Method for constructing oral insulin nano drug delivery system based on deoxycholic acid modified probiotic spores and application of oral insulin nano drug delivery system

Abstract

The invention discloses a method for constructing an oral insulin nano drug delivery system based on deoxycholic acid modified probiotic spores and application, and the method comprises the following specific preparation steps: dissolving deoxycholic acid in absolute ethyl alcohol, then mixing with EDC and NHS to activate carboxyl, adding purified spores, and carrying out freeze drying to obtain the oral insulin nano drug delivery system based on deoxycholic acid modified probiotic spores. Stirring and reacting at room temperature to obtain a deoxycholic acid modified probiotic spore carrier DA-Spore; in a weak acid environment, insulin and DA-Spore are mixed and stirred, and the oral insulin nano drug delivery system DA-Spore / Ins is synthesized through physical adsorption. The DA-Spore / Ins drug delivery system constructed by the invention can overcome the degradation of gastrointestinal tract protease, strides across intestinal mucous and intestinal epithelial cell barriers, realizes efficient intestinal delivery of oral insulin, improves the oral bioavailability of insulin, and realizes a better oral hypoglycemic effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of oral insulin nanoformulations, and specifically relates to a method and application of constructing an oral insulin nanoformulation drug delivery system based on deoxycholic acid modified probiotic spores. Background Art

[0002] Diabetes is a chronic metabolic disease. About 415 million people suffer from diabetes worldwide, and it is expected to grow exponentially in the next few years. Chronic hyperglycemia in diabetes is associated with long-term damage, dysfunction and failure of different organs, especially the eyes, kidneys, nerves, heart and blood vessels. Diabetes is clinically divided into two types: type 1 diabetes and type 2 diabetes. According to statistics, among all diabetic patients, about 95% are mainly type 2 diabetes, and less than 5% are type 1 diabetes. The incidence rate is also gradually increasing with age. Insulin (Ins) is not only the first choice for type 1 diabetes, but also a must-have drug for some late-stage type 2 diabetes. Although subcutaneous injection of insulin has a fast onset and high bioavailability, long-term injection brings great pain to patients and is also expensive. Oral insulin has the advantages of convenience, painlessness, simulation of endogenous insulin pathway, protection of pancreatic β cells, reduction of adverse reactions of long-term injection and improvement of patient compliance.

[0003] The bioavailability of oral insulin is less than 1% due to the barrier effects of gastric acid, gastrointestinal enzyme environment, intestinal mucosa and intestinal epithelial cells. Therefore, oral insulin delivery still faces great challenges. Studies have shown that nanoparticles can effectively overcome the gastrointestinal barrier, protect protein or peptide drugs from the effects of the gastrointestinal environment, and improve their oral bioavailability by enhancing drug internalization and permeation.

[0004] Bacillus cululans (BC) is a type of lactic acid bacteria recognized as "generally recognized as safe" by the U.S. Food and Drug Administration. In addition, the surface of Bacillus coagulans spores has a protein coating that can withstand high temperature, acid, alkali, ultraviolet light and a variety of chemical substances. As a dormant life form of probiotics, Bacillus spores have the characteristics of resisting the harsh environment of the gastrointestinal tract, penetrating the mucus layer, shedding the amphiphilic protein shell, and colonizing intestinal epithelial cells to produce probiotics. Inspired by the physiological characteristics of spores, the present invention constructs a new type of deoxycholic acid (DA) modified Bacillus oral insulin nano-drug delivery system, which is used as a spontaneous nano "generator" to solve the absorption barrier problem of oral insulin. There are currently no related reports in this regard. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a method for constructing an oral insulin nano-drug delivery system based on deoxycholic acid-modified probiotic spores. The oral insulin nano-drug delivery system prepared by this method can overcome the enzyme barrier, intestinal mucus barrier and intestinal epithelial cell barrier in the gastrointestinal tract, thereby improving the bioavailability of oral insulin and exerting a better hypoglycemic effect. Therefore, the prepared oral insulin nano-drug delivery system can be used to prepare oral drugs for treating and / or preventing diabetes.

[0006] The present invention adopts the following technical solution to solve the above technical problems, and is a method for constructing an oral insulin nano drug delivery system based on deoxycholic acid-modified probiotic spores, characterized in that the specific steps are: Step S1, dissolving deoxycholic acid in anhydrous ethanol, and then mixing with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) to activate the carboxyl group, and then adding purified spores (Spore), stirring and reacting at room temperature, dialyzing after the reaction is completed, centrifuging to obtain a precipitate, washing the precipitate with ultrapure water, and freeze-drying to obtain a deoxycholic acid modified probiotic spore carrier DA-Spore; Step S2, in a weakly acidic environment, insulin and the deoxycholic acid-modified probiotic spore carrier DA-Spore are mixed and stirred to synthesize the oral insulin nano-drug delivery system DA-Spore / Ins by physical adsorption. The oral insulin nano-drug delivery system DA-Spore / Ins spontaneously forms deoxycholic acid-modified insulin nanoparticles DA-Spore / InsNPs in the intestinal environment.

[0007] It is further defined that the specific preparation process of the purified spores in step S1 is: incubating Bacillus coagulans at 37°C for 48 hours, detecting the spore production degree of Bacillus by scanning electron microscopy, and after the spore production rate reaches 90%, heating the suspension at 80°C for 30 minutes, then centrifuging at 4°C for 30 minutes, and then washing with 1M KCl solution and deionized water, and freeze-drying to obtain purified spores.

[0008] It is further defined that the weight percentage ratio of deoxycholic acid to spores in the deoxycholic acid modified probiotic spore carrier DA-Spore in step S1 is 25wt%~70wt%:30wt%~75wt%.

[0009] It is further defined that the weight percentage ratio of deoxycholic acid, spores and insulin in the oral insulin nano-drug delivery system DA-Spore / Ins in step S2 is 20wt%~60wt%: 30wt%~65wt%: 5wt%~45wt%.

[0010] The oral insulin nano drug delivery system of the present invention is used in the preparation of oral drugs for treating and / or preventing diabetes.

[0011] It is further defined that the oral insulin nano-drug delivery system is used to protect insulin from the strong acid environment of the stomach and the destruction of pepsin and trypsin in the gastrointestinal tract, and maintain the secondary spatial active structure of insulin in the oral insulin nano-drug delivery system; the oral insulin nano-drug delivery system uses spores to colonize and germinate in the intestine to generate probiotics, and sheds the protein shell to spontaneously form deoxycholic acid-modified insulin nanoparticles DA-Spore / Ins NPs; the deoxycholic acid-modified insulin nanoparticles DA-Spore / Ins NPs overcome the intestinal mucus and intestinal epithelial cell barriers with the active transport of spore surface functional proteins and apical sodium-dependent bile acid transporter (ASBT), thereby improving the oral bioavailability of insulin and achieving the hypoglycemic effect of insulin.

[0012] It is further defined that the diabetes is type 1 diabetes or type 2 diabetes.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention constructs an oral insulin nano-drug delivery system DA-Spore / Ins based on deoxycholic acid-modified probiotic spores. The DA-Spore / Ins drug delivery system can overcome the degradation of gastrointestinal proteases, cross the intestinal mucus and intestinal epithelial cell barriers, achieve efficient intestinal delivery of oral insulin, improve the oral bioavailability of insulin, and achieve a better oral hypoglycemic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a scanning electron micrograph of Bacillus coagulans and spores (bar value: 10 μm).

[0015] Figure 2 FTIR graphs of the preparations in Example 1.

[0016] Figure 3 The spore activity of blank spores and DA-Spore / Ins drug delivery system.

[0017] Figure 4 TEM images of the morphological changes of the DA-Spore / Ins drug delivery system before and after incubation in intestinal perfusion fluid and the spontaneous formation of nanoparticles.

[0018] Figure 5 The efficacy and pharmacokinetics of DA-Spore / Ins drug delivery system were studied by intragastric administration in type 1 diabetic rats. DETAILED DESCRIPTION

[0019] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention. Example 1

[0020] Preparation of Spore-DA / Ins drug delivery system: The coagulant Bacillus was incubated at 37°C and 150 r / min for 48 h, and the spore production of the Bacillus was detected by scanning electron microscopy. When the spore production rate reached 90%, the suspension was heated at 80°C for 30 min, then centrifuged for 30 min (10000g, 4°C), washed with 1M KCl solution and deionized water, and freeze-dried to obtain purified spores for storage.

[0021] 7 mg of deoxycholic acid was dissolved in anhydrous ethanol, and then mixed with 8 mg of EDC and 8 mg of NHS for 1 hour to activate the carboxyl group, and then 5 mg of purified spores were added and stirred at room temperature for 24 hours. After the reaction was completed, it was dialyzed for 24 hours, and the water was changed every 2 hours. The precipitate was obtained by centrifugation, and the precipitate was washed with ultrapure water and freeze-dried to obtain the deoxycholic acid modified probiotic spore carrier DA-Spore. The product was characterized by infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and thermogravimetric analysis (TGA), and the results showed that DA-Spore was successfully prepared.

[0022] In a weak acid environment, 3 mg of insulin was mixed with 12 mg of Spore and 12 mmg of DA-Spore for 24 hours, and oral insulin nano-drug delivery systems Spore / Ins and DA-Spore / Ins were synthesized by physical adsorption. The products were characterized by infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and thermogravimetric analysis (TGA), and the results showed that Spore-DA / Ins was successfully prepared.

[0023] The freeze-dried DA-Spore / Ins drug delivery system was added to the simulated intestinal solution and incubated in a constant temperature shaker at 37°C for 8 hours at a shaker speed of 65r / min. After incubation, the mixed preparation was centrifuged (10000g, 5min), the supernatant was taken, and freeze-dried to obtain DA-Spore / Ins NPs. The simulated intestinal fluid incubation experiment showed that the DA-Spore / Ins drug delivery system can be successfully self-assembled into nanoparticles. TEM showed that the nanoparticles were uniformly spherical. Combined with the Malvern particle size analyzer, the particle size of DA-Spore / Ins NPs was confirmed to be 193 ± 2.3nmnm.

[0024] Figure 1This is a scanning electron microscope image of Bacillus coagulans and spores (bar value is 10μm). It can be seen from the image that Bacillus coagulans is rod-shaped, while one end of the spores produced by Bacillus coagulans is spherical or elliptical.

[0025] Figure 2 The FTIR images of each preparation are shown in Figure 1. As can be seen from the figure, the stretching vibration of the –C=O bond in the DA molecule is 1715 cm -1 The stretching vibration of –C=O bond in the synthetic product DA-Spore is 1654cm -1 DA-Spore / Ins at 3346cm -1 There is a new broad peak at , indicating that DA-Spore is bound to insulin via hydrogen bonds.

[0026] Figure 3 The spore activity of blank spores and DA-Spore / Ins drug delivery system. As shown in the figure, compared with blank spores, DA-Spore / Ins drug delivery system has better activity, indicating that it can germinate under appropriate conditions, and insulin and deoxycholic acid do not affect the activity of spores.

[0027] Figure 4 The TEM images of the DA-Spore / Ins drug delivery system before and after incubation in intestinal perfusion fluid and the spontaneous formation of nanoparticles. As can be seen from the figure, after incubation in situ intestinal perfusion fluid for 8 hours, the DA-Spore / Ins drug delivery system can spontaneously generate DA-Spore / Ins nanoparticles with a particle size of 193 ± 2.3nm (red is the DA-Spore / Ins system, and yellow is the spontaneously formed nanoparticles).

[0028] Figure 5 This is a study on the efficacy and pharmacokinetics of the DA-Spore / Ins drug delivery system in rats with type 1 diabetes. As shown in the figure, the relative bioavailability of free insulin after oral administration is only 1.4%, while the relative bioavailability of the DA-Spore / Ins drug delivery system reaches 15.1%; the efficacy results show that free insulin after oral administration has no obvious hypoglycemic effect, while subcutaneous insulin can take effect immediately, but the duration is short, while the DA-Spore / Ins drug delivery system has a better long-term hypoglycemic effect. Example 2

[0029] Preparation of Spore-DA / Ins drug delivery system: The coagulant Bacillus was incubated at 37°C and 150 r / min for 48 h, and the spore production of the Bacillus was detected by scanning electron microscopy. When the spore production rate reached 90%, the suspension was heated at 80°C for 30 min, then centrifuged for 30 min (10000g, 4°C), washed with 1M KCl solution and deionized water, and freeze-dried to obtain purified spores for storage.

[0030] 5 mg of deoxycholic acid was dissolved in anhydrous ethanol, and then mixed with 8 mg of EDC and 8 mg of NHS for 1 hour to activate the carboxyl group. Then 8 mg of purified spores were added and stirred at room temperature for 24 hours. After the reaction was completed, dialyzed for 24 hours, and the water was changed every 2 hours. The precipitate was obtained by centrifugation. The precipitate was washed with ultrapure water and freeze-dried to obtain the deoxycholic acid modified probiotic spore carrier DA-Spore.

[0031] In a weakly acidic environment, 3 mg of insulin was mixed with 13 mg of DA-Spore and stirred for 24 h to synthesize the oral insulin nano-drug delivery system DA-Spore / Ins by physical adsorption.

[0032] The freeze-dried DA-Spore / Ins drug delivery system was added to the simulated intestinal solution and incubated in a constant temperature shaker at 37°C for 8 hours at a shaker speed of 65 r / min. After incubation, the mixed preparation was centrifuged (10,000 g, 5 min), the supernatant was taken, and freeze-dried to obtain DA-Spore / Ins NPs. Example 3

[0033] Preparation of Spore-DA / Ins drug delivery system: The coagulant Bacillus was incubated at 37°C and 150 r / min for 48 h, and the spore production of the Bacillus was detected by scanning electron microscopy. When the spore production rate reached 90%, the suspension was heated at 80°C for 30 min, then centrifuged for 30 min (10000g, 4°C), washed with 1M KCl solution and deionized water, and freeze-dried to obtain purified spores for storage.

[0034] 8 mg of deoxycholic acid was dissolved in anhydrous ethanol, and then mixed with 8 mg of EDC and 8 mg of NHS for 1 hour to activate the carboxyl group. Then 12 mg of purified spores were added and stirred at room temperature for 24 hours. After the reaction was completed, dialyzed for 24 hours, and the water was changed every 2 hours. The precipitate was obtained by centrifugation. The precipitate was washed with ultrapure water and freeze-dried to obtain the deoxycholic acid modified probiotic spore carrier DA-Spore.

[0035] In a weakly acidic environment, 4 mg of insulin was mixed with 20 mg of DA-Spore and stirred for 24 h to synthesize the oral insulin nano-drug delivery system DA-Spore / Ins by physical adsorption.

[0036] The freeze-dried DA-Spore / Ins drug delivery system was added to the simulated intestinal solution and incubated in a constant temperature shaker at 37°C for 8 hours at a shaker speed of 65 r / min. After incubation, the mixed preparation was centrifuged (10,000 g, 5 min), the supernatant was taken, and freeze-dried to obtain DA-Spore / Ins NPs.

[0037] The basic principles, main features and advantages of the present invention are shown and described above. Without departing from the spirit and scope of the present invention, the present invention may also be subject to various changes and improvements, which all fall within the scope of the present invention to be protected.

Claims

1. A method for constructing an oral insulin nano-drug delivery system based on deoxycholic acid-modified probiotic spores, characterized in that The specific steps are: Step S1, dissolving deoxycholic acid in anhydrous ethanol, and then mixing with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to activate the carboxyl group, and then adding the purified spores, stirring and reacting at room temperature, dialyzing after the reaction is completed, centrifuging to obtain a precipitate, washing the precipitate with ultrapure water, and freeze-drying to obtain a deoxycholic acid modified probiotic spore carrier DA-Spore; Step S2, in a weakly acidic environment, mixing and stirring insulin and the deoxycholic acid-modified probiotic spore carrier DA-Spore, synthesizing an oral insulin nano-drug delivery system DA-Spore / Ins by physical adsorption, wherein the oral insulin nano-drug delivery system DA-Spore / Ins spontaneously forms deoxycholic acid-modified insulin nanoparticles DA-Spore / Ins NPs in an intestinal environment.

2. The method for constructing an oral insulin nano-drug delivery system based on deoxycholic acid-modified probiotic spores according to claim 1, characterized in that The specific preparation process of the purified spores in step S1 is as follows: incubate Bacillus coagulans at 37°C for 48h, detect the spore production degree of Bacillus by scanning electron microscopy, and after the spore production rate reaches 90%, heat the suspension at 80°C for 30min, then centrifuge at 4°C for 30min, wash with 1M KCl solution and deionized water, and freeze-dry to obtain purified spores.

3. The method for constructing an oral insulin nano-drug delivery system based on deoxycholic acid-modified probiotic spores according to claim 1, characterized in that: In step S1, the weight percentage ratio of deoxycholic acid to spores in the deoxycholic acid modified probiotic spore carrier DA-Spore is 25wt%~70wt%:30wt%~75wt%.

4. The method for constructing an oral insulin nano-drug delivery system based on deoxycholic acid-modified probiotic spores according to claim 1, characterized in that: In step S2, the weight percentage ratio of deoxycholic acid, spores and insulin in the oral insulin nano-drug delivery system DA-Spore / Ins is 20wt%~60wt%: 30wt%~65wt%: 5wt%~45wt%.

5. Use of the oral insulin nano drug delivery system prepared according to the method of any one of claims 1 to 4 in the preparation of oral drugs for the treatment and / or prevention of diabetes.

6. The use according to claim 5, characterized in that: The oral insulin nano drug delivery system is used to protect insulin from the strong acid environment of the stomach and the destruction of pepsin and trypsin in the gastrointestinal tract, and maintain the secondary spatial active structure of insulin in the oral insulin nano drug delivery system; The oral insulin nano-drug delivery system relies on spores to colonize and germinate in the intestine to generate probiotics, and then shed the protein shell to spontaneously form deoxycholic acid-modified insulin nanoparticles DA-Spore / Ins NPs; the deoxycholic acid-modified insulin nanoparticles DA-Spore / Ins NPs overcome the intestinal mucus and intestinal epithelial cell barriers with the active transport of spore surface functional proteins and apical sodium-dependent bile acid transporters, thereby improving the oral bioavailability of insulin and achieving the hypoglycemic effect of insulin.

7. The use according to claim 5, characterized in that: The diabetes mellitus is type I diabetes mellitus or type II diabetes mellitus.

Images