-administered smegglutide formulation according to claim 1, characterized in that, The mass ratio of the glycosylated smegglutinin to wheat lectin is 1:0.5-4.

4. The preparation method according to claim 1, characterized in that, The molar ratio of EDC·HCl to NHS is 1:0.8-1.2, and the activation reaction time is 0.5-2 h.

5. A nasal administration formulation of smegglutide, characterized in that, It is prepared using the preparation method described in any one of claims 1-4.

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