Dendritic lipopeptide molecules for promoting oral absorption of macromolecular drugs, and preparation method and application thereof

Abstract

This invention discloses a class of dendritic lipopeptide molecules that promote the oral absorption of macromolecular drugs, their preparation method, and applications. The dendritic lipopeptide molecules are formed by combining a hydrophilic head and a hydrophobic tail. The hydrophilic head consists of lysine and glutamic acid linked by amide bonds, while the hydrophobic tail is composed of organic carbon chains or fluorinated chains. The oral absorption enhancer used in this invention exhibits amphoteric characteristics, possessing excellent anti-protein adsorption capabilities, superior mucus penetration and small intestinal permeability, and good biocompatibility due to its amino acid element construction. The oral absorption enhancer used in this invention can cross multiple barriers, including the mucus layer and intestinal epithelial cells, without opening tight junctions, facilitating efficient and safe oral encapsulation and delivery of proteins.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to dendritic lipopeptide molecules that promote the oral absorption of macromolecular drugs, their preparation methods, and applications. Background Technology

[0002] Glucose and lipid metabolism disorders, such as type 1 and type 2 diabetes, obesity, non-alcoholic fatty liver disease, and atherosclerotic cardiovascular disease, have become major global public health problems. These diseases are typically accompanied by common pathophysiological features such as insulin resistance, hyperglycemia, dyslipidemia (e.g., high triglycerides, high LDL-C), chronic inflammation, and energy metabolism imbalance. Currently, drug therapy is one of the core means of managing these diseases, including the use of hypoglycemic drugs such as GLP-1 receptor agonists, SGLT2 inhibitors, biguanides, and insulin, as well as lipid-lowering drugs such as statins and fibrates.

[0003] Despite the availability of several effective drugs, numerous challenges remain in clinical treatment, particularly regarding the limitations of oral administration, such as: (a) poor gastrointestinal stability: Many potentially potent active molecules (such as certain peptides, proteins, or acid-sensitive small molecules) are easily degraded by gastric acid or hydrolyzed by pancreatic enzymes as they pass through the upper digestive tract, resulting in extremely low bioavailability or even complete inactivation. For example, GLP-1, a highly promising hypoglycemic drug, is degraded in its natural form by dipeptidyl peptidase-4 (DPP-4) within minutes in vivo, making direct oral administration difficult. (b) low absorption efficiency: drug absorption depends on its solubility and permeability. Many poorly water-soluble drugs (BCS Class II and IV) dissolve slowly and incompletely in gastrointestinal fluids, limiting their absorption. Furthermore, large molecule drugs and hydrophilic drugs have difficulty penetrating the lipid membrane barrier of intestinal epithelial cells. The tight junctions between the intestinal mucus layer and epithelial cells also constitute physical and biochemical barriers to drug absorption. (c) Significant first-pass effect: After being absorbed from the gastrointestinal tract, some drugs must first pass through the portal vein system to enter the liver, where they undergo extensive metabolic breakdown under the action of hepatic metabolic enzymes. This results in a significant reduction in the actual amount of drug entering the systemic circulation, thus decreasing efficacy. (d) Patient compliance challenges: Although oral administration is the most acceptable method for patients, some existing drugs require multiple daily doses or have side effects such as gastrointestinal irritation, affecting long-term medication adherence.

[0004] To overcome the aforementioned obstacles, existing delivery technologies employ enteric coating, oral absorption enhancers, and lipid-based delivery systems to promote lymphatic transport. While current oral drug delivery technologies have made some progress, a comprehensive delivery platform that can synergistically address the three core challenges of gastric acid / enzyme degradation, absorption barriers, and first-pass effect, while simultaneously possessing good biocompatibility, high drug loading capacity, stable and controllable delivery, and high absorption efficiency, is still lacking. This is crucial for fully leveraging the efficacy of the increasing number of novel glucose and lipid metabolism therapies, reducing side effects, and improving patients' quality of life. Therefore, there is an urgent need in this field to develop a novel, efficient, and safe oral delivery system to better meet the clinical treatment needs of glucose and lipid metabolism diseases. Summary of the Invention

[0005] Purpose of the invention: To address the problems existing in the prior art, this invention provides a class of dendritic lipopeptide molecules that can significantly improve the oral absorption of macromolecular drugs. These dendritic lipopeptide molecules are characterized by high efficiency of intestinal mucus penetration, excellent cellular uptake efficiency, and high oral bioavailability enhancement.

[0006] This invention also relates to methods for preparing dendritic lipopeptide molecules that promote the oral absorption of macromolecular drugs, and their applications in the encapsulation and delivery of polypeptide drugs such as insulin, GLP-1 structured liraglutide, dulaglutide, smegglutide, benaglutide, exendin-4 exenatide, exenatide, lixisenatide, and polyethylene glycol loxenatide.

[0007] Technical Solution: To achieve the above solution, this invention provides a dendritic lipopeptide molecule that promotes the oral absorption of macromolecular drugs. The dendritic lipopeptide molecule is formed by the combination of a hydrophilic head and a hydrophobic tail. The hydrophilic head is composed of lysine and glutamic acid linked by an amide bond, and the hydrophobic tail is composed of an organic carbon chain or a fluorinated chain. The hydrophilic head is selected from... , or The hydrophobic tail section is selected from or ;

[0008] Where: R1 is -C n H 2n-1 n is 2~20; R2 is -C m F 2m+1 m is 2~9.

[0009] Where R1 is C2~C 20 An unsaturated alkane chain; wherein R2 is selected from the following structures:

[0010] .

[0011] The method for preparing dendritic lipopeptide molecules that promote oral absorption of macromolecular drugs according to the present invention includes the following steps:

[0012] Lys-NHR2 was obtained by sequentially esterifying Boc-Lys(Boc)-OH and R1NH2 and then deprotecting it with Boc. Lys-NHR2 was then reacted with Boc-L-Glu-1-OtBu by sequentially amide condensation, silica gel column purification, deprotection of Boc and protection with OtBu to obtain E2K-NHR2.

[0013] Alternatively, Lys-NHR2 can be obtained by sequentially amide condensation and deBoc protection of Boc-Lys(Boc)-OH and R1NH2, and then Lys-NHR2 can be obtained by sequentially amide condensation, silica gel column purification, deBoc protection and OtBu protection of Boc-Gly.

[0014] Alternatively, Lys-OCH2R2 can be obtained by sequentially esterifying Boc-Lys(Boc)-OH and R2CH2OH and removing Boc protection. Lys-OCH2R2 can then be reacted with Boc-L-Glu-1-OtBu by sequentially amide condensation, silica gel column purification, and removal of Boc and OtBu protection to obtain E2K-OCH2R2.

[0015] R1 is -C n H 2n-1 n is 2~20; R2 is -C m F 2m+1 m is 2~9.

[0016] The present invention provides a method for preparing nanomedicines based on the aforementioned dendritic lipopeptide molecules, comprising the following steps:

[0017] (1) Dissolve dendritic lipopeptide molecules in an organic solvent to obtain mixed solution A. Inject mixed solution A into a solution containing the drug and stir magnetically to remove the organic solvent to obtain mixed solution B.

[0018] (2) The mixed solution B is ultrafiltered and centrifuged to remove impurities and unencapsulated drugs, thus obtaining dendritic lipopeptide nanomedicine.

[0019] The organic solvent mentioned in step (1) is tetrahydrofuran, methanol, chloroform, acetone, dichloromethane, ethanol, etc.; the organic solvent is preferably tetrahydrofuran.

[0020] Wherein, the concentration of the mixed solution A in step (1) is 1~10 mg / mL; the concentration of the mixed solution A is preferably 10 mg / mL.

[0021] In step (2), the drugs are insulin, GLP-1 structured liraglutide, dulaglutide, smegglutide, benaglutide, exendin-4 (exendin-4) exenatide, exenatide, lixisenatide, polyethylene glycol loxenatide, etc.; the preferred drugs are insulin and GLP-1 structured liraglutide.

[0022] In step (2), the solution of the drug is prepared using dilute sodium bicarbonate solution, dilute sodium hydroxide solution, sodium citrate, pure water, etc. as solvents; the solution of the drug is preferably dilute sodium bicarbonate solution and dilute sodium hydroxide solution.

[0023] In step (2), the concentration of the drug solution is 1~10 mg / mL; the preferred concentration of the drug solution is 10 mg / mL.

[0024] In step (2), the mass ratio of the dendritic lipopeptide molecule to the drug is 1~10:1~10; the preferred mass ratio is 1:4.

[0025] In the preparation of insulin, zinc chloride is added, and the mass ratio of zinc chloride to insulin is 1~10:1~10.

[0026] The application of the dendritic lipopeptide molecule of the oral absorption enhancer described in this invention in the preparation of drugs for delivering insulin, liraglutide, and treating glucose and lipid metabolism disorders.

[0027] The aforementioned oral absorption enhancer consists of two parts: a hydrophilic head and a hydrophobic tail. The hydrophilic head is an amphoteric polypeptide formed by lysine and glutamic acid or glycine in a certain proportion through an amide bond, or by lysine and glutamic acid in a certain proportion through an ester bond. The amphoteric ions can penetrate the intestinal mucosa. The hydrophobic tail is a long-chain hydrocarbon or a long fluorinated chain.

[0028] Preferably, the amphoteric polypeptide is a dendritic polypeptide composed of lysine and glutamic acid or glycine in a certain proportion via amide or ester bonds. (n represents lysine and glutamic acid or glycine, forming a dendritic network via amide or ester bonds), where the outer surface of the dendritic network needs to have amino and carboxyl groups. The preferred number of amino acids in the constituent dendritic amphoteric peptides is 3, with 1 lysine and 2 glutamic or glycine. Using low-generation dendritic peptides simplifies and controls material synthesis, meeting the requirements for high reproducibility and high yield in industrial production. Furthermore, materials constructed from lower-generation dendritic peptides can still exhibit the advantages of higher-generation dendritic molecules through chemical self-assembly processes, while reducing the toxicity of higher-generation molecules and improving safety in use. Its head structure is as follows:

[0029] , .

[0030] The preferred structure of the dendritic lipopeptide molecule of this invention is as follows:

[0031] Compound 1

[0032]

[0033] Compound 2

[0034]

[0035] Compound 3

[0036]

[0037] Compound 4

[0038]

[0039] The above R1 is: -C 18 H 35 R2 is -C m F 2m+1 m can be 3, 6, or 8.

[0040] Preferably, the method for preparing the oral absorption enhancer includes the following steps:

[0041] (1) Preparation of hydrophobic tail Lys-OA

[0042] Protected lysine and oleylamine were amide condensed in a 1:1 ratio under the catalysis of HOBT, HBTU condensing agent and DIPEA, and then deprotected to obtain tail-end Lys-OA.

[0043] (2) Preparation of hydrophobic tail Lys-OCH2R2

[0044] Protected lysine and R2CH2OH were esterified in a 1:1 ratio under the catalysis of EDCI and DMAP, followed by deprotection to obtain the tail-end Lys-OCH2R2 (R2 is -C). n F 2n+1 (where n is 3, 6, or 8).

[0045] (3) Preparation of E2K-OA-5 by connecting the tail and head

[0046] Protected glutamic acid and the tail of (1) synthesis were amide condensed in a 2:1 ratio under the catalysis of HOBT, HBTU condensing agent and DIPEA, and then deprotected to obtain the amphoteric material E2K-OA-5.

[0047] (4) Connecting the tail and head to prepare E2K-OA-1

[0048] Protected glutamic acid and the tail of (1) synthesis were amide condensed in a 2:1 ratio under the catalysis of HOBT, HBTU condensing agent and DIPEA, and then deprotected to obtain the amphoteric material E2K-OA-1.

[0049] (5) Connecting the tail and head to prepare G2K-OA

[0050] Protected glycine and the tail of (1) were synthesized into an amide in a 2:1 ratio under the catalysis of HOBT, HBTU condensing agent and DIPEA, and then deprotected to obtain the amphoteric material G2K-OA.

[0051] (6) Preparation of E2K-OCH2R2 by connecting the tail and head

[0052] The protected glutamic acid head and the tail synthesized in (2) were amide condensed in a 2:1 ratio under the catalysis of HOBT, HBTU condensing agent and DIPEA, and then deprotected to obtain the amphoteric material E2K-OCH2R2.

[0053] More preferably, the dendritic lipopeptide molecule is selected from any one of the following:

[0054]

[0055] The application of the above-mentioned oral absorption enhancers in the preparation of protein drug formulations.

[0056] In one embodiment of the present invention, insulin is used as a model drug, and a delivery carrier is prepared using the above-mentioned dendritic lipopeptide molecules.

[0057] Specifically: an oral absorption enhancer was added to an insulin solution and stirred. Then, a zinc chloride solution was added dropwise to the solution containing insulin and the oral absorption enhancer. The solution was purified by ultrafiltration to obtain drug-loaded amphoteric micelles.

[0058] Invention Principle: This invention proposes using zwitterionic fluorinated materials to encapsulate insulin. Zwitterionic materials, with their unique charge-balanced structure, superhydrophilicity, and biocompatibility, can effectively reduce non-specific protein adsorption and immune system recognition, reduce adsorption by gastrointestinal mucus and digestive enzymes, prevent premature drug degradation, and promote drug penetration in intestinal mucus. The fluorinated hydrophobic chains, due to their excellent stability, low surface tension, and controllable hydrophobicity, significantly enhance the stability of drug delivery and the penetration into intestinal epithelial cells.

[0059] This invention addresses the problems of low biocompatibility, insufficient safety, and limited absorption enhancement efficiency in existing oral delivery systems for protein and peptide drugs. This innovative technology achieves a triple synergistic mechanism through multi-dimensional optimization of the molecular structure: (1) Based on the amphiphilic material properties, the near-neutral charge of its hydrophilic head effectively enhances the drug's penetration efficiency in the intestinal mucus layer; (2) By introducing a specific tripeptide domain at the hydrophilic end, it promotes transmembrane transport pathways mediated by intestinal epithelial cell membrane transporters; (3) The use of a fluorinated, hydrophobic tail structure significantly improves the drug's uptake efficiency within intestinal epithelial cells. Compared to traditional oral delivery systems, the novel promoter exhibits optimization in key parameters such as mucus penetration, transmembrane transport efficiency, and oral absorption effect, providing an innovative solution for the development of oral formulations of protein and peptide drugs.

[0060] The dendritic lipopeptide molecule prepared by this invention is an amino acid synthetic peptide with a tripeptide head; the overall material is nearly neutral; it can penetrate the mucus layer, enter the cell, and exert its function.

[0061] Beneficial results: Compared with the prior art, the present invention has the following advantages:

[0062] (1) The dendritic lipopeptide molecule of the present invention is a hybrid molecule that combines dendritic macromolecules and lipid structures. Its core is composed of a branched peptide backbone and is modified with hydrophobic fatty acid chains / fluorinated chains to form a unique amphiphilic structure that can effectively balance hydrophilicity and hydrophobicity. The dendritic lipopeptide molecule combines the high branching degree and nanoscale size of dendritic molecules, while inheriting the membrane affinity of lipopeptides, giving it unique advantages in drug delivery such as multivalent effects, high biocompatibility and convenient synthesis.

[0063] (2) The dendritic lipopeptide molecule preparation process of the present invention is simple, convenient and easy to carry out, and has a high yield.

[0064] (3) The present invention uses dendritic lipopeptide molecules to encapsulate peptide drugs and delivers them orally, which has superior patient compliance, convenience and flexibility compared with injection administration.

[0065] (4) The dendritic lipopeptide molecules prepared in this invention have amphoteric characteristics, excellent resistance to protein adsorption, excellent mucus penetration and small intestinal permeability, and good biocompatibility due to the construction of amino acid elements.

[0066] (5) The dendritic lipopeptide molecules prepared in this invention can cross multiple barriers, including the mucus layer and intestinal epithelial cells, without opening tight junctions, which helps to achieve efficient and safe oral encapsulation and delivery of proteins. Attached Figure Description

[0067] Figure 1 A schematic diagram of the synthesis route for G2K-OA;

[0068] Figure 2 A schematic diagram of the synthesis route for E2K-OA-5;

[0069] Figure 3 A schematic diagram of the synthesis route for E2K-OA-1;

[0070] Figure 4 A schematic diagram of the synthesis route for E2K-OF7;

[0071] Figure 5 E2K-OF 13 A schematic diagram of the synthesis route;

[0072] Figure 6 E2K-OF 17 A schematic diagram of the synthesis route;

[0073] Figure 7 The mass spectrum of G2K-OA;

[0074] Figure 8 The mass spectrum of E2K-OA-5;

[0075] Figure 9 The mass spectrum of E2K-OA-1;

[0076] Figure 10 The mass spectrum of E2K-OF7;

[0077] Figure 11 E2K-OF 13 The mass spectrum;

[0078] Figure 12 E2K-OF 17 The mass spectrum;

[0079] Figure 13 The particle size distribution and potential of different drug-loaded nanoparticles;

[0080] Figure 14 The particle size distribution, potential, encapsulation efficiency and drug loading of liraglutide were determined by different nanoparticles.

[0081] Figure 15 Transwell mucus permeation diagrams for different drug-loaded nanoparticles;

[0082] Figure 16 Diagram showing the anti-protein adsorption of different drug-loaded nanoparticles;

[0083] Figure 17 Quantitative flow cytometry diagram of cellular uptake of different drug-loaded nanoparticles;

[0084] Figure 18Transwell transmembrane transport diagrams for different drug-loaded nanoparticles;

[0085] Figure 19 Figures from in vitro inverted intestinal sac experiments with different drug-loaded nanoparticles;

[0086] Figure 20 The hypoglycemic and weight-reducing effects of long-term oral administration of different drug-loaded nanoparticles in type II diabetic rats.

[0087] Figure 21 The hypoglycemic effect and duration of hypoglycemia, normoglycemia and hyperglycemia of orally administered drug-loaded nanoparticles in type 1 diabetic miniature pigs. Detailed Implementation

[0088] To make the present invention easier to understand, the present invention will be further described below with reference to specific embodiments. These embodiments are not intended to limit the present invention in any way. They are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any modifications or changes to the present invention that are easily implemented by those skilled in the art without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

[0089] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0090] Example 1

[0091] The oral absorption enhancer of this invention can serve as a lipopeptide dendritic molecule (G2K-OA, E2K-OA-5, E2K-OA-1, E2K-OF7, E2K-OF) for use as a macromolecular drug carrier. 13 and E2K-OF 17 Its structural formula is as follows:

[0092]

[0093] The preparation method of G2K-OA is as follows, and its synthetic route is as follows: Figure 1 As shown:

[0094] Step 1: Accurately weigh 2.00 g of Boc-Lys(Boc)-OH (CAS: 2483-46-7), 1.15 g of HOBT (CAS: 2592-95-2), and 3.22 g of HBTU (CAS: 619-076-7) into a three-necked round-bottom flask. Add a stir bar to the flask, and then seal the flask with a rubber stopper and sealing film. Under nitrogen protection, evacuate the round-bottom flask using a water-based vacuum pump, repeating this process three times until the flask is in a near-vacuum state. Then, fix the round-bottom flask on a stirrer (500 rpm) and slowly add 50 mL of DMF (CAS: 200-679-5) to the flask under ice bath conditions until the reactants are fully dissolved. Subsequently, 2.79 mL of oleylamine (CAS: 112-90-3) and 7.90 mL of DIPEA (CAS: 230-392-0) were added to the round-bottom flask, and the reaction was carried out at room temperature for 12 hours. A small amount of the reaction liquid was taken from the round-bottom flask with a sampling needle for TLC monitoring until the reaction was complete.

[0095] Step 2: Pour all the reaction solution into a separatory funnel, add an equal volume of ethyl acetate as the organic phase for extraction, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain crude Boc-Lys(Boc)-OA. Purify the crude Boc-Lys(Boc)-OA by column chromatography (petroleum ether:ethyl acetate = 4:1, V:V) to obtain Boc-Lys(Boc)-OA, yield: 95.28%. Dry the obtained product in a vacuum drying oven for 24 hours to remove the organic solvent.

[0096] Step 3: Accurately weigh 2.00 g of the reaction product Boc-Lys(Boc)-OA and place it in a round-bottom flask. Add a stir bar and seal the flask with a rubber stopper. Vacuum the flask, then purge with nitrogen, repeating this process three times. Place the flask under nitrogen protection in an ice bath and add 12 mL of a trifluoroacetic acid / dichloromethane mixed solvent (trifluoroacetic acid:dichloromethane = 1:1, V:V). Stir the mixture at 500 rpm at room temperature for 4 h. Use a sampling needle to take a small amount of the reaction liquid from the round-bottom flask for TLC monitoring until the reaction is complete. After the reaction is complete, concentrate the reaction solution under reduced pressure and repeatedly evaporate to dryness. Seal the round-bottom flask with plastic wrap and poke small holes, then place it in a fume hood for vacuum drying for 24 hours to obtain the intermediate product Lys-OA.

[0097] Step 4: Accurately weigh 1.00 g of Lys-OA, 0.98 g of Boc-Gly (CAS:4530-20-5), 1.00 g of HOBT (CAS:2592-95-2), and 2.88 g of HBTU (CAS:619-076-7) into a three-necked round-bottom flask. Add a stir bar to the flask, and then seal the flask with a rubber stopper and sealing film. Under nitrogen protection, evacuate the round-bottom flask using a water-based vacuum pump, repeating this process three times until the flask is in a near-vacuum state. Then, fix the round-bottom flask on a stirrer (500 rpm) and slowly add 50 mL of DMF (CAS:200-679-5) to the flask under ice bath conditions until the reactants are fully dissolved. Then, 3.96 mL of DIPEA (CAS: 200-679-5) was added to the round-bottom flask, and the reaction was carried out at room temperature for 12 hours. A small amount of the reaction liquid was taken from the round-bottom flask with a sampling needle for TLC monitoring until the reaction was complete.

[0098] Step 5: Pour all the reaction solution into a separatory funnel, add an equal volume of ethyl acetate as the organic phase for extraction, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain crude (Boc-Gly)2Lys-OA. Purify the crude (Boc-Gly)2Lys-OA by column chromatography (dichloromethane:methanol = 40:1, V:V) to obtain (Boc-Gly)2Lys-OA. Place the obtained product in a vacuum drying oven and dry for 24 hours to remove the organic solvent.

[0099] Step 6: Accurately weigh 1.00 g of the above reaction product (Boc-Gly)2Lys-OA and place it in a round-bottom flask. Add a stir bar and seal the flask with a rubber stopper. Vacuum the flask, then purge with nitrogen, repeating this process three times. Place the flask under nitrogen protection in an ice bath, add 8 mL of a trifluoroacetic acid / dichloromethane mixed solvent (trifluoroacetic acid:dichloromethane = 1:1, V:V), and stir at 500 rpm at room temperature for 4 h. Use a sampling needle to take a small amount of the reaction liquid into the round-bottom flask for TLC monitoring until the reaction is complete. After the reaction is complete, concentrate the reaction solution under reduced pressure and repeatedly evaporate to dryness. Seal the round-bottom flask with plastic wrap and poke a small hole, then place it in a fume hood and vacuum dry for 24 hours to obtain the final product G2K-OA. Its mass spectrum is shown below. Figure 7 [M + H] obtained by mass spectrometry analysis +The molecular weight was 510.6, which is consistent with the theoretical molecular weight and the measured molecular weight, indicating that G2K-OA was successfully synthesized.

[0100] The preparation method of E2K-OA-5 is as follows, and its synthetic route is as follows: Figure 2 As shown:

[0101] Step 1: Accurately weigh 2.00 g of the intermediate product Lys-OA, 3.14 g of Boc-Glu-5-OtBu-OH (cas:13726-84-6), 2.90 g of EDCI, and 2.06 g of HOBT into a three-necked round-bottom flask. Add a stir bar to the flask, and then seal the flask with a rubber stopper and sealing film. Under nitrogen protection, evacuate the round-bottom flask using a water-based vacuum pump, repeating this process three times until the flask is in a near-vacuum state. Then, fix the round-bottom flask on a stirrer (500 rpm) and slowly add 50 mL of DCM to the flask under ice bath conditions until the reactants are fully dissolved. After the solids in the flask are completely dissolved, slowly add 7.06 mL of DIPEA dropwise to the round-bottom flask in batches. Remove the ice bath and allow the reaction system to react at room temperature for 24 hours. A small amount of the reaction liquid was taken into the round-bottom flask using a sampling needle and monitored by TLC until the reaction was complete.

[0102] Step 2: Pour all the reaction solution into a separatory funnel, add an equal volume of ethyl acetate as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40.0℃ and 100 rpm to obtain the crude intermediate Boc-E2K(OtBu)-OA-5. The crude Boc-E2K(OtBu)-OA-5 is purified by column chromatography (dichloromethane:methanol = 60:1, V:V) to obtain Boc-E2K(OtBu)-OA-5. The obtained product is dried in a vacuum drying oven for 24 hours to remove moisture for subsequent experiments and research.

[0103] Step 3: Accurately weigh 1.00 g of the above reaction product Boc-E2K(OtBu)-OA-5 and place it in a round-bottom flask. Add a stir bar and seal the flask with a rubber stopper. Vacuum the flask and purge with nitrogen, repeating this process three times. Place the nitrogen-protected round-bottom flask in an ice bath and add 9 mL of a mixed solvent of trifluoroacetic acid / dichloromethane / triethylsilane (trifluoroacetic acid:dichloromethane:triethylsilane = 4:4:1, V:V:V). Stir the mixture at 500 rpm at room temperature for 4 h. Use a sampling needle to take a small amount of the reaction liquid in a round-bottom flask for TLC monitoring until the reaction is complete. After the reaction is complete, concentrate the reaction solution under reduced pressure and repeatedly evaporate to dryness. Then, add 5.0 mL of pre-cooled ice-cold diethyl ether to the round-bottom flask, repeatedly stir, and let it stand until a white precipitate forms. Remove the supernatant and repeat the above operation twice. The mixture of the above reactants was concentrated under reduced pressure and evaporated to dryness. The flask was then sealed with plastic wrap with small holes and placed in a vacuum drying oven overnight. After the organic reagents were completely dried, the final product E2K-OA-5 was collected. Its mass spectrum is shown below. Figure 8 [M + H] obtained by mass spectrometry analysis + The molecular weight was 654.48, which is consistent with the theoretical molecular weight and the measured molecular weight, indicating that E2K-OA-5 was successfully synthesized.

[0104] The preparation method of E2K-OA-1 is as follows, and its synthetic route is as follows: Figure 3 As shown:

[0105] Step 1: Accurately weigh 2.00 g of the intermediate product Lys-OA, 3.14 g of Boc-Glu-1-OtBu-OH (cas:24277-39-2), 2.90 g of EDCI, and 2.06 g of HOBT into a three-necked round-bottom flask. Add a stir bar to the flask, and then seal the flask with a rubber stopper and sealing film. Under nitrogen protection, evacuate the round-bottom flask using a water-based vacuum pump, repeating this process three times until the flask is in a near-vacuum state. Then, fix the round-bottom flask on a stirrer (500 rpm) and slowly add 50 mL of DCM to the flask under ice bath conditions until the reactants are fully dissolved. After the solid in the flask is completely dissolved, slowly add 7.06 mL of DIPEA dropwise to the round-bottom flask in batches. Remove the ice bath and allow the reaction system to react at room temperature for 24 hours. A small amount of the reaction liquid was taken into the round-bottom flask using a sampling needle and monitored by TLC until the reaction was complete.

[0106] Step 2: Pour all the reaction solution into a separatory funnel, add an equal volume of ethyl acetate as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40.0℃ and 100 rpm to obtain the crude intermediate Boc-E2K(OtBu)-OA-1. The crude Boc-E2K(OtBu)-OA-1 is purified by column chromatography (dichloromethane:methanol = 60:1, V:V) to obtain Boc-E2K(OtBu)-OA-1. The obtained product is dried in a vacuum drying oven for 24 hours to remove moisture for subsequent experiments and research.

[0107] Step 3: Accurately weigh 1.00 g of the above reaction product Boc-E2K(OtBu)-OA-1 and place it in a round-bottom flask. Add a stir bar and seal the flask with a rubber stopper. Vacuum the flask, then purge with nitrogen, repeating this process three times. Place the flask under nitrogen protection in an ice bath and add 9 mL of a trifluoroacetic acid / dichloromethane / triethylsilane mixed solvent (trifluoroacetic acid:dichloromethane:triethylsilane = 4:4:1, V:V:V). Stir the mixture at 500 rpm for 4 h. Use a sampling needle to take a small amount of the reaction liquid in a round-bottom flask for TLC monitoring until the reaction is complete. After the reaction is complete, concentrate the reaction solution under reduced pressure and repeatedly evaporate to dryness. Then add 5.0 mL of pre-cooled ice-cold diethyl ether to the flask, repeatedly stir, and let it stand until a white precipitate forms. Remove the supernatant and repeat the above operation twice. Concentrate the flask containing the above reaction mixture under reduced pressure and evaporate to dryness. Seal the flask with plastic wrap and poke a small hole, then place it in a vacuum drying oven to dry overnight. After the organic reagents had completely dried, the final product E2K-OA-1 was collected. Its mass spectrum is shown below. Figure 9 [M + Na] obtained by mass spectrometry analysis + The molecular weight was 676.72, which is consistent with the theoretical molecular weight and the measured molecular weight, indicating that E2K-OA-1 was successfully synthesized.

[0108] The preparation method of E2K-OF7 is as follows, and its synthetic route is as follows: Figure 4 As shown:

[0109] Step 1: Accurately weigh 0.90 g of Boc-Lys(Boc)-OH, 0.64 g of F7-OH (perfluorobutanol) (CAS: 375-01-9), 2.02 g of EDCI, and 1.90 g of DMAP into a 250 mL three-necked round-bottom flask. Add a stir bar to the flask, and then seal the flask with a rubber stopper and sealing film. Under nitrogen protection, evacuate the round-bottom flask using a water-based vacuum pump, repeating this process three times until the flask is in a near-vacuum state. Then, fix the round-bottom flask on a stirrer (500 rpm) and slowly add 70 mL of anhydrous dichloromethane to the flask under ice bath conditions until the reactants are fully dissolved. React for 4 hours. Use a sampling needle to take a small amount of the reaction liquid from the round-bottom flask for TLC monitoring until the reaction is complete.

[0110] Step 2: Pour all the reaction mixture into a separatory funnel, add dichloromethane as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain crude Boc-Lys(Boc)-OF7. Purify the crude Boc-Lys(Boc)-OF7 by column chromatography (dichloromethane:methanol = 60:1, V:V) to obtain Boc-Lys(Boc)-OF7, yield: 97.56%. Dry the obtained product in a vacuum drying oven for 24 hours to remove the organic solvent.

[0111] Step 3: Accurately weigh 1.00 g of the reaction product Boc-Lys(Boc)-OF7 and place it in a round-bottom flask. Add a stir bar and seal the flask with a rubber stopper. Vacuum the flask, then purge with nitrogen, repeating this process three times. Place the nitrogen-protected flask in an ice bath and add 12 mL of a trifluoroacetic acid / dichloromethane mixed solvent (trifluoroacetic acid:dichloromethane = 1:1, V:V). Stir the reaction at 500 rpm for 4 h, using a sampling needle to take a small amount of the reaction liquid into the round-bottom flask for TLC monitoring until the reaction is complete. After the reaction is complete, concentrate the reaction solution under reduced pressure and repeatedly evaporate to dryness. Seal the round-bottom flask with plastic wrap and poke small holes, then place it in a fume hood for vacuum drying for 24 hours to obtain the intermediate product Lys-OF7.

[0112] Step 4: Accurately weigh 1.52 g of Lys-OF7, 3.23 g of Boc-Glu-1-OtBu-OH, 1.88 g of HOBT, and 5.28 g of HBTU into a 500 mL three-necked round-bottom flask. Add a stir bar to the flask, and then seal the flask with a rubber stopper and sealing film. Under nitrogen protection, evacuate the round-bottom flask using a water-based vacuum pump, repeating this process three times until the flask is in a near-vacuum state. Then, fix the round-bottom flask on a stirrer (500 rpm) and slowly add 80 mL of DMF to the flask under ice bath conditions until the reactants are fully dissolved. Next, add 7.27 mL of DIPEA to the flask and react for 6 hours. Use a sampling needle to take a small amount of the reaction liquid from the round-bottom flask for TLC monitoring until the reaction is complete.

[0113] Step 5: Pour all the reaction solution into a separatory funnel, add an equal volume of ethyl acetate as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain crude Boc-E2K(OtBu)-OF7. Purify the crude Boc-E2K(OtBu)-OF7 by column chromatography (dichloromethane:methanol = 40:1, V:V) to obtain Boc-E2K(OtBu)-OF7. Place the obtained product in a vacuum drying oven and dry for 24 hours to remove the organic solvent.

[0114] Step 6: Accurately weigh 1.00 g of the above reaction product Boc-E2K(OtBu)-OF7 into a 50 mL round-bottom flask, add a stir bar, and seal with a rubber stopper. Vacuum the flask, then purge with nitrogen, repeating this process three times. Place the nitrogen-protected flask in an ice bath, add 9 mL of a trifluoroacetic acid / dichloromethane / triethylsilane mixed solvent (trifluoroacetic acid:dichloromethane:triethylsilane = 4:4:1, V:V:V), and stir at 500 rpm for 4 h. Use a sampling needle to take a small amount of the reaction liquid in a round-bottom flask for TLC monitoring until the reaction is complete. After the reaction is complete, concentrate the reaction solution under reduced pressure and repeatedly evaporate to dryness. Then add 5.0 mL of pre-cooled ice-cold diethyl ether to the above flask, repeatedly stir, and let stand until a white precipitate forms. Remove the supernatant and repeat the above operation twice. The mixture of the above reactants was concentrated under reduced pressure and evaporated to dryness. The flask was then sealed with plastic wrap with small holes and placed in a vacuum drying oven overnight. After the organic reagents were completely dried, the final product E2K-OF7 was collected. Its mass spectrum is shown below. Figure 10 [M-H] obtained by mass spectrometry analysis+ The molecular weight was 585.18, which is consistent with the theoretical molecular weight and the measured molecular weight, indicating that E2K-OF7 was successfully synthesized.

[0115] E2K-OF 13 The preparation method is as follows, and its synthetic route is as follows: Figure 5 As shown:

[0116] Step 1: Accurately weigh 0.60 g of Boc-Lys(Boc)-OH, F 13 -OH (1H,1H-tridecylfluoro-1-heptanol) (CAS: 375-82-6) 0.77 g, EDCI 1.34 g, DMAP 1.27 g were added to a 250 mL three-necked round-bottom flask. A stir bar was added to the flask, and then the flask was sealed with a rubber stopper and sealing film. Under nitrogen protection, the round-bottom flask was evacuated using a water-based vacuum pump, and this process was repeated three times until a near-vacuum state was achieved. Then, the round-bottom flask was fixed to a stirrer (500 rpm), and 70 mL of anhydrous dichloromethane was slowly added to the flask under ice bath conditions until the reactants were fully dissolved. The reaction was allowed to proceed for 4 hours. A small amount of the reaction liquid was taken from the round-bottom flask using a sampling needle for TLC monitoring until the reaction was complete.

[0117] Step 2: Pour all the reaction solution into a separatory funnel, add dichloromethane as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain Boc-Lys(Boc)-OF. 13 Crude product. Boc-Lys(Boc)-OF 13 The crude product was purified by column chromatography (dichloromethane:methanol = 60:1, V:V) to obtain Boc-Lys(Boc)-OF 13 Yield: 97.86%. The obtained product was dried in a vacuum drying oven for 24 hours to remove organic solvents.

[0118] Step 3: Accurately weigh the above reaction product Boc-Lys(Boc)-OF 131.00 g was placed in a 50 mL round-bottom flask, a stir bar was added, and the flask was sealed with a rubber stopper. Vacuum was applied, and nitrogen was introduced; this process was repeated three times. The flask, under nitrogen protection, was placed in an ice bath, and 12 mL of a trifluoroacetic acid / dichloromethane mixed solvent (trifluoroacetic acid:dichloromethane = 1:1, V:V) was added. The mixture was stirred at 500 rpm for 4 h. A small amount of the reaction liquid was taken into the round-bottom flask using a sampling needle for TLC monitoring until the reaction was complete. After the reaction was complete, the reaction solution was concentrated under reduced pressure and repeatedly evaporated to dryness. The round-bottom flask was sealed with plastic wrap with small holes and placed in a fume hood for vacuum drying for 24 hours to obtain the intermediate product Lys-OF. 13 .

[0119] Step 4: Accurately weigh the above product Lys-OF 13 1.50 g of Boc-Glu-1-OtBu-OH, 3.18 g of HOBT, 1.85 g of HBTU, and 5.20 g of HBTU were placed in a 500 mL three-necked round-bottom flask. A stir bar was added to the flask, and the flask was then sealed with a rubber stopper and sealing film. Under nitrogen protection, the flask was evacuated using a water-based vacuum pump, and this process was repeated three times until a near-vacuum was achieved. The flask was then fixed to a stirrer (500 rpm), and 80 mL of DMF was slowly added to the flask under ice bath conditions until the reactants were fully dissolved. Then, 7.16 mL of DIPEA was added to the flask, and the reaction was allowed to proceed for 6 hours. A small amount of the reaction liquid was taken from the flask using a sampling needle for TLC monitoring until the reaction was complete.

[0120] Step 5: Pour all the reaction solution into a separatory funnel, add an equal volume of ethyl acetate as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain Boc-E2K(OtBu)-OF. 13 Crude product. Boc-E2K(OtBu)-OF 13 The crude product was purified by column chromatography (dichloromethane:methanol = 40:1, V:V) to obtain Boc-E2K(OtBu)-OF 13 The obtained product was placed in a vacuum drying oven and dried for 24 hours to remove the organic solvent.

[0121] Step 6: Accurately weigh the above reaction product Boc-E2K(OtBu)-OF 131.00 g was placed in a 50 mL round-bottom flask, a stir bar was added, and the flask was sealed with a rubber stopper. Vacuum was applied, and nitrogen was introduced; this process was repeated three times. The flask, under nitrogen protection, was placed in an ice bath, and 9 mL of a trifluoroacetic acid / dichloromethane / triethylsilane mixed solvent (trifluoroacetic acid:dichloromethane:triethylsilane = 4:4:1, V:V:V) was added. The mixture was stirred at 500 rpm for 4 h. A small amount of the reaction liquid was taken from the round-bottom flask using a sampling needle for TLC monitoring until the reaction was complete. After the reaction was complete, the reaction solution was concentrated under reduced pressure and repeatedly evaporated to dryness. Then, 5.0 mL of pre-cooled ice-cold diethyl ether was added to the flask, and the mixture was repeatedly stirred and allowed to stand until a white precipitate formed. The supernatant was removed, and the above operation was repeated twice. The flask containing the above reaction mixture was concentrated under reduced pressure and evaporated to dryness, sealed with plastic wrap with small holes, and placed in a vacuum drying oven to dry overnight. After the organic reagents were completely dried, the final product E2K-OF was collected. 13 Its mass spectrum is shown below. Figure 11 [M-H] obtained by mass spectrometry analysis + The molecular weight was 735.17, consistent with the theoretical and measured molecular weights, indicating that E2K-OF... 13 It was successfully synthesized.

[0122] E2K-OF 17 The preparation method is as follows, and its synthetic route is as follows: Figure 6 As shown:

[0123] Step 1: Accurately weigh 0.80 g of Boc-Lys(Boc)-OH, F 17 1.07 g of -OH(1H,1H-perfluoro-1-nonanol) (CAS: 423-56-3), 1.79 g of EDCI, and 1.69 g of DMAP were placed in a 250 mL three-necked round-bottom flask. A stir bar was added to the flask, and then the flask was sealed with a rubber stopper and sealing film. Under nitrogen protection, the flask was evacuated using a water-based vacuum pump, and this process was repeated three times until a near-vacuum state was achieved. The flask was then fixed to a stirrer (500 rpm), and 70 mL of anhydrous dichloromethane was slowly added to the flask under ice bath conditions until the reactants were fully dissolved. The reaction was allowed to proceed for 4 hours. A small amount of the reaction liquid was taken from the flask using a sampling needle for TLC monitoring until the reaction was complete.

[0124] Step 2: Pour all the reaction solution into a separatory funnel, add dichloromethane as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain Boc-Lys(Boc)-OF. 17 Crude product. Boc-Lys(Boc)-OF 17 The crude product was purified by column chromatography (dichloromethane:methanol = 60:1, V:V) to obtain Boc-Lys(Boc)-OF 17 Yield: 97.96%. The obtained product was dried in a vacuum drying oven for 24 hours to remove organic solvents.

[0125] Step 3: Accurately weigh the above reaction product Boc-Lys(Boc)-OF 17 1.50 g of the solution was placed in a 50 mL round-bottom flask, a stir bar was added, and the flask was sealed with a rubber stopper. Vacuum was applied, and nitrogen gas was introduced; this process was repeated three times. The round-bottom flask, under nitrogen protection, was placed in an ice bath, and 12 mL of a trifluoroacetic acid / dichloromethane mixed solvent (trifluoroacetic acid:dichloromethane = 1:1, V:V) was added. The mixture was stirred at 500 rpm for 4 h. A small amount of the reaction liquid was taken from the round-bottom flask using a sampling needle for TLC monitoring until the reaction was complete. After the reaction was complete, the reaction solution was concentrated under reduced pressure and repeatedly evaporated to dryness. The round-bottom flask was sealed with plastic wrap with small holes punched in it and placed in a fume hood for vacuum drying for 24 hours to obtain the intermediate product Lys-OF. 17 .

[0126] Step 4: Accurately weigh the above product Lys-OF 17 1.85 g of Boc-Glu-1-OtBu-OH, 2.23 g of HOBT, 1.30 g of HBTU, and 3.64 g of HBTU were added to a 500 mL three-necked round-bottom flask. A stir bar was added to the flask, and then the flask was sealed with a rubber stopper and sealing film. Under nitrogen protection, the flask was evacuated using a water-based vacuum pump, and this process was repeated three times until a near-vacuum state was achieved. The flask was then fixed to a stirrer (500 rpm), and 80 mL of DMF was slowly added to the flask under ice bath conditions until the reactants were fully dissolved. Then, 5.00 mL of DIPEA was added to the flask, and the reaction was allowed to proceed for 6 hours. A small amount of the reaction liquid was taken from the flask using a sampling needle for TLC monitoring until the reaction was complete.

[0127] Step 5: Pour all the reaction solution into a separatory funnel, add an equal volume of ethyl acetate as the organic phase, and wash the organic phase successively with saturated sodium bicarbonate solution, 0.1 mol / L hydrochloric acid solution, and saturated sodium chloride solution. Dry the washed organic phase with an appropriate amount of anhydrous sodium sulfate solid for 6 hours. After drying, filter the organic phase to remove the anhydrous sodium sulfate solid, and then concentrate the filtered organic phase under reduced pressure at 40℃ and 100 rpm to obtain Boc-E2K(OtBu)-OF. 17 Crude product. Boc-E2K(OtBu)-OF 17 The crude product was purified by column chromatography (dichloromethane:methanol = 40:1, V:V) to obtain Boc-E2K(OtBu)-OF 17 The obtained product was placed in a vacuum drying oven and dried for 24 hours to remove the organic solvent.

[0128] Step 6: Accurately weigh the above reaction product Boc-E2K(OtBu)-OF 17 1.00 g was placed in a 50 mL round-bottom flask, a stir bar was added, and the flask was sealed with a rubber stopper. Vacuum was applied, and nitrogen was introduced; this process was repeated three times. The flask, under nitrogen protection, was placed in an ice bath, and 9 mL of a trifluoroacetic acid / dichloromethane / triethylsilane mixed solvent (trifluoroacetic acid:dichloromethane:triethylsilane = 4:4:1, V:V:V) was added. The mixture was stirred at 500 rpm for 4 h. A small amount of the reaction liquid was taken from the round-bottom flask using a sampling needle for TLC monitoring until the reaction was complete. After the reaction was complete, the reaction solution was concentrated under reduced pressure and repeatedly evaporated to dryness. Then, 5.0 mL of pre-cooled ice-cold diethyl ether was added to the flask, and the mixture was repeatedly stirred and allowed to stand until a white precipitate formed. The supernatant was removed, and the above operation was repeated twice. The flask containing the above reaction mixture was concentrated under reduced pressure and evaporated to dryness, sealed with plastic wrap with small holes, and placed in a vacuum drying oven to dry overnight. After the organic reagents were completely dried, the final product E2K-OF was collected. 17 Its mass spectrum is shown below. Figure 12 [M-H] obtained by mass spectrometry analysis + The molecular weight was 835.17, consistent with the theoretical and measured molecular weights, indicating that E2K-OF... 17 It was successfully synthesized.

[0129] Example 2

[0130] Preparation of FITC-labeled insulin (INS):

[0131] Accurately weigh FITC (5 mg) and insulin (100 mg) (Xuzhou Wanbang Jinqiao Pharmaceutical Co., Ltd., batch number: 1903A10), and dissolve them separately in 2 mL DMSO and 4 mL 0.1 mol / mL sodium bicarbonate solution. Transfer the insulin solution to a stirrer-equipped flask, and add the FITC solution dropwise to the stirred insulin solution. Incubate at room temperature in the dark for 12 hours. After the reaction is complete, transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 3500 Da, clamp the dialysis bag tightly, and dialyze with ultrapure water as the dialysis medium for 48 hours, changing the dialysate every 12 hours. Pre-freeze the dialyzed FITC-insulin solution at -20°C for 24 hours, and then freeze-dry it for 48 hours. After freeze-drying, an orange-yellow powder of FITC-insulin is obtained.

[0132] Example 3

[0133] Preparation of different blank nanoparticles and drug-loaded nanoparticles:

[0134] The lipopeptide dendritic molecules prepared in Example 1 were dissolved in tetrahydrofuran. 0.2 mL of the above tetrahydrofuran lipopeptide dendritic molecule solution (10 mg / mL) was slowly added dropwise to 1.5 mL of deionized water while stirring at 800 rpm. The mixture was stirred until the solvent evaporated to obtain blank nanomicelles.

[0135] The lipopeptide dendritic molecules prepared in Example 1 were dissolved in tetrahydrofuran. 0.2 mL of the above tetrahydrofuran lipopeptide dendritic molecule solution (10 mg / mL) was slowly added dropwise to 2 mL of insulin solution (0.25 mg / mL, dissolved in 0.1 mol / mL sodium bicarbonate) while stirring at 800 rpm. Then, 100 μL of zinc chloride solution (2 mg / mL) was added dropwise to the solution containing insulin and dendritic lipopeptide material. The mixture was stirred at room temperature for 24 h until the solvent evaporated. The resulting solution was ultrafiltered using a 10 kDa ultrafiltration tube to remove free insulin, yielding a solution of dendritic lipopeptide nanomicelles containing insulin (INS).

[0136] The dendritic lipopeptide molecules prepared in Example 1 were dissolved in tetrahydrofuran. 0.2 mL of the above tetrahydrofuran lipopeptide dendritic molecule solution (10 mg / mL) was slowly added dropwise to 2 mL of liraglutide solution (0.25 mg / mL, dissolved in 0.1 mol / mL sodium bicarbonate) while stirring at 800 rpm, and the mixture was stirred. After stirring until the solvent evaporated, the resulting solution was ultrafiltered using a 10 kDa ultrafiltration tube to remove free liraglutide, yielding a solution of dendritic lipopeptide nanomicelles containing liraglutide (Lira).

[0137] Example 4

[0138] Measurement of particle size distribution and potential of drug-loaded nanoparticles:

[0139] 0.2 mL of the drug-loaded nanoparticles prepared in Example 3 were transferred to a suitable centrifuge tube, and the total volume was diluted to 2 mL with ultrapure water. Subsequently, the diluted dendritic lipopeptide nanoparticle solution was transferred to a quartz particle size distribution measuring dish, and its particle size distribution was determined using a laser particle size analyzer at 25°C.

[0140] 0.2 mL of the drug-loaded nanoparticles prepared in Example 3 was transferred to a suitable centrifuge tube, and the total volume was diluted to 2 mL using PBS solution at pH 6.8. Subsequently, the diluted dendritic lipopeptide nanoparticle solution was transferred to a quartz particle size analyzer, and its potential distribution was measured using a laser particle size analyzer at 25°C.

[0141] The results are as follows Figure 13 As shown, the particle size of each drug-loaded nanoparticle is around 80 nm. The amphoteric head nanoparticles have a near-neutral surface charge, while the pure amino head nanoparticles have a positive surface charge. Their near-neutral hydrophilic surface is expected to enhance the mucus penetration effect of the nanoparticles.

[0142] The results are as follows Figure 14 As shown, if the drug is liraglutide, the particle size of the drug-loaded nanoparticles is 75 nm-100 nm, and they have a near-neutral potential. Both the encapsulation efficiency and drug loading are high, meeting the requirements.

[0143] Example 5

[0144] Transwell mucus permeation experiments with different drug-loaded nanoparticles:

[0145] Following Example 3, different dendritic lipopeptide molecules were prepared to encapsulate FITC-insulin, and the concentration of FITC-insulin was uniformly adjusted to 500 μg / mL according to the fluorescence standard curve. Fresh rat intestinal mucus was collected and appropriately diluted with ultrapure water at pH 6.8. During the experiment, 100 μL of mucus solution was added to the upper chamber of a 24-well Transwell plate, and the plate was placed in a constant temperature environment of 37°C for 15 minutes for equilibration. After equilibration, 100 μL of dendritic lipopeptide nanomedicine encapsulated with FITC-insulin was added above the mucus layer, while 800 μL of ultrapure water at pH 6.8 was added to the lower chamber of the plate as the receiving solution. Three replicates were set for each experiment to ensure data reliability. The 24-well plate was placed in a constant temperature plate shaker and incubated at 37°C and 100 rpm for 2 hours. After incubation, 200 μL of the receiving solution was aspirated from the lower chamber, and its fluorescence intensity was measured using a microplate reader. Finally, the permeability percentage of the nanomedicine was determined by calculating the ratio of the FITC-INS content in the receiving fluid to the FITC-INS content above the initially added mucus.

[0146] The results are as follows Figure 15 As shown in Figure A), A) is a schematic diagram of the experiment. From B), it can be seen that the amphoteric surface nanoparticles (E2K-OA-5 / INS, E2K-OA-1 / INS, E2K-OF7 / INS, E2K-OF7 / INS, E2K-OF7 / INS, E2K-OF7 / INS) are... 13 / INS、E2K-OF 17 Due to their near-neutral charge, / INS nanoparticles exhibit significantly better Transwell mucus penetration than G2K-OA nanoparticles with a purely amino positively charged surface.

[0147] Example 6

[0148] Experiments on the anti-protein adsorption of different blank nanoparticles:

[0149] Blank nanoparticles of different dendritic lipopeptide molecules were prepared according to Example 3, with a concentration of 0.05 mg / mL. Porcine gastric mucin was selected as a model protein to evaluate the anti-adsorption performance of different dendritic lipopeptide nanoparticles on mucin. Each blank carrier was mixed with a 0.05 mg / mL mucin solution in simulated intestinal fluid at pH 6.8, and then incubated in a water bath at 37°C for 2 hours with gentle shaking. After incubation, the mixture was centrifuged at 10000 g for 15 minutes. The supernatant was then collected, and its absorbance was measured at 280 nm using a UV-Vis spectrophotometer. The amount of mucin adsorbed on the sample was calculated using a standard curve of mucin. Three parallel measurements were performed for each sample.

[0150] The results are as follows Figure 16As shown, amphoteric surface nanoparticles (E2K-OA-5, E2K-OA-1, E2K-OF7, E2K-OF7) 13 E2K-OF 17 The anti-adsorption effect of E2K-OF7 is significantly better than that of pure amino positively charged surface nanoparticles (G2K-OA). Furthermore, under the same head conditions, E2K-OF7 and E2K-OF... 13 E2K-OF 17 Due to the presence of fluorinated hydrophobic chains, the nanoparticles exhibit slightly better anti-adsorption effects on sticky proteins than alkane chain nanoparticles (G2K-OA, E2K-OA-5, E2K-OA-1).

[0151] Example 7

[0152] Cellular uptake experiments of different drug-loaded nanoparticles:

[0153] Caco-2 and HT-29 cells in the logarithmic growth phase were selected, digested, resuspended, and counted using a cell counting chamber. The cells were then diluted appropriately to adjust the cell suspension concentration to 10 × 10⁻⁶. 4 Cells / mL. The diluted cell suspension was seeded at 1 mL per well in 12-well plates and incubated at 37°C with 5% CO2 for 24 hours. During incubation, dendritic lipopeptide nanomedicines encapsulating FITC-insulin (INS) were prepared in advance according to Example 3, and diluted to the same concentration (10 μg / mL) using DMEM basal medium. After the cells showed good condition under a microscope, the original medium was discarded, and the cells were washed three times with PBS to remove residual medium components. Next, the diluted drug solution was added, and the 12-well plates were placed in a 37°C, 5% CO2 incubator for another 4 hours, with three replicates per group (n=3). The drug solution was then discarded, and the cells were washed three more times with PBS to remove any untaken drug. Subsequently, 200 μL of trypsin was added to each well to digest the cells. After incubation for 3-4 minutes, 800 μL of complete medium was added to terminate the digestion process. Gently pipette the cells to detach them from the well walls, then transfer the collected cell suspension to a 1.5 mL centrifuge tube. Centrifuge the tube at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in 1 mL of PBS. Centrifuge again under the same conditions, discard the supernatant, and resuspend the cells in 500 μL of PBS. Finally, perform quantitative fluorescence analysis of the cells using flow cytometry to accurately assess the cells' drug uptake.

[0154] The results are as follows Figure 17As shown, A) represents the uptake results of Caco-2 cells, and B) represents the uptake results of HT-29 cells. Due to the negative charge of the cell membrane, positively charged nanoparticles (G2K-OA / INS) exhibit significantly higher uptake efficiency compared to alkane-chain near-neutral surface nanoparticles (E2K-OA-5 / INS, E2K-OA-1 / INS). However, after fluorination modification (E2K-OF7 / INS, E2K-OF...), the uptake efficiency of these nanoparticles is significantly improved. 13 / INS、E2K-OF 17 / INS), the near-neutral surface fluorinated hydrophobic chains significantly enhanced cellular uptake, exhibiting the highest average fluorescence intensity. Among them, E2K-OF 17 / / INS works best.

[0155] Example 8

[0156] Transwell transmembrane transport experiments with different drug-loaded nanoparticles:

[0157] Caco-2 cells and HT-29 cells were selected and co-cultured at a ratio of 9:1, with the cell suspension concentration adjusted to 3×10⁻⁶. 4 Cells / mL were used to construct a cell monolayer model. After 21 days of culture, cells with a TEER value greater than 300 Ω·cm were selected. - Transmembrane transport experiments were performed on the cell monolayer membrane of ². Before the experiment, the culture medium in the Transwell chamber (AP side) and the basal side (BL side) was discarded, and the cells were washed three times with HBSS buffer preheated to 37°C. Then, HBSS buffer at 37°C was added to both the AP and basal sides, and the cells were incubated for 20 minutes to equilibrate. Dendritic lipopeptide nanomedicine loaded with FITC-insulin was prepared according to the method in Example 3 and diluted to a uniform concentration of 100 μg / mL with HBSS solution. The culture plate was removed from the incubator, and the buffer solutions on both sides were discarded. Then, 200 μL (100 μg / mL) of free insulin and 200 μL (100 μg / mL) of the diluted drug solution were added to the AP side, and 800 μL of HBSS buffer was added to the BL side. The culture plate was returned to the incubator at 37°C and 5% CO2 and incubated for 2 hours. After incubation, the receiving solution was collected from the BL side, and the fluorescence intensity of the AP and BL sides was measured using a microplate reader to calculate the amount of drug transported and the apparent permeability coefficient (P). app In the experiment, each group had 3 duplicate wells (n = 3). The apparent permeability (P) of each sample was... app ) is calculated using the following formula:

[0158]

[0159] dQ / dt is the drug transport rate per unit time (μg / s); A is the effective membrane area of ​​the 24-well Transwell chamber (0.33 cm²). 2 C0 represents the initial concentration of the drug solution (μg / mL).

[0160] The results are as follows Figure 18 As shown, in a simulated transmembrane transport experiment of a single intestinal epithelial layer, the E2K-OF of both sexes... 17 / INS apparent permeability coefficient (P app (The best)

[0161] Example 9

[0162] Experiments on rat in vitro inverted intestinal sacs with different drug-loaded nanoparticles:

[0163] Rats were fasted overnight and then anesthetized and euthanized. A 6 cm segment of jejunum was quickly removed and rinsed with physiological saline until almost no contents were present. The jejunum was then transferred to oxygen-saturated Krebs-Ringer buffer (KR solution) to remove surface blood vessels and fat. The intestinal lumen was everted using a glass rod and both ends were ligated to create an everted intestinal pouch approximately 5 cm long. 1 mL of blank KR liquid was instilled, and the pouch was equilibrated in KR solution with 95% O2 and 5% CO2 for 10 minutes. Subsequently, the intestinal pouch was placed in different dendritic lipopeptide nanomedicine KR dilutions (25 μg / mL) prepared according to the method in Example 3. 150 μL of liquid was sampled from the intestinal pouch at 20, 40, 60, 80, 100, and 120 minutes, and 150 μL of blank KR liquid was quickly added. Finally, the fluorescence intensity of the sampled liquid was measured using an ELISA reader. The FITC-INS content ingested in the intestinal pouch was quantitatively analyzed by the change in fluorescence intensity to assess the drug absorption in the intestine. The cumulative absorption (Q) was calculated using a formula.

[0164]

[0165] C n C represents the actual detected mass concentration (mg / mL) of the intestinal fluid at time point n. i V0 represents the actual detection concentration (mg / mL) of the sample taken at time point i (i≤n-1), V0 is the initial volume (mL) of blank KR solution added to the intestinal pouch, and A is the intestinal segment area (cm²). 2 ).

[0166] The results are as follows Figure 19 As shown, E2K-OF 17 / INS has the best intestinal mucosal penetration effect.

[0167] As can be seen from Examples 7-9, the dendritic lipopeptide molecules that promote oral absorption of macromolecular drugs prepared in this invention have significantly better performance than the previously reported E2K-OA-5, especially E2K-OF. 17 It performed best in cell uptake, Transwell transmembrane transport, and in vitro inverted intestinal sac experiments, and its performance far exceeded that of other materials.

[0168] Example 10

[0169] The hypoglycemic and weight-reducing effects of long-term oral administration of different drug-loaded nanoparticles in type 2 diabetic rats:

[0170] Male SD rats weighing approximately 230 g were selected to establish a type 2 diabetes model. Before modeling, rats were fed a high-fat diet of XTHF60 (Synerbio) and a high-fat diet of XTHF45 (Synerbio) for 14 days, and this high-fat diet was continued throughout the subsequent experiments. Modeling began when rats reached a weight of 380-400 g. Rats were fasted overnight but allowed water before modeling. Nicotinamide (NA) at 110 mg / kg was prepared using physiological saline (pH 7.4), and STZ at 65 mg / kg was prepared using citrate buffer (pH 4.2). Rats were first injected intraperitoneally with NA solution, followed by an intraperitoneal injection of STZ solution 0.5 h later. Two hours after NA and STZ administration, food and sucrose water were provided. Blood glucose levels were monitored at 4, 8, 24, 48, and 72 hours after modeling; a fasting blood glucose level exceeding 16.7 mmol / L was considered a successful model establishment.

[0171] The nanomedicines Lira / E2K-OA-1 and Lira / E2K-OF, loaded with liraglutide (Lira), were prepared according to the method in Example 3. 17 The freeze-dried Lira / E2K-OA-1 and Lira / E2K-OF 17 Nanomedicines were encapsulated in enteric-coated capsules, and type 2 diabetic rats were randomly divided into 5 groups, with 6 rats in each group. The groups were: subcutaneous injection of liraglutide, oral administration of liraglutide enteric-coated capsules, oral administration of Lira / E2K-OA-1 enteric-coated capsules, and oral administration of Lira / E2K-OF. 17Enteric-coated capsule group (preparation method same as in Example 3). Before the experiment, rats were fasted overnight but allowed water access, and their tails were cut off to measure blood glucose concentration before administration. Subsequently, rats in different groups were administered liraglutide subcutaneously (0.3 mg / kg) and orally (3.0 mg / kg), respectively, for 20 days. Blood was collected from the tails of each group to measure blood glucose concentration, and a rat blood glucose concentration-time curve was plotted. Simultaneously, the rats were weighed daily, and weight changes were recorded over the 20 days of administration. The weight of the rats before administration (day 1) was set as 100%, and the percentage of weight at each time point relative to the initial weight was calculated, and a rat weight percentage-time curve was plotted.

[0172] The results are as follows Figure 20 As shown in the figures, A) weight changes in type 2 diabetic rats during long-term treatment, and B) the hypoglycemic effect in type 2 diabetic rats during long-term treatment. The blue arrows indicate drug administration; the rats were fed a normal high-fat diet for the remaining time. From these two figures, it can be seen that, in terms of blood glucose, while long-term oral administration of Lira / E2K-OA-1 enteric-coated capsules lowered blood glucose, it did not effectively maintain blood glucose within the normal range (11.1 ~ 16.7 mmol / L). In contrast, long-term oral administration of Lira / E2K-OF... 17 The enteric-coated capsule group maintained blood sugar within the normal range; in terms of weight loss, the Lira / E2K-OA-1 enteric-coated capsule group only reduced blood sugar to 97.48% of the initial value, while the Lira / E2K-OF group... 17 The enteric-coated capsule group effectively reduced the body weight of type II diabetic rats to 90.32% of the initial value, demonstrating the best hypoglycemic control and weight reduction effects.

[0173] Example 11

[0174] Hypoglycemic effects and duration of hypoglycemia, normoglycemia, and hyperglycemia after oral administration of different drug-loaded nanoparticles in type 1 diabetic miniature pigs:

[0175] Six-month-old male Bama miniature pigs were induced to develop diabetes via intravenous streptozotocin (STZ, 150 mg / kg) after overnight fasting. STZ was dissolved in freshly prepared citrate buffer (pH 4.5) to a concentration of 75 mg / ml and administered intravenously over 10 minutes. After a 7-day recovery period, glucose levels were monitored using a continuous glucose monitoring system (CGMS, range 39.6–500.0 mg dL). −1 Blood glucose levels were monitored to confirm the successful establishment of the type 1 diabetes model. Once blood glucose levels stabilized, diabetic miniature pigs were used for subsequent research.

[0176] The insulin (INS)-encapsulated nanomedicines E2K-OA-1 / INS and E2K-OF were prepared according to the method in Example 3. 17 / INS, freeze-dried E2K-OA-1 / INS and E2K-OF 17 / INS nanomedicine is encapsulated in enteric-coated capsules and administered in the same dose as insulin, at a dose of 5 IU / kg. −1 The control group received enteric-coated free insulin capsules and subcutaneous injections of 0.5 IU / kg. −1 Free insulin. During treatment, the miniature pigs are allowed free access to food and water.

[0177] The results are as follows Figure 21 As shown, A) the hypoglycemic effect of a single dose in type 1 diabetic miniature pigs, and B) the duration of hypoglycemia, normoglycemia, and hyperglycemia after a single dose in type 1 diabetic miniature pigs. No significant change in blood glucose levels was observed in miniature pigs treated with insulin capsules. In contrast, oral administration of E2K-OA-1 and E2K-OF... 17 After administration of enteric-coated capsules, blood glucose levels in miniature pigs gradually decreased and returned to normal within 4 hours. Compared to the short-acting hypoglycemic effect of subcutaneous insulin injection, oral administration of E2K-OA-1 / INS enteric-coated capsules maintained normal blood glucose levels for approximately 7.6 hours, while oral administration of E2K-OF... 17 / INS enteric-coated capsules can maintain their effect for up to 12 hours, and both exhibit postprandial blood glucose fluctuations. These results indicate that E2K-OF 17 It has a superior ability to lower and control blood sugar.

Claims

1. A class of dendritic lipopeptide molecules that promote the oral absorption of macromolecular drugs, characterized in that, The dendritic lipopeptide molecule is formed by the combination of a hydrophilic head and a hydrophobic tail. The hydrophilic head consists of lysine and glutamic acid linked by amide or ester bonds, while the hydrophobic tail is composed of organic carbon chains or fluorinated chains. The hydrophilic head is selected from...

2. Where: R1 is -C n H 2n-1 n is 2~20; R2 is -C m F 2m+1 m is 2~9.

3. The dendritic lipopeptide molecule for promoting oral absorption of macromolecular drugs according to claim 1, characterized in that, R1 is C2-C 20 The unsaturated alkyl group; R2 is selected from any of the following structures: 。 4. The dendritic lipopeptide molecule for promoting oral absorption of macromolecular drugs according to claim 1, characterized in that, The dendritic lipopeptide molecule is preferably selected from any one of the following: 。 5. A method for preparing a dendritic lipopeptide molecule that promotes oral absorption of macromolecular drugs as described in claim 1, characterized in that, Includes the following steps: Lys-NHR2 was obtained by sequentially esterifying Boc-Lys(Boc)-OH and R1NH2 and then deprotecting it with Boc. Lys-NHR2 was then reacted with Boc-L-Glu-1-OtBu by sequentially amide condensation, silica gel column purification, deprotection of Boc and protection with OtBu to obtain E2K-NHR2. Alternatively, Lys-NHR2 can be obtained by sequentially amide condensation and deBoc protection of Boc-Lys(Boc)-OH and R1NH2, and then Lys-NHR2 can be obtained by sequentially amide condensation, silica gel column purification, deBoc protection and OtBu protection of Boc-Gly. Alternatively, Lys-OCH2R2 can be obtained by sequentially esterifying Boc-Lys(Boc)-OH and R2CH2OH and removing Boc protection. Lys-OCH2R2 can then be reacted with Boc-L-Glu-1-OtBu by sequentially amide condensation, silica gel column purification, and removal of Boc and OtBu protection to obtain E2K-OCH2R2. Where: R1 is -C n H 2n-1 n is 2~20; R2 is -C m F 2m+1 m is 2~9.

6. A method for preparing a nanomedicine based on dendritic lipopeptide molecules that promote oral absorption of macromolecular drugs as described in claim 1, characterized in that, Includes the following steps: (1) Dissolve dendritic lipopeptide molecules in an organic solvent to obtain mixed solution A, inject mixed solution A into a solution containing the drug, and stir to remove the organic solvent to obtain mixed solution B; (2) The mixed solution B is ultrafiltered and centrifuged to remove impurities and unencapsulated drugs, thus obtaining dendritic lipopeptide nanomedicine.

7. The method for preparing nanomedicine according to claim 5, characterized in that, The organic solvent is tetrahydrofuran, methanol, chloroform, acetone, dichloromethane, or ethanol, etc.

8. The method for preparing nanomedicine according to claim 5, characterized in that, The drug is insulin, liraglutide with GLP-1 structure, dulaglutide, smegglutide, benaglutide, exendin-4 exenatide, exenatide, lixisenatide, or polyethylene glycol loxenatide, and the solution of the drug is in the form of dilute sodium bicarbonate solution, dilute sodium hydroxide solution, sodium citrate, or pure water.

9. The method for preparing nanomedicine according to claim 5, characterized in that, The concentration of the mixed solution A is 1~10 mg / mL, the concentration of the drug solution is 1~10 mg / mL, and the mass ratio of the dendritic lipopeptide molecule to the drug is 1~10:1~10.

10. The method for preparing nanomedicine according to claim 7, characterized in that, Zinc chloride is added during the preparation of insulin, and the mass ratio of zinc chloride to insulin is 1~10:1~10.

11. The use of a dendritic lipopeptide molecule as described in claim 1 for promoting oral absorption of macromolecular drugs or the nanomedicine as described in claim 5 in the preparation of a medicament for delivering insulin and treating diabetes.

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