Polypeptide compound and application thereof in resisting obesity

Abstract

The invention discloses a polypeptide compound. The polypeptide compound is formed by covalently connecting small-molecule polypeptide with an amino acid sequence of Cys-Ser-Phe with gold nanoparticles through sulfydryl of cysteine residues, the polypeptide compound disclosed by the invention can be used for remarkably improving the stability and bioavailability of peptide medicines, effectively inhibiting appetite, reducing food intake, reducing body weight and improving glycometabolism and insulin sensitivity. In a diet-induced obesity (DIO) mouse model, the polypeptide compound shows an excellent anti-obesity effect, fat accumulation can be remarkably reduced, abnormal glucose tolerance of a mouse induced by high fat diet can be improved, the tolerance of an organism to glucose and insulin sensitivity are enhanced, and the polypeptide compound is simple and convenient in preparation process, suitable for industrial production and suitable for industrial production. The method can be widely applied to development and preparation of anti-obesity special medical foods, health care products and medicines.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanobiotechnology, specifically to a polypeptide complex modified with gold nanoparticles and its application in the prevention and / or treatment of obesity and related metabolic diseases. Background Technology

[0002] Obesity is closely associated with a variety of metabolic diseases, especially type 2 diabetes, cardiovascular disease, fatty liver, and various malignant tumors, seriously endangering human health. Currently available anti-obesity drugs, such as semaglutide, tirzepatide, and retatrutide, while having some efficacy, generally have side effects such as nausea, vomiting, pancreatitis, the risk of thyroid C-cell tumors, and internal bleeding, limiting their long-term use.

[0003] Recent studies have found that exercise can induce the production of an endogenous dipeptide called Lac-Phe (N-lactyl-phenylalanine). This metabolite can effectively suppress appetite, reduce food intake, and decrease weight, providing a new approach for anti-obesity treatment. However, as a polypeptide, Lac-Phe suffers from poor stability, a short half-life, easy enzymatic degradation, and low bioavailability in vivo, which severely limits its clinical application.

[0004] The development of nanotechnology has provided an effective strategy for improving the delivery efficiency and stability of peptide drugs. Among them, gold nanoparticles (AuNPs) are widely regarded as ideal drug carriers due to their excellent biocompatibility, ease of synthesis, flexible surface functionalization modification, and stable physicochemical properties, which can significantly improve the solubility, stability, and bioavailability of loaded drugs.

[0005] Therefore, developing a novel anti-obesity peptide delivery system based on nanocarrier technology is of great research value and application potential in overcoming the side effects of existing drugs and the inherent limitations of natural bioactive peptides. Summary of the Invention

[0006] This invention provides a polypeptide complex based on modified gold nanoparticles, which is composed of a tripeptide with the amino acid sequence Cys-Ser-Phe (CSF) covalently linked to gold nanoparticles through the thiol groups of cysteine ​​residues.

[0007] Another objective of this invention is to apply peptide complexes in the preparation of drugs for the prevention and / or treatment of obesity. These peptide complexes can significantly enhance the stability and bioavailability of peptides, effectively suppress food intake, reduce weight, and improve glucose and lipid metabolism disorders associated with obesity.

[0008] The objective of this invention is achieved through the following solution: 1. Using the natural anti-obesity dipeptide Lac-Phe (lactic acid-phenylalanine) as the parent compound, the lactic acid group in its molecular structure was replaced with serine (Ser), and cysteine ​​(Cys) was linked to the N-terminus to design a tripeptide with the amino acid sequence Cys-Ser-Phe. 2. Dissolve the small molecule peptide in DMF, then add N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), mix well, and then add chloroauric acid solution dropwise to the above mixture. React at 20-30℃ for more than 24 hours. After dialysis of the reaction product, the peptide complex is obtained. The molar ratio of small molecule peptides to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid is 1:400-600, and the molar ratio of small molecule peptides to chloroauric acid is 1:1-3. 3. The polypeptide complex was injected intraperitoneally into DIO (diet-induced obesity) mice for one week. The food intake and body weight of the DIO mice were observed daily, and various obesity-related indicators of the DIO mice before and after treatment were further tested. The polypeptide complex of this invention can significantly improve the stability and bioavailability of peptide drugs, effectively suppress appetite, reduce food intake, lower body weight, and improve glucose metabolism and insulin sensitivity. In a diet-induced obesity (DIO) mouse model, the polypeptide complex exhibits excellent anti-obesity effects, significantly reducing fat accumulation, improving glucose tolerance abnormalities induced by a high-fat diet, and enhancing the body's glucose tolerance and insulin sensitivity. The polypeptide complex of this invention has a simple preparation process, is suitable for industrial production, and can be widely used in the development and preparation of anti-obesity medical foods, health products, and drugs. Attached Figure Description

[0009] Figure 1 These are the characteristic absorption peaks produced by a UV spectrophotometer on the dialyzed polypeptide complex solution. Figure 2 This is a morphology image of the polypeptide complex under TEM. Figure 3 The results are from X-ray photoelectron spectroscopy (XPS). Figure 4 The results show the food intake of each experimental group within 12 hours on the first day of injection; Figure 5 The results show the daily weight changes of each experimental group during the 9 consecutive days of injection. Figure 6 Micro-CT scans of abdominal fat in mice from each experimental group; Figure 7 The results of the intraperitoneal glucose tolerance test (IPGTT) for mice in each experimental group are shown in the left figure, which is the glucose tolerance curve; the right figure is the statistical results of the area under the glucose tolerance curve. Figure 8 The left figure shows the insulin sensitivity (ITT) test results of mice one week after injection of the nano-gold drug. The right figure shows the blood glucose curve and the quantitative results of the blood glucose curve. Detailed Implementation

[0010] The technical solution of the present invention will be further described in detail below through embodiments, but the content of the present invention is not limited thereto. Unless otherwise specified, the methods in this embodiment are conventional methods, and the materials and reagents used are obtained commercially unless otherwise specified.

[0011] Example 1: Design and Synthesis of Small Molecule Peptide CSF Using the natural anti-obesity dipeptide Lac-Phe (lactic acid-phenylalanine) as the parent compound, the lactic acid group in its molecular structure was replaced with serine (Ser), and cysteine ​​(Cys) was linked to the N-terminus. The tripeptide (CSF) with the amino acid sequence Cys-Ser-Phe was synthesized by Jier Biochemical (Shanghai) Co., Ltd. The thiol group (-SH) of cysteine ​​is used to covalently bind to gold nanoparticles, and the hydroxyl group (-OH) of serine retains the active site of Lac-Phe, ensuring the appetite-suppressing function.

[0012] Example 2: Preparation and Characterization of Peptide Complexes The tripeptide CSF (0.25 mM) was dissolved in DMF (50 µL), and then HEPES (950 µL, 100 mM, pH 7.5) was added. HAuCl4·3H2O solution was slowly added dropwise to the peptide solution until the final concentration of HAuCl4 was 0.5 mM. The reaction was carried out at 25 °C with continuous stirring for 8 days. The reaction product was separated and purified by dialysis using a dialysis bag (15000 Da). The dialysis product was freeze-dried to obtain the polypeptide complex (Au-CSF). The reaction products on days 1, 3, 5, 6, 7, and 8 of the reaction were scanned using a WPA Biowave II UV-Vis spectrophotometer within the wavelength range of 300-800 nm, and the positions of the surface plasmon resonance (SPR) peaks were recorded. The results are shown in [Figure number missing]. Figure 1 The SPR peak of the complex was located near 532 nm and increased with increasing reaction time, which is consistent with the positive correlation between the particle size of gold nanoparticles and the position of the SPR peak, proving that the peptide complex was successfully prepared.

[0013] The particle size of the polypeptide complex was observed using transmission electron microscopy (TEM), and the results are shown in the figure. Figure 2 The polypeptide complex is a uniform spherical shape with a particle size of about 15 nm.

[0014] The linkage status of the peptide complex was detected by X-ray photoelectron spectroscopy (XPS), and the results are shown in [Figure number missing]. Figure 3The presence of Au-S bonds in the figure confirms the covalent connection between the gold nanoparticles and the thiol groups in the peptide.

[0015] Example 3: Detection of the anti-obesity activity of the polypeptide complex 1. DIO mouse modeling and experimental grouping Five-week-old male C57BL / 6J mice (purchased from Spiford (Beijing) Biotechnology Co., Ltd.) were selected and acclimatized for one week. They were then fed a high-fat diet containing 60% fat for 4-6 weeks to establish a diet-induced obesity (DIO) mouse model. After successful model establishment, the mice were randomly divided into four groups according to the following groupings: model group (Vehicle group, injected with saline), positive control group (intraperitoneal injection of smegglutinide, 0.25 mg / kg), CSF group (intraperitoneal injection of Cys-Ser-Phe, 50 mg / kg), Lac-Phe group (intraperitoneal injection of Lac-Phe, 50 mg / kg), and Au-CSF complex group (intraperitoneal injection of polypeptide complex, 5 mg / kg). Injections were administered once daily at 19:00 for 9 consecutive days. 2. Food intake and body weight On the first day after injection, the hourly food intake of each experimental group was observed during the 12-hour nighttime period. Figure 4 ), and recorded the mice's daily weight over 9 days ( Figure 5 ); from Figure 4 The results show that the food consumption of mice in the Vehicle group increased continuously with the extension of time after injection, reaching a high level at 12 hours, indicating that the control group mice ate normally and their food intake increased over time. The food consumption of mice in the Lac-Phe group also increased over time after injection, but the increase was slightly lower than that in the Vehicle group, indicating that Lac-Phe had a certain inhibitory effect on the appetite of mice, but it was not significant. The food intake of mice in the CSF group was similar to that in the Lac-Phe group, indicating that CSF and Lac-Phe had similar inhibitory effects on the appetite of mice. The food consumption of mice in the Au-CSF complex group increased to some extent after injection, but the increase was smaller than that in the Vehicle group, and it tended to stabilize in the later stage without increasing significantly. This corresponds to the result that the weight of mice in this group decreased to some extent, indicating that the Au-CSF complex inhibited the appetite of mice or affected the amount of food intake to some extent. The food consumption of mice in the Semaglutide group remained at a very low level throughout the observation period, with almost no significant food intake.

[0016] from Figure 5The weight results showed that the vehicle group mice maintained a relatively stable weight change, with no significant increase or decrease, indicating that the saline solution itself had no effect on mouse weight, thus eliminating interference from solvent factors during the experiment. The semaglutide group mice showed a continuous and significant decrease in weight, gradually decreasing by 6g from an initial change close to zero. This suggests that semaglutide effectively promotes weight loss in mice, possibly related to its mechanisms of regulating metabolism and suppressing appetite. The Au-CSF complex group mice showed a slow decrease in weight, reducing by approximately 2g, indicating that a 5mg / kg dose of Au-CSF complex has a weight-reducing effect, but the effect is weaker than that of the semaglutide group.

[0017] 3. Fat content and tissue morphology Nine days after injection, the abdominal fat weight of mice was measured using Micro-CT. The results are shown below. Figure 6 In the Vehicle group, there was no significant change in fat mass before and after injection of saline. However, in the Au-CSF complex group and the Semaglutide group, fat mass decreased significantly after administration, indicating that the Au-CSF complex has a good anti-obesity effect. Fat mass statistics are shown in the table below: ; 4. Glucose metabolism and insulin sensitivity testing Nine days after injection, an intraperitoneal glucose tolerance test (IPGTT) was performed. The experiment was divided into three groups: a normal diet group (ND group), a high-fat diet group (HFD group), and an Au-CSF treatment group (HFD+Au-CSF group). After removing the mice's food and giving them water overnight, the mice were intraperitoneally injected with a 50% high-glucose solution at a dose of 2 mg / g. Blood glucose levels in the tail vein of the mice were measured using blood glucose test strips at 0, 30, 60, 90, and 120 minutes after injection.

[0018] See results Figure 7 As shown in the figure, the blood glucose levels in the normal diet (ND) group rose and fell relatively steadily; the blood glucose levels in the high-fat diet (HFD) group rose sharply and reached a high peak during the experiment, followed by a slow decline; while the high-fat diet combined with the gold nanoparticle complex (HFD+Au-CSF) group showed higher blood glucose changes than the ND group, the rise was smaller and the decline was more rapid compared to the HFD group. In the area under the glucose tolerance curve (right figure), the AUC of the HFD group was significantly higher than that of the ND group, indicating that the high-fat diet led to impaired glucose tolerance in mice; the AUC of the HFD+Au-CSF group was significantly lower than that of the HFD group, indicating that Au-CSF intervention can improve high-fat diet-induced glucose intolerance in mice and enhance the body's tolerance to glucose.

[0019] Simultaneously, an intraperitoneal insulin sensitivity test (ITT) was conducted, divided into three groups: a normal diet group (ND group), a high-fat diet group (HFD group), and an Au-CSF treatment group (HFD+Au-CSF group). Mice were deprived of their food and fasted for approximately 4-5 hours before being intraperitoneally injected with a calculated dose of Novolin insulin solution (1.5 U / kg). Blood glucose levels in the tail vein were measured using blood glucose test strips at 0, 30, 60, 90, and 120 minutes post-injection.

[0020] See results Figure 8 As shown in the figure, the ND group (normal diet group) had relatively stable and low blood glucose levels, indicating that under normal dietary conditions, the body's insulin sensitivity was normal, and insulin could effectively regulate blood glucose within the normal range. The HFD group (high-fat diet group) had higher initial blood glucose levels, and these levels remained relatively high for a longer period. This is because a long-term high-fat diet leads to insulin resistance, i.e., reduced insulin sensitivity. Insulin resistance decreases the efficiency with which insulin promotes cellular glucose uptake and utilization, resulting in inefficient glucose utilization and persistently high blood glucose levels. The Au-CSF treatment group showed a significant reduction in blood glucose levels compared to the HFD group, indicating that Au-CSF intervention improved the body's insulin sensitivity.

Claims

1. A polypeptide complex, characterized in that: It is composed of a small molecule polypeptide with the amino acid sequence Cys-Ser-Phe, which is covalently linked to gold nanoparticles through the thiol group of cysteine ​​residues.

2. The polypeptide complex according to claim 1, characterized in that: The polypeptide complex is prepared by dissolving a small molecule polypeptide in DMF, adding N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, mixing well, adding chloroauric acid solution dropwise to the mixture, reacting at 20-30°C for 1-7 days, and dialysis of the reaction product.

3. The polypeptide complex according to claim 2, characterized in that: The molar ratio of the small molecule peptide to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid is 1:400-600, and the molar ratio of the small molecule peptide to chloroauric acid is 1:1-3.

4. The use of the polypeptide complex of claim 1 in the preparation of a drug for the prevention and / or treatment of obesity.

Images