Liraglutide nasal delivery nano preparation as well as preparation method and application thereof

Abstract

The invention belongs to the field of medicines, and particularly relates to a liraglutide nasal delivery nano preparation as well as a preparation method and application thereof. The technical problem to be solved by the invention is to provide a brand new liraglutide nasal delivery nano preparation. According to the liraglutide nasal delivery nano preparation, chitosan serves as a nano carrier material, liraglutide nanoparticles are prepared through an ionic crosslinking method, in order to improve the stability of the nanoparticles, a surfactant poloxamer188 is added into the preparation, and finally protamine is added into the liraglutide nanoparticles to improve the transmembrane transport capacity of the nanoparticles. The liraglutide nasal delivery nano preparation disclosed by the invention can effectively play a role in reducing blood sugar through a nasal delivery way, and the pharmacological bioavailability is improved; the preparation is a stable and long-acting nasal administration hypoglycemic preparation, the patient compliance is improved, and a new choice is provided for a non-injection administration route of protein polypeptide drugs.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a liraglutide nasal administration nano - preparation, its preparation method and application. Background Art

[0002] Liraglutide (LIRA) is a GLP - 1RAs composed of 31 amino acid residues, with a molecular weight of 3751.20 daltons. This drug was developed by Novo Nordisk of Denmark for many years and was approved by the FDA for marketing in 2010. It is mainly used for the clinical treatment of type 2 diabetes, and its structural formula is shown in Formula 1.

[0003] NH2 - His - Ala - Glu - Gly - Thr - Phe - Thr - Ser - Asp - Val - Ser - Ser - Tyr - Leu - Glu - Gly - Gln - Ala - Ala - Lys(N - ε-(N - α - Palmitoyl - L - γ - glutamyl)-Glu - Phe - Ile - Ala - Trp - Leu - Val - Arg - Gly - Arg - Gly - COOH

[0004] Formula 1

[0005] Liraglutide has 97% homology with endogenous glucagon - like peptide - 1 (GLP - 1). It is obtained by replacing lysine with arginine at position 34 of endogenous GLP - 1 and adding a C16 fatty acid to the ε - amino group of lysine at position 26 through a glutamate spacer. And due to the reduction of high - protein binding and enzymatic degradation after subcutaneous administration, the plasma half - life is extended to 13 hours.

[0006] The currently marketed dosage form is a subcutaneous injection. Long - term daily subcutaneous injection will cause pain to patients, resulting in poor compliance of patients. Therefore, in order to meet the needs of patients and improve the compliance of patients during medication, new liraglutide administration routes need to be explored. Due to the instability of the properties of protein and polypeptide drugs, the currently marketed drug administration routes mainly rely on injection. In non - injection drug delivery systems, drug delivery can mainly be achieved through four methods: oral route, transdermal route, mucosal route and pulmonary route.

[0007] In this application background, the team where the inventor belongs has been exploring the research and development of liraglutide dosage forms, such as CN114452258A, liraglutide liposome preparation, its preparation method and application, CN117084971A, liraglutide nasal administration in - situ thermosensitive gel preparation, its preparation method and use. Different aspects have been explored from excipients to dosage forms. This time, an attempt is made to further explore the improvement of the preparation system on the basis of nasal administration. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a novel nano - preparation of liraglutide for nasal administration. The nano - preparation of liraglutide for nasal administration of the present invention uses chitosan as the nano - carrier material, and prepares liraglutide nanoparticles by the ionic cross - linking method. In order to increase the stability of the nanoparticles, the surfactant poloxamer 188 (Poloxamer188) is added to the preparation. Finally, protamine is added to the liraglutide nanoparticles to improve the transmembrane transport ability of the nanoparticles.

[0009] The nano - preparation of liraglutide for nasal administration of the present invention, per 10 mL, comprises the following components in the following weight ratio:

[0010]

[0011] Preferably, the nano - preparation of liraglutide for nasal administration of the present invention, per 10 mL, comprises the following components in the following weight ratio:

[0012]

[0013]

[0014] Most preferably, the nano - preparation of liraglutide for nasal administration of the present invention, per 10 mL, comprises the following components in the following weight ratio:

[0015]

[0016] Furthermore, the components of the nano - preparation of liraglutide for nasal administration of the present invention preferably satisfy the following weight ratio relationship:

[0017] The weight ratio of chitosan to liraglutide is 5:1 to 1:1, and the weight ratio of chitosan to poloxamer 188 is 15:1 to 5:1.

[0018] Preferably, the weight ratio of chitosan to liraglutide is 4:1 to 2:1, and the weight ratio of chitosan to poloxamer 188 is 15:1 to 5:1.

[0019] Most preferably, the weight ratio of chitosan to liraglutide is 2.95:1, and the weight ratio of chitosan to poloxamer 188 is 10.55:1.

[0020] The ionic cross - linker of the nano - preparation of liraglutide for nasal administration of the present invention is sodium tripolyphosphate (TPP).

[0021] The nano - preparation of liraglutide for nasal administration of the present invention is prepared by the following method:

[0022] A. Prepare a suspension of liraglutide - chitosan nanoparticles by the ionic cross - linking method:

[0023] (1) Weigh the liraglutide raw material drug, and add it to PBS7.40 buffer solution to prepare a liraglutide solution;

[0024] (2) Weigh chitosan and poloxamer 188 according to the weight ratio of dry matter components. After swelling them sufficiently with a solvent, adjust the pH value to obtain a chitosan solution;

[0025] (3) Dropwise add the liraglutide solution obtained in step (1) to the chitosan solution obtained in step (2), and stir until fully mixed;

[0026] (4) Dropwise add an ionic cross-linking agent to the mixed solution in step (3) until the solution shows a light blue opalescence. After ultrasonic dispersion, a liraglutide chitosan nanoparticle suspension is obtained;

[0027] B. Preparation of protamine-modified liraglutide chitosan nanoparticles: Dropwise add a protamine solution to the liraglutide chitosan nanoparticle suspension obtained in step A, and stir until fully mixed to obtain protamine-modified liraglutide chitosan nanoparticles.

[0028] In the above technical solution, in step (1) of step A: The solvent of the liraglutide solution is PBS7.40 buffer solution, water, or sodium acetate solution.

[0029] Preferably, in step (1) of step A: The solvent of the liraglutide solution is PBS7.40 buffer solution.

[0030] In step (1) of step A: The concentration of the liraglutide solution is 4.3 - 8.6 mg·mL -1 .

[0031] Preferably, in step (1) of step A: The concentration of the liraglutide solution is 4.3 mg·mL -1 .

[0032] In the above technical solution, in step (2) of step A, chitosan is used as a carrier material, and poloxamer 188 is used as a surfactant.

[0033] In step (2) of step A, chitosan and poloxamer 188 are weighed according to the weight ratio of chitosan to liraglutide being 5:1 to 1:1, and the weight ratio of chitosan to poloxamer 188 being 15:1 to 5:1.

[0034] Preferably, in step (2) of step A, chitosan and poloxamer 188 are weighed according to the weight ratio of chitosan to liraglutide being 4:1 to 2:1, and the weight ratio of chitosan to poloxamer 188 being 15:1 to 5:1.

[0035] Most preferably, in step (2) of step A, chitosan and poloxamer 188 are weighed according to the weight ratio of chitosan to liraglutide being 2.95:1 and the weight ratio of chitosan to poloxamer 188 being 10.55:1.

[0036] In step (2) of step A, the solvent for chitosan and poloxamer 188 is an aqueous acetic acid solution of 0.1-2%. Chitosan is insoluble in water and common organic solvents and dissolves at pH < 6.5. As a weak acid, acetic acid can provide a large amount of H + to protonate the amino groups on chitosan, further promoting dissolution. At the same time, the acidity is mild and is not likely to cause degradation of the chitosan chain.

[0037] Preferably, in step (2) of step A, the solvent is an aqueous acetic acid solution of 1%.

[0038] The sufficient swelling in step (2) of step A means that chitosan and poloxamer 188 are mixed with the aqueous acetic acid solution by stirring and absorb the aqueous acetic acid solution to an equilibrium state.

[0039] The conditions for stirring in step (2) of step A are stirring for 3-5 hours until sufficient swelling.

[0040] Preferably, the conditions for stirring in step (2) of step A are stirring for 4 hours.

[0041] The pH value is adjusted to 5.0-5.5 in step (2) of step A.

[0042] Preferably, the pH value is adjusted to 5.25 in step (1) of step A.

[0043] The pH value is adjusted using an alkaline solution in step (2) of step A.

[0044] The alkaline solution in step (2) of step A is NaOH solution, KOH solution, phosphate buffer solution, ammonia water.

[0045] Preferably, the alkaline solution in step (2) of step A is NaOH solution.

[0046] The concentration of the NaOH solution in step (2) of step A is 1-3 M.

[0047] Preferably, the concentration of the NaOH solution in step (2) of step A is 2 M.

[0048] If the chitosan solution prepared by step (2) of step A is not used immediately, it can be refrigerated for standby at 1-8°C. The preferred refrigeration condition is 4°C.

[0049] In the above technical solution, in step (3) of step A, taking the dosage of liraglutide as 8.6 mg, a chitosan solution containing 8.6 - 43 mg of chitosan and 0.57 - 8.6 mg of poloxamer 188 is added;

[0050] Preferably, in step (3) of step A, taking the dosage of liraglutide as 8.6 mg, a chitosan solution containing 17.2 - 34.4 mg of chitosan and 1.15 - 6.88 mg of poloxamer 188 is added;

[0051] Most preferably, in step (3) of step A, taking the dosage of liraglutide as 8.6 mg, a chitosan solution containing 25.37 mg of chitosan and 2.40 mg of poloxamer 188 is added;

[0052] In step (3) of step A, the stirring time of the chitosan solution and the liraglutide solution is 1 - 3 h.

[0053] Preferably, in step (3) of step A, the stirring time of the chitosan solution and the liraglutide solution is 1.5 h.

[0054] In the above technical solution, in step (4) of step A: The ionic cross - linker used is sodium tripolyphosphate (TPP). Commonly used ionic cross - linkers mainly include sodium tripolyphosphate, sodium hexametaphosphate, sodium alginate, etc. Since the cation concentration of sodium tripolyphosphate is relatively high, the degree of cross - linking is relatively high and the stability is better. Therefore, the present invention selects sodium tripolyphosphate as the ionic cross - linker.

[0055] During the experiment, it was found that when the liraglutide solution was added to the chitosan solution, the system became a milky white and slightly transparent colloidal solution. By measuring the charge of the liraglutide solution, it was found that it carried a negative charge. After adding it to the chitosan solution, it cross - linked with the positively charged chitosan. However, the encapsulation efficiency of the nanoparticles obtained by this method was relatively low. Therefore, a small amount of ionic cross - linker still needs to be added subsequently to improve the encapsulation efficiency of the nanoparticles.

[0056] In step (4) of step A: The sodium tripolyphosphate (TPP) is added in the form of a sodium tripolyphosphate solution.

[0057] Further, in step (4) of step A: The concentration of the sodium tripolyphosphate (TPP) solution is 0.5 - 5 mg·mL -1 ;

[0058] Preferably, in step (4) of step A: The concentration of the sodium tripolyphosphate (TPP) solution is 0.5 - 1 mg·mL -1 ;

[0059] Most preferably, in step (4) of step A: the concentration of the sodium tripolyphosphate (TPP) solution is 0.95 mg·mL -1 .

[0060] In step (4) of step A, the dosage relationship between the sodium tripolyphosphate (TPP) and liraglutide is as follows: for every 8.6 mg of liraglutide dosage, 0.5 - 5 mg of sodium tripolyphosphate is used;

[0061] Preferably, in step (4) of step A, the dosage relationship between the sodium tripolyphosphate (TPP) and liraglutide is as follows: for every 8.6 mg of liraglutide dosage, 0.5 - 1 mg of sodium tripolyphosphate is used;

[0062] Preferably, in step (4) of step A, the dosage relationship between the sodium tripolyphosphate (TPP) and liraglutide is as follows: for every 8.6 mg of liraglutide dosage, 0.95 mg of sodium tripolyphosphate is used;

[0063] In step (4) of step A: the low-temperature ultrasonic condition is: ultrasonic under the condition of an ice-water bath.

[0064] In step (4) of step A: the low-temperature ultrasonic condition is: ultrasonic for 2 - 10 min, intermittent for 5 s / 5 s, 900 W × 20 - 40%.

[0065] Preferably, in step (4) of step A: the low-temperature ultrasonic condition is: ultrasonic for 6 min, intermittent for 5 s / 5 s, 900 W × 30%.

[0066] In the above technical solution, the protamine in step B is protamine sulfate.

[0067] The solvent of the protamine in step B is water or PBS 7.40 buffer solution.

[0068] Preferably, the solvent of the protamine in step B is water.

[0069] The protamine in step B is added with water to prepare a protamine solution with a concentration of 0.5 - 3 mg·mL -1 of the protamine solution.

[0070] Preferably, the protamine in step B is added with water to prepare a protamine solution with a concentration of 1 mg·mL -1 of the protamine solution.

[0071] The dosage relationship between the protamine and liraglutide in step B is as follows: for every 8.6 mg of liraglutide dosage, 0.5 - 3 mg of protamine is used;

[0072] Preferably, the dosage relationship between the protamine and liraglutide in step B is as follows: for every 8.6 mg of liraglutide dosage, 1 mg of protamine is used;

[0073] The time for stirring the protamine with water in step B is 0.5 - 2 h.

[0074] Preferably, the time for stirring the protamine with water in step B is 1.5 h.

[0075] The present invention also provides new uses of the liraglutide nasal administration nanopharmaceutics of the present invention. Specifically, it relates to: (1) the application of the liraglutide nasal administration nanopharmaceutics in the preparation of drugs for nasal administration; (2) the application of the liraglutide nasal administration nanopharmaceutics in the preparation of drugs for treating diabetes by nasal administration; in particular, the application of the liraglutide nasal administration nanopharmaceutics in the preparation of drugs for treating type 2 diabetes by nasal administration; and (3) the application of the liraglutide nasal administration nanopharmaceutics in the preparation of drugs for weight loss by nasal administration.

[0076] In the above preparation method, the inventors of the present invention focused on investigating the ratio relationship of the main raw materials, liraglutide, chitosan, poloxamer 188, and protamine, as well as parameters such as the concentration of key reagents, pH value, and reaction conditions during the preparation process, and obtained preparation parameters that meet the requirements of nanoparticles for key quality control indicators such as morphology, particle size, zeta potential, encapsulation efficiency, and drug loading. The liraglutide nasal administration nanopharmaceutics of the present invention has a high in vitro release amount. For example, the in vitro release amount of liraglutide chitosan nanoparticles can reach up to 73% of the total drug amount within 5 h, while the in vitro release amount of protamine-modified liraglutide chitosan nanoparticles can reach up to 79% of the total drug amount at 4 h. At the same time, cell experiments confirmed that protamine-modified liraglutide chitosan nanoparticles have excellent cell uptake ability and reliable safety for nasal drug use.

[0077] For the nasal drug delivery system, due to the large molecular weight of polypeptide drugs, the clearance of nasal mucosa cilia and the continuous secretion of nasal mucus limit the drug delivery in the nasal cavity. Compared with traditional preparations, the liraglutide nasal administration nanopharmaceutics of the present invention can protect biomacromolecular drugs from being destroyed during the drug delivery process by the nanocarrier, improve the drug transport efficiency, and have a sustained release effect at the same time. The liraglutide nasal administration nanopharmaceutics of the present invention uses chitosan as a common nanocarrier, which has good peptide loading ability and low toxicity. Its mucosal adhesion characteristics can increase the interaction time between the preparation and the nasal mucosa, thereby reducing the clearance rate of the drug in the nasal cavity. Modified with protamine sulfate (PS), it can increase the absorption of the drug in the nasal cavity. In vivo pharmacodynamic experiments have proved that protamine-modified liraglutide chitosan nanoparticles can increase the transmembrane ability of the drug and can effectively play a hypoglycemic role through the nasal drug administration route, with improved pharmacological bioavailability; it is a stable and long-acting nasal hypoglycemic preparation, improving patient compliance, and providing a new choice for the non-injection administration route of protein polypeptide drugs. Description of the Drawings

[0078] Figure 1 Preparation flow chart of liraglutide chitosan nanoparticles.

[0079] Figure 2 Preparation flow chart of protamine-modified liraglutide nanoparticles.

[0080] Figure 3 Appearance diagram of protamine-modified liraglutide nanoparticles.

[0081] Among them, Figure 3 A is the initially prepared nanoparticles; Figure 3 B is after the nanoparticles are placed in a 4°C refrigerator for two weeks.

[0082] Figure 4 Particle size distribution diagram of protamine-modified liraglutide nanoparticles.

[0083] Figure 5 Zeta potential diagram of protamine-modified liraglutide nanoparticles.

[0084] Figure 6 Transmission electron microscopy image of protamine-modified liraglutide nanoparticles.

[0085] Among them, Figure 6 The scale of A is 200 nm, Figure 6 The scale of B is 100 nm.

[0086] Figure 7 Circular dichroism spectrum of liraglutide.

[0087] Figure 8 In vitro release curve.

[0088] Figure 9 Survival rate of RPMI 2650 cells after treatment with blank nanocarriers at different concentrations for 24 h (n = 5, mean ± SD).

[0089] Figure 10 Survival rate of RPMI 2650 cells after treatment with liraglutide preparations at different concentrations for 24 h (n = 5, mean ± SD).

[0090] Figure 11 Fluorescence microscopic images of the uptake of different preparations by RPMI 2650 cells

[0091] Figure 12 Relative fluorescence intensity.

[0092] Among them, C6: Coumarin 6; CS-C6 NPs: Coumarin 6 nanoparticles; PS-CS-C6 NPs: Protamine-modified Coumarin 6 nanoparticles; n = 3, ***P < 0.001, **P < 0.01.

[0093] Figure 13 Flow cytometry was used to measure the uptake of RPMI 2650 cells by different formulations.

[0094] Among them, C6: Coumarin 6; CS-C6 NPs: Coumarin 6 nanoparticles; PS-CS-C6 NPs: Protamine-modified Coumarin 6 nanoparticles.

[0095] Figure 14 Quantitative analysis chart of cell uptake.

[0096] Among them, C6: Coumarin 6; CS-C6 NPs: Coumarin 6 nanoparticles; PS-CS-C6 NPs: Protamine-modified Coumarin 6 nanoparticles; n = 3, ***P < 0.001, *P < 0.05.

[0097] Figure 15 Blood glucose percentage-time curve.

[0098] Among them, Group A: Nasal administration of normal saline; Group B: Nasal administration of LIRA solution; Group C: Nasal administration of CS-LIRA NPs; Group D: Nasal administration of PS-CS-LIRA NPs; Group E: Subcutaneous injection of LIRA solution.

[0099] Figure 16 Relative bioavailability and analysis of variance results from 0 to 12 (n = 6, ***P < 0.001, **P < 0.01, *P < 0.05).

[0100] Figure 17 Relative bioavailability and analysis of variance results from 0 to 24 (n = 6, ***P < 0.001, **P < 0.01, *P < 0.05). Detailed implementation manners

[0101] The following will explain and illustrate the solution of the present invention in combination with embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For reagents or instruments not specified with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0102] The following are the interpretations of English abbreviations and the main noun explanations that appear in the present invention:

[0103] Diabetes Mellitus is abbreviated as DM; Glucagon-like peptide-1 is abbreviated as GLP-1; Exenatide, Semaglutide, Dulaglutide, and Liraglutide are abbreviated as LIRA; Nanoparticles are abbreviated as NPs; Chitosan-Liraglutide nanoparticles are abbreviated as CS-LIRA NPs; Protamine-modified chitosan-liraglutide nanoparticles are abbreviated as PS-CS-LIRA NPs; Chitosan is abbreviated as CS; Protamine; Protamine Sulfate is abbreviated as PS; Poloxamer 188 is abbreviated as P188; Sodium tripolyphosphate is abbreviated as TPP; Entrapping Efficiency is abbreviated as EE.

[0104] The research group where the inventors are located has been conducting research on liraglutide preparations. And some technical attempts have been made in the nasal administration route. The technical direction of this attempt is to increase the transmembrane effect in order to expect a more stable and long-acting hypoglycemic effect of liraglutide. The inventors made liraglutide into a chitosan nano-preparation and added a cell-penetrating peptide to improve the transmembrane transport ability of the drug, which is used for nasal drug delivery. It can change the invasive administration method of the marketed liraglutide injection and improve the poor compliance of patients during subcutaneous injection. At the same time, it avoids the instability, easy decomposition by gastrointestinal enzymes, and first-pass effect of the liver in oral administration. It is also possible to modify the chitosan-liraglutide nanoparticles by adding the cell-penetrating peptide protamine to improve the nasal mucosa permeability of the drug, enhance the trans-nasal mucosa transport ability of the drug, increase the bioavailability of the drug, and improve the drug efficacy.

[0105] I. Before preparing the protamine-modified chitosan-liraglutide nanoparticles of the present invention, the inventors also established a content analysis method with strong specificity and system adaptability and a method for determining the encapsulation efficiency of nanoparticles:

[0106] 1. Content analysis method

[0107] (1) The chromatographic conditions are as follows: Chromatographic column: C12 Jupiter 4μm proteo (250×4.6mm, 5μm); Mobile phase A: 0.05mol·L -1 Ammonium dihydrogen phosphate (pH adjusted to 3.70 with phosphoric acid)-acetonitrile = 9:1; Mobile phase B: Acetonitrile-water = 85:15; Detection wavelength: 215nm; Column temperature: 35°C; Flow rate: 1mL·min -1 ; Injection volume: 20μL; Isocratic elution program: Mobile phase A-Mobile phase B = 1:1, 0-18min.

[0108] (2) Pore size of the microporous membrane: The membrane with a pore size of 0.80 μm has the least influence on the content determination of liraglutide, and finally a 0.80 μm microporous membrane is selected.

[0109] (3) Characteristic peak: The peak elution time is about 13 min. The main peak and impurity peaks can be well separated, and the peak shape is good. There is a good linear relationship between the peak area and the concentration in the range of 21.5 - 150.5 μg·mL -1 The standard curve is A = 18.574C - 64.186 (r = 0.9999), with good precision and repeatability, and the sample addition recovery rate meets the requirements.

[0110] 2. Encapsulation efficiency determination method:

[0111] The Sephadex column method, ultrafiltration centrifugation method, flocculation centrifugation method and low-temperature centrifugation method were investigated. Finally, the low-temperature centrifugation method was selected to determine the encapsulation efficiency, and methanol was selected as the demulsifier. This encapsulation efficiency determination method is simple to operate, has a good recovery rate and high repeatability.

[0112] Low-temperature centrifugation method: Take two 1.5 mL centrifuge tubes, add 1 mL of liraglutide nanoparticles to each of them, centrifuge at 4 °C and 12,000 rpm for 15 min. Take 0.5 mL of the supernatant respectively, mix them, add 6 mL of methanol, and ultrasonically demulsify in an ice-water bath for 10 min. Dilute to 10 mL with PBS 7.40 buffer solution, filter through a 0.80 μm filter membrane, and inject the sample for detection according to the chromatographic conditions under "1. Content analysis method" to calculate the amount of free drug; in addition, take 1 mL of nanoparticles, demulsify with 6 mL of methanol for 10 min, dilute to 10 mL with PBS 7.40 buffer solution, filter through 0.80 μm, and inject the sample for detection according to the chromatographic conditions under "1. Content analysis method" to calculate the total amount of drug. Then calculate the encapsulation efficiency (Entrapping Efficiency, EE) and drug loading (Drug Loading, DL) according to formulas (1.1) and (1.2):

[0113]

[0114] II. Under the optimized investigation conditions mentioned above, the preparation methods of chitosan nanoparticles include: ionic cross-linking method, emulsion cross-linking method, spray drying method, reverse micelle method, co-precipitation method and self-assembly method, etc. During the experiment, it was found that when the liraglutide solution was added to the chitosan solution, the system became a milky white and slightly transparent colloidal solution. By measuring the charge of the liraglutide solution, it was found that it was negatively charged. After adding it to the chitosan solution, it cross-linked with the positively charged chitosan. Therefore, only a small amount of ionic cross-linking agent needs to be added subsequently. So the ionic cross-linking method was selected to prepare liraglutide chitosan nanoparticles.

[0115] Chitosan was selected as the nanocarrier material, poloxamer 188 as the surfactant, and liraglutide nanoparticles were prepared by the ionic cross-linking method. Sodium tripolyphosphate (TPP) was used as the ionic cross-linking agent, and protamine was added as a cell-penetrating peptide to improve the cell-penetrating ability of liraglutide nasal delivery. In the preliminary experiment, it was found that the Zeta potential of the nanoparticles did not change much, probably because chitosan itself has a strong positive charge, making the charge of the nanoparticles stable and the charge density distribution uniform on the surface of the nanoparticles. Therefore, the ratio of chitosan to liraglutide, the concentration of TPP, the pH of chitosan, the ratio of chitosan to poloxamer 188, the stirring time of liraglutide, and the ultrasonic fragmentation time were investigated by single-factor with particle size and encapsulation efficiency as the evaluation indexes.

[0116] 1. Preparation of liraglutide nanoparticles

[0117] Nanoparticles were prepared by the ionic cross-linking method. Weighed the prescription amounts of chitosan and P188 according to the weight ratio of chitosan to liraglutide and chitosan to P188, dissolved them in an acetic acid aqueous solution and stirred on a magnetic stirrer (stirring time 4 h) until they were fully swollen, and then adjusted the pH of the solution with 2M NaOH to obtain the chitosan solution. Take 6 mL of the above chitosan solution, slowly drop 2 mL of 4.3 mg·mL -1 liraglutide solution, with PBS 7.40 buffer as the solvent, continue to stir on a magnetic stirrer (stirring time 1.5 h), slowly drop 1 mL of sodium tripolyphosphate (TPP) solution and stir for 20 min until the system shows a light blue opalescence. After taking it out, probe ultrasound at low temperature (ultrasound time 6 min) (intermittent 5 s / 5 s, 900 W×30%), and make up to 10 mL to obtain the liraglutide chitosan nanoparticle suspension (CS-LIRA NPs). The preparation flow chart of liraglutide chitosan nanoparticles is shown in Figure 1 .

[0118] 2. Screening of the prescription process of liraglutide nanoparticles

[0119] (1) Investigation of the chitosan / liraglutide ratio

[0120] Fix the pH of the chitosan solution at 5.50, the weight ratio of chitosan to P188 at 10:1, the concentration of the TPP solution at 1 mg·mL -1 , the ultrasonic fragmentation time at 4 min (intermittent 5 s / 5 s, 900 W×30%), and the stirring time after adding liraglutide to the chitosan solution at 2 h. According to the method in "1. Preparation of liraglutide nanoparticles", fix the use of 6 mL of chitosan solution and 2 mL of 4.3 mg·mL -1 liraglutide solution as the raw material, and prepare nanoparticles with the weight ratio of chitosan to liraglutide of 1:1, 2:1, 3:1, 4:1, and 5:1 respectively. With encapsulation efficiency, particle size, and PDI as the evaluation indexes, the results are shown in Table 1.

[0121] Table 1 Evaluation Results of Chitosan / Liraglutide Ratio

[0122]

[0123] When the weight ratio of chitosan to liraglutide is 1:1, due to the low concentration of chitosan, precipitation occurs during the preparation process, and it is impossible to encapsulate the prescribed amount of liraglutide. When the weight ratio of chitosan to liraglutide is 5:1, due to the excessive concentration of chitosan, flocculates will be produced after adding TPP. It can be seen from the results in Table 1 that as the amount of chitosan used increases, the particle size and encapsulation efficiency also increase. When the weight ratio of chitosan to liraglutide is 2:1, the nanoparticles are unstable and will settle after being placed in a 4°C refrigerator for two days. The encapsulation efficiencies of the weight ratios of 3:1 and 4:1 are not very different, but the particle size and PDI value of 4:1 are larger, and due to the high concentration of the chitosan solution, it is difficult to adjust the pH. Therefore, the subsequent investigation tentatively determines the weight ratio of chitosan to liraglutide as 3:1.

[0124] (2) Investigation of TPP Solution Concentration

[0125] The principle of the ionic cross-linking method is that the positively charged chitosan solution cross-links with the negatively charged ionic cross-linking agent to form nanoparticles. TPP is a commonly used cross-linking agent. Since the isoelectric point of liraglutide is 4.90 and it is easily soluble under weakly alkaline conditions, it is negatively charged after being dissolved in PBS7.40 buffer solution. When liraglutide is added to the chitosan solution, the positively charged chitosan will bind to the negatively charged liraglutide. Therefore, only a small amount of TPP needs to be added subsequently. Fix the weight ratio of chitosan to liraglutide as 3:1, the stirring time of liraglutide as 2 h, the pH of the chitosan solution as 5.50, the weight ratio of chitosan to P188 as 10:1, and the ultrasonic fragmentation time as 4 min (intermittent 5 s / 5 s, 900 W×30%). According to the method in "1. Preparation of Liraglutide Nanoparticles", add TPP solutions with concentrations of 0, 0.5, 1, 3, 5 mg·mL -1 to prepare nanoparticles, and use the encapsulation efficiency, particle size, and PDI value as evaluation indicators. The results are shown in Table 2.

[0126] Table 2 Evaluation Results of TPP Solution Concentration

[0127]

[0128] Since the concentrations of the TPP solutions of 3 mg·mL -1 and 5 mg·mL -1 are too high, the prepared nanoparticles are prone to sedimentation, so the particle size and encapsulation efficiency are not measured for them. It can be seen from the results in Table 2 that the encapsulation efficiency of the nanoparticles is low without adding TPP. As the concentration of TPP increases, the particle size and encapsulation efficiency also increase. When the concentration of the added TPP solution is 1 mg·mL -1The nanoparticles with a concentration of 0.5 mg mL -1 The stability of the nanoparticles was poor. Considering the subsequent investigation, the concentration of TPP solution was tentatively set to 0.5 mg mL -1 .

[0129] (3) pH investigation of chitosan solution

[0130] The pH of the nasal environment is generally in the range of 5.0 to 6.5. It was found that the chitosan solution was unstable when the pH was higher than 5.5, and flocculent precipitation would occur. Therefore, the weight ratio of chitosan to liraglutide was fixed at 3:1, the stirring time of liraglutide was 2h, the weight ratio of chitosan to P188 was 10:1, and the concentration of TPP solution was 0.5mg·mL -1 The ultrasonic crushing time was 4 min (interval 5s / 5s, 900W×30%). Nanoparticles with chitosan solution pH of 5.0, 5.25, and 5.5 were prepared according to the method in “1. Preparation of liraglutide nanoparticles”. The encapsulation efficiency, particle size and PDI value were used as evaluation indicators. The results are shown in Table 3.

[0131] Table 3 Chitosan solution pH evaluation results

[0132]

[0133] As shown in the results in Table 3, as the pH of the chitosan solution increases, the encapsulation efficiency gradually increases, and the particle size first decreases and then increases. The encapsulation efficiency is the lowest when the pH of the chitosan solution is 5.0, and the encapsulation efficiency at pH 5.25 and 5.5 is not much different. However, when the pH of the chitosan solution is 5.5, the particle size is larger and the stability is poor. Therefore, the pH value of the chitosan solution is adjusted to 5.25.

[0134] (4) Investigation of the ratio of chitosan to poloxamer 188

[0135] The addition of surfactant can increase the stability of nanoparticles. In this experiment, poloxamer 188 was selected as the surfactant. The weight ratio of chitosan to liraglutide was fixed at 3:1, the stirring time of liraglutide was 2 h, the pH of the chitosan solution was 5.25, and the concentration of the TPP solution was 0.5 mg·mL -1 The ultrasonic crushing time was 4 min (interval 5s / 5s, 900W×30%). Nanoparticles with a weight ratio of chitosan to P188 of 5:1, 10:1, and 15:1 were prepared according to the method in “1. Preparation of liraglutide nanoparticles”. The encapsulation efficiency, particle size and PDI value were used as evaluation indicators. The results are shown in Table 4.

[0136] Table 4 Evaluation results of chitosan and P188

[0137]

[0138] As can be seen from the results in Table 4, the PDI values of the nanoparticles under different ratios of chitosan to P188 changed little. When the weight ratio of chitosan to P188 was 5:1, the particle size was the largest, and as the amount of P188 added decreased, the particle size also decreased. The encapsulation efficiency showed a trend of first increasing and then decreasing. When the weight ratio of chitosan to P188 was 10:1, the encapsulation efficiency was the largest and the particle size was the smallest. Therefore, the subsequent investigation tentatively set the weight ratio of chitosan to P188 at 10:1.

[0139] (5) Investigation of the stirring time of liraglutide

[0140] After liraglutide was added to the chitosan solution, it was necessary to stir to make liraglutide and chitosan fully mixed and uniform. Through preliminary experiments, it was found that when the stirring time of liraglutide was greater than 3 h, liraglutide would precipitate, affecting the preparation of nanoparticles. Therefore, the weight ratio of chitosan to liraglutide was fixed at 3:1, the pH of the chitosan solution was 5.25, the weight ratio of chitosan to P188 was 10:1, and the TPP concentration was 0.5 mg·mL -1 , the ultrasonic fragmentation time was 4 min (intermittent 5 s / 5 s, 900 W×30%), and nanoparticles were prepared according to the method in "(1) Preparation of liraglutide nanoparticles". The encapsulation efficiency, particle size, and PDI value of the nanoparticles were investigated when the stirring time of liraglutide was 1, 1.5, and 2 h, respectively. The results are shown in Table 5.

[0141] Table 5 Evaluation results of the stirring time of liraglutide

[0142]

[0143] As can be seen from the results in Table 5, the overall influence of the stirring time on the encapsulation efficiency, particle size, and PDI value was not significant. However, if the stirring time was too short, the drug and the carrier material might not be fully mixed, and if the stirring time was too long, the drug would precipitate, affecting the preparation of nanoparticles. When the stirring time was 1.5 h, the encapsulation efficiency of the nanoparticles was the largest. Therefore, the subsequent selection of the stirring time of liraglutide was 1.5 h.

[0144] (6) Investigation of the ultrasonic fragmentation time

[0145] Ultrasonic fragmentation of the nanoparticles was beneficial to optimizing the particle size and encapsulation efficiency. With the ultrasonic power fixed, the influence of different ultrasonic fragmentation times on the nanoparticles was investigated. The weight ratio of chitosan to liraglutide was fixed at 3:1, the stirring time of liraglutide was 2 h, the pH of the chitosan solution was 5.25, the weight ratio of chitosan to P188 was 10:1, and the concentration of the TPP solution was 0.5 mg·mL -1 , and nanoparticles were prepared according to the method in "(1) Preparation of liraglutide nanoparticles". The encapsulation efficiency, particle size, and PDI value of the nanoparticles were investigated when the ultrasonic fragmentation time was 2, 4, 6, and 10 min (intermittent 5 s / 5 s, 900 W×30%), respectively. The results are shown in Table 6.

[0146] Table 6 Evaluation Results of Ultrasonic Disruption Time

[0147]

[0148] When the ultrasonic time was 10 min, the nano-system was damaged and the solution became turbid. As can be seen from the results in Table 6, with the increase of ultrasonic time, the particle size decreased, the encapsulation efficiency gradually increased, and the PDI value gradually decreased, indicating that the longer the ultrasonic disruption time, the more uniform the distribution of nanoparticles and no aggregation phenomenon. Considering comprehensively, the ultrasonic time of 6 min when the encapsulation efficiency was the largest was finally selected for subsequent investigation.

[0149] 3. Process Optimization of Liraglutide Nanoparticles

[0150] Nasal administration has low requirements for particle size. Therefore, the encapsulation efficiency of nanoparticles is an important evaluation index. According to the results of single-factor investigation, the encapsulation efficiency of the finally obtained liraglutide nanoparticles was not high, all less than 80%. Taking the encapsulation efficiency as the evaluation index, the Box-Behnken response surface method was used to optimize the formulation of liraglutide nanoparticles.

[0151] Based on the results of single-factor experiments, the ratio of chitosan to liraglutide and the concentration of TPP had the greatest influence on the encapsulation efficiency. Therefore, the ratio of chitosan to liraglutide (A), the concentration of TPP solution (B), and the ratio of chitosan to P188 (C) were selected as the optimization factors, and the encapsulation efficiency was selected as the response value (Y) for the optimization experiment of three factors and three levels. The BBD design parameter table is shown in Table 7, and the experimental design and results of formulation optimization are shown in Table 8.

[0152] Table 7 Box-Behnken Design Parameter Table

[0153]

[0154] Table 8 Experimental Design and Results of Formulation Optimization

[0155]

[0156] Using Design Expert 10.0 statistical analysis software, based on the BBD experimental design principle, the experimental data in Table 8 were regressively fitted by a binary multiple equation to establish a quantitative relationship model between the encapsulation efficiency (Y) of liraglutide nanoparticles and each investigated factor. The quadratic polynomial fitting regression equation was:

[0157] Y = 80.26 - 4.30A + 8.32B - 2.78C + 0.38AB - 1.81AC - 0.055BC - 9.36A 2 - 0.54B 2 - 5.22C 2 (P = 0.0027, R2 = 0.9310)

[0158] The experimental data of the formulation optimization of liraglutide nanoparticles were statistically processed by analysis of variance (ANOVA), and the results are shown in Table 9 below.

[0159] Table 9 Results of analysis of variance and statistical tests

[0160]

[0161] According to the above results, it can be seen that the model fitting degree of the BBD response surface design is good and has significant differences, indicating that this model can be used for the analysis and prediction of the optimal formulation of liraglutide nanoparticles. As shown in Table 9, A (P = 0.0133), B (P = 0.0004), A 2 (P = 0.0013), C 2 (P = 0.0231) have a significant statistical impact on the encapsulation efficiency Y. The F value of the lack-of-fit term is 0.64, and the P value is 0.6275 (P > 0.05), indicating that the impact is not significant, suggesting that the above model has good accuracy and reliability in the prediction of liraglutide nanoparticle preparation technology.

[0162] Using Design Expert 10.0 software, a three-dimensional response surface diagram of the encapsulation efficiency of three different factors, namely the ratio of chitosan to liraglutide (A), the concentration of TPP solution (B), and the ratio of chitosan to P188 (C), was plotted. The encapsulation efficiency of liraglutide nanoparticles first increases and then decreases with the increase of the ratio of chitosan to liraglutide, increases with the increase of TPP concentration, and the change of the ratio of chitosan to P188 has a relatively small impact on it. By limiting the encapsulation efficiency of liraglutide nanoparticles, the optimized formulation was obtained as A = 2.95, B = 0.95, C = 10.55, and the predicted encapsulation efficiency was Y = 87.68%.

[0163] The optimal formulation predicted after BBD response surface optimization is as follows: nanoparticles with a liraglutide content of 0.86 mg·mL -1 in which the ratio of chitosan to liraglutide (A) is 2.95, the concentration of TPP solution (B) is 0.95 mg·mL -1 , and the ratio of chitosan to P188 (C) is 10.55. According to the optimal formulation, three batches of liraglutide nanoparticles were prepared in parallel, and the encapsulation efficiency was measured. The results are shown in Table 10 below. The results indicate that the optimization scheme of liraglutide nanoparticles designed by the BBD response surface method is scientifically feasible, and the established model can effectively predict the optimal formulation. After three repeated experiments, the average encapsulation efficiency of the obtained nanoparticles is 87.04%, and the error is 0.73%.

[0164] Table 10 Results of verification of the prescription process

[0165]

[0166] That is, the present invention prepares the liraglutide chitosan nanoparticle suspension by the ion cross-linking method, and the optimization method includes the following steps:

[0167] (1) Prepare a liraglutide solution by adding liraglutide to PBS 7.40 buffer solution, with a concentration of 4.3 mg·mL -1 ;

[0168] (2) Based on the liraglutide dosage of 8.6 mg, weigh chitosan and poloxamer 188 according to the dry matter weight ratio of chitosan to liraglutide of 2.95:1 and the dry matter weight ratio of chitosan to poloxamer 188 of 10.55:1. Use 1% acetic acid aqueous solution as the solvent, with a solvent volume of 6 mL, stir for 4 hours, and after sufficient swelling, adjust the pH value to 5.25 to obtain a chitosan solution;

[0169] (3) Drop the liraglutide solution obtained in step (1) into the chitosan solution obtained in step (2) dropwise in an ice bath, and stir for 1.5 h to obtain a mixed solution;

[0170] (4) Drop 1 mL of 0.95 mg·mL -1 of TPP solution into the mixed solution in step (3) until the solution shows a light blue opalescence, then react for 20 min, and then ultrasonicate at low temperature for 6 min (intermittent 5 s / 5 s, 900 W×30%), that is, obtain the liraglutide chitosan nanoparticle suspension.

[0171] III. Under the above-mentioned optimized investigation conditions, modify the nanoparticles of the optimal formulation with protamine by simple physical mixing, investigate the particle size, potential and appearance of the nanoparticles after adding protamine, and finally determine the addition conditions of protamine.

[0172] 1. Preparation of protamine-modified liraglutide nanoparticles

[0173] Prepare liraglutide nanoparticles according to the optimal formulation: accurately weigh 25.37 mg of chitosan and 2.40 mg of P188, dissolve them in 6 mL of 1% acetic acid aqueous solution, stir on a magnetic stirrer for 4 h, and after sufficient swelling, adjust the pH to 5.25 with 2M NaOH. While stirring the above chitosan solution in an ice bath on a magnetic stirrer, slowly drop 2 mL of 4.3 mg·mL -1 liraglutide solution, continue to stir for 1.5 h, and then slowly add 0.95 mg·mL -11 mL of the TPP solution until the solution showed a faint blue opalescence, reacted for 20 min, and sonicated with a probe at low temperature for 6 min (intermittent 5 s / 5 s, 900 W × 30%). The obtained liraglutide nanoparticles were slowly added to 1 mL of the protamine solution with the prescribed concentration on a magnetic stirrer, and the stirring was continued to fully mix the protamine and the liraglutide nanoparticles, thus obtaining protamine-modified liraglutide nanoparticles (PS-CS-LIRA NPs). The preparation flow chart is shown in Figure 2 .

[0174] 2. Selection of protamine solvent

[0175] According to the results of preliminary experiments, the concentration of the protamine solution was fixed at 0.5 mg·mL -1 , the stirring time was 1 h, and the changes in particle size, PDI value, Zeta potential and nanoparticle phenomenon were investigated when protamine was used as the solvent with water and PBS 7.40 buffer respectively. The results are shown in Table 11.

[0176] Table 11 Results of protamine solvent selection

[0177]

[0178] As can be seen from Table 11, the overall potential did not differ much after the addition of protamine. The potential of the protamine solution was measured to be (4.62 ± 1.58 mV) through experiments, while the chitosan nanoparticles themselves carried a strong positive charge and the amount of protamine added was limited. Therefore, there was no obvious upward trend in the potential after the addition of protamine. At the same time, the addition of protamine decreased the PDI values. The smaller the PDI value, the more uniform the distribution of the nanoparticles. When protamine was used with PBS 7.40 buffer as the solvent, the particle size increased slightly compared with the liraglutide nanoparticles without protamine addition, but the difference was not significant. However, the solution was unstable when using PBS 7.40 buffer as the solvent. It was analyzed that this might be because the chitosan nanoparticles were sensitive to pH and ions, and PBS 7.40 buffer contained more ions, which would cause the liraglutide nanoparticles to be unstable and settle. Therefore, PBS 7.40 buffer was not selected as the solvent in the follow-up. When using water as the solvent, the particle size of the nanoparticles decreased significantly. In order to exclude the interaction between liraglutide and protamine, blank nanoparticles were prepared, and an aqueous protamine solution was added to them to investigate the changes in particle size, PDI value and Zeta potential. The results are shown in Table 12. After adding the protamine solution to the blank nanoparticles, the particle size also decreased significantly, while the potential changed little. Therefore, the interaction between protamine and liraglutide was excluded. It was speculated that the addition of positively charged protamine increased the entanglement with negatively charged TPP, making the nanoparticles become denser particles, thus resulting in a decrease in the particle size of the nanoparticles.

[0179] Table 12 Results of investigating the addition of protamine to blank nanoparticles

[0180]

[0181] 3. Investigation of stirring time

[0182] Using water as the solvent and fixing the concentration of protamine at 0.5 mg·mL -1 , the particle size, PDI value, and potential changes of the nanoparticles were investigated when the stirring time of protamine was 0.5, 1, 1.5, and 2 h, respectively. The results are shown in Table 13.

[0183] Table 13 Results of the investigation of protamine stirring time

[0184]

[0185] As can be seen from Table 13, the PDI values of the liraglutide nanoparticles modified with protamine are all less than 0.2, and with the increase of the protamine stirring time, the particle size of the nanoparticles shows a downward trend. The particle size decrease is not obvious when the stirring time is 0.5 - 1.5 h, while when the stirring time is 2 h, the particle size of the nanoparticles decreases significantly. This may be due to the fact that the excessive stirring time leads to the rupture of some nanoparticles. Therefore, the finally selected stirring time is 1.5 h.

[0186] 4. Investigation of protamine concentration

[0187] Using water as the solvent and fixing the stirring time at 1.5 h, the particle size, PDI value, and potential changes of the nanoparticles were investigated when the protamine concentrations were 0.5, 1, 2, and 3 mg·mL -1 , respectively. The results are shown in Table 14.

[0188] Table 14 Results of the investigation of protamine concentration

[0189]

[0190] As can be seen from Table 14, the PDI values of the liraglutide nanoparticles modified with protamine at all concentrations are less than 0.2, indicating that the nanoparticles are well-distributed. With the increase of the protamine concentration, the particle size and potential of the nanoparticles show a trend of first decreasing and then increasing. When the protamine concentration is 1 mg·mL -1 , the particle size of the nanoparticles is the smallest. With the increase of the protamine content, the particle size and potential of the nanoparticles also gradually increase, indicating that the 1 mg·mL -1 protamine solution can better bind to the nanoparticles, making the dispersion of the nanoparticles better. However, the protamine solution concentrations of 2 mg·mL -1 and 3 mg·mL -1 are relatively high, resulting in more free protamine, and the particle size and potential of the nanoparticles also increase accordingly.

[0191] 5. Formulation of Protamine-modified Liraglutide Nanoparticles

[0192] Since liraglutide is a macromolecular polypeptide drug, it is thermally unstable and has better stability at low temperatures. The preparation process needs to be carried out under an ice-water bath condition. Therefore, based on the results of the above single-factor experiments, the final formulation of the protamine-modified nanoparticles is as follows: the concentration of protamine is 1 mg·mL -1 , the solvent is water, and the stirring time is 1.5 h under the ice-water bath condition.

[0193] 6. Verification of the Encapsulation Efficiency of Protamine-modified Liraglutide Nanoparticles

[0194] Three batches of protamine-modified liraglutide nanoparticles were prepared in parallel according to the final formulation, and the encapsulation efficiency was measured. The results are shown in Table 15. The encapsulation efficiency of the protamine-modified nanoparticles decreased compared with that of the unmodified nanoparticles. It is speculated that the continued stirring after the addition of protamine caused a small number of liraglutide nanoparticles to rupture, resulting in a slight decrease in the encapsulation efficiency. However, the encapsulation efficiency of the nanoparticles after the decrease was still greater than 80%, indicating that the addition of protamine had little effect on the encapsulation efficiency of liraglutide nanoparticles.

[0195] Table 15 Results of the Encapsulation Efficiency of Protamine-modified Liraglutide Nanoparticles

[0196]

[0197] 7. Protamine-modified Liraglutide Nanoparticles Prepared According to the Preferred Formula of the Present Invention

[0198] (1) Appearance

[0199] The protamine-modified liraglutide nanoparticles are a milky white suspension with a semi-transparent appearance and a faint blue opalescence, and the Tyndall effect is significant, as shown in Figure 3 A. After the nanoparticles were placed in a 4°C refrigerator for two weeks, as shown in Figure 3 B, the appearance of the nanoparticles did not change significantly compared with that at the initial preparation.

[0200] (2) Particle Size and Distribution

[0201] Take 1 mL of each of the freshly prepared three batches of PS-CS-LIRA NPs suspension samples, add 4 mL of ultrapure water for dilution, and then analyze the particle size distribution by dynamic light scattering (DLS) in a laser particle size analyzer. The results are shown in Table 16, and the particle size distribution diagram is shown in Figure 4 . It can be seen from Table 16 and Figure 4 that the particle sizes of the three batches of nanoparticles are all about 295 nm, and the PDI values are all less than 0.2.

[0202] Table 16 Results of the Particle Size Measurement of Three Batches of Protamine-modified Liraglutide Nanoparticles

[0203]

[0204] (3) Potential

[0205] Take 0.5 mL of each of the newly prepared three batches of PS-CS-LIRA NPs suspensions, dilute it to 5 mL with ultrapure water, and measure the Zeta potential in a laser particle size analyzer. The results are shown in Table 17, and the potential diagram is shown in Figure 5 . From Table 17 and Figure 5 it can be seen that the protamine-modified liraglutide nanoparticles are positively charged, and the average value of the Zeta potential of the three batches of nanoparticles is 31.59 mV.

[0206] Table 17 Zeta potential results of three batches of protamine-modified liraglutide nanoparticles

[0207]

[0208] (4) Morphology characterization

[0209] Prepare PS-CS-LIRA NPs according to the optimal formula. Take 20 μL of the nanoparticles and place them on a sample plate. Place the positive side of the copper mesh on the surface of the nanoparticle droplet. After drying at room temperature, perform negative staining with 2% phosphotungstic acid, and blot the excess staining solution with filter paper. Observe the morphology characterization of the nanoparticles under a transmission electron microscope (TEM). As Figure 6 shown, the protamine-modified nanoparticles are spherical or quasi-spherical with relatively regular shapes under TEM. The particle size is slightly smaller compared to the particle size measured by DLS. This is because the principles of the particle size measurement methods are different. The requirements for the sample in DLS are liquid, and thousands of particles can be analyzed simultaneously during the measurement, which is more used for quantitative analysis. While the requirements for the sample in TEM are dry samples, and the visualization of nanoparticles is carried out under TEM, which is more used for qualitative analysis. At the same time, the nanoparticles may shrink after drying, resulting in a smaller particle size observed under TEM.

[0210] (5) pH value determination

[0211] Take three batches of PS-CS-LIRA NPs suspensions and measure the pH value of the nanoparticles according to the pH measurement method in the Chinese Pharmacopoeia (2020 Edition). The results are shown in Table 18, and the pH values are 5.39, 5.43, and 5.41 respectively, within the pH range of 5.0 - 6.5 in the normal nasal cavity environment.

[0212] Table 18 pH value determination results of three batches of protamine-modified liraglutide nanoparticles

[0213]

[0214] (6) Encapsulation efficiency

[0215] The entrapment efficiency of three newly prepared batches of PS-CS-LIRA NPs was determined. The results are shown in Table 19, and the entrapment efficiencies were 83.37%, 82.10%, and 80.30% respectively.

[0216] Table 19 Results of the entrapment efficiency of three batches of protamine-modified liraglutide nanoparticles

[0217]

[0218] (7) Drug loading

[0219] Three batches of PS-CS-LIRA NPs suspensions were prepared, and the drug loading of the nanoparticles was determined. The results are shown in Table 20, and the drug loadings were 16.83%, 16.88%, and 16.29% respectively.

[0220] Table 20 Results of the drug loading of three batches of protamine-modified liraglutide nanoparticles

[0221]

[0222] (8) Yield

[0223] Take 1 mL of the suspensions of three newly prepared batches of PS-CS-LIRA NPs, break the emulsion, and detect the total drug amount in the liquid phase. Calculate the content compared with the input amount of liraglutide. The results are shown in Table 21, and the yields were 98.47%, 98.14%, and 97.92% respectively, all within the range of 95% - 105%, meeting the requirements.

[0224] Table 21 Results of the yield determination of three batches of protamine-modified liraglutide nanoparticles

[0225]

[0226] (9) Circular dichroism measurement

[0227] The secondary structure of proteins plays a crucial role in their conformation and stability. Circular dichroism is usually used to detect the secondary structure of proteins or polypeptides. To investigate whether the preparation process of the nanoparticles and the presence of protamine would cause changes in the folding structure of liraglutide, CS-LIRA NPs and PS-CS-LIRA NPs were prepared respectively, and they were diluted to a concentration of liraglutide of 0.172 μg·mL -1 , and a liraglutide buffer solution with the same concentration was prepared as a control. At the same time, a blank sample solution was prepared to deduct the absorption of excipients in the nanoparticles. The circular dichroisms of the LIRA solution, CS-LIRA NPs, and PS-CS-LIRANPs were measured, the scanning wavelength was 190 - 260 nm, and the nitrogen flow rate was 2 - 3 L·min -1, with a spectral bandwidth of 1 nm, a resolution of 1 nm, scanning 500 nm per minute, and scanning 3 times in total, the ellipticity θ is obtained. Calculate the mean residue ellipticity (MRE, [θ]) of amino acid residues according to the formula. Plot with wavelength as the abscissa and [θ] as the ordinate, and the result is shown by Figure 7 As shown. Liraglutide solution has a negative absorption peak near 233 nm, and CS-LIRA NPs and PS-CS-LIRA NPs have a negative absorption peak near 235 nm. The slight change in the wavelength of the negative absorption peak may be affected by the nanoparticles. However, the typical negative band CD spectrum indicates that both the liraglutide solution and the nanoparticles exhibit a β-sheet conformation, indicating that the folding structure of liraglutide is not changed after being prepared into nanoparticles, and the addition of protamine does not affect the secondary structure of liraglutide.

[0228]

[0229] Among them, θ: ellipticity, M: molar mass of liraglutide (g·mol -1 ), l: cell path length (mm); c: concentration of liraglutide (mg·mL -1 ), n: number of amino acids in liraglutide.

[0230] Summary: The quality evaluation of protamine-modified liraglutide nanoparticles was carried out. The appearance of PS-CS-LIRA NPs is a milky white suspension with slight transparency and a faint blue opalescence, and it has an obvious Tyndall effect. The morphology is spherical or quasi-spherical, the particle size is about 295 nm, the PDI values are all less than 0.2, the average Zeta potential value is (31.59 ± 1.97) mV, the average encapsulation efficiency is (81.92 ± 1.54)%, the average drug loading is (16.66 ± 0.33)%, the pH value conforms to the normal nasal environment of humans, and the circular dichroism spectrum shows that the secondary structure of liraglutide is not affected after being prepared into nanoparticles, and the addition of protamine does not affect the result. Both PS-CS-LIRA NPs and CS-LIRA NPs should be stored in a low-temperature environment at 4°C.

[0231] IV. The prepared liraglutide nanoparticles were subjected to pharmaceutical evaluation, in vitro cell evaluation, and pharmacodynamic evaluation.

[0232] 1. In vitro release investigation

[0233] In vitro release experiments were carried out on CS-LIRA NPs and PS-CS-LIRA NPs, and the sample separation method was used for investigation: 1 mL of the suspension of CS-LIRA NPs and PS-CS-LIRA NPs was taken respectively, and added to 10 mL with PBS 7.40 buffer solution. It was placed in a thermostatic magnetic stirrer, and the temperature of the thermostatic magnetic stirrer was set at 37 °C and the rotation speed was 40 rpm. 1 mL of the sample was taken at the time points of 15, 30, 60, 90, 120, 180, 240, and 300 min, and the same volume of PBS 7.40 buffer solution at 37 °C was immediately replenished. The sample was centrifuged at 4 °C and 12,000 rpm for 15 min to separate the nanoparticles and the free drug. After ultrasonic demulsification of the supernatant, HPLC injection analysis was carried out on the sample solution to be measured according to the chromatographic conditions. Taking the release amount of liraglutide at the initial moment as 0, the release amount of liraglutide at each sampling time point was calculated by the difference method, and the release degree-time relationship curve was constructed, as Figure 8 shown. The ordinate is the cumulative release degree, and the abscissa is the time t.

[0234] As can be seen from Figure 8 , the release trends of the two kinds of nanoparticles are generally consistent. The cumulative release amount of PS-CS-LIRA NPs at each time point is greater than that of CS-LIRA NPs. 32% of liraglutide is released from PS-CS-LIRA NPs at 15 min, while only 23% of liraglutide is released from CS-LIRA NPs at 15 min. According to the determination results of the encapsulation efficiency, the encapsulation efficiency of PS-CS-LIRA NPs is smaller than that of CS-LIRA NPs, and there are more free drugs. Therefore, the burst release phenomenon is stronger than that of CS-LIRA NPs. CS-LIRA NPs release 73% of the total drug amount within 300 min. The release amount of PS-CS-LIRA NPs reaches the highest at 240 min, which is 79% of the total drug amount, while the cumulative release amount decreases to 74% at 300 min. It is speculated that the reason may be related to the preparation method of the nanoparticles. The negatively charged liraglutide will cross-link with the positively charged chitosan through positive and negative charge cross-linking before adding the ionic cross-linking agent. Therefore, in the later stage of nanoparticle release, some free liraglutide in the system combines with chitosan to form nanoparticles again, resulting in a decrease in the cumulative release amount.

[0235] In order to deeply explore the drug release characteristics of CS-LIRA NPs and PS-CS-LIRA NPs, the in vitro release curves of CS-LIRA NPs and PS-CS-LIRA NPs were fitted with the zero-order, first-order, and Higuchi equations using Origin 2022 software, and the best drug release model was determined according to the correlation coefficient. The results are shown in Tables 22 and 23. As can be seen from Table 22, the drug release rule of CS-LIRA NPs conforms to the Higuchi equation drug release model, Y = 3.9602x1 / 2 +2.6428, R 2 = 0.9681. It can be seen from Table 23 that the drug release pattern of PS-CS-LIRANPs also conforms to the Higuchi equation drug release model, Y = 4.6428x 1 / 2 +8.8820, R 2 = 0.9517, indicating that both CS-LIRA NPs and PS-CS-LIRA NPs have good sustained-release effects.

[0236] Table 22 Fitting of in vitro drug release model of liraglutide nanoparticles

[0237]

[0238] Table 23 Fitting of in vitro drug release model of protamine-modified liraglutide nanoparticles

[0239]

[0240] Summary:

[0241] 1) The precision of using PBS 5.50, PBS 6.50, and PBS 7.40 buffers as release media was investigated in the in vitro release preliminary experiment, simulating the nasal environment and in vivo environment conditions respectively. Since liraglutide is unstable in PBS 5.50 and PBS 6.50 buffers, and considering that the residence time of the drug in the nasal cavity is very short after nasal administration, PBS 7.40 buffer was selected as the release medium for the nanoparticles in this experiment. Also, because liraglutide itself is negatively charged and can bind to positively charged chitosan, in the later stage of release, the free liraglutide in the system binds to chitosan again to form nanoparticles, resulting in the cumulative release of the nanoparticles first increasing and then decreasing when the in vitro release time was 0 - 12 h. Therefore, only the in vitro release of the nanoparticles at 0 - 5 h was finally investigated, and the cumulative release degree in this time period can reach more than 70%.

[0242] 2) Liraglutide nanoparticles were prepared by the ionic cross-linking method, and the encapsulation efficiency and drug loading of the nanoparticles were determined by the low-temperature direct centrifugation method. Through single-factor, BBD response surface optimization, and screening of the addition conditions of protamine, the final preparation method of protamine-modified liraglutide nanoparticles was obtained as follows: According to the ratio of chitosan to liraglutide of 2.95:1 and the ratio of chitosan to P188 of 10.55:1, accurately weigh 25.37 mg of chitosan and 2.40 mg of P188, dissolve them in 6 mL of 1% acetic acid aqueous solution, stir on a magnetic stirrer for 4 h to fully swell, then adjust the pH to 5.25 with 2M NaOH, and slowly drop 2 mL of 4.3 mg·mL -1Liraglutide solution, continue stirring for 1.5 h, then slowly dropwise add 1 mL of TPP solution with a concentration of 0.95 mg·mL -1 until the solution shows a faint blue opalescence, react for 20 min, probe sonicate at low temperature for 6 min (intermittent 5 s / 5 s, 900 W × 30%), after taking out, add 1 mL of protamine aqueous solution with a concentration of 1 mg·mL -1 , continue stirring for 1.5 h, take out and make up the volume to 10 mL to obtain.

[0243] The prepared nanoparticles were evaluated for quality. The morphology of the protamine-modified liraglutide nanoparticles was spherical or near-spherical, with a particle size of about 295 nm, a Zeta potential of (31.59 ± 1.97) mV, an encapsulation efficiency of (81.92 ± 1.54)%, a drug loading of (16.66 ± 0.33)%, the pH value was in line with the normal human nasal cavity environment, and the prepared nanoparticles should be stored at 4 °C. In vitro release showed that the liraglutide nanoparticles released 73% of the total drug amount within 5 h, and the release amount of the protamine-modified liraglutide nanoparticles reached the highest at 4 h, which was 79% of the total drug amount, and their drug release rules all conform to the Higuchi equation drug release model.

[0244] 2. In vitro cell evaluation of liraglutide nanoparticles

[0245] Using RPMI 2650 cells as the cell model, the cytotoxicity and cellular uptake of liraglutide nanoparticles before and after protamine modification in RPMI 2650 cells were studied. The CCK-8 reagent was used to investigate the cytotoxicity of free LIRA, CS-LIRA NPs and PS-CS-LIRA NPs, and the blank nanocarrier. At the same time, nanoparticles loaded with the fluorescent probe coumarin 6 were prepared, and their cellular uptake was qualitatively and quantitatively evaluated to investigate the transmembrane ability of the nanoparticles before and after protamine modification.

[0246] Experimental methods and results

[0247] 1) Cytotoxicity experiment

[0248] To investigate the safety of the blank nanocarrier, LIRA solution, CS-LIRA NPs and PS-CS-LIRA NPs, the CCK-8 kit method was used for the cytotoxicity experiment. The main component of CCK-8 is WST-8. In the presence of the electron-coupling reagent 1-Methoxy PMS, a specific reduction reaction occurs through the catalysis of dehydrogenase in mitochondria to generate a water-soluble chromogenic product formazan with characteristic light absorption. The number of live cells is linearly related to it, and it has been widely used for the investigation of cytotoxicity.

[0249] (1) Cell seeding

[0250] When the cell density of RPMI 2650 cells reaches 80 - 90%, inoculation treatment can be carried out. Take out the culture flask from the cell incubator, digest the cells in the flask with 0.25% trypsin, centrifuge and then resuspend with 2 mL of complete medium to form a uniformly dispersed cell suspension. Then aspirate 100 μL of the cell suspension, dilute it to 1 mL with PBS 7.40 buffer, aspirate 10 μL onto a cell counting chamber, count the cells under an inverted microscope, and after counting, dilute the cell suspension with complete medium to a certain concentration. Take a 96-well plate and inoculate RPMI 2650 cells at 5×10 4 cells / well, add 100 μL of the cell suspension to each well, and add 100 μL of PBS 7.40 buffer to the outermost layer to prevent evaporation. After inoculation, place the 96-well plate in a cell incubator at 37°C and 5% CO2 for 48 h to allow the cells to adhere to the wall.

[0251] (2) Sample preparation and administration

[0252] Weigh an appropriate amount of liraglutide, dissolve it with PBS 7.40 buffer, and prepare a LIRA solution with a concentration of 0.86 mg·mL -1 . At the same time, prepare CS-LIRA NPs, PS-CS-LIRA NPs, and blank nanocarrier solutions. After filtering the above solutions through a sterile filter membrane on the operating table, dilute the free LIRA, CS-LIRA NPs, and PS-CS-LIRA NPs suspensions with basal medium to a series of solutions with concentrations of 8.6, 21.5, 43, 86, 215 μg·mL -1 , and at the same time dilute the blank nanocarrier to a series of solutions with chitosan concentrations of 25.8, 64.5, 129, 258, 645 μg·mL -1 for standby.

[0253] Take out the 96-well plate inoculated with RPMI 2650 cells from the incubator, remove the medium, add 100 μL of the prepared series of sample solutions with gradient concentrations to each well, and at the same time set up a control group and a blank group (n = 5), and continue to culture in the cell incubator for 24 h.

[0254] (3) CCK-8 cytotoxicity test

[0255] Dilute CCK-8 with basal medium to obtain a 10% CCK-8 solution. Take out the 96-well plate from the cell incubator, aspirate the supernatant, and add 100 μL of the 10% CCK-8 solution to each well. Culture in an incubator at 37°C and 5% CO2 for 1 h until obvious orange-yellow appears, read the absorbance at a wavelength of 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. The calculation method is shown in formula 2.1. The cytotoxicity results of the blank carrier on RPMI 2650 are as Figure 9As shown, the cytotoxicity results of free drug and drug-loaded nanoparticles against RPMI 2650 are as Figure 10 shown.

[0256]

[0257] As Figure 9 can be seen, the cell viability of blank chitosan nanoparticles is greater than 80% in the range of chitosan concentration from 0 to 645 μg·mL -1 , indicating that blank chitosan nanoparticles are non-toxic in all concentration ranges and are a safe and non-toxic carrier material. The cell viability of the blank nanoparticles modified with protamine is greater than 90% in the range of chitosan concentration from 0 to 258 μg·mL -1 , indicating that the nanocarrier is non-toxic in this concentration range. When the chitosan concentration is 645 μg·mL -1 and the protamine concentration is 25 μg·mL -1 , the cell viability of the blank nanoparticles modified with protamine is only 74.13%, indicating that the presence of protamine will reduce the cell viability at this concentration. Therefore, the protamine concentration needs to be controlled in subsequent experiments to avoid a large number of cell deaths. As Figure 10 can be seen, free LIRA, CS-LIRANPs, and PS-CS-LIRA NPs have no obvious cytotoxicity in the concentration range of 0 to 215 μg·mL -1 , and the cell viability is greater than 90%. Moreover, as the LIRA concentration increases, the cell viability of the free LIRA group and the CS-LIRANPs group shows an increasing trend, while the cell viability of the PS-CS-LIRANPs shows little difference, which may be due to the presence of protamine. Generally speaking, this preparation is considered non-toxic within a certain range.

[0258] 2) Cell uptake experiment

[0259] As a cell-penetrating peptide, protamine has a strong transmembrane ability and can promote the uptake of nanoparticles by cells. To investigate the cell uptake of nanoparticles before and after protamine modification, coumarin 6 (C6), a fluorescent probe, was used instead of the model drug liraglutide to prepare CS-C6 NPs and PS-CS-C6 NPs. The uptake of free C6, CS-C6 NPs, and PS-CS-C6 NPs by RPMI 2650 cells was analyzed qualitatively by fluorescence microscopy and quantitatively by flow cytometry.

[0260] (1) Qualitative investigation of cell uptake

[0261] When the cell density of RPMI 2650 cells reaches 80 - 90%, plate seeding treatment is carried out. Take out the culture flask from the cell incubator, digest the cells in the flask. The digestion process is the same as that in cell passage during the cell cytotoxicity experiment (2). After centrifugal separation, resuspend with 2 mL of complete medium to make the cells evenly dispersed, and count the cells using a cell counting plate. Take a 96-well plate, inoculate RPMI 2650 cells at a density of 5×10 4 cells / well, add 100 μL of cell suspension to each well, and add PBS 7.40 buffer to the outermost layer to prevent evaporation. After inoculation, place the 96-well plate in the cell incubator and culture for 36 h.

[0262] Weigh a certain amount of coumarin 6, dissolve it with DMSO, then dilute it with PBS 7.40 buffer and solubilize it with Tween 80. At the same time, prepare chitosan nanoparticles containing coumarin 6 and chitosan nanoparticles modified with protamine according to the optimal formulation, so that the final concentration of C6 in free C6, CS-C6 NPs and PS-CS-C6 NPs is 20 μg·mL -1 . Place the prepared free C6, CS-C6 NPs and PS-CS-C6 NPs on the operating table, filter them using a sterile filter membrane, and then dilute them to a sample solution with a concentration of 2 μg·mL -1 using the basal medium.

[0263] Take out the 96-well plate inoculated with RPMI 2650 cells from the cell incubator, aspirate the supernatant, wash it twice with PBS 7.40 buffer, and respectively administer 100 μL of free C6, CS-C6 NPs and PS-CS-C6 NPs sample solutions. Place it in the cell incubator and continue to culture for 2 h, then take it out, aspirate the sample solution in the 96-well plate, and wash it with PBS 7.40 buffer. First, fix it with 4% paraformaldehyde for 15 min, then stain the nuclei with DAPI stain for 10 min. After washing with PBS 7.40 buffer, observe the cell uptake under an inverted fluorescence microscope, and operate in the dark throughout the process. The results are as Figure 11 shown. And use Image J software to quantitatively analyze the fluorescence intensity, and the results are as Figure 12 shown.

[0264] From Figure 11 and Figure 12It can be seen that the green fluorescence represents the entry of coumarin 6 into cells and into the cytoplasm, and the blue fluorescence represents the nucleus after DAPI staining. In the control group, since C6 was not added, no green fluorescence was observed; the green fluorescence intensity in the free C6 group was weak, indicating that only a small part of free C6 entered the cells; the green fluorescence intensities of CS-C6 NPs and PS-CS-C6 NPs were significantly stronger than those in the free C6 group, and the green fluorescence intensity of PS-CS-C6 NPs was stronger than that of CS-C6 NPs. This shows that the cellular uptake of C6 increased after encapsulation into nanoparticles, and the addition of protamine further increased the uptake of nanoparticles, indicating the cell-penetrating effect of protamine.

[0265] (2) Quantitative analysis of cellular uptake

[0266] Cells with a growth density of 80 - 90% were taken, digested and counted according to the steps in "3.2.1(2)". At the same time, a 6-well plate was taken and seeded at 2×10 6 cells / well, and 2 mL of cell suspension was added to each well. After inoculation, the 6-well plate was placed in a cell culture incubator and cultured for another 36 h.

[0267] Free C6, CS-C6 NPs and PS-CS-C6 NPs solutions were prepared by the same method as in the qualitative investigation of cellular uptake, so that the final concentration of C6 was 20 μg·mL -1 . The prepared free C6, CS-C6 NPs and PS-CS-C6 NPs were placed on the operating table, filtered through a sterile filter membrane, and then diluted to a sample solution with a concentration of 1 μg·mL -1 using basal medium.

[0268] The 6-well plate was taken out of the cell culture incubator, the old medium was discarded, and it was rinsed twice with PBS 7.40 solution. Free C6, CS-C6 NPs, and PS-CS-C6 NPs were administered respectively, 2 mL per well, and 3 parallel replicates were made for each group. After administration, it was placed back in the cell culture incubator for further incubation. After 2 h, it was taken out, the supernatant was discarded, and it was rinsed twice with PBS 7.40 buffer. 0.5 mL of trypsin was added to each well for digestion and then centrifuged to remove the medium. It was gently rinsed twice with PBS 7.40 buffer to completely discard the medium. 500 μL of precooled PBS 7.40 buffer was added, and the cells in the sample were resuspended using a pipette gun. Finally, it was transferred to a flow tube for flow cytometry detection. The flow uptake results are as Figure 13 shown, and the quantitative analysis is as Figure 14 shown.

[0269] From Figure 13 and Figure 14It can be seen that the cellular uptake of RPMI 2650 cells for the CS-C6 NPs and PS-CS-C6 NPs groups was significantly greater than that of the free C6 group, and the cellular uptake of the PS-CS-C6 NPs group was greater than that of the CS-C6 NPs group. There were significant differences in the cellular uptake among the three groups of free C6, CS-C6 NPs, and PS-CS-C6 NPs. This indicates that the cellular uptake of free C6 increased significantly after being encapsulated in nanoparticles, and the cellular uptake was the highest after modification with protamine. It shows that encapsulating the drug in chitosan nanoparticles can protect the drug and improve the cellular uptake ability of the drug, and protamine also has strong cellular penetration ability, which is consistent with the conclusion of the qualitative investigation of cellular uptake.

[0270] 3. In vivo pharmacodynamic study of liraglutide nanoparticles

[0271] Healthy male SD rats (weighing 220 ± 10 g) were selected as experimental animals to establish a diabetic rat model. The glucose concentration in the serum of diabetic rats was measured by the glucose oxidase method to investigate the hypoglycemic effects of liraglutide nanoparticles (CS-LIRANPs) and protamine-modified liraglutide nanoparticles (PS-CS-LIRA NPs) in rats. The pharmacodynamic differences among CS-LIRANPs, PS-CS-LIRA NPs, and liraglutide solution administered intranasally were compared. At the same time, subcutaneous injection of liraglutide was used as a positive control to calculate the relative bioavailability and evaluate the feasibility of CS-LIRA NPs and PS-CS-LIRANPs studied in this project as intranasal administration preparations of liraglutide, providing a basis for the non-injection administration route of polypeptide drugs.

[0272] Experimental methods and results

[0273] 1) Establishment of a rat diabetic model

[0274] In this experiment, alloxan was used to establish a rat diabetic model. Thirty male SD rats weighing 220 ± 10 g were adaptively fed for one week, fasted but not water-deprived for 12 hours before modeling. Each experimental rat was rapidly injected with a 5% alloxan solution through the tail vein, and the dosage was 50 mg·kg -1 . In addition, according to the drug characteristics of alloxan, in order to prevent rats from dying due to low blood sugar, 2 mL of 30% glucose solution was gavaged to each rat for intervention 2.5 hours after alloxan administration. Then the rats were normally raised. On the 3rd and 5th days, blood was collected from the tail vein of the rats, and 0.1 - 0.2 mL of whole blood samples were collected, incubated in a water bath at 37°C for 30 min, and then centrifuged at 5000 r·min -1Centrifuge at a rotational speed for 10 min, take the supernatant, and measure the random blood glucose value of the rats by the glucose oxidase method. If the random blood glucose values measured twice are both greater than 16.7 mmol·L -1 , it indicates that the diabetic rat model is successfully established.

[0275] 2) Administration of drugs to diabetic rats

[0276] (1) Dosage

[0277] According to the results of the preliminary experiment, liraglutide solution at a dose of 17.2 μg·kg -1 has a significant hypoglycemic effect on rats when administered subcutaneously. Calculated based on the absorption after nasal administration being 10% of subcutaneous injection absorption

[58] , the final subcutaneous injection dose of liraglutide is 17.2 μg·kg -1 , and the nasal administration dose of liraglutide is 172 μg·kg -1 .

[0278] (2) Preparation of solutions

[0279] PBS 7.40 buffer solution: Take a 250 mL volumetric flask, weigh 1.7000 g of potassium dihydrogen phosphate and place it in it, dissolve it with water. Then weigh 0.3900 g of sodium hydroxide in a 25 mL beaker, dissolve it with water, transfer it to the above 250 mL volumetric flask, and then make up the volume to the calibration line with water. Adjust the pH to 7.40 with phosphoric acid or sodium hydroxide to obtain it.

[0280] 0.86 mg·mL -1 Liraglutide solution: Take a 10 mL volumetric flask, accurately weigh 8.60 mg of liraglutide raw material and place it in it, dissolve and make up the volume with PBS 7.40 buffer solution to obtain it.

[0281] 17.2 μg·mL -1 Liraglutide subcutaneous injection solution: Take a 10 mL volumetric flask, pipette 0.2 mL of the above 0.86 mg·mL -1 liraglutide solution into it, dilute and make up the volume with PBS 7.40 buffer solution to obtain it.

[0282] (3) Experimental grouping and drug administration

[0283] According to preliminary experiments, it is difficult to ensure that all rats maintain a consistent food intake and fasting time point during the experiment. Moreover, if rats are not fasted after drug administration, it will lead to large and irregular fluctuations in rat blood glucose levels, making it impossible to judge accurate experimental results. Therefore, in order to accurately evaluate the hypoglycemic effect of the drug, rats were allowed free access to food before drug administration, fasted but given water for 12 h after drug administration, and then allowed free access to food after 12 h. At the same time, to avoid hypoglycemic symptoms in experimental rats due to a sharp drop in blood glucose levels, each rat was intragastrically administered a 30% glucose solution at a dose of 10 mg·kg -1 . Using the blood glucose value before drug administration (0 h) as a control, the hypoglycemic effect of the drug was evaluated by calculating the percentage of blood glucose decrease in each group.

[0284] The successfully modeled diabetic rats were randomly divided into 5 experimental groups, with 6 rats in each group. Each group was numbered A, B, C, D, and E, and were nasally administered normal saline, LIRA solution, CS-LIRA NPs suspension, PS-CS-LIRA NPs suspension, and subcutaneous injection of LIRA solution in sequence. After administration, 0.1 - 0.2 mL of blood was collected from the tail vein at 0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h, incubated in a 37 °C water bath for 30 min, and then centrifuged at a speed of 5000 r·min -1 for 10 min to separate the serum. The serum was transferred to a clean centrifuge tube and stored at -20 °C in a refrigerator for later measurement.

[0285] (4) Determination of rat serum glucose concentration

[0286] The serum sample to be measured was taken out from the -20 °C refrigerator and thawed at room temperature. The OD value was measured by an enzyme-labeled instrument using the glucose oxidase method, and the glucose concentration in the serum was calculated.

[0287] 3) Experimental results

[0288] (1) Blood glucose percentage-time curve

[0289] Taking the glucose concentration in the rat serum before drug administration (0 h) as 100%, the blood glucose concentrations measured at each time point were converted into blood glucose percentages (W%). With time as the abscissa (T / h) and blood glucose percentage as the ordinate, a blood glucose percentage-time relationship curve was plotted. The blood glucose percentage values and serum percentage-time curves of different experimental groups at each time point are as Figure 15 shown. Among them, group A was nasally administered normal saline, group B was nasally administered LIRA solution, group C was nasally administered CS-LIRA NPs, group D was nasally administered PS-CS-LIRA NPs, and group E was subcutaneously injected with LIRA solution.

[0290] The serum concentration change rate was calculated according to formula 3.1:

[0291]

[0292] It can be seen from Figure 15 that the hypoglycemic effect of the subcutaneous injection group takes effect the fastest, and the blood glucose shows an obvious downward trend at 1 h. While the blood glucose of the nasal administration of LIRA solution group and the normal saline group shows an upward trend at 1 h. This is because glucose was intragastrically administered immediately after drug administration. Generally, there is a certain hypoglycemic effect when comparing the nasal administration of LIRA solution group with the normal saline group. The hypoglycemic effect of the nasal administration of nanoparticles group is stronger than that of the solution group, and the hypoglycemic effect of the nanoparticles modified with protamine is significantly stronger than that of the unmodified nanoparticles group after 4 h, indicating that the addition of protamine promotes the absorption of liraglutide nanoparticles by nasal mucosa.

[0293] (2) Pharmacological relative bioavailability

[0294] To evaluate the pharmacodynamic characteristics of liraglutide in animal models, the area above the blood glucose percentage-time curve (Area Above the Curve, AAC) was used as the evaluation index, and the pharmacological relative bioavailability (F%) of the drug was reflected by calculating this parameter. The calculation of AAC uses the trapezoidal method, and the calculation formula is shown in Formula 3.2.

[0295] AAC = SUM{(T i+1 - T i )[(100 - C i ) + (100 - C i+1 )]÷2} (3.2)

[0296] Among them, T i represents the i-th blood sampling time point, and C i represents the blood glucose percentage corresponding to the i-th blood sampling point.

[0297] The calculation formula of relative pharmacological bioavailability (F%) is shown in Formula 3.3:

[0298]

[0299] Among them, AAC nasal is the area above the blood glucose percentage-time curve after nasal administration; AAC ih is the area above the blood glucose percentage-time curve after subcutaneous injection; (dose) nasal is the dose of nasal administration; (dose) ih is the dose of subcutaneous injection.

[0300] The relative bioavailability (F%) of nasal administration of each sample calculated by the above formula was analyzed by variance using IBM SPSS Statistics 27 software, and a statistical difference comparison was made with the nasal administration LIRA solution group. The results are shown in Tables 24, 25 and Figure 16 , Figure 17 as shown. (In the table, group A was given normal saline by nasal cavity; group B was given LIRA solution by nasal cavity; group C was given CS-LIRA NPs by nasal cavity; group D was given PS-CS-LIRA NPs by nasal cavity; group E was given LIRA solution by subcutaneous injection).

[0301] Table 24 Results of relative bioavailability from 0 to 12 h (n = 6, Mean±SD)

[0302]

[0303] Table 25 Results of relative bioavailability and variance analysis from 0 to 24 h (n = 6, Mean±SD)

[0304]

[0305] From Tables 25 - 26 and Figure 16 - 17 it can be seen that the pharmacological relative bioavailabilities of the solution group at 0 - 12 h and 0 - 24 h were 5.44% and 6.02% respectively. The pharmacological relative bioavailabilities of the nasal administration CS-LIRA NPs group at 0 - 12 h and 0 - 24 h were 8.25% and 8.40% respectively. The pharmacological relative bioavailabilities of the nasal administration PS-CS-LIRA NPs group at 0 - 12 h and 0 - 24 h were 9.91% and 10.15% respectively. The results of variance analysis (P<0.05) showed statistical significance, indicating that preparing liraglutide into nanoparticles can improve the absorption of the drug in the nasal mucosa, and the absorption ability of the nasal mucosa after protamine modification is stronger.

[0306] Summary: The glucose oxidase method was used to determine the glucose concentration in serum, and the blood glucose percentage-time curve was plotted. The bioavailability was calculated based on the blood glucose percentage of each group of rats at each time point, and an analysis of variance was performed. The research results showed that the positive control group injected subcutaneously with LIRA solution had the most obvious effect on reducing blood glucose. The hypoglycemic effects of nasal administration of CS-LIRA NPs and PS-CS-LIRA NPs were stronger than those of the nasal administration of LIRA solution group. The pharmacological relative bioavailability of the nasal administration of CS-LIRA NPs group at 0-12h and 0-24h were 8.25% and 8.40% respectively, and the pharmacological relative bioavailability of the nasal administration of PS-CS-LIRA NPs group at 0-12h and 0-24h were 9.91% and 10.15% respectively, both of which were significantly different from the nasal administration of LIRA solution group, and there were also significant differences between the PS-CS-LIRA NPs group and the CS-LIRA NPs group. It was shown that the preparation of liraglutide into nanoparticles could improve the absorption of the drug in the nasal mucosa, and the nasal mucosa absorption ability was stronger after modification with protamine.

[0307] In summary, in the present invention, chitosan was selected as the nanocarrier material, poloxamer 188 as the surfactant, and the ion cross-linking method was used to prepare liraglutide nanoparticles. Sodium tripolyphosphate (TPP) was used as the ion cross-linking agent, and protamine was added as a cell-penetrating peptide to improve the cell-penetrating ability of nasal delivery of liraglutide. The optimized preparation method of liraglutide nanoparticles was as follows: According to the ratio of chitosan to liraglutide of 2.95:1 and the ratio of chitosan to P188 of 10.55:1, 25.37 mg of chitosan and 2.40 mg of P188 were accurately weighed and dissolved in 6 mL of 1% acetic acid aqueous solution and stirred on a magnetic stirrer for 4 h. After sufficient swelling, the pH was adjusted to 5.25 with 2M NaOH. While stirring the above chitosan solution in an ice-water bath on a magnetic stirrer, 2 mL of 4.3 mg·mL -1 liraglutide solution was slowly added dropwise, and stirring was continued for 1.5 h. Then, 1 mL of 0.95 mg·mL -1 TPP solution was slowly added until the solution showed a light blue opalescence, and the reaction was carried out for 20 min. The probe was ultrasonically treated at a low temperature for 6 min (intermittent 5 s / 5 s, 900 W×30%), and liraglutide chitosan nanoparticles were obtained. The condition for adding protamine to liraglutide chitosan nanoparticles was: the concentration of protamine was 1 mg·mL -1, the solvent is water, and the stirring time is 1.5 h under the condition of an ice-water bath. The obtained protamine-modified liraglutide chitosan nanoparticles are a milky white suspension with a slightly transparent appearance and a faint blue opalescence, and have an obvious Tyndall effect. The morphology is spherical or quasi-spherical, the particle size is about 295 nm, the PDI value is less than 0.2, the average Zeta potential value is (31.59 ± 1.97) mV, the average encapsulation efficiency is (81.92 ± 1.54)%, the average drug loading is (16.66 ± 0.33)%, the pH value conforms to the normal nasal cavity environment of humans. The circular dichroism spectrum shows that preparing liraglutide into nanoparticles will not affect the secondary structure of liraglutide, and the addition of protamine will not affect the results. The in vitro release results show that there is an initial burst release phenomenon at the beginning of the release of CS-LIRA NPs and PS-CS-LIRA NPs. CS-LIRA NPs released 73% of the total drug amount at 5 h, and the release amount of PS-CS-LIRA NPs reached the highest at 4 h, which was 79% of the total drug amount; both CS-LIRA NPs and PS-CS-LIRA NPs have good sustained-release effects. The qualitative and quantitative investigation experiments of cell uptake show that the cell uptake ability of the protamine-modified nanoparticles is stronger than that of the unmodified nanoparticles. The rat diabetes model shows that the pharmacological relative bioavailability of the CS-LIRA NPs group at 12 h and 24 h after nasal administration is 8.25% and 8.40% respectively, and the pharmacological relative bioavailability of the PS-CS-LIRA NPs group at 12 h and 24 h after nasal administration is 9.91% and 10.15% respectively, which are significantly different compared with the LIRA solution group given nasally, indicating that preparing LIRA into nanoparticles can improve the absorption of the drug in the nasal mucosa, and the nasal mucosa absorption ability of the protamine-modified nanoparticles is stronger.

Claims

1. Liraglutide nasal administration nano - preparation, characterized in that: Each 10 mL contains components in the following weight ratios:

2. The liraglutide nasal administration nano - preparation according to claim 1, wherein: Each 10 mL contains components in the following weight ratios:

3. The liraglutide nasal administration nano - preparation according to claim 1, characterized in that: Each 10 mL contains components in the following weight ratios:

4. The liraglutide nasal administration nano - preparation according to any one of claims 1 - 3, characterized in that: At least meet any one of the following: The components of the liraglutide nasal administration nanopreparation meet the following weight ratio relationship: (1) The weight ratio of chitosan to liraglutide is 5:1 to 1:1, and the weight ratio of chitosan to poloxamer 188 is 15:1 to 5:1; (2) Or preferably, the weight ratio of chitosan to liraglutide is 4:1 to 2:1, and the weight ratio of chitosan to poloxamer 188 is 15:1 to 5:1; (3) Or most preferably, the weight ratio of chitosan to liraglutide is 2.95:1, and the weight ratio of chitosan to poloxamer 188 is 10.55:1; The ionic crosslinking agent is sodium tripolyphosphate.

5. Preparation method of liraglutide nasal administration nano - preparation, characterized in that: It includes the following steps: A. Prepare a liraglutide chitosan nanoparticle suspension by the ionic crosslinking method: (1) Weigh the liraglutide raw material drug and add it to PBS7.40 buffer solution to prepare a liraglutide solution; (2) Weigh chitosan and poloxamer 188 according to the weight ratio relationship of the dry matter of the components. After swelling sufficiently with a solvent, adjust the pH value to obtain a chitosan solution; (3) Drop the liraglutide solution obtained in step (1) into the chitosan solution obtained in step (2), and stir until fully mixed; (4) Drop the ionic crosslinking agent into the mixed solution in step (3) until the solution shows a light blue opalescence. After ultrasonic dispersion, the liraglutide chitosan nanoparticle suspension is obtained; B. Prepare protamine-modified liraglutide chitosan nanoparticles: Drop the protamine solution into the liraglutide chitosan nanoparticle suspension obtained in step A, and stir until fully mixed to obtain protamine-modified liraglutide chitosan nanoparticles.

6. The preparation method of the liraglutide nasal administration nano - preparation according to claim 5, characterized in that: At least meet any one of the following: In step (1) of step A: The solvent of the liraglutide solution is PBS7.40 buffer solution, water, or sodium acetate solution; Preferably, in step (1) of step A: The solvent of the liraglutide solution is PBS7.40 buffer solution; In step (1) of step A: the concentration of the liraglutide solution is 4.3 - 8.6 mg·mL -1 ; Preferably, in step (1) of step A: the concentration of the liraglutide solution is 4.3 mg·mL -1 ; In step (2) of step A, chitosan is used as a carrier material and poloxamer 188 is used as a surfactant; In step (2) of step A, chitosan and poloxamer 188 are weighed according to the relationship that the weight ratio of chitosan to liraglutide is 5:1 to 1:1, and the weight ratio of chitosan to poloxamer 188 is 15:1 to 5:1; Preferably, in step (2) of step A, chitosan and poloxamer 188 are weighed according to the relationship that the weight ratio of chitosan to liraglutide is 4:1 to 2:1, and the weight ratio of chitosan to poloxamer 188 is 15:1 to 5:1; Most preferably, in step (2) of step A, chitosan and poloxamer 188 are weighed according to the relationship that the weight ratio of chitosan to liraglutide is 2.95:1, and the weight ratio of chitosan to poloxamer 188 is 10.55:1; In step (2) of step A, the solvent of chitosan and poloxamer 188 is an aqueous acetic acid solution of 0.1-2%; chitosan is poorly soluble in water and common organic solvents and dissolves at pH < 6.

5. As a weak acid, acetic acid can provide a large amount of H + to protonate the amino groups on chitosan, further promoting dissolution. At the same time, the acidity is mild and not likely to cause degradation of the chitosan chain; Preferably, the solvent in step (2) of step A is 1% acetic acid aqueous solution; The so-called sufficient swelling in step (2) of step A means that chitosan and poloxamer 188 are mixed with acetic acid aqueous solution by stirring and absorb the acetic acid aqueous solution to the equilibrium state; In step (2) of step A, the conditions for stirring are that the stirring time is 3 - 5 hours until sufficient swelling occurs; Preferably, in step (2) of step A, the condition for stirring is that the stirring time is 4 hours; In step (2) of step A, the pH value is adjusted to 5.0 - 5.5; Preferably, in step (1) of step A, the pH value is adjusted to 5.25; In step (2) of step A, the pH value is adjusted using an alkaline solution; In step (2) of step A, the alkaline solution is NaOH solution, KOH solution, phosphate buffer solution, ammonia water; Preferably, in step (2) of step A, the alkaline solution is NaOH solution; In step (2) of step A, the concentration of the NaOH solution is 1 - 3M; Preferably, in step (2) of step A, the concentration of the NaOH solution is 2M; For the chitosan solution prepared by step (2) of step A, if it is not used immediately, it can be refrigerated at 1 - 8°C for standby; preferably, the refrigeration condition is 4°C; In step (3) of step A, based on the dosage of liraglutide being 8.6 mg, a chitosan solution containing 8.6 - 43 mg of chitosan and 0.57 - 8.6 mg of poloxamer 188 is added; Preferably, in step (3) of step A, based on the dosage of liraglutide being 8.6 mg, a chitosan solution containing 17.2 - 34.4 mg of chitosan and 1.15 - 6.88 mg of poloxamer 188 is added; Most preferably, in step (3) of step A, based on the dosage of liraglutide being 8.6 mg, a chitosan solution containing 25.37 mg of chitosan and 2.40 mg of poloxamer 188 is added; In step (3) of step A, the stirring time of the chitosan solution and the liraglutide solution is 1 - 3 h; Preferably, in step (3) of step A, the stirring time of the chitosan solution and the liraglutide solution is 1.5 h; In step (4) of step A: The ionic cross - linker used is sodium tripolyphosphate (TPP); common ionic cross - linkers mainly include sodium tripolyphosphate, sodium hexametaphosphate, sodium alginate, etc. Since the cation concentration of sodium tripolyphosphate is relatively high, the degree of cross - linking is relatively high and the stability is better. Therefore, in this invention, sodium tripolyphosphate is selected as the ionic cross - linker; In step (4) of step A: The sodium tripolyphosphate (TPP) is added in the form of a sodium tripolyphosphate solution; Further, in step (4) of step A: the concentration of the sodium tripolyphosphate (TPP) solution is 0.5 - 5 mg·mL -1 ; Preferably, in step (4) of step A: the concentration of the sodium tripolyphosphate (TPP) solution is 0.5-1 mg·mL -1 ; Most preferably, in step (4) of step A: the concentration of the sodium tripolyphosphate (TPP) solution is 0.95 mg·mL -1 ; In step (4) of step A, the dosage relationship between sodium tripolyphosphate (TPP) and liraglutide: For every 8.6 mg of liraglutide dosage, 0.5 - 5 mg of sodium tripolyphosphate is used; Preferably, in step (4) of step A, the dosage relationship between sodium tripolyphosphate (TPP) and liraglutide: For every 8.6 mg of liraglutide dosage, 0.5 - 1 mg of sodium tripolyphosphate is used; Preferably, in step (4) of step A, the dosage relationship between sodium tripolyphosphate (TPP) and liraglutide: For every 8.6 mg of liraglutide dosage, 0.95 mg of sodium tripolyphosphate is used; In step (4) of step A: The low - temperature ultrasonic condition is: ultrasonic under the condition of an ice - water bath; In step (4) of step A: The low-temperature ultrasonic conditions are as follows: ultrasonic treatment for 2 to 10 min, intermittent for 5 s / 5 s, 900 W × 20 to 40%; Preferably, in step (4) of step A: The low-temperature ultrasonic conditions are as follows: ultrasonic treatment for 6 min, intermittent for 5 s / 5 s, 900 W × 30%.

7. The preparation method of the liraglutide nasal administration nano - preparation according to claim 5, characterized in that: At least meet any one of the following: The protamine in step B is protamine sulfate; The solvent of the protamine in step B is water or PBS 7.40 buffer solution; Preferably, the solvent of the protamine in step B is water; The protamine described in step B is added with water to prepare a protamine solution with a concentration of 0.5 - 3 mg·mL -1 ; Preferably, the protamine described in step B is added with water to prepare a protamine solution with a concentration of 1 mg·mL -1 ; The dosage relationship between the protamine and liraglutide in step B: For every 8.6 mg of liraglutide dosage, 0.5 - 3 mg of protamine is used; Preferably, the dosage relationship between the protamine and liraglutide in step B: For every 8.6 mg of liraglutide dosage, 1 mg of protamine is used; The stirring time of the protamine with water in step B is 0.5 - 2 h; Preferably, the stirring time of the protamine with water in step B is 1.5 h.

8. Application of liraglutide nasal administration nanon preparation in the preparation of drugs for nasal administration.

9. Application of liraglutide nasal administration nanon preparation in the preparation of drugs for treating diabetes by nasal administration; Or, application of liraglutide nasal administration nanon preparation in the preparation of drugs for treating type 2 diabetes by nasal administration.

10. Application of liraglutide nasal administration nanon preparation in the preparation of drugs for weight loss by nasal administration.

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