Targeted self-driven nanomotor hydrogel and preparation method and application thereof

Abstract

The application provides a kind of targeted self-driven nanomotor hydrogel and its preparation method and application, belong to the field of biological medicine. Including the following steps: Janus mesoporous silica platinum nanomotor is synthesized by Pickering emulsion method, first loaded liraglutide by amination reaction, then grafted rapeseed DPP-IV inhibiting peptide by amide method, and encapsulated in sodium alginate microspheres, to obtain self-driven nanomotor hydrogel. The self-driven nanomotor described in the application can actively overcome the intestinal mucus barrier, pass through the mucus barrier into the specific site by autonomous navigation, significantly enhance the drug efficiency, avoid degradation in harsh stomach environment, reverse insulin resistance, reduce inflammatory complications such as diabetes, and enhance the safety and bioavailability of drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a targeted self-driven nanomotor hydrogel, its preparation method, and its application. Background Technology

[0002] Currently, diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia. Its pathogenesis mainly involves insulin resistance and insulin secretion defects. Its incidence is rising globally, posing a serious threat to human health. Currently, the main treatment for type 2 diabetes includes injectable exogenous drugs, which are rapid and reliable. However, long-term subcutaneous injections can cause safety problems such as hypoglycemia, and even be fatal. In contrast, oral medications offer better adherence and convenience, greatly improving the quality of life for diabetic patients. However, oral medications such as metformin and sulfonylureas are limited due to their side effects and eventual drug resistance. Therefore, choosing naturally derived active substances as an alternative treatment strategy is essential. In recent years, food protein peptides with anti-diabetic properties have also received increasing attention from researchers.

[0003] Rapeseed, the world's second-largest oilseed producer, possesses immense nutritional value. Rapeseed protein, extracted from rapeseed, is considered a suitable source of dietary protein due to its excellent amino acid balance and high bioavailability. The potential DPP-IV enzyme inhibitory peptide in rapeseed protein is a key component in the treatment of type 2 diabetes (T2D), with few side effects. It improves insulin levels by delaying GLP-1 degradation, resulting in more stable efficacy and a longer duration of action. Unfortunately, oral administration faces the extreme gastrointestinal environment, such as gastric acid and enzyme degradation, leading to poor absorption and hindering the bioavailability and therapeutic effect of oral drugs. While sodium alginate hydrogel microspheres can effectively control the specific release of nanoparticles at the target site, the ability of passive nanoparticles to penetrate the intestinal barrier remains limited. To improve the permeability of the mucus barrier, nanomotors, as an emerging drug carrier, can convert energy from the local disease environment or external sources into mechanical energy, thereby achieving self-propelled movement and penetrating the physiological barrier to reach the target site through autonomous motion.

[0004] Currently, various technologies have been developed to activate nanomotors, including the application of external light and magnetic fields. However, these physical stimuli may have adverse effects on organs. Therefore, utilizing platinum (Pt)-associated nanozymes as catalysts to activate existing fuels in a biological environment to drive nanomotors to achieve autonomous movement, thereby inhibiting and clearing ROS, is crucial for the treatment of type 2 diabetes (T2D). Summary of the Invention

[0005] The purpose of this invention is to provide an oral delivery system that can be loaded with edible rapeseed DPP-IV inhibitory peptide and enteropancreatin-like peptide (GLP-1) with good biosafety and high bioavailability.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a targeted, self-driven oral nanomotor hydrogel, comprising the following steps: (1) Mesoporous silica nanoparticles were dispersed in an aqueous solution containing short-chain alcohols and cationic surfactants, heated, and solid wax was added. Pickering emulsion was obtained by shearing. (2) Cool the emulsion, add a silane coupling agent solution containing thiol, filter, wash, and further disperse in a platinum nanoparticle colloidal solution for adsorption; (3) After adsorption, the solid was filtered and resuspended in ethanol to remove the solid wax. The solid was then centrifuged and the precipitate was washed with ethanol and chloroform in sequence to obtain Janus mesoporous silica platinum nanomotors. (4) First, liraglutide was loaded onto Janus mesoporous silica platinum nanomotors using an amination reaction, and then rapeseed DPP-IV inhibitory peptide was grafted onto them by an amide method to obtain nanomotors loaded with active peptides.

[0007] Furthermore, in step (1), the ratio of the mesoporous silica nanoparticles, the aqueous solution containing short-chain alcohols and cationic surfactants, and the solid wax is 160~200 mg: 8~12 mL: 1 g; and the heating temperature is 65~85℃.

[0008] Furthermore, in step (1), the volume fraction of the short-chain alcohol in the aqueous solution containing the short-chain alcohol and the cationic surfactant is 6.0~7.0%; and the content of the cationic surfactant is 0.8~1.2 μM.

[0009] Furthermore, the cutting conditions in step (1) are: 20000~30000 rpm, 10~15 min.

[0010] Furthermore, in step (2), the ratio of the mercapto-containing silane coupling agent solution to the mesoporous silica nanoparticles is 180~220 μL: 150~200 mg; The ratio of the platinum nanoparticle colloidal solution to the mesoporous silica nanoparticles is 25~35 mL: 150~200 mg.

[0011] Further, the method for loading liraglutide in step (4) is as follows: prepare Janus MSN-Pt toluene solution, add APTES, react for 20-25 h, centrifuge, wash the precipitate, dry to obtain Janus MSN-Pt–NH2, disperse it in water, add liraglutide Lira solution to react, centrifuge and wash, freeze dry to obtain Janus MSN-Pt@Lira; The ratio of toluene solution, Janus MSN-Pt, and APTES was 18-22 mL: 55-65 mg: 1-3 mL. The ratio of Janus MSN-Pt–NH2, water, and liraglutide Lira solution is: 2~6 mg: 2~6 mL: 1 mL; The concentration of liraglutide is 0.8~1.2 mg / mL.

[0012] Further, step (4) involves grafting rapeseed DPP-IV inhibitory peptides using the amide method as follows: after loading liraglutide, 2-(N-morpholine) ethanesulfonic acid solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added sequentially, stirred at 2~5℃ for 60 min, and finally rapeseed DPP-IV inhibitory peptides are added, stirred for 3~5 h, centrifuged and washed to obtain nanomotors loaded with active peptides; The ratio of the liraglutide-loaded nanomotor to 2-(N-morpholine) ethanesulfonic acid solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and rapeseed DPP-IV inhibitory peptide was 5 mg: 4-6 mL: 5-9 mg: 1-3 mg: 4-6 mg.

[0013] Furthermore, step (5) involves encapsulating the nanomotor loaded with active peptides in sodium alginate microspheres using a microfluidic method to obtain the self-driven nanomotor hydrogel.

[0014] This invention provides a targeted, self-driven oral nanomotor hydrogel.

[0015] The present invention also provides the use of the oral nanomotor hydrogel in the preparation of drugs that promote insulin release or are used to treat diabetes.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: 1. In this invention, the nanomotor generates oxygen bubbles by consuming local reactive oxygen species. These bubbles serve as the driving force for the autonomous movement of the nanomotor, enabling it to navigate autonomously through the mucus barrier and enter specific locations to exert its function.

[0017] 2. The nanomotor of the present invention is loaded with natural rapeseed DPP-IV inhibitory peptide, which can reduce oxidative stress, reverse insulin resistance, alleviate inflammatory complications such as diabetes, and enhance drug safety and bioavailability.

[0018] 3. The self-driven nanomotor hydrogel loaded with active peptides of the present invention can actively overcome the intestinal mucus barrier, significantly enhance drug delivery efficacy, and avoid degradation by the harsh gastric environment, thereby reaching the target site to exert its effect. Attached Figure Description

[0019] Figure 1 TEM images of self-driven nanomotors loaded with rapeseed peptides and different carriers (a: MSNs; b: Janus MSN-Pt; c: Janus MSN-Pt@Lira; d: Janus MSN-Pt@Lira / IP).

[0020] Figure 2 Mapping diagrams of different elements in self-driven nanomotors and nanomotors loaded with rapeseed peptides (a: JanusMSN-Pt; b: Si; c: O; d: Pt; e: Janus MSN-Pt@Lira / IP; f: Si; g: O; h: Pt; i: C; g: N; k: S; l: P).

[0021] Figure 3 (a) Zeta potential diagram and (b) particle size distribution diagram of self-driven nanomotors loaded with rapeseed peptides at different preparation stages.

[0022] Figure 4 Particle size variation of self-driven nanomotor loaded with rapeseed peptides in different media over 48 h.

[0023] Figure 5 Fourier transform infrared spectra of self-driven nanomotors loaded with rapeseed peptides at different preparation stages.

[0024] Figure 6 The catalase-like activity of self-driven nanomotors: (a) hydrogen peroxide scavenging capacity and (b) oxygen generation capacity.

[0025] Figure 7 In vitro motion behavior of self-driven nanomotors: (a) Trajectory diagram of nanomotors moving under different concentrations of H2O2; (b) Diffusion coefficient diagram of nanomotors moving under different concentrations of H2O2; (c) Mean square displacement diagram of nanomotors moving under different concentrations of H2O2; and (d) Motion velocity diagram of nanomotors moving under different concentrations of H2O2.

[0026] Figure 8Biosafety evaluation of self-driven nanomotors loaded with rapeseed peptides: Effects of different concentrations of the motor on the survival rate of Ins-1(a) and Caco-2(b) cells.

[0027] Figure 9 : Intracellular reactive oxygen species scavenging ability of self-driven nanomotors, 20μm.

[0028] Figure 10 Digestion of self-driven nanomotor sodium alginate hydrogel in in vitro gastrointestinal fluids at different pH values.

[0029] Figure 11 Analysis of in vivo and in vitro DPP-IV enzyme, GLP-1 and insulin content in rapeseed peptide-loaded self-driven nanomotor. Detailed Implementation

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the examples are conventional techniques well known to those skilled in the art, and the reagents used are commercially available.

[0031] Among them, hexadecyltrimethylammonium bromide, tetraethyl orthosilicate, sodium hydroxide, chloroplatinic acid hexahydrate, polyvinylpyrrolidone, ascorbic acid, 3-mercaptopropyltrimethoxysilane, and anhydrous ethanol (all analytical grade) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; paraffin and chloroform were purchased from Sinopharm Group Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; liraglutide was purchased from MCE Corporation, USA; and the active peptide was synthesized by Nanjing Jietai Co., Ltd.

[0032] Caco-2 cells were purchased from the Shanghai Cell Bank; Ins-1 cells from Shanghai Jinyuan Biotechnology Co., Ltd.; sterile phosphate-buffered saline (PBS), 0.25% trypsin, and 1% penicillin-streptomycin were purchased from Gibco, Inc., USA; fetal bovine serum (FBS) was purchased from Suzhou Yikesai Biotechnology Co., Ltd.; CCK-8 assay kit, live / dead cell assay kit, DCFH-DA fluorescence assay kit, and JC-1 fluorescent probe kit were purchased from Beyotime Biotechnology Co., Ltd., Shanghai; hydrogen peroxide assay kit was purchased from Nanjing Jiancheng Biotechnology Institute; dimethyl sulfoxide (DMSO) cell culture grade and sterile glucose solution were purchased from Beijing Solarbio Biotechnology Co., Ltd.; Cy5 dye was purchased from Shanghai Pengshuo Biotechnology Co., Ltd.

[0033] DPP-4 enzyme (electrophoretic purity > 95%, specific enzyme activity ≥ 200 units / mg protein) was purchased from ProSpec-Tany Ltd., Israel; glycyl-prolyl-p-nitroaniline hydrochloride (Gly-Pro-pNA), Tris-HCl buffer, and acetate-sodium acetate buffer were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; sitagliptin was purchased from Shanghai Maclean Biotechnology Co., Ltd.; GLP-1 and insulin enzyme-linked immunosorbent assay (ELISA) kits were purchased from Wuhan Yilairuit Biotechnology Co., Ltd.; human recombinant insulin and benzyl sulfonyl fluoride (PMSF) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) cell culture grade and sterile glucose solution were purchased from Beijing Solarbio Biotechnology Co., Ltd.

[0034] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1: Fabrication of a self-driven nanomotor

[0036] (1) The preparation of mesoporous silica nanocarriers includes the following steps: 0.5 g of hexadecyltrimethylammonium bromide (CTAB), 240 mL of deionized water, and 48 mL of anhydrous ethanol were placed in a beaker, and 2 mL of NaOH (1 mol / L) was added. The mixture was then dispersed evenly by sonication. The mixture was then stirred at 37 °C for 30 min. The temperature was raised to 70 °C, and 3 mL of TEOS was added. The reaction was allowed to proceed for 3 h. After the reaction was complete, the resulting solution was allowed to stand at room temperature and then cooled to room temperature. The solution was centrifuged and washed. After washing, the solution was vacuum dried at 100 °C for 6 h, ground, and passed through a 200-mesh sieve to obtain a white product. Finally, the white sample was calcined in a muffle furnace at 550 °C for 6 h at a heating rate of 1.5 °C / min to obtain mesoporous silica nanocarriers (MSN).

[0037] (2) The preparation of platinum nanoparticles includes the following steps: 164 mg of chloroplatinic acid hexahydrate (H₂PtCl₆) and 20 mg of polyvinylpyrrolidone (PVP) were dissolved in 20 mL of deionized water. Then, 10 mL of ascorbic acid aqueous solution (35 mg / mL) was added dropwise to the H₂PtCl₆ solution. The reaction was carried out at 45 °C for 6 h with magnetic stirring. Platinum nanoparticles were successfully synthesized when the solution changed from an initial pale yellow to a black color.

[0038] (3) The fabrication of a self-driven nanomotor includes the following steps: 180 mg of mesoporous silica nanoparticles were uniformly dispersed in an aqueous solution of CTAB (10 mL, 1.0 μM) containing 6.7% ethanol. After heating at 75 °C for a period of time, 1 g of paraffin was added. Once the paraffin melted, the mixture was vigorously stirred at 25,000 rpm for 10 min using a homogenizer. The resulting emulsion was then further heated in a 75 °C water bath for 1 h. The Pickering emulsion was then cooled to room temperature, mixed with 10 mL of methanol solution, and treated with 200 μL of 3-mercaptopropyltrimethoxysilane. After 3 hours of magnetic stirring, the silanized emulsion was filtered, washed three times with methanol, and further dispersed in 30 mL of platinum nanoparticles. The mixture was stirred overnight, then filtered and washed. The solid was suspended in ethanol, centrifuged, and washed three times each with ethanol and chloroform to obtain the Janus mesoporous silica platinum nanomotor (Janus MSN-Pt).

[0039] (4) Preparation of liraglutide loaded with a self-driven nanomotor: 60 mg of JanusMSN-Pt was added to 20 mL of toluene solution and stirred for 1 h. 2 mL of APTES was added and reacted at room temperature for 24 h. After centrifugation, the product was washed three times with ethanol and dried at 60 °C to obtain the aminated product Janus MSN-Pt–NH2. Subsequently, 4 mg of the aminated product was dispersed in 4 mL of water, and 1 mL of liraglutide Lira solution (1 mg / mL) was added. The mixture was reacted at 1000 rpm for 24 h at room temperature, centrifuged, washed, and freeze-dried. 1 mL of the original (1 mg / mL) Lira solution was mixed with 4 mL of Janus MSN-Pt suspension (1 mg / mL) and reacted at 4 °C for 24 h to obtain Janus MSN-Pt@Lira.

[0040] (5) Preparation of self-driven nanomotor loaded with liraglutide and rapeseed peptide: A solution of nanomotor loaded with liraglutide (5 mg) was introduced into MES solution (5 mL, pH=5.5). Subsequently, 7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 mg of N-hydroxysuccinimide (NHS) were added. The solution was stirred at 4 °C for 60 min, and then 5 mg of rapeseed DPP-IV inhibitory peptide IPQVS was added to the mixture. The mixture was stirred for 4 h, and finally the nanomotor loaded with active peptide (Janus MSN-Pt@Lira / IP) was obtained by centrifugation and washing.

[0041] Example 2 Characterization of self-driven nanomotors loaded with rapeseed peptides and liraglutide

[0042] Transmission electron microscopy image of the self-driven nanomotor prepared in Example 1 is shown below. Figure 1As shown. Electron microscopy results show that the self-driven nanomotor synthesized in this invention has a significantly asymmetric structure, which is characteristic of Janus nanomotors (Janus nanomotors). Figure 1 Pt particles are distributed on one side of the nanomotor. After loading liraglutide and rapeseed DPP-IV inhibitory peptide, significant changes occurred in the pore structure, indicating that the drugs were successfully loaded onto the mesoporous silica support.

[0043] Mapping results show ( Figure 2 In addition to Si, O, and Pt elements in the asymmetric structure, common elements of peptide drugs such as C, N, and S were also detected in the drug-loaded nanomotors, indicating the successful synthesis of the Janus structure and the successful loading of the peptide drug.

[0044] The particle size and potential distribution of the self-driven nanomotor prepared in Example 1 were determined by scanning electron microscopy and dynamic light scattering, as follows: Figure 3 As shown: the change in Zeta potential confirms the successful modification at each step ( Figure 3 a). After thiol modification, the surface charge changed to -19.5 mV, and the potential became more negative, indicating successful loading of platinum particles. Amino modification led to an increase in surface charge from -19.5 mV to +27.5 mV, further proving the successful amino functionalization. After drug loading, the potential became negative again, and the gradually changing zeta potential further proved the synthesis of the nanomotor. As shown in the figure, the particle size of different nanomaterials is in the range of 100-180 nm. Each step of modification led to a continuous increase in the particle size of the nanomotor. Figure 3 (b) indicates successful drug loading.

[0045] The kinetic diameter variation in different solvents (water, PBS, and RPMI-1640) was measured using DLS. Figure 4 The results showed that the particle size of the nanomotors remained stable within 48 h in water, PBS, and RPMI-1640 media, indicating that the nanomotors have good stability in biological environments. Moreover, the particle size is less than 200 nm, which is beneficial for the efficient penetration of biological barriers such as the blood-brain barrier by the drug delivery system and enhances the drug delivery efficiency.

[0046] Infrared spectroscopy analysis was performed on motors (MSN, Janus MSN-Pt, Janus MSN-Pt-NH2, Janus MSN-Pt@Lira / IP) of different materials and preparation stages using the potassium bromide pellet method. Figure 5 As shown, the above materials exhibit the characteristic infrared absorption bands of silicon-based materials. 967 cm -1 The peak at 813 cm⁻¹ is attributed to the Si-OH bond. -1 The band at that location corresponds to a SiO4 tetrahedron, 1056 cm⁻¹. -1 and 1245 cm-1 The band at 2925 cm⁻¹ is attributed to Si-O-Si bonds. The FT-IR spectrum of Janus MSN-Pt shows a band at 2925 cm⁻¹. -1 The antisymmetric and symmetric stretching vibrations of CH2 confirmed the modification of MSN with (3-mercaptopropyl)trimethoxysilane. (Per 1568 cm⁻¹) -1 The torsional vibration of -NH at the α-hydroxyl group confirms the successful modification of the amino group. These absorption peaks were all observed in the nanomotor, and another peak was observed at 1666 cm⁻¹. -1 The detection of the characteristic peak of the -NH-CO-amide I band at the site confirms the successful synthesis of the self-driven nanomotor loaded with rapeseed peptides.

[0047] Example 3: Characterization of the motility of self-driven nanomotors loaded with rapeseed peptides and liraglutide

[0048] (1) Hydrogen peroxide removal and oxygen generation capabilities of self-driven nanomotors

[0049] Different materials were added to 10 mM H2O2. The H2O2 concentration was determined using an H2O2 detection kit (Nanjing Jiancheng), and the oxygen dissolution rate was measured using a dissolved oxygen meter. The oxygen removal efficiency was compared with that of hydrogen peroxide. Figure 6 a) and oxygen production capacity ( Figure 6 (b) It can be seen that the self-driven nanomotor has good catalytic activity, and the catalytic ability is time- and concentration-dependent. As the concentration and reaction time increase, more bubbles are generated, which is also the key to self-drive.

[0050] (2) Motion performance of self-driven nanomotors

[0051] The motion trajectory of nanoparticles was analyzed using a Nanosight NS300 instrument. Aqueous solutions of nanomotors were mixed with 1 mM H₂O₂ solutions of different mass fractions (0, 5, 15, 20, 25%) to achieve final concentrations of 0, 50, 150, 200, and 250 μM. The mixture was injected into the chamber using a 1 mL syringe, and the x and y coordinates of the nanoparticles were extracted at a rate of 25 frames / s. The motion trajectory, MSD, effective diffusion coefficient, and velocity were then analyzed and compared using Origin software. The calculation formulas are as follows: ² Figure 7 As shown, in H2O2 solutions of different concentrations, the trajectory of the particle gradually increases with the increase of H2O2 concentration. Figure 7 a). MSD increases over time ( Figure 7 (b) Furthermore, the slope gradually increases with increasing H2O2 concentration, indicating that the nanomotor exhibits enhanced motion behavior under oxidative stress conditions. Figure 7c). Under higher concentrations of H₂O₂, the nanomotors exhibited enhanced motion behavior ( Figure 7 (d) helps to improve its diffusion and transport capabilities in oxidative stress microenvironments.

[0052] Example 4 Biosafety assessment of self-driven nanomotors loaded with rapeseed peptides and liraglutide

[0053] (1) Effects of self-driven nanomotors on the viability of Caco-2 and Ins-1 cells

[0054] This example illustrates the safety of nanomotors. Two types of cells were evenly seeded in 96-well plates and incubated overnight in a cell culture incubator. After the cells adhered and grew to a certain density, different concentrations (0, 10, 20, 50, 75, 100, 200 μg / mL) of nanomotors were added and incubated for 24 h. After incubation, cell viability of each group was measured using the CCK8 method.

[0055] The results showed that even at a concentration of 200 μg / mL, no significant cytotoxicity was observed, indicating that the nanomotors have good biocompatibility. Figure 8 This technology can effectively deliver drugs to target cells, achieving a therapeutic effect. Based on the CCK8 results, a nanomotor with a concentration of 100 μg / mL was selected for subsequent experiments.

[0056] (2) Self-driven nanomotor's ability to scavenge reactive oxygen species

[0057] This example illustrates the reactive oxygen species (ROS) scavenging ability of nanomotors. Cells were seeded in confocal microarrays and incubated with different materials (H2O2, MSN, MSN-Pt, and Motor). Intracellular ROS levels were analyzed using DCFH-DA. Cells were incubated with H2O2 (300 μM) and the sample (100 μg / mL) for 12 h. Untreated cells served as a control. Cells were incubated with 10 μM DCFH-DA for 30 min and washed with PBS. Finally, intracellular DCF fluorescence images were acquired using laser confocal microscopy. Figure 9 The results showed that the motor group exhibited a decrease in fluorescence intensity due to the consumption of reactive oxygen species. The Pt-modified nanomotor can catalyze the decomposition of H2O2 to produce O2, thereby exhibiting H2O2-responsive motility behavior and helping to improve its migration ability in oxidative stress environment, achieving the inhibition and scavenging of ROS, which is crucial for the treatment of T2D.

[0058] Example 5: In vitro gastrointestinal simulation and pH-responsive release of sodium alginate hydrogel microspheres loaded with self-driven nanomotors.

[0059] Peptides have poor chemical stability due to the presence of amide bonds that are sensitive to biological enzymes, the C-terminus / N-terminus of residues, and their susceptibility to oxidation and racemization. After oral administration, they are easily degraded by acids, alkalis, and enzymes in the gastrointestinal tract.

[0060] Motor@SAM was prepared using a microfluidic method: an aqueous phase (phosphate buffer containing 5 wt.% sodium alginate, 2.5 wt.% nanomotors, and 0.5 wt.% light stabilizer) and an oil phase (paraffin oil containing 5 wt.% Span 80) were introduced into a microfluidic device connected to a syringe pump. The flow rates of the aqueous and oil phases were controlled at 20 μL / min and 15 μL / min, respectively. The resulting monodisperse emulsion droplets underwent photocrosslinking under 365 nm UV light for 1 min. Subsequently, the underlying fluorinated oil layer was removed, and the sample was washed three times with physiological saline to finally obtain sodium alginate microspheres encapsulating nanomotors.

[0061] Sodium alginate (SAM) has been reported to exhibit colon-specific drug release capabilities in response to pH changes. This example was used to evaluate the stability of SAM in the gastric and colonic environments. Motor@SAM was placed in different digestive fluids (simulated gastric fluid pH 3.0 and simulated intestinal fluid pH 6.8) for 48 hours, and its morphological changes were observed. Figure 10 As shown, after immersion in gastric juice for 48 hours, the morphological changes of Motor@SAM were negligible, indicating that Motor@SAM is stable in the gastric environment. Conversely, Motor@SAM rapidly swelled after storage in artificial intestinal fluid, exhibiting numerous cracks and compromised integrity within 12 hours, followed by extensive fragmentation within 24 hours, and complete degradation within 48 hours. This is due to the excellent pH response of sodium alginate, which endows the microspheres with pH-responsive gastrointestinal protection and intestinal release properties. This contributes to improving the release and local delivery efficiency of nanomotors in the gastrointestinal environment, representing a crucial functional characteristic of oral delivery systems.

[0062] Example 6: Therapeutic effects of self-driven nanomotors loaded with rapeseed peptides and liraglutide

[0063] Two in vitro cell models, Caco-2 and pancreatic β-cells, were used to evaluate the GLP-1 secretory activity, DPP-4 inhibitory activity, and insulin-releasing activity of the motor. In vitro and in vivo DPP-IV enzyme activity was measured using a substrate luminescence assay. A Transwell bilayer co-culture cell model was then established. Two hours after drug administration (0.5 mL of drug at a concentration of 100 μg / mL in complete culture medium in the upper chamber), the culture medium from the BL side was collected to determine the levels of active GLP-1 and insulin.

[0064] Figure 11The results showed that IPQVS can reduce endogenous GLP-1 degradation by inhibiting DPP-IV enzyme activity, while liraglutide, as a GLP-1 receptor agonist, further promotes insulin secretion, thereby exerting a synergistic hypoglycemic effect and effectively reversing insulin resistance, revealing its potential efficacy in the clinical treatment of type 2 diabetes.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a targeted self-driven nanomotor hydrogel, characterized in that, Includes the following steps: (1) Mesoporous silica nanoparticles were dispersed in an aqueous solution containing short-chain alcohols and cationic surfactants, heated, and solid wax was added. Pickering emulsion was obtained by shearing. (2) Cool the emulsion, add a silane coupling agent solution containing thiol, filter, wash, and further disperse in a platinum nanoparticle colloidal solution for adsorption; (3) After adsorption, the solid was filtered and resuspended in ethanol to remove the solid wax. The solid was then centrifuged and the precipitate was washed with ethanol and chloroform in sequence to obtain Janus mesoporous silica platinum nanomotors. (4) First, liraglutide is loaded onto the Janus mesoporous silica platinum nanomotor by amination reaction, and then rapeseed DPP-IV inhibitory peptide is grafted onto it by amide method to obtain a self-driven nanomotor loaded with active peptide.

2. The preparation method according to claim 1, characterized in that, The ratio of the mesoporous silica nanoparticles, the aqueous solution containing short-chain alcohols and cationic surfactants, and the solid wax in step (1) is: 160~200 mg: 8~12 mL: 1 g; The heating temperature is 65~85℃.

3. The preparation method according to claim 2, characterized in that, In step (1), the volume fraction of the short-chain alcohol in the aqueous solution containing the short-chain alcohol and the cationic surfactant is 6.0~7.0%; the content of the cationic surfactant is 0.8~1.2 μM.

4. The preparation method according to claim 3, characterized in that, The cutting conditions in step (1) are: 20000~30000rpm, 10~15min.

5. The preparation method according to claim 1, characterized in that, In step (2), the ratio of the mercapto-containing silane coupling agent solution to the mesoporous silica nanoparticles is 180~220 μL: 150~200 mg; The ratio of the platinum nanoparticle colloidal solution to the mesoporous silica nanoparticles is 25~35 mL: 150~200 mg.

6. The preparation method according to claim 1, characterized in that, The method for loading liraglutide in step (4) is as follows: prepare Janus MSN-Pt toluene solution, add APTES, react for 20-25 h, centrifuge, wash the precipitate, dry to obtain Janus MSN-Pt–NH2, disperse it in water, add liraglutide Lira solution to react, centrifuge and wash, freeze dry to obtain Janus MSN-Pt@Lira; The ratio of toluene solution, Janus MSN-Pt, and APTES was 18-22 mL: 55-65 mg: 1-3 mL. The ratio of Janus MSN-Pt–NH2, water, and liraglutide Lira solution is: 2~6 mg: 2~6 mL: 1 mL; The concentration of liraglutide is 0.8~1.2 mg / mL.

7. The preparation method according to claim 1, characterized in that, Step (4) The method of grafting rapeseed DPP-IV inhibitory peptide by amide method is as follows: After the loading of liraglutide is completed, 2-(N-morpholine) ethanesulfonic acid solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added in sequence, stirred at 2~5℃ for 60 min, and finally rapeseed DPP-IV inhibitory peptide is added, stirred for 3~5 h, centrifuged and washed to obtain nanomotor loaded with active peptide; The ratio of the liraglutide-loaded nanomotor to 2-(N-morpholine)ethanesulfonic acid solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and rapeseed DPP-IV inhibitory peptide was 5 mg: 4~6 mL: 5~9 mg: 1~3 mg: 4~6 mg.

8. The preparation method according to claim 1, characterized in that, It also includes step (5) using microfluidics to encapsulate the nanomotor loaded with active peptides in sodium alginate microspheres to obtain a self-driven nanomotor hydrogel.

9. The targeted self-driven nanomotor hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the targeted self-driven nanomotor hydrogel of claim 9 in the preparation of drugs that promote insulin release or are used to treat diabetes.

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