Double-stimulus response type sodium alginate grafted metformin / chitosan nanoparticle as well as preparation method and application thereof

Abstract

The invention relates to the technical fields of biological material technologies, medicinal chemistry and medicinal preparations, in particular to a double-stimulation response type sodium alginate grafted metformin / chitosan nanoparticle as well as a preparation method and application thereof. Comprising the following steps: (1) synthesizing sodium alginate grafted metformin: preparing sodium alginate grafted metformin through an amidation condensation reaction catalyzed by EDC / NHS; and (2) preparing the sodium alginate grafted metformin / chitosan nanoparticles: carrying out polyelectrolyte complexation reaction to obtain the nanoparticles. The synthesis method is simple in path, and the prepared nanoparticles are good in dispersity, uniform in particle size and good in stability and have the pH and intestinal flora double-stimulation response characteristic. The metformin oral delivery system can overcome the gastric acid barrier and prolong the residence time of metformin in an intestinal flora enrichment area during oral delivery, greatly improves the intestinal delivery efficiency, can be applied to metformin oral targeting delivery of various metabolic diseases including type 2 diabetes and metabolic syndrome, and enhances the treatment effect of metformin.

Description

Technical Field

[0001] The present invention belongs to the fields of biomaterial technology, medicinal chemistry and pharmaceutical preparation technology, and particularly relates to dual-stimulus-responsive sodium alginate-grafted metformin / chitosan nanoparticles and a preparation method and application thereof. Background Art

[0002] Metformin is a cornerstone treatment for type 2 diabetes and metabolic syndrome, yet the precise mechanisms underlying its therapeutic effects remain elusive. Recent studies have revealed a profound impact of the intestinal environment and its dynamic interactions with the gut microbiota on metformin's therapeutic effects. Specifically, the gut microbiota and its underlying microbiota play a key role in mediating metformin's clinical benefits, particularly its glucose-lowering and immune-modulating effects. To explore the extent to which the gut and its microbiota mediate metformin's clinical benefits, it is essential to develop novel drug delivery systems that can target metformin to the gut and microbiota-rich areas to enhance their regulatory effects on both, thereby maximizing metformin's efficacy by leveraging the complex intestinal environment. Oral delivery of metformin to the gut and microbiota-rich areas faces two major challenges: maintaining drug activity while navigating the extreme pH and digestive enzyme conditions of the upper gastrointestinal tract, particularly the stomach; and ensuring precise, targeted release of the drug within microbiota-rich areas.

[0003] To overcome these challenges, it is necessary to design an oral nano-drug delivery system that meets the following specific criteria: maintaining integrity and stability during passage through the upper gastrointestinal tract to protect metformin from enzymatic degradation and the harsh gastric environment, and slowly and precisely releasing the drug in the lower gastrointestinal tract, where intestinal flora are enriched. These characteristics help maximize metformin's regulatory effects on the intestine and intestinal flora, thereby clarifying the extent to which the intestine and intestinal flora mediate metformin's clinical benefits. This system can also be applied to the oral targeted intestinal flora delivery of metformin in metabolic diseases such as type 2 diabetes, metabolic syndrome, and non-alcoholic fatty liver disease. Summary of the Invention

[0004] The purpose of the present invention is to provide a dual stimulus responsive sodium alginate grafted metformin / chitosan nanoparticle and its preparation method, comprising (1) synthesizing sodium alginate grafted metformin: sodium alginate is fully dissolved in water, and EDC and NHS are added in sequence, metformin hydrochloride is added to the system after a certain period of time, and triethylamine is used to control the pH value of the reaction system to 6-7, and the system is subjected to an amidation condensation reaction at room temperature in the dark and with gentle magnetic stirring. After the reaction is completed, the system is dialyzed and freeze-dried to obtain sodium alginate grafted metformin. Finally, the product is subjected to 1H-NMR, MALDI-TOF and IR characterization were used to determine its grafting rate, molecular weight range and characteristic functional groups. (2) Preparation of sodium alginate grafted metformin / chitosan nanoparticles: Sodium alginate grafted metformin and chitosan were dissolved in water and adjusted to an appropriate pH using hydrochloric acid and sodium hydroxide solutions respectively. After filtration through a filter membrane, sodium alginate grafted metformin and chitosan were subjected to polyelectrolyte complexation reaction to prepare nanoparticles. The nanoparticles have dual response characteristics of pH and intestinal flora, prolonging the intestinal retention time of metformin and promoting the slow, continuous and precise release of metformin in areas rich in intestinal flora. The dual stimulus responsive sodium alginate grafted metformin / chitosan nanoparticles can achieve oral targeted delivery of metformin to intestinal flora, thereby answering the question of the degree to which the intestine and intestinal flora mediate the clinical benefits of metformin, and are used to treat a variety of metabolic diseases including type 2 diabetes, metabolic syndrome, non-alcoholic fatty liver disease, and improve the therapeutic effect of metformin.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a sodium alginate-grafted metformin derivative having a structure shown in formula (I):

[0007]

[0008] The present invention also provides a method for synthesizing the sodium alginate-grafted metformin derivative, comprising the following steps:

[0009] (1) Weigh an appropriate amount of sodium alginate and disperse it in an aqueous solution under magnetic stirring in a warm water bath. After 1 hour, remove the warm water bath and stir at room temperature for 24 hours to fully dissolve the sodium alginate.

[0010] (2) adding a certain amount of EDC·HCl to the completely dissolved sodium alginate solution (1) to activate the carboxyl groups on the sodium alginate, and after a certain period of time, adding a certain amount of NHS to the above system, and continuing the reaction for a certain period of time;

[0011] (3) Weighing a certain amount of metformin hydrochloride and adding it to the solution system (2) under continuous stirring;

[0012] (4) slowly adding triethylamine dropwise to the solution system (3) to adjust the pH of the solution system, while monitoring the pH of the reaction system to be within the optimal reaction range to ensure the optimal activity of the amidation condensation reaction between the amino group and the carboxyl group, and then continuing the reaction at room temperature in the dark with gentle magnetic stirring for a certain period of time to obtain the sodium alginate grafted metformin derivative;

[0013] (5) The reaction-completed solution system is placed in a dialysis bag for dialysis operation to remove small molecular weight compounds as much as possible. Finally, the dialyzed system is collected and the solvent is removed by freeze drying to obtain the target product, sodium alginate grafted with metformin. The product is subjected to 1 H-NMR, MALDI-TOF and IR characterization were used to determine its grafting rate, molecular weight range and characteristic functional groups.

[0014] Furthermore, in step (1), the sodium alginate is preferably low-viscosity sodium alginate.

[0015] Furthermore, in step (2), the time for activating the carboxyl groups of sodium alginate using EDC·HCl was 15 minutes, and the time for continuing the reaction after adding NHS was 15 minutes.

[0016] Furthermore, in steps (1), (2) and (3), metformin, EDC and NHS need to be added in excess, and sodium alginate-grafted metformin derivatives with different grafting rates are prepared by controlling the molar ratio of sodium alginate (structural unit): metformin: EDC: NHS.

[0017] Furthermore, in step (4), triethylamine is used to adjust the pH of the solution system and the pH is monitored to be in the range of 6 to 7.

[0018] Furthermore, in step (4), the amidation condensation reaction time of the amino group and the carboxyl group is 12 to 24 hours.

[0019] On the other hand, the present invention provides a dual stimulus responsive sodium alginate grafted metformin / chitosan nanoparticle and a preparation method thereof, comprising the following steps:

[0020] (1) Weigh an appropriate amount of sodium alginate grafted metformin derivative and disperse it in a magnetically stirred aqueous solution in a warm water bath to prepare a solution of a certain concentration. After 2 to 3 hours, remove the warm water bath, sonicate for 30 minutes, and stir at room temperature for 24 hours to fully dissolve it.

[0021] (2) Weigh an appropriate amount of chitosan and disperse it in a certain concentration of hydrochloric acid to prepare a chitosan solution of a certain concentration. After ultrasonication for 30 minutes, stir at room temperature for 1 to 2 hours to fully dissolve it.

[0022] (3) Using a certain concentration of sodium hydroxide and hydrochloric acid to adjust the pH values ​​of chitosan solution and sodium alginate grafted metformin solution to 4.5-5.0 respectively;

[0023] (4) Filter the sodium alginate and chitosan solutions using a 0.22 μm filter membrane to remove insoluble matter;

[0024] (5) The chitosan solution in (4) was measured in a certain proportion and added dropwise to the sodium alginate grafted metformin solution in (4) under magnetic stirring. The reaction was continued at room temperature under gentle magnetic stirring for 1 hour to obtain the sodium alginate grafted metformin / chitosan nano-solution.

[0025] Furthermore, in step (1), the temperature of the warm water bath is 30-40°C.

[0026] Furthermore, in step (1), the concentration of the sodium alginate grafted metformin aqueous solution of a certain concentration is 0.005% w / v.

[0027] Furthermore, in step (2), the concentration of the hydrochloric acid is 1.0M.

[0028] Furthermore, in step (2), the concentration of the chitosan aqueous solution of a certain concentration is 1% w / v.

[0029] Furthermore, in step (3), the concentrations of the certain concentrations of sodium hydroxide and hydrochloric acid are both 1.0M.

[0030] Furthermore, in step (3), the pH value of the sodium alginate grafted metformin solution is 4.9, and the pH value of the chitosan solution is 4.6.

[0031] Furthermore, in step (5), the volume ratio of the sodium alginate grafted metformin solution to the chitosan nanoparticle solution is 100:1.

[0032] Furthermore, the dual-stimulus-responsive sodium alginate grafted metformin / chitosan nanoparticles can achieve oral targeted delivery of metformin to intestinal flora, and are used to treat various metabolic diseases including type 2 diabetes, metabolic syndrome, non-alcoholic fatty liver disease, etc.

[0033] The beneficial effects of the present invention are:

[0034] (1) Because our nanoformulations are designed for biological applications, we established a facile synthetic route to synthesize sodium alginate-grafted metformin derivatives, avoiding lengthy experimental steps or the use of toxic chemicals or harsh chemical conditions. The reaction conditions are aqueous solution, which is in line with the concept of green chemistry. The amidation grafting reaction has a high yield, ensuring the successful grafting of metformin onto the molecular chain of sodium alginate.

[0035] (2) Sodium alginate grafted metformin / chitosan nanoparticles prepared by electrostatic complexation can achieve oral targeted delivery of metformin to intestinal flora, achieving slow, precise and sustained targeted release of metformin in the intestine and intestinal flora-rich areas, thereby enhancing the regulatory effect of metformin on the intestine and intestinal flora. It can be used as a media tool to clarify the degree to which intestinal flora mediates the clinical benefits of metformin, and can be further used in the treatment of various metabolic diseases including type 2 diabetes, metabolic syndrome, non-alcoholic fatty liver disease, etc., to improve the therapeutic effect of metformin. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Figure 1 This is a schematic diagram of the synthesis reaction of the sodium alginate grafted metformin derivative of the present invention;

[0038] Figure 2 IR spectra of the sodium alginate grafted metformin derivative prepared in Example 1 of the present invention and the raw materials metformin and sodium alginate;

[0039] Figure 3 This is a MALDI-TOF spectrum of the sodium alginate grafted metformin derivative prepared in Example 1 of the present invention and the raw materials metformin and sodium alginate;

[0040] Figure 4 The sodium alginate grafted metformin derivative prepared in Example 1 of the present invention and the raw materials metformin and sodium alginate are 1 H-NMR spectrum;

[0041] Figure 5 The sodium alginate grafted metformin derivative prepared in Example 2 of the present invention and the raw materials metformin and sodium alginate are 1 H-NMR spectrum;

[0042] Figure 6 This is a transmission electron microscopy characterization image of sodium alginate grafted metformin / chitosan nanoparticles prepared in Example 3 of the present invention;

[0043] Figure 7 Statistical graph of particle sizes of sodium alginate grafted metformin, chitosan, and sodium alginate grafted metformin / chitosan nanoparticles prepared in Example 3 of the present invention;

[0044] Figure 8 This is a Zeta potential characterization graph of sodium alginate grafted metformin, chitosan, and sodium alginate grafted metformin / chitosan nanoparticles prepared in Example 3 of the present invention;

[0045] Figure 9This is a graph showing the particle size stability of sodium alginate grafted metformin / chitosan nanoparticles prepared in Example 3 of the present invention;

[0046] Figure 10 3 is the release behavior curve of sodium alginate grafted metformin / chitosan nanoparticles in Example 4 of the present invention in simulated gastric fluid, simulated small intestinal fluid and simulated large intestinal fluid;

[0047] Figure 11 It is a transmission electron microscopy characterization image and a particle size change image of the pH and intestinal flora dual stimulation responsive drug release performance test of the sodium alginate grafted metformin / chitosan nanoparticles in Example 4 of the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] It should also be noted here that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or technical steps closely related to the technical solution of the present invention are shown in the accompanying drawings, while other details that are not closely related to the present invention are omitted.

[0050] Of course, the relevant description is not a limitation of the present invention, and the present invention is not limited to the following examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the scope of protection of the present invention.

[0051] Example 1

[0052] A method for synthesizing a sodium alginate-grafted metformin derivative comprises the following steps:

[0053] (1) Weigh 1.08 g of very low viscosity sodium alginate (based on the monomer molecular weight Mw = 216, of which the active carboxyl groups are 50%) and disperse it in a 50°C warm water bath under magnetic stirring. After 1 hour, remove the warm water bath and stir at room temperature for 24 hours to fully dissolve the sodium alginate.

[0054] (2) 1.1736 g of EDC·HCl (Mw=191.70, purity 98%) was added to the completely dissolved sodium alginate solution (1) to activate the carboxyl groups on the sodium alginate. After activation for 15 min, 0.8808 g of NHS (Mw=115.0874, purity 98%) was added to the above system and the reaction was continued for 15 min.

[0055] (3) Weigh 1.2676 g of metformin hydrochloride (Mw = 165.62, of which metformin Mw = 129.16, purity 98%) and add it to the above solution system (2) under continuous stirring;

[0056] (4) Triethylamine is slowly added dropwise to the above solution system (3) to adjust the pH of the solution system, while monitoring the pH of the reaction system to be 6 to 7 (optimally 6 to 6.5) to ensure the optimal activity of the amidation condensation reaction between the amino group and the carboxyl group, and then the reaction is continued under gentle magnetic stirring at room temperature in the dark for 12 to 24 hours to obtain the sodium alginate grafted metformin derivative.

[0057] (5) The reaction-completed solution system is placed in a dialysis bag for dialysis operation to remove small molecular weight compounds, and finally the dialyzed system is collected and the solvent is removed by freeze drying to obtain the target product sodium alginate grafted metformin. 1 H-NMR, MALDI-TOF and IR characterization were used to determine its grafting rate, molecular weight range and characteristic functional groups.

[0058] Among them, the molar ratio of sodium alginate (structural unit): metformin: EDC: NHS is 1:1.5:1.2:1.5.

[0059] The schematic diagram of the synthetic reaction of sodium alginate grafted metformin derivatives is shown in Figure 1 As shown, it is formed by amidation condensation reaction of sodium alginate and metformin.

[0060] In order to prove that the method of Example 1 was used to successfully synthesize sodium alginate grafted metformin derivatives, IR, MALDI-TOF and 1 The results were characterized by H-NMR.

[0061] The IR spectra of the sodium alginate grafted metformin derivative prepared in Example 1 and the raw materials sodium alginate and metformin are as follows: Figure 2 Sodium alginate is at about 3373cm -1 A broad band appears at 2923 cm, which belongs to the stretching vibration mode of the OH group. -1 The weak peaks at 1613.8 and 1411.2 cm-1 belong to the asymmetric stretching vibration of CH. -1 The two peaks at 3370.4 and 3297.3 cm-1 belong to C═O and COOH, respectively. -1 The peaks at 3157.6 cm are the asymmetric and symmetric stretching vibrations of NH2. -1 The peak at 1626.2 cm is the stretching vibration of NH. -1 The absorption peaks at 1475.6 and 1448.5 cm are the stretching vibrations of C═N. -1The double absorption peak near the methyl CH peak is the symmetrical bending vibration. In the spectrum of sodium alginate grafted metformin derivatives, the characteristic peaks of sodium alginate and metformin disappeared, and a new amide peak was generated, while the physical mixture of sodium alginate and metformin retained the characteristic peak. In short, compared with sodium alginate, sodium alginate grafted metformin has a better absorption peak at 3386cm -1 The most significant difference is in the position of the broad singlet, which is considered to be the NH vibration of CO-NH, and the characteristic double singlet of metformin primary amine (3370.4 cm -1 and 3297.3cm -1 ) and fingerprint area (1300-400cm -1 ) has disappeared. The above changes prove that metformin has been successfully grafted onto the molecular chain of sodium alginate.

[0062] The MALDI-TOF spectra of the sodium alginate grafted metformin derivative prepared in Example 1 and the raw materials sodium alginate and metformin are as follows: Figure 3 Among them, the molecular weight of the sodium alginate grafted metformin derivative increased from 805.31 to 935.37Da, from 827.30 to 957.82Da, from 821.27 to 951.37Da, and from 843.27 to 973.34Da, all of which shifted to the right by the same molecular weight (metformin ~130.1), proving that metformin was successfully grafted onto the molecular chain of sodium alginate.

[0063] Sodium alginate grafted metformin derivatives prepared in Example 1 and raw materials sodium alginate and metformin 1 H-NMR spectrum Figure 4 As shown. 1 The H-NMR spectrum shows peaks specific for the two methyl groups (6H, (-CH3)2, "α") in metformin and the glycosidic group (H) (5H, "β") in sodium alginate, appearing at 3.0 and 3.1-4.0 ppm, respectively. Furthermore, the degree of substitution (≈41.2%) of the amide bond was determined by integrating the methyl peak ("α", shaded in blue) relative to the glycosidic (H) ("α-H") ("β", shaded in gray) in sodium alginate.

[0064] Example 2

[0065] A method for synthesizing a sodium alginate-grafted metformin derivative comprises the following steps:

[0066] (1) Weigh 1.08 g of very low viscosity sodium alginate (based on the monomer molecular weight Mw = 216, of which the active carboxyl groups are 50%) and disperse it in a 50°C warm water bath under magnetic stirring. After 1 hour, remove the warm water bath and stir at room temperature for 24 hours to fully dissolve the sodium alginate.

[0067] (2) 1.1736 g of EDC·HCl (Mw=191.70, purity 98%) was added to the completely dissolved sodium alginate solution (1) to activate the carboxyl groups on the sodium alginate. After activation for 15 min, 0.8808 g of NHS (Mw=115.0874, purity 98%) was added to the above system and the reaction was continued for 15 min.

[0068] (3) Weigh 1.014 g of metformin hydrochloride (Mw = 165.62, of which metformin Mw = 129.16, purity 98%) and add it to the above solution system (2) under continuous stirring;

[0069] (4) Triethylamine is slowly added dropwise to the above solution system (3) to adjust the pH of the solution system, while monitoring the pH of the reaction system to be 6 to 7 (optimally 6 to 6.5) to ensure the optimal activity of the amidation condensation reaction between the amino group and the carboxyl group, and then the reaction is continued under gentle magnetic stirring at room temperature in the dark for 12 to 24 hours to obtain the sodium alginate grafted metformin derivative.

[0070] (5) The reaction-completed solution system is placed in a dialysis bag for dialysis operation to remove small molecular weight compounds, and finally the dialyzed system is collected and the solvent is removed by freeze drying to obtain the target product sodium alginate grafted metformin. 1 H-NMR, MALDI-TOF and IR characterization were used to determine its grafting rate, molecular weight range and characteristic functional groups.

[0071] Among them, the molar ratio of sodium alginate (structural unit): metformin: EDC: NHS is 1:1.2:1.2:1.5.

[0072] Sodium alginate grafted metformin derivatives prepared in Example 2 and raw materials sodium alginate and metformin 1 H-NMR spectrum Figure 5 As shown. 1 The H-NMR spectrum shows peaks specific for the two methyl groups (6H, (-CH3)2, "α") in metformin and the glycosidic group (H) (5H, "β") in sodium alginate, appearing at 3.0 and 3.1-4.0 ppm, respectively. Furthermore, the degree of substitution (≈35.4%) of the amide bond was determined by integrating the methyl peak ("α", shaded in blue) relative to the glycosidic (H) ("α-H") ("β", shaded in gray) in sodium alginate.

[0073] Example 3

[0074] A dual-stimulus responsive sodium alginate grafted metformin / chitosan nanoparticle and a preparation method thereof, specifically comprising the following steps:

[0075] (1) Weigh 0.005 g of sodium alginate-grafted metformin derivative and disperse it in 100 mL of aqueous solution under magnetic stirring in a 30-40°C warm water bath to prepare a 0.005% w / v solution. After 2-3 hours, remove the warm water bath, sonicate for 30 minutes, and stir at room temperature for 24 hours to fully dissolve the solution.

[0076] (2) Weigh 0.5 g of chitosan and disperse it in 1.0 M hydrochloric acid to prepare a 1% w / v chitosan solution. After ultrasonication for 30 min, stir at room temperature for 1 to 2 h to fully dissolve the chitosan solution.

[0077] (3) Use 1.0 M sodium hydroxide and 1.0 M hydrochloric acid to adjust the pH of the sodium alginate grafted metformin solution to 4.9 and the pH of the chitosan solution to 4.6.

[0078] (4) Filter the sodium alginate and chitosan solutions using a 0.22 μm filter membrane to remove insoluble matter;

[0079] (5) According to the volume ratio of sodium alginate grafted metformin:chitosan nanoparticle solution of 100:1, the chitosan solution in (4) was measured and added dropwise to the sodium alginate grafted metformin solution in (4) under magnetic stirring. The reaction was continued at room temperature under gentle magnetic stirring for 1 hour to obtain the sodium alginate grafted metformin / chitosan nanoparticle solution.

[0080] Subsequently, the sodium alginate grafted metformin / chitosan nanoparticles prepared in Example 3 were characterized by transmission electron microscopy, particle size and potential, and the stability of the nanoparticles was tested.

[0081] Transmission electron microscopy images of sodium alginate grafted metformin / chitosan nanoparticles are shown in Figure 2. Figure 6 As shown, the sodium alginate grafted metformin / chitosan nanoparticles have a typical spherical structure, a smooth and round surface, a particle size of about 150 nm, and a relatively uniform particle size distribution, which proves that the preparation method of the nanoparticles is better.

[0082] The particle size statistics of sodium alginate grafted metformin, chitosan and sodium alginate grafted metformin / chitosan nanoparticles are shown in Figure 2. Figure 7 The Zeta potential changes of sodium alginate grafted metformin / chitosan nanoparticles, sodium alginate and chitosan are shown in Figure 2. Figure 8As shown. The particle size distribution and Zeta potential of the three solutions were characterized by dynamic light scattering. The results showed that the average particle size and Zeta potential values ​​of sodium alginate grafted metformin / chitosan nanoparticles were 140.20nm and +8.27mV, respectively. The average particle sizes of chitosan and sodium alginate grafted metformin were 233.10nm and 107.03nm, respectively. The Zeta potential value of chitosan was +9.69mV, and the Zeta potential value of sodium alginate grafted metformin was -23.10mV. The decrease in the average particle size and the reversal of the Zeta potential of sodium alginate grafted metformin / chitosan nanoparticles proved that the negatively charged sodium alginate grafted metformin and the positively charged chitosan in the solution underwent a polyelectrolyte condensation reaction, further indicating the successful preparation of the nanoparticles.

[0083] In order to test the stability of sodium alginate grafted metformin / chitosan nanoparticles, we used dynamic light scattering (DLS) to test the PDI and particle size changes of the nanoparticle solution at 4°C. Figure 9 As shown in Figure 3, the results showed that sodium alginate grafted metformin / chitosan nanoparticles were stable in water at 4°C for 30 days.

[0084] Example 4

[0085] The dialysis diffusion technique was used to study the in vitro release behavior of sodium alginate grafted metformin / chitosan nanoparticles in a simulated gastrointestinal environment. The specific steps involved were:

[0086] (1) First, 1 mL of sodium alginate-grafted metformin / chitosan nanoparticles was added to a dialysis bag suspended in simulated gastric fluid (SGF; 0.32% pepsin, pH 1.2, 20 mL) for 2 h to simulate the conditions of gastric fluid transport;

[0087] (2) Then, the dialysis bag was transferred to simulated intestinal fluid (SIF; 1% trypsin, pH 6.8 PBS, 20 mL) and soaked for 4 h to simulate the state of small intestinal fluid;

[0088] (3) Finally, the dialysis bag was suspended in simulated colonic fluid (SCF; 1% trypsin, pH 7.4 PBS, 20 mL) for 30 h, which contained 10% intestinal microbiota (collected from C57BL / 6 mice);

[0089] At predetermined time points, an appropriate volume of sample liquid (0.5 mL) was removed and replaced with an equal volume of fresh dissolution medium. The solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected. The metformin content in the sample solution was then determined by HPLC to test its in vitro drug release behavior. Based on the sensitivity of the sodium alginate-grafted metformin / chitosan nanoparticles to pH conditions and intestinal flora, their particle size distribution and morphology were evaluated using DLS and transmission electron microscopy to characterize their stability in simulated gastrointestinal fluid.

[0090] like Figure 10 As shown, the sustained release of metformin from sodium alginate grafted metformin / chitosan nanoparticles in simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8), and simulated colonic fluid (SCF, pH 7.4, containing 10% intestinal flora) in vitro was demonstrated. No burst release was observed from sodium alginate grafted metformin / chitosan nanoparticles in SIF and SGF, which may be due to the strong electrostatic interaction between chitosan and sodium alginate grafted metformin, which inhibited the release of metformin from chitosan and sodium alginate grafted metformin / chitosan polyelectrolyte complex nanoparticles. The intestinal delivery system can protect metformin from degradation and premature release in the low pH environment of the stomach, which is crucial for delivering substances through the gastrointestinal tract. At the same time, it can prolong the retention time of metformin in the intestine and enhance the regulatory effect of metformin on the intestine and intestinal flora.

[0091] like Figure 11 As shown, transmission electron microscopy images and particle size distribution of the alginate-grafted metformin / chitosan nanoparticles were subsequently evaluated in various simulated biological fluids, confirming the delivery system's sensitivity to pH and dual-stimulus responsiveness to intestinal microbiota. At low pH (pH 1.2-2.5), the alginate-grafted metformin / chitosan nanoparticles maintained their original or even smaller size. At higher pH (6.5-7.4), the electrostatic interaction between chitosan and the alginate-grafted metformin weakened, allowing water to intercalate through the pores of the polymer matrix, causing the particles to swell and subsequently degrade. Furthermore, hydrolases produced by intestinal microbiota also triggered degradation of the amide bonds of the alginate-grafted metformin. This phenomenon suggests that the alginate-grafted metformin / chitosan nanoparticles remain stable and protect metformin at low gastric pH, but swell and release the drug upon reaching the neutral pH and microbial-rich areas of the lower gastrointestinal tract. These characteristics help delay metformin release and increase local intestinal concentration, thereby maximizing metformin's regulatory effects on the intestine and intestinal flora. This approach can be applied to oral targeted delivery of metformin to the intestinal flora in a variety of metabolic diseases, including type 2 diabetes, metabolic syndrome, and non-alcoholic fatty liver disease, thereby enhancing metformin's therapeutic efficacy.

[0092] The above embodiments are only used to illustrate the design ideas and technical solutions of the present invention and are not limiting. The present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the scope of protection of the present invention.

Claims

1. A sodium alginate-grafted metformin derivative having the structure shown in formula (I):

2. A method for synthesizing a sodium alginate-grafted metformin derivative according to claim 1, characterized in that: The following steps are involved: (1) Weigh an appropriate amount of sodium alginate and disperse it in an aqueous solution under magnetic stirring in a warm water bath. After 1 hour, remove the warm water bath and stir at room temperature for 24 hours to fully dissolve the sodium alginate. (2) adding a certain amount of EDC·HCl to the completely dissolved sodium alginate solution (1) to activate the carboxyl groups on the sodium alginate, and after a certain period of time, adding a certain amount of NHS to the above system, and continuing the reaction for a certain period of time; (3) Weighing a certain amount of metformin hydrochloride and adding it to the solution system (2) under continuous stirring; (4) slowly adding triethylamine dropwise to the solution system (3) to adjust the pH of the solution system, while monitoring the pH of the reaction system within the optimal reaction range to ensure the optimal activity of the amidation condensation reaction between the amino group and the carboxyl group, and then continuing the reaction at room temperature in the dark with gentle magnetic stirring for a certain period of time to obtain the sodium alginate grafted metformin derivative; (5) The reaction-completed solution system is placed in a dialysis bag for dialysis operation to remove small molecular weight compounds as much as possible, and finally the dialyzed system is collected and the solvent is removed by freeze drying to obtain the target product, sodium alginate grafted metformin derivative, and the product is subjected to 1 H-NMR, MALDI-TOF and IR characterization were used to determine its grafting rate, molecular weight range and characteristic functional groups.

3. The method for synthesizing a sodium alginate-grafted metformin derivative according to claim 2, wherein: In step (1), the sodium alginate is preferably low-viscosity sodium alginate.

4. The method for synthesizing a sodium alginate-grafted metformin derivative according to claim 3, wherein: In step (2), the activation time of the carboxyl groups of sodium alginate using EDC·HCl was 15 min, and the reaction time after adding NHS was also 15 min.

5. The method for synthesizing a sodium alginate-grafted metformin derivative according to claim 4, wherein: In steps (1), (2) and (3), metformin, EDC and NHS need to be added in excess, and sodium alginate-grafted metformin derivatives with different grafting rates are prepared by controlling the molar ratio of sodium alginate (structural unit): metformin: EDC: NHS.

6. The method for synthesizing a sodium alginate-grafted metformin derivative according to claim 5, wherein: In step (4), triethylamine is used to adjust the pH of the solution system and the pH is monitored in the range of 6 to 7.

7. The method for synthesizing a sodium alginate-grafted metformin derivative according to claim 6, wherein: In step (4), the amidation condensation reaction time of the amino group and the carboxyl group is 12 to 24 hours.

8. A dual stimulus responsive sodium alginate grafted metformin / chitosan nanoparticle and a preparation method thereof, characterized in that: The following steps are involved: (1) Weigh an appropriate amount of sodium alginate grafted metformin derivative and disperse it in a magnetically stirred aqueous solution under a warm water bath to prepare a solution of a certain concentration. After 2 to 3 hours, remove the warm water bath, sonicate for 30 minutes, and stir at room temperature for 24 hours to fully dissolve it. (2) Weigh an appropriate amount of chitosan and disperse it in a certain concentration of hydrochloric acid to prepare a chitosan solution of a certain concentration. After ultrasonication for 30 minutes, stir at room temperature for 1 to 2 hours to fully dissolve it. (3) Using a certain concentration of sodium hydroxide and hydrochloric acid to adjust the pH values ​​of chitosan solution and sodium alginate grafted metformin solution to 4.5-5.0 respectively; (4) Filtering the sodium alginate and chitosan solutions with a 0.22 μm filter membrane to remove insoluble matter; (5) Adding the chitosan solution in (4) dropwise according to a certain ratio to the sodium alginate-grafted metformin solution in (4) under magnetic stirring, and continuing the reaction at room temperature under gentle magnetic stirring for 1 hour to obtain a sodium alginate-grafted metformin / chitosan nano-solution.

9. The method for preparing sodium alginate grafted metformin / chitosan nanoparticles according to claim 8, characterized in that: In step (1), the temperature of the warm water bath is 30-40°C.

10. The method for preparing sodium alginate grafted metformin / chitosan nanoparticles according to claim 9, characterized in that: In step (1), the concentration of the sodium alginate grafted metformin aqueous solution of a certain concentration is 0.005% w / v.

11. The method for preparing sodium alginate grafted metformin / chitosan nanoparticles according to claim 10, characterized in that: In step (2), the concentration of the hydrochloric acid is 1.0M.

12. The method for preparing sodium alginate grafted metformin / chitosan nanoparticles according to claim 11, characterized in that: In step (2), the concentration of the chitosan aqueous solution of a certain concentration is 1% w / v.

13. The method for preparing sodium alginate grafted metformin / chitosan nanoparticles according to claim 12, characterized in that: In step (3), the concentrations of the certain concentrations of sodium hydroxide and hydrochloric acid are both 1.0M.

14. The method for preparing sodium alginate grafted metformin / chitosan nanoparticles according to claim 13, characterized in that: In step (3), the pH value of the sodium alginate grafted metformin solution is 4.9, and the pH value of the chitosan solution is 4.

6.

15. The method for preparing sodium alginate grafted metformin / chitosan nanoparticles according to claim 14, characterized in that: In step (5), the volume ratio of the sodium alginate grafted metformin solution to the chitosan nanoparticle solution is 100:

1.

16. The dual stimulus responsive sodium alginate grafted metformin / chitosan nanoparticles according to claims 8 to 15 are used for the treatment of various metabolic diseases such as type 2 diabetes, metabolic syndrome, and non-alcoholic fatty liver disease.

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