An amphoteric bacterial cellulose-encapsulated insulin micelle and its preparation method

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to an amphoteric bacterial cellulose-encapsulated insulin micelle and a preparation method thereof. The amphoteric bacterial cellulose carrier described in the present invention is obtained by grafting glabridin through an esterification reaction and cocamidopropyl betaine through an acyl chloride substitution reaction as the hydrophobic and hydrophilic ends respectively onto bacterial cellulose, and can form a micelle structure with the hydrophobic end inward and the hydrophilic end outward in water, and encapsulate insulin through a self-assembly reaction. The oral insulin nano-micelle described in the present invention has the ability to bind to non-specific proteins, can effectively penetrate the cell membrane phospholipids and has P-gp efflux effect, so that insulin can achieve a sustained release effect in the gastrointestinal tract, which is beneficial to be absorbed by intestinal epithelial cells, enter the blood to the greatest extent and play a good hypoglycemic effect, improve the oral bioavailability of insulin, and has potential value for the research and application of insulin oral delivery systems.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to an amphoteric bacterial cellulose-loaded insulin micelle and a preparation method thereof. Technical Background

[0002] Diabetes is a metabolic disease characterized by increased blood glucose levels, manifested as defects in insulin secretion in the pancreas and reduced sensitivity of peripheral tissues to insulin, resulting in the inability of glucose in the blood to be converted into glycogen by tissues such as the liver and muscles. The metabolic disorder of diabetes can cause functional and structural abnormalities in various tissues and organs throughout the body, and can be fatal in severe cases. Diabetes is the main cause of diseases such as blindness, renal failure, heart disease, stroke, and lower limb amputation. Insulin (INS) is the only protein hormone secreted by pancreatic islet cells that regulates blood glucose levels. It has a relative molecular mass of 5807.69 and is composed of 51 amino acids, mainly including two peptide chains (chain A and chain B). Among them, chain A contains 21 amino acids, and chain B contains 30 amino acids. The intra-chain (chain A) and inter-chain are connected by disulfide bonds. Insulin is a biopolymer drug widely used in diabetic patients. It has a short half-life, poor lipid solubility, and is not easily penetrable through biological membranes.

[0003] At present, the only way to clinically apply insulin is subcutaneous injection. Although it has a fast onset and high bioavailability, long-term injection brings great pain, inconvenience, and poor compliance to patients, and is often accompanied by the occurrence of complications. Therefore, in order to meet the needs of diabetic patients, extend the action time of insulin in the body, and better treat diabetes, the development of non-injection administration routes has become a research hotspot. Currently, the main non-injection administration routes under study include pulmonary administration, nasal administration, transdermal administration, oral mucosal administration, and oral administration, etc. Although the above administration methods can improve the bioavailability of insulin to a certain extent, long-term administration is likely to cause complications such as pulmonary edema, skin inflammation, and hematoma. Among all non-injection administration routes, oral administration is the safest, most convenient, and has the best patient compliance. Its greatest advantage is to avoid complications at the administration site. After oral administration, insulin enters the systemic circulation through the hepatic portal vein and can directly participate in the metabolism of sugar in the liver, simulating the action of endogenous insulin secreted by the pancreas, which can reduce the fluctuation of blood glucose in the body, thereby reducing damage to blood vessels. It is the best and preferred administration route for insulin.

[0004] Natural polymer materials have the characteristics of low cost, easy modification, high safety, good biocompatibility, etc., and have become the main materials for preparing nanocarriers. In this invention, the natural polysaccharide polymer material bacterial cellulose (BC) is used as the main body, and after chemical modification, an amphiphilic bacterial cellulose carrier with the ability to penetrate cell membrane phospholipids and the function of resisting P-gp efflux is obtained. Insulin is encapsulated by the ultrasonic cavitation self-assembly method to construct an oral insulin nanomicelle. This nanomicelle has a high encapsulation efficiency and drug loading capacity, and a good sustained-release effect. It can be absorbed by the blood to the greatest extent to achieve a good hypoglycemic effect and improve the oral bioavailability of insulin. In vitro cell and in vivo safety evaluations confirm that the nanomicelle has no toxic or side effects, can enhance the convenience and compliance of patients' medication, and can also reflect the physiological advantages of oral administration, having important scientific research application value and clinical significance. Summary of the Invention

[0005] The purpose of the present invention is to provide an amphiphilic bacterial cellulose-encapsulated insulin micelle and its preparation method. The amphiphilic bacterial cellulose-encapsulated insulin micelle provided by the present invention can avoid the irritation of drugs to the gastrointestinal tract and the destruction of drugs by the complex environment in the gastrointestinal tract during oral administration, effectively protect the drug to pass safely through the gastrointestinal tract environment and be absorbed by the mucus layer of the small intestine, realize the improvement of bioavailability, and can be effectively applied to the treatment of abnormal elevation of the body's blood glucose level.

[0006] In order to achieve the above purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an amphiphilic bacterial cellulose-encapsulated insulin micelle, which includes a micelle carrier and an active drug encapsulated in the micelle carrier; the active drug is insulin; the micelle carrier is an amphiphilic bacterial cellulose carrier, and the micelle carrier is obtained by grafting glabridin (GLD) through an esterification reaction and cocamidopropyl betaine (BP) through an acyl chloride substitution reaction as the hydrophobic and hydrophilic ends respectively onto bacterial cellulose; the amphiphilic bacterial cellulose-encapsulated insulin micelle is obtained through an ultrasonic cavitation self-assembly reaction.

[0008] Preferably, the preparation method of the amphiphilic bacterial cellulose carrier includes the following steps, and the synthetic route diagram is as follows:

[0009]

[0010] Under certain temperature conditions, succinic anhydride is used as a linker to derivatize glabridin (GLD) into compound A; the tetrabutylammonium chloride-dimethyl sulfoxide (TBAC-DMSO) solution of bacterial cellulose, the solution of compound A, and a condensation reagent are mixed to carry out an esterification reaction for a certain time to obtain compound B as the hydrophobic end of the amphoteric bacterial cellulose carrier; cocamidopropyl betaine and oxalyl chloride are subjected to an acyl chloride nucleophilic substitution reaction to obtain compound C as the hydrophilic end of the amphoteric bacterial cellulose carrier; the solution of compound C, the solution of compound B, and a condensation reagent are mixed to carry out an acyl chloride electrophilic substitution reaction for a certain time to obtain the amphoteric bacterial cellulose carrier.

[0011] The present invention provides a method for preparing an amphoteric bacterial cellulose-encapsulated insulin micelle according to the above technical solution, comprising the following steps:

[0012] The aqueous solution of the amphoteric bacterial cellulose carrier and the aqueous solution of insulin acid are mixed and subjected to ultrasonic self-assembly for drug loading to obtain the amphoteric bacterial cellulose-encapsulated insulin micelle.

[0013] Preferably, the pH of the aqueous acid solution of insulin is 1.2 to 3.0.

[0014] Preferably, the concentration of the aqueous solution of the amphoteric bacterial cellulose carrier is 0.1 to 2.5 mg / mL.

[0015] Preferably, the concentration of the aqueous acid solution of insulin is 1 to 3 mg / mL.

[0016] Preferably, the mass ratio of the amphoteric bacterial cellulose carrier to insulin is 10:(1 to 5).

[0017] Preferably, the time for ultrasonic drug loading is 5 to 30 min, and the ultrasonic power for ultrasonic drug loading is 90 to 900 W.

[0018] Preferably, after the ultrasonic drug loading, a drug-loaded solution is obtained, and the method further includes centrifuging and separating the drug-loaded solution to obtain a supernatant, and freeze-drying the supernatant to obtain the amphoteric bacterial cellulose-encapsulated insulin micelle; the rotation speed for centrifuging and separating is 5000 to 10000 r / min, and the time for centrifuging and separating is 5 to 15 min.

[0019] Preferably, when the active drug is insulin, the mass content of insulin in the micelle is 5 to 20 wt%.

[0020] Preferably, the particle size of the oral insulin nano-micelle is 50 to 240 nm.

[0021] The present invention provides the use of the amphoteric bacterial cellulose carrier according to the above technical solution or the amphoteric bacterial cellulose-encapsulated insulin micelle prepared by the preparation method according to the above technical solution in the preparation of a drug for treating diabetes.

[0022] The present invention provides an amphoteric bacterial cellulose-encapsulated insulin micelle, which comprises a micelle carrier and an active drug in the micelle carrier; the active drug is insulin; the micelle carrier is an amphoteric bacterial cellulose carrier, which is obtained by grafting glabridin onto bacterial cellulose through an esterification reaction and cocamidopropyl betaine onto bacterial cellulose through an acyl chloride substitution reaction as the hydrophobic and hydrophilic ends respectively, and can form a micelle structure with the hydrophilic end (cocamidopropyl betaine) facing outward and the hydrophobic end (glabridin) facing inward in water, and can effectively encapsulate the drug. The amphoteric bacterial cellulose carrier has strong biocompatibility and safety, and can effectively protect the drug from being destroyed by the gastrointestinal fluid environment as a micelle carrier, which is beneficial to the drug being transported to the small intestine and realizing the improvement of bioavailability; moreover, bacterial cellulose is a natural polymer polysaccharide material, which has high biological safety and certain immunomodulatory effects itself. The present invention uses the amphoteric bacterial cellulose carrier as a drug micelle carrier to encapsulate insulin and act on the body, which can stabilize the blood sugar balance in the body, can, to a certain extent, ensure that the drug is inactivated due to the destruction by the complex environment in the gastrointestinal tract when taking the oral drug, enables the drug to safely reach the absorption site in the body, and is beneficial to the long-term treatment of diabetic patients with the drug.

[0023] The present invention provides a preparation method of the amphoteric bacterial cellulose-encapsulated insulin micelle described in the above technical solution. The obtained amphoteric bacterial cellulose-encapsulated insulin micelle has a high drug loading amount and a good encapsulation efficiency, and the preparation method provided by the present invention is simple, the reaction is mild, the materials and solvents used in the preparation are non-toxic, and it has good environmental benefits and can be applied to industrial production. Description of the Drawings

[0024] Figure 1 FTIR diagrams of compound B (BC-GLD) and amphoteric bacterial cellulose carrier (BP-BC-GLD);

[0025] Figure 2 For GLD and BC-GLD 1 1H-NMR spectra;

[0026] Figure 3 Critical micelle concentration diagram of BP-BC-GLD;

[0027] Figure 4 Cell viability diagram of BP-BC-GLD;

[0028] Figure 5 Freeze-dried sample diagram of amphoteric bacterial cellulose-encapsulated insulin (BP-BC-GLD-INS), state of the aqueous solution of the BP-BC-GLD-INS freeze-dried sample;

[0029] Figure 6For the average particle size distribution diagrams of BP-BC-GLD and BP-BC-GLD-INS;

[0030] Figure 7 For the transmission electron microscope images of BP-BC-GLD and BP-BC-GLD-INS;

[0031] Figure 8 For the scanning electron microscope images of BP-BC-GLD and BP-BC-GLD-INS;

[0032] Figure 9 For the scanning energy spectrum point diagrams and C, N element analysis diagrams of BP-BC-GLD and BP-BC-GLD-INS;

[0033] Figure 10 For the FTIR diagrams of BP-BC-GLD, INS, BP-BC-GLD-INS and physical mixture;

[0034] Figure 11 For the XRD diagrams of BP-BC-GLD, INS, BP-BC-GLD-INS and physical mixture;

[0035] Figure 12 For the DSC diagrams of BP-BC-GLD, INS, BP-BC-GLD-INS and physical mixture;

[0036] Figure 13 For the TG curve diagrams of BP-BC-GLD, INS, BP-BC-GLD-INS and physical mixture;

[0037] Figure 14 For the dissolution curve diagrams of INS and BP-BC-GLD-INS in artificial gastric juice and artificial intestinal juice;

[0038] Figure 15 For the hypoglycemic effect diagrams of INS, BP-BC-GLD-INS and injected insulin;

[0039] Figure 16 For the blood drug concentration-time diagrams of INS, BP-BC-GLD-INS and injected insulin;

[0040] Figure 17 For the H&E staining diagrams of the main organ sections of mice in different administration groups; Specific implementation manners

[0041] The present invention provides an amphoteric bacterial cellulose-loaded insulin, which includes a micelle carrier and an active drug loaded in the micelle carrier; the active drug is insulin; the micelle carrier is an amphoteric bacterial cellulose carrier, and the micelle carrier is obtained by grafting glabridin through an esterification reaction and cocamidopropyl betaine through an acyl chloride substitution reaction as the hydrophobic and hydrophilic ends respectively onto bacterial cellulose; the amphoteric bacterial cellulose-loaded insulin is obtained through an ultrasonic cavitation self-assembly reaction.

[0042] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well-known to those skilled in the art.

[0043] In the present invention, the preparation method of the amphoteric bacterial cellulose preferably includes the following steps:

[0044] Under certain temperature conditions, a bacterial cellulose solution, a condensation reagent, and a glabridin solution are mixed for an esterification reaction for a certain period of time to obtain a bacterial cellulose-glabridin polymer as the hydrophobic end of the amphoteric bacterial cellulose micelle carrier; the bacterial cellulose-glabridin polymer solution, an acyl chloride substituting agent, a cocamidopropyl betaine solution, and a condensation reagent are mixed for an acyl chloride electrophilic substitution reaction for a certain period of time as the hydrophilic end of the amphoteric bacterial cellulose micelle carrier, and the solution is post-treated to obtain the amphoteric bacterial cellulose micelle carrier.

[0045] In the present invention, the bacterial cellulose solution is specifically preferably a TBAC-DMSO solution.

[0046] In the present invention, the glabridin solution is specifically preferably a DMSO solution.

[0047] In the present invention, the condensation reagent is specifically preferably a first cross-linking agent, a second cross-linking agent, and a third cross-linking agent.

[0048] In the present invention, the first cross-linking agent is specifically preferably succinic anhydride.

[0049] In the present invention, the second cross-linking agent is specifically preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC·Hcl).

[0050] In the present invention, the third cross-linking agent is specifically preferably 4-dimethylaminopyridine (DMAP).

[0051] In the present invention, the acyl chloride substituting agent is specifically preferably oxalyl chloride.

[0052] In the present invention, the cocamidopropyl betaine solution is specifically preferably methanol.

[0053] In the present invention, the specific implementation method of the esterification reaction is preferably:

[0054] At room temperature, bacterial cellulose was added to the TBAC-DMSO solution and completely dissolved, with no visible colloidal substances to the naked eye.

[0055] Under certain temperature conditions, glabridin and the first crosslinking agent were mixed and added to the DMSO solution for carboxyl activation to obtain an activation solution.

[0056] Under the same temperature conditions, the second crosslinking agent and the third crosslinking agent were added to the activation solution for continued activation.

[0057] Under the same temperature conditions, the activation solution was added to the TBAC-DMSO solution of bacterial cellulose for an esterification reaction.

[0058] In the present invention, the molar ratio of the bacterial cellulose to glabridin is preferably 1:(1 - 3), and most preferably 1:2.

[0059] In the present invention, the molar ratio of the bacterial cellulose to the second crosslinking agent and the third crosslinking agent is preferably 1:1.

[0060] In the present invention, the volume ratio of the solution of the bacterial cellulose to glabridin is preferably 2:1.

[0061] In the present invention, the molar ratio of glabridin to the first crosslinking agent is preferably 1:1, the activation temperature is preferably 40 °C, and the activation time is preferably 16 h.

[0062] In the present invention, the molar ratio of the second crosslinking agent to the third crosslinking agent is preferably 1:1, the activation temperature is preferably 40 °C, and the activation time is preferably 30 min.

[0063] In the present invention, the temperature of the esterification reaction is preferably 40 °C, and the time of the esterification reaction is preferably 24 h.

[0064] In the present invention, the specific implementation method of the acyl chloride electrophilic substitution reaction is preferably as follows:

[0065] At room temperature, cocamidopropyl betaine was added to the methanol solution and completely dissolved.

[0066] Under certain temperature conditions, oxalyl chloride solution was slowly added dropwise for an acyl chloride nucleophilic substitution reaction. After completion, the unreacted acyl chloride was removed and dried to obtain a light yellow solid.

[0067] Under the same temperature conditions, a small amount of the third crosslinking agent was added to the system after the esterification reaction for activation.

[0068] Under the same temperature condition, the coconut oil amide phosphate betaine after acyl chloride reaction substitution is dissolved in methanol and slowly added dropwise into the activated system for acyl chloride electrophilic substitution reaction.

[0069] In the present invention, the molar ratio of the bacterial cellulose to the coconut oil amide phosphate betaine is preferably 1:1.

[0070] In the present invention, the molar ratio of the coconut oil amide phosphate betaine to the oxalyl chloride is preferably 1:2.

[0071] In the present invention, the reaction temperature of the acyl chloride nucleophilic substitution reaction is preferably 0 °C, and the reaction time is preferably 6 h.

[0072] In the present invention, the method for removing the unreacted acyl chloride is preferably rotary evaporation drying.

[0073] In the present invention, the activation temperature of the acyl chloride electrophilic substitution reaction is preferably 40 °C, and the activation time is preferably 30 min.

[0074] In the present invention, the temperature of the acyl chloride nucleophilic substitution reaction is preferably 40 °C, and the reaction time is preferably 24 h.

[0075] In the present invention, the esterification reaction and the acyl chloride substitution reaction are preferably carried out under the condition of stirring in a water bath, and the present invention has no special requirements for the specific implementation process of the stirring in the water bath.

[0076] In the present invention, after the synthesis reaction of the amphoteric bacterial cellulose micelle carrier, an amphoteric bacterial cellulose micelle carrier reaction solution is obtained. The present invention preferably performs post-treatment on the amphoteric bacterial cellulose micelle carrier reaction solution to obtain an amphoteric bacterial cellulose micelle carrier. In the present invention, the post-treatment preferably includes: successively adding an alcohol solvent to remove the reaction agent and the condensation reagent, solid-liquid separation, adding water to the solid product obtained by solid-liquid separation for re-dissolution to remove the unreacted bacterial cellulose, and freeze-drying at low temperature. In the present invention, the alcohol solvent is preferably anhydrous ethanol. In the present invention, the volume ratio of the alcohol solvent to the amphoteric bacterial cellulose micelle carrier reaction solution is preferably 10:1. In the present invention, the solid-liquid separation is preferably centrifugal separation, the rotation speed of the centrifugal separation is preferably 10000 r / min, and the centrifugal separation time is preferably 10 min.

[0077] The oral insulin nano-micelles provided by the present invention include an active drug encapsulated in the micelle carrier; the active drug is insulin.

[0078] In the present invention, the insulin is preferably encapsulated in the hydrophobic inner shell of the micelle carrier.

[0079] In the present invention, the mass content of insulin in the oral insulin nanomicelles is preferably 5-20 wt%, more preferably 10-20 wt%, and further preferably 15-20 wt%.

[0080] In the present invention, the particle size of the oral insulin nanomicelles is preferably 50-240 nm, more preferably 200-240 nm.

[0081] The oral insulin nanomicelles provided by the present invention select natural polymer polysaccharide material bacterial cellulose as the main body, and use glabridin through an esterification reaction and cocamidopropyl betaine through an acyl chloride substitution reaction as the hydrophobic and hydrophilic ends respectively to be grafted onto the bacterial cellulose together to synthesize an amphoteric bacterial cellulose micelle carrier, and insulin is encapsulated through an ultrasonic cavitation self-assembly reaction. The micelle carrier, as the shell of the drug, can avoid the destruction of insulin by gastrointestinal proteases, effectively protect insulin to safely pass through the gastrointestinal environment and be absorbed by the mucus layer of the small intestine, realize the improvement of the water solubility and bioavailability of insulin, and be effectively applied to the treatment of diabetes.

[0082] The present invention provides a preparation method of the oral insulin nanomicelles described in the above technical solution, including the following steps:

[0083] Mix an aqueous solution of the amphoteric bacterial cellulose micelle carrier and an acidic aqueous solution of insulin for ultrasonic drug loading to obtain the oral insulin nanomicelles.

[0084] In the present invention, the aqueous solution of the amphoteric bacterial cellulose micelle carrier is specifically preferably a distilled aqueous solution of the amphoteric bacterial cellulose micelle carrier.

[0085] In the present invention, the acidic aqueous solution of insulin is specifically preferably a hydrochloric acid aqueous solution.

[0086] In the present invention, the pH of the hydrochloric acid aqueous solution of insulin is preferably 1.2-3.0, more preferably 2.0-3.0.

[0087] In the present invention, the concentration of the aqueous solution of the amphoteric bacterial cellulose micelle carrier is preferably 0.1-2.5 mg / mL, more preferably 0.5-1.5 mg / mL.

[0088] In the present invention, the concentration of the hydrochloric acid aqueous solution of insulin is preferably 1-3 mg / mL, preferably 1.5-2.5 mg / mL.

[0089] In the present invention, the mass ratio of the amphoteric bacterial cellulose micelle carrier to insulin is preferably 10:(1-5), more preferably 10:(2-4).

[0090] In the present invention, the ultrasonic power of the ultrasonic drug loading is preferably 90-900 W, more preferably 100-300 W.

[0091] In the present invention, the time for ultrasonic drug loading is preferably 5 - 30 min, more preferably 15 - 25 min.

[0092] In the present invention, the ultrasonic drug loading is specifically preferably the probe ultrasonic method.

[0093] In the present invention, after ultrasonic drug loading, a drug-loaded solution is obtained. The present invention preferably further includes removing the solvent from the drug-loaded solution, centrifuging to obtain a supernatant, and freeze-drying the supernatant to obtain the oral insulin nanomicelles.

[0094] In the present invention, the rotation speed of the centrifugation is 5000 - 10000 r / min, more preferably 6300 r / min.

[0095] In the present invention, the time for centrifugation is preferably 8 - 15 min, more preferably 10 min.

[0096] The preparation method provided by the present invention is simple, the reaction is mild, the materials and solvents used in the preparation are non-toxic, and it has good environmental benefits and can be applied to industrial production.

[0097] The present invention provides the application of the amphoteric bacterial cellulose micelle carrier described in the above technical solution or the oral insulin nanomicelles prepared by the preparation method described in the above technical solution in the preparation of drugs for treating diabetes.

[0098] For the oral insulin nanomicelles prepared by the present invention using this method, the drug loading amount is preferably 15 - 20%, and the encapsulation efficiency is preferably 70 - 90%.

[0099] In the present invention, insulin has poor water solubility. After drug loading, the insulin is encapsulated in the zwitterionic micelles based on bacterial cellulose, which can improve its solubility in water.

[0100] The present invention provides a drug, which includes a drug active ingredient and a drug excipient. The drug active ingredient is the oral insulin micelles prepared by the preparation method described in the above technical solution.

[0101] In the present invention, the drug excipient is a pharmaceutically acceptable excipient.

[0102] The present invention has no special limitation on the types of the pharmaceutically acceptable excipients, and the excipients well-known to those skilled in the art can be used.

[0103] In the present invention, the drug is a drug for treating diabetes.

[0104] In the present invention, the dosage form of the drug is preferably an oral preparation.

[0105] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0106] Example 1

[0107] Succinic anhydride and glabridin were added to a DMSO solution at a molar ratio of 1:1. After dissolution, EDC·HCl and DMAP (molar ratio 1:1) were added to activate the carboxyl group for 30 min. Then it was added to a TBAC-DMSO solution of bacterial cellulose (molar ratio with glabridin 1:1), and an esterification reaction was carried out at 40 °C for 24 h. A small amount of DMAP was added to activate for 30 min, and the solution of cocamidopropyl betaine phosphate after acyl chloride reaction substitution was slowly dropped into the TBAC-DMSO system, and an acyl chloride electrophilic substitution reaction was continued at 40 °C for 24 h. The reaction solution after the reaction was dropped into an ethanol solution with a volume 10 times that of the reaction solution. After stirring, it was centrifuged at 8000 rpm for 15 min and repeated once to remove the reaction agent and condensation reagent. The centrifuged precipitate was redissolved in water and freeze-dried to obtain an amphoteric bacterial cellulose micelle carrier;

[0108] The freeze-dried BP-BC-GLD was redissolved in distilled water, and insulin was dissolved in a hydrochloric acid aqueous solution with a pH of 1.2. The insulin hydrochloric acid aqueous solution with a concentration of 1 mg / mL was slowly added to the BP-BC-GLD aqueous solution with a concentration of 0.1 mg / mL. The mass ratio of BP-BC-GLD to insulin was 10:1. The probe ultrasonic method was used to enable BP-BC-GLD to encapsulate insulin. The ultrasonic drug loading time was 30 min, and the ultrasonic drug loading power was 90 W;

[0109] After ultrasonic drug loading, it was centrifuged at 10000 rpm for 15 min, and the supernatant was taken and freeze-dried to obtain an oral insulin nanomicelle;

[0110] The content of insulin in BP-BC-GLD-INS was detected by HPLC method, and the drug loading amount and encapsulation efficiency of insulin were calculated. The drug loading amount and encapsulation efficiency were 12.85% and 55.71% respectively.

[0111] Example 2

[0112] Succinic anhydride and glabridin were added to a DMSO solution at a molar ratio of 1:1. After dissolution, EDC·HCl and DMAP (molar ratio 1:1) were added to activate the carboxyl group for 30 min. Then it was added to a TBAC-DMSO solution of bacterial cellulose (molar ratio with glabridin 1:3), and esterification reaction was carried out at 40 °C for 24 h. A small amount of DMAP was added to activate for 30 min, and the solution of cocamidopropyl betaine after acyl chloride reaction substitution was slowly added dropwise into the TBAC-DMSO system, and acyl chloride electrophilic substitution reaction was continued at 40 °C for 24 h. The reaction solution after reaction was added dropwise to an ethanol solution with a volume 10 times that of the reaction solution, stirred and centrifuged at 12000 rpm for 5 min to remove the reactants and condensation reagents. The centrifuged precipitate was redissolved in water and freeze-dried to obtain an amphoteric bacterial cellulose micelle carrier;

[0113] The freeze-dried BP-BC-GLD was redissolved in distilled water, and insulin was dissolved in a hydrochloric acid aqueous solution with a pH of 3.0. The hydrochloric acid aqueous solution of insulin with a concentration of 3 mg / mL was slowly added to the aqueous solution of BP-BC-GLD with a concentration of 2.5 mg / mL. The mass ratio of BP-BC-GLD to insulin was 10:4. The probe ultrasonic method was used to make BP-BC-GLD encapsulate insulin. The ultrasonic drug loading time was 5 min, and the ultrasonic drug loading power was 900 W;

[0114] After ultrasonic drug loading, it was centrifuged at 5000 rpm for 5 min, and the supernatant was taken and freeze-dried to obtain oral insulin nanomicelles;

[0115] The content of insulin in BP-BC-GLD-INS was detected by HPLC method, and the drug loading amount and encapsulation efficiency of insulin were calculated. The drug loading amount and encapsulation efficiency were 15.67% and 73.33% respectively.

[0116] Example 3

[0117] Succinic anhydride and glabridin were added to a DMSO solution at a molar ratio of 1:1. After dissolution, EDC·HCl and DMAP (molar ratio 1:1) were added to activate the carboxyl group for 30 min. Then it was added to a TBAC-DMSO solution of bacterial cellulose (molar ratio with glabridin 1:2), and esterification reaction was carried out at 40 °C for 24 h. A small amount of DMAP was added to activate for 30 min, and the solution of cocamidopropyl betaine after acyl chloride reaction substitution was slowly added dropwise into the TBAC-DMSO system, and acyl chloride electrophilic substitution reaction was continued at 40 °C for 24 h. The reaction solution after reaction was added dropwise to an ethanol solution with a volume 10 times that of the reaction solution, stirred and centrifuged at 10000 rpm for 10 min, repeated 3 times, to remove the reactants and condensation reagents. The centrifuged precipitate was redissolved in water and freeze-dried to obtain an amphoteric bacterial cellulose micelle carrier;

[0118] The freeze-dried BP-BC-GLD was redissolved in distilled water, and insulin was dissolved in hydrochloric acid aqueous solution with a pH of 2.0. The hydrochloric acid aqueous solution of insulin with a concentration of 3 mg / mL was slowly added to the aqueous solution of BP-BC-GLD with a concentration of 1 mg / mL. The mass ratio of BP-BC-GLD to insulin was 10:3. The probe ultrasonic method was used to load insulin with BP-BC-GLD. The ultrasonic drug loading time was 20 min, and the ultrasonic drug loading power was 360 W;

[0119] After ultrasonic drug loading, it was centrifuged at 6300 rpm for 15 min, and the supernatant was taken for freeze-drying to obtain oral insulin nanomicelles;

[0120] The content of insulin in BP-BC-GLD-INS was detected by HPLC method, and the drug loading amount and encapsulation efficiency of insulin were calculated. The drug loading amount and encapsulation efficiency were 16.16% and 78.70% respectively.

[0121] Example 4

[0122] Succinic anhydride and glabridin were added to the DMSO solution at a molar ratio of 1:1. After dissolution, EDC·HCl and DMAP (molar ratio of 1:1) were added, and the carboxyl group was activated for 30 min. Then it was added to the TBAC-DMSO solution of bacterial cellulose (molar ratio of 1:2 to glabridin), and the esterification reaction was carried out at 40 °C for 24 h. A small amount of DMAP was added for activation for 30 min, and the solution of cocamidopropyl betaine after acyl chloride reaction substitution was slowly added dropwise into the TBAC-DMSO system, and the acyl chloride electrophilic substitution reaction was continued at 40 °C for 24 h. The reaction solution after the reaction was dropped into an ethanol solution with a volume 10 times that of the reaction solution, stirred and then centrifuged at 10000 rpm for 10 min to remove the reaction agent and condensation agent. The centrifuged precipitate was redissolved in water and freeze-dried to obtain an amphoteric bacterial cellulose micelle carrier;

[0123] The freeze-dried BP-BC-GLD was redissolved in distilled water, and insulin was dissolved in hydrochloric acid aqueous solution with a pH of 2.0. The hydrochloric acid aqueous solution of insulin with a concentration of 2 mg / mL was slowly added to the aqueous solution of BP-BC-GLD with a concentration of 1 mg / mL. The mass ratio of BP-BC-GLD to insulin was 10:3. The probe ultrasonic method was used to load insulin with BP-BC-GLD. The ultrasonic drug loading time was 25 min, and the ultrasonic drug loading power was 180 W;

[0124] After ultrasonic drug loading, it was centrifuged at 6300 rpm for 10 min, and the supernatant was taken for freeze-drying to obtain oral insulin nanomicelles;

[0125] The content of insulin in BP-BC-GLD-INS was detected by HPLC method, and the drug loading amount and encapsulation efficiency of insulin were calculated. The drug loading amount and encapsulation efficiency were 16.88% and 80.58% respectively.

[0126] Test Example 1

[0127] The physicochemical structure of the amphoteric bacterial cellulose micelle carriers prepared in Examples 1 to 4 was characterized. Figure 1 and Figure 2 As shown:

[0128] Figure 1 a is the FTIR graph of BC-GLD. Figure 1 b is the FTIR graph of BP-BC-GLD; Figure 2 a is GLD 1 H-NMR spectrum, Figure 2 Where b is BC-GLD 1 H-NMR spectrum.

[0129] Figure 1 It can be seen that in the infrared spectrum of BC-GLD, in addition to maintaining the original characteristic peak of BC, the peak at 1647cm -1 and 1566cm -1 The infrared characteristic peaks of GLD appeared at two locations, and at 1734 cm -1 The new characteristic peak at 2964cm is due to the OC=O characteristic peak formed after BC combines with succinic anhydride. -1 The peak at 1187 cm-1 is more obvious, indicating that BC forms a hydrogen bond after combining with GLD. Therefore, after coupling with succinic anhydride, BC and GLD are successfully chemically combined to form the hydrophobic end of the bacterial cellulose zwitterionic micelle. -1 and 1030cm -1 The peaks of BP-BC-GLD are 1719cm-1 and 1729cm-2, which are caused by the stretching vibration of P=O and PO in the phosphate group. -1 The characteristic peak of C=O generated by the acyl chloride substitution reaction appears at 1032 cm -1 The PO characteristic peak appeared at , and the peak shape near the P=O characteristic peak was more obvious. This showed that BP, as the hydrophilic end, chemically combined with BC-GLD through acyl chloride substitution reaction in structure, and successfully constructed the amphoteric bacterial cellulose micelle carrier.

[0130] Figure 2 It can be seen that GLD 1 H-NMR spectrum, where the chemical shift of each hydrogen position of GLD is represented by letters, 1H-NMR (δ, ppm): 1.34 (a + b), 2.50 (g), 3.34 (h), 3.95 (i), 5.66 (c), 6.19 (m), 6.55 (e), 6.85 (d + f + k), 9.11 (n), 9.38 (i). For the 1 H-NMR spectrum results of BC-GLD polymer Figure 2 as shown in b of, it can be seen that the hydrogen of bacterial cellulose mainly appears between 2.0 - 4.0 ppm, and the peaks appearing between 6.5 - 8.5 ppm correspond to the proton signals of n, l, d, f, k, and c of GLD, and affected by the polymer, the chemical shift of hydrogen undergoes a chemical shift, proving that GLD has been successfully combined with BC.

[0131] Test Example 2

[0132] Characterize the performance of the amphoteric bacterial cellulose micelle carriers prepared in Examples 1 - 4. The specific method is as follows:

[0133] 1. Critical micelle concentration (CMC) test, specifically as follows:

[0134] Using pyrene as a hydrophobic fluorescent probe, the CMC of BP-BC-GLD was determined by the fluorescence probe method. Add 200 μL of a pyrene solution made of acetone with a concentration of 1.2x10 -4 M to a 10 mL brown tube, and dry and volatilize it in an oven to remove acetone. Add BP-BC-GLD solutions with various concentrations (3.125×10 -4 ~1.28 mg / mL) to the tube containing pyrene. Ultrasonic for 30 min and equilibrate at 37 °C in an oven for 24 h. Measure it with a fluorescence spectrophotometer. Use 340 nm as the excitation wavelength, and the emission wavelength is 350 - 600 nm. Pyrene is easily affected by external conditions, and its main characteristics are I1 = 373 nm and I3 = 391 nm. Therefore, determine the CMC value of BP-BC-GLD with the value of I1 / I3. The test results are as Figure 3 shown;

[0135] Figure 3 It can be seen that when BP is used as the hydrophilic end and coupled with BC-GLD, its CMC value is 0.0049 mg / mL. When the CMC value is less than 0.135 mg / mL, the micelles are sufficient to resist rapid dissociation after oral administration. The smaller the CMC value, when the drug is mixed with a large amount of water, gastric juice, etc. after oral administration, it is still in the micelle form and transports the drug to the intestinal epithelial cells.

[0136] 2. Cytotoxicity test of BP-BC-GLD, specifically as follows:

[0137] The LLC cells of mice were selected as a model to evaluate the cytotoxicity of the zwitterionic micelles of BP-BC-GLD. LLC was cultured with 10% fetal bovine serum + 1% penicillin-streptomycin + 90% high-glucose DMEM medium. After digestion with trypsin containing 0.25% EDTA and centrifugation at 1500 r / min for 10 min, subculture or preparation of the suspension was carried out. The MTT method was used to determine the cytotoxic effect of the zwitterionic micelles of BP-BC-GLD on mouse LLC cells. The centrifuged cells were added to the fresh and complete medium, and seeded into a 96-well plate at a density of 5×10 3 / well, with a volume of 100 μL / well, and incubated at 37 °C and 5% CO2 for 12 h. The BP-BC-GLD solution was formulated into different concentrations (1 μg / mL, 5 μg / mL, 15 μg / mL, 30 μg / mL, 60 μg / mL, 120 μg / mL, 250 μg / mL, 500 μg / mL) with blank medium. 100 μL of each concentration sample was added to the 96-well plate respectively to replace the original medium, with 3 replicates, and blank medium was used as the control. Then, 10 μL of MTT solution was added to each well, and 100 μL of Formazan solubilization solution was added to each well. After proper mixing, the incubation was continued for 3 - 4 h in the incubator until all Formazan was dissolved under an ordinary optical microscope. The absorbance was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm. The cell survival rate formula is as follows:

[0138]

[0139] ODe is the average absorbance of the experimental group, and ODc is the average absorbance of the control group.

[0140] Figure 4 It can be seen that after co-incubation of the BP-BC-GLD micelles with cells, the cell viability did not decrease significantly with the increase of the micelle concentration. Even when the polymer concentration reached 500 μg / mL, no cytotoxicity was shown, and the survival rate of LLC cells was still above 90%. The results indicate that the amphiphilic ionic micelles formed by BP-BC-GLD have no obvious cytotoxicity to normal cells and have high biocompatibility.

[0141] Test Example 3

[0142] The morphologies of the BP-BC-GLD and BP-BC-GLD-INS prepared in Examples 1 - 4 were tested, as specifically shown in Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 and Figure 9 shown below:

[0143] Figure 5a is the freeze-dried sample of BP-BC-GLD-INS prepared in the example, Figure 5 b is the state of the BP-BC-GLD-INS freeze-dried sample prepared in the example after being reconstituted in water; Figure 6 a is the average particle size distribution diagram of BP-BC-GLD prepared in the example, Figure 6 b is the average particle size distribution diagram of BP-BC-GLD-INS prepared in the example; Figure 7 Figure a is a transmission electron microscope image of BP-BC-GLD prepared in the example. Figure 7 b is a transmission electron microscopy image of BP-BC-GLD-INS prepared in the example; Figure 8 a, b, and c are scanning electron microscope images of BP-BC-GLD prepared in the example. Figure 8 d, e, and f are scanning electron micrographs of BP-BC-GLD-INS prepared in the examples; Figure 9 a is the scanning energy spectrum point diagram of BP-BC-GLD prepared in the example, Figure 9 b is the scanning energy spectrum point diagram of BP-BC-GLD-INS prepared in the example, Figure 9 Figure c is the C and N elemental analysis diagram of BP-BC-GLD and BP-BC-GLD-INS prepared in the examples.

[0144] Figure 5 , Figure 6 and Figure 7 It can be seen that after BP-BC-GLD-INS is redissolved in distilled water, it has good water solubility, and the nanoparticles are evenly dispersed and relatively stable. In the transmission electron microscope, Figure a clearly shows that the blank micelles are black and uniformly spherical structures with a particle size of about 100nm. Figure b shows that the micelles after drug loading are core-shell structures, with the core being a black solid part tightly wrapped by a transparent shell, and the particle size distribution is about 200nm, which is consistent with the particle size of the average particle size distribution diagram measured by DLS.

[0145] Figure 8 and Figure 9It can be seen that at scales of 10 μm and 5 μm (a, b), the fibrous structure of BC can be observed in BP-BC-GLD in the dry state. At a scale of 1 μm (c), the surface shows a smooth porous structure. In the case of BP-BC-GLD-INS, in all three states, spherical structures of various sizes and irregular shapes can clearly be seen distributed on the surface of the fibrous structure of the carrier (d, e), and they are tightly wrapped by the carrier, showing a swollen state with plump drug particles (f). Elemental analysis of C and N before and after micelle drug loading was carried out by the EDS point scanning mode of scanning electron microscopy. Insulin is a macromolecular protein polypeptide drug, and its elemental composition mainly consists of five elements: C, H, N, O, and S. The proportions of C and H elements are relatively large, while the proportion of N element is relatively small. By calculating the theoretical C / N mass ratio and atomic number ratio of BP-BC-GLD and BP-BC-GLD-INS compounds before and after loading INS, it was found that the theoretical C / N mass ratio and atomic number ratio of the BP-BC-GLD carrier are significantly higher than those of the C / N mass and atomic number ratio measured for the BP-BC-GLD-INS nanomicelles, indicating indirectly that INS was successfully loaded into the BP-BC-GLD carrier.

[0146] Test Example 4

[0147] The BP-BC-GLD and BP-BC-GLD-INS prepared in Examples 1 to 4 were subjected to physical and chemical characterization tests, specifically as Figure 10 、 Figure 11 、 Figure 12 and Figure 13 shown:

[0148] Figure 10 is the FTIR diagram of BP-BC-GLD, INS, BP-BC-GLD-INS, and physical mixture prepared in the examples; Figure 11 is the XRD diagram of BP-BC-GLD, INS, BP-BC-GLD-INS, and physical mixture prepared in the examples; Figure 12 is the DSC diagram of BP-BC-GLD, INS, BP-BC-GLD-INS, and physical mixture prepared in the examples; Figure 13 is the TG curve diagram of BP-BC-GLD, INS, BP-BC-GLD-INS, and physical mixture prepared in the examples.

[0149] Figure 10 、 Figure 11 、 Figure 12 and Figure 13 It can be seen that in the infrared spectrum of BP-BC-GLD-INS, corresponding characteristic peaks also appear at 1651 cm -1 and 1537 cm -1 . After drug loading, the structural characteristics of the micelles themselves were not changed. At 2967 cm-1 The hydrogen bonding force between micelle molecules has not weakened. In the physical mixing state, the characteristic peaks are similar to those of INS, but the peak shape is weakened, and there is no characteristic peak of micelles, indicating that the carrier has successfully encapsulated insulin without changing its own structure. From the XRD, DSC and TG diagrams, it can be seen that INS is a crystalline structure. After drug loading, the diffraction peak of INS completely disappears in the drug-loaded micelles, and it changes from a crystalline state to an amorphous state. It is successfully encapsulated in BP-BC-GLD micelles and presents an amorphous state in the hydrophobic core of the micelle, which is conducive to the oral absorption of INS.

[0150] Test Example 5

[0151] The BP-BC-GLD-INS prepared in Examples 1 to 4 was subjected to functional evaluation test, and the specific method was as follows:

[0152] 1. Dissolution test in artificial gastric juice and artificial intestinal juice. The specific steps are as follows:

[0153] Prepare 2 portions of 25 mL of artificial gastric juice and artificial intestinal juice, respectively. Dissolve 5 mg of INS and 29.62 mg of BP-BC-GLD (including 5 mg of INS) in a small amount of deionized water, pour into a cellulose dialysis bag with a molecular weight cutoff of 50,000 Da, and then put into artificial gastric juice and artificial intestinal juice. Put 4 beakers into a 37°C water bath at a speed of 100 r / min. Take out 1 mL of the dissolution solution at 12 time points of 0.083, 0.167, 0.25, 0.333, 0.5, 1, 2, 4, 6, 8, 12 and 24 h, and add 1 mL of fresh artificial gastric juice and artificial intestinal juice to the system. The taken INS and BP-BC-GLD-INS are filtered through a 0.22 μm membrane and then tested by HPLC for INS content. Calculate the cumulative release percentage (%) per unit time and draw the drug release curve.

[0154] Figure 14 a in the figure is the dissolution curve of INS and BP-BC-GLD-INS in artificial gastric juice. Figure 14In Figure b, the dissolution curves of INS and BP-BC-GLD-INS in artificial intestinal fluid are shown. The in vivo environment is extremely complex. To investigate the influence of different environments in the digestive system on the release of INS from micelles, dissolution experiments of INS and BP-BC-GLD-INS were carried out in artificial gastric fluid and artificial intestinal fluid. The release rate and cumulative release amount of INS released from the nanodrug through the dialysis bag were detected. The results showed that the cumulative release rate of the original INS drug was higher than that of BP-BC-GLD-INS in both artificial gastric fluid and artificial intestinal fluid. This indicates that the micelle shell protected INS from being released in the gastric environment, playing a protective and sustained-release role. Similar results were obtained in artificial intestinal fluid. This behavior is beneficial to the application of oral drugs. The main reason for the sustained release is that INS is encapsulated in the core by the micelles. Therefore, the amphoteric bacterial cellulose micelle carrier is a good carrier for hydrophobic oral drugs, increasing the dissolution of the original drug and reducing the side effects of the drug. At this time, insulin overcomes the biochemical barrier of the gastrointestinal tract and enters the mucus layer of the small intestine, where it is released and transported into the blood circulation.

[0155] 2. Hypoglycemic test in rats, the specific steps are as follows:

[0156] The SD rats were fasted for 10 h without water restriction overnight before the experiment, weighed, and their fasting blood glucose values were measured by tail vein blood sampling. Then, 1% STZ solution (dose: 60 mg / kg) was injected intraperitoneally. After 2 h of injection, the rats were fed normally and drank water normally before and after modeling. After 72 h, the weights of the modeled rats were measured and their fasting blood glucose values were measured (fasted for 10 h the previous night). If the body weight of the rats decreased abnormally and the blood glucose value was greater than 16.7 mmol / L, the modeling was successful.

[0157] The modeled SD rats were evenly divided into 4 groups, with 5 rats in each group, namely the blank group, the insulin group (50 IU / kg), the micelle-encapsulated insulin group (50 IU / kg), and the injected insulin group (5 IU / kg). Before the experiment, the rats were fasted for 12 h, and their fasting body weights and fasting blood glucose were measured. The dosage of gavage or subcutaneous injection for each rat was calculated according to the fasting body weight for drug administration. Blood was taken from the tail vein at different time intervals (0.5, 1, 2, 3, 4, 6, 8, 12, 24 h), and the blood glucose value was measured with a blood glucose meter to evaluate the hypoglycemic effect.

[0158] Figure 15The hypoglycemic effect diagrams of INS, BP-BC-GLD-INS and injected insulin are shown. The blood glucose level in the subcutaneous insulin injection group decreased rapidly within 2 h, reaching a minimum of 5.3 mmol / L, but then started to rise, and approached the blood glucose level before modeling after 6 h. This indicates that although subcutaneous injection can quickly restore the blood glucose level to normal, it cannot maintain the blood glucose stability in the body for a long time, mainly because the half-life of injected insulin in the body is relatively short. The large fluctuations in subcutaneous insulin injection may also cause harm to the body and cannot meet the long-term and stable requirements of blood glucose levels in diabetic patients. However, in the BP-BC-GLD-INS group, the increase in blood glucose level before 1 h may be due to the stress response in rats caused by gavage and tail vein blood sampling, and the insulin was not effectively absorbed in a short time, resulting in an increase in blood glucose. After 1 h, insulin overcame multiple barriers in the body and was effectively absorbed by the body, achieving a slow release effect. The blood glucose level dropped to 7.8 mmol / L after 24 h, approaching the normal range. Combining with the in vitro release data, it can be found that BP-BC-GLD-INS nanomicelles have a slow release effect in the body. This is mainly because after insulin is encapsulated by BP-BC-GLD, it has a resistance to gastrointestinal proteases. The formed nanomicelles have a small particle size (about 200 nm), can effectively overcome the full contact with the intestinal mucosa, prolong the residence time in the intestine, and thus facilitate the absorption by intestinal epithelial cells, achieving a slow release in the blood, which plays a key role in maintaining blood glucose stability.

[0159] 3. Bioavailability test, the specific steps are as follows:

[0160] Female SD rats with an average body weight of 200 - 220 g were fasted overnight before the experiment and allowed free access to water. Fifteen rats were randomly divided into 3 groups, with 5 rats in each group, namely the insulin group (50 IU / kg), the insulin-loaded micelle group (50 IU / kg), and the injected insulin group (5 IU / kg). After weighing the rats, gavage and subcutaneous injection were administered. Orbital blood sampling was performed on anesthetized rats at 0.083, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after administration. After centrifuging the blood samples at 4°C and 12,000 r for 10 min, the serum was transferred to a new centrifuge tube and stored at -20°C for later use. The INS concentration in the serum at different time points was measured using a human insulin kit. And the pharmacokinetic analysis of the blood drug concentration was performed using DAS 2.0 software.

[0161] Figure 16 The blood drug concentration-time diagrams of INS, BP-BC-GLD-INS and injected insulin are shown; Tables 1, 2 and 3 are the pharmacokinetic parameter tables of INS, BP-BC-GLD-INS and injected insulin. The C max value of INS is 114.25 nIU / mL, and the AUC 0-24his 1.22 nIU / mL*h. While the C value of BP-BC-GLD-INS max is 234.71 nIU / mL and the AUC 0-24h is 3.98 nIU / mL, which are 2.05 and 3.26 times that of INS respectively. This is because the BP-BC-GLD-INS nanomicelles provide INS with a smaller particle size and a hydrophilic outer shell, having a higher saturation solubility in gastrointestinal fluid, increasing the drug concentration gradient between the gastrointestinal tract and the blood, and improving the absorption of INS. The T max value of BP-BC-GLD-INS is 12 h. The reason for the delay may be that the micelles can inhibit the efflux of P-gp. At the same time, the bioadhesion of the BP-BC-GLD micelles allows INS to stay in the intestine for a longer time, greatly improving the bioavailability of INS. While the C max value of subcutaneous insulin injection is 450.66 nIU / mL, and the AUC 0-24h is 2.28 nIU / mL*h, which are 3.94 and 1.87 times that of the original drug respectively, but the T max value is 2 h. Compared with the high-efficient sustained-release effect of the nanomicelles, it is easily metabolized in the body and loses its drug activity.

[0162] Table 1 Pharmacokinetic parameters of the original insulin drug

[0163]

[0164] Table 2 Pharmacokinetic parameters of amphoteric bacterial cellulose-encapsulated insulin micelles

[0165]

[0166]

[0167] Table 3 Pharmacokinetic parameters of injected insulin

[0168]

[0169] 4. In vivo safety test, the specific steps are as follows:

[0170] Continuous administration for 15 days, collecting the main organs of mice for histopathological examination to evaluate the in vivo biosafety of BP-BC-GLD-INS. The operation process is specifically divided into two parts: paraffin embedding and H&E staining.

[0171] Paraffin embedding: (1) Tissue fixation: The tissue specimens are fixed thoroughly with 10% formaldehyde. (2) Running water rinsing: The fixed tissue specimens are rinsed in running water for 30 min to remove the excess fixative. (3) Trimming: The tissue is cut into appropriate-sized pieces. (4) Dehydration: The tissue is successively immersed in 70%, 80%, 90% and 100% ethanol for 1 hour each to gradually dehydrate the tissue. (5) Clearing: The tissue is immersed in xylene:ethanol (v / v = 1:1) and pure xylene for 20 minutes each to make the tissue clear. (6) Wax infiltration: The cleared tissue is successively placed in melted paraffin (I) (xylene:paraffin (v / v = 2:1)), paraffin (II) (xylene:paraffin (v / v = 1:1)) and pure paraffin for 1.5 hours each. (7) Embedding: The infiltrated tissue is embedded into blocks in sequence for subsequent sectioning.

[0172] H&E staining: (1) Sectioning: The tissues embedded in conventional paraffin are cut into sections with a thickness of 6 μm. (2) Treatment: The sections are treated with xylene twice for 5 minutes each, absolute ethanol twice for 5 minutes each, 90%, 80%, 70% ethanol for 5 minutes each, and immersed in distilled water for 5 minutes. (3) Staining: The sections are successively stained with hematoxylin for 5 minutes, differentiated in hydrochloric acid-ethanol solution for 20 seconds, and stained with eosin for 5 minutes. (4) Washing: The sections are immersed in distilled water for 5 minutes, 70%, 80% alcohol for 5 minutes each, absolute ethanol twice for 5 minutes each, and xylene twice for 5 minutes each. (5) Mounting: Finally, the sections are mounted with neutral balsam and photographed under a microscope at 200× magnification.

[0173] Figure 17 These are H&E staining images of the main organ sections of mice in different dosing groups. After continuously gavaging mice with blank micelles, INS raw drug, and BP-BC-GLD-INS nanomicelles for 15 days, sections of the main organs (heart, liver, spleen, intestine, kidney) were taken for H&E staining to observe pathological changes. In the blank micelle group, the low-dose (50 IU / kg) and high-dose (200 IU / kg) INS raw drug dosing groups, and the low-dose (50 IU / kg) and high-dose (200 IU / kg) BP-BC-GLD-INS nanomicelle dosing groups, compared with the normal saline group, there was an obvious boundary between the cytoplasm and nucleus of all organs, the cells were arranged compactly and regularly, and no obvious organ damage was found. This may be because insulin is a protein drug and will be decomposed and inactivated in the body when taken orally directly, and is metabolized out of the body by the body without any toxic and side effects. It further verifies that BP-BC-GLD-INS, as a relatively safe and non-toxic drug delivery system, has no toxic and side effects on normal tissues and can also improve the hypoglycemic effect, providing great possibilities for future clinical use.

Claims

1. An amphoteric bacterial cellulose-encapsulated insulin micelle, characterized in that The preparation method comprises the following steps: (1) Using succinic anhydride as a linker to derivatize glabridin (GLD) into compound A, and then grafting it with bacterial cellulose (BC) through an esterification reaction to obtain compound B. The synthesis route is as follows: (2) Cocamidopropyl betaine (BP) reacts with oxalyl chloride through an acyl chloride nucleophilic substitution reaction to obtain compound C. The synthesis route is as follows: (3) Compound C and compound B are coupled through an acyl chloride electrophilic substitution reaction to obtain compound D, which is an amphoteric bacterial cellulose carrier. The synthesis route is as follows: (4) Dissolve the amphoteric bacterial cellulose carrier in distilled water to prepare an aqueous solution of the amphoteric bacterial cellulose carrier; (5) Dissolve insulin in hydrochloric acid aqueous solution to prepare an insulin hydrochloric acid aqueous solution; (6) Dropwise add the insulin hydrochloric acid aqueous solution obtained in step (5) to the aqueous solution of the amphoteric bacterial cellulose carrier obtained in step (4) under an ice bath. The obtained material is ultrasonically treated under an ice bath for a self-assembly reaction. After the reaction, the material is transferred to a room temperature environment and centrifuged at 5000 - 10000 r / min for 5 - 15 min. The supernatant solution is separated and freeze-dried to obtain an amphoteric bacterial cellulose-encapsulated insulin micelle; The preparation method of compound B described in step (1) is as follows: Cut the dried bacterial cellulose into small pieces of 0.5 cm 2 and dissolve them in a tetrabutylammonium chloride-dimethyl sulfoxide (TBAC-DMSO) solution. Stir until no gelatinous substances agglomerate to obtain bacterial cellulose solution A; Weigh glabridin and succinic anhydride and add them to the DMSO solution to obtain a mixed solution with a molar ratio of glabridin to succinic anhydride of 1:

1. Heat and stir in a 40 °C water bath for 24 h to obtain solution B; Add 4-dimethylpyridine (DMAP) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to solution B, control the molar ratio of GLD:DMAP:EDC = 1:1:1 in the system, and continue stirring for 30 min for activation to obtain solution C; Take solution C and add it to solution A and mix evenly. Control the molar ratio of BC:GLD = 1:1 to 1:3 in the system, and continue heating and stirring at 40 °C for 24 h to obtain solution D; The preparation method of compound C described in step (2) is as follows: Add cocamidopropyl betaine and oxalyl chloride to a methanol solution to obtain a mixed solution with a molar ratio of cocamidopropyl betaine to oxalyl chloride of 1:

2. Stir at 0 °C for 6 h, then rotary evaporate to remove the unreacted oxalyl chloride at 40 °C to obtain an acyl chloride compound C, and redissolve it with methanol to obtain solution E; The preparation method of compound D described in step (3) is as follows: Add DMAP to solution D and continue stirring for 30 min for activation, then add solution E to the system, controlling the ratio of BC:BP in the system to be 1:

1. Heat and stir in a 40 °C water bath for 24 h to obtain solution F; Dropwise add solution F to anhydrous ethanol with a volume ratio of 10 times its own volume, stir for 30 min, centrifuge at 8000 - 12000 r / min for 5 - 15 min to remove the supernatant, wash the precipitate with anhydrous ethanol 1 - 3 times, centrifuge, dissolve the remaining precipitate in distilled water, ultrasonically treat to obtain a homogeneous and clear solution, centrifuge at 8000 - 12000 r / min for 5 - 15 min, take the supernatant, and freeze-dry to obtain the amphoteric bacterial cellulose carrier.

2. According to the amphoteric bacterial cellulose-encapsulated insulin micelle described in claim 1, wherein: The concentration of the amphoteric bacterial cellulose carrier in the aqueous solution in step (4) is 0.1 - 2.5 mg / mL, the pH of the hydrochloric acid aqueous solution of insulin in step (5) is 1.2 - 3.0, and the concentration of insulin in hydrochloric acid water is 1 - 3 mg / mL.

3. According to the amphoteric bacterial cellulose-encapsulated insulin micelle described in claim 1, wherein: The mass ratio of the amphoteric bacterial cellulose carrier to insulin in step (6) is 10:1 - 10:4, the ultrasonic time of the ultrasonic treatment is 5 - 30 min, and the ultrasonic power is 90 - 900 W.

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