RVG29 peptide modified tripterine transfersome nasal in-situ gel as well as preparation method and application of RVG29 peptide modified tripterine transfersome nasal in-situ gel

Abstract

The invention discloses RVG29 peptide modified tripterine transfersome nasal in-situ gel and a preparation method and application thereof.The in-situ gel is mainly prepared from tripterine, soybean phospholipid, cholesterol, mPEG2000-DSPE, mPEG2000-DSPE-RVG29, sodium deoxycholate and DGG, the RVG29 peptide modified tripterine transfersome nasal in-situ gel can increase the solubility of indissolvable drugs, and the RVG29 peptide modified tripterine transfersome nasal in-situ gel has the advantages that the solubility of the indissolvable drugs is improved; and the RVG29 peptide which can be specifically recognized with a dopaminergic neuron high-expression acetylcholine receptor is modified on the surface of a carrier, so that efficient active targeting endocytosis can be effectively realized, and the RVG29 peptide can be used for treating the Parkinson's disease.

Description

Technical Field

[0001] The invention relates to an RVG29 peptide-modified tripterygium wilfordii transfersome in-situ gel for nose use, a preparation method and application thereof, and belongs to the field of medicine. Background Art

[0002] Parkinson's disease (PD), also often referred to as "tremor paralysis", is a neurodegenerative disease. The main cause of this disease is due to the degeneration and death of dopaminergic neurons in the substantia nigra, which is related to multiple factors such as genetics, environmental factors and aging of the nervous system. It has become a disease that seriously endangers the health of the elderly after tumors and cardiovascular and cerebrovascular diseases. PD is a disease of the central nervous system, but the existence of the blood-brain barrier limits the efficacy of most drugs in the brain. Recent studies have found that after nasal administration, drugs can bypass the blood-brain barrier and be directly transported into the brain along the olfactory pathway and trigeminal nerve pathway, providing a new delivery strategy for drug treatment of PD. However, the rapid clearance of nasal cilia and the low permeability of the nasal mucosa greatly reduce intranasal drug delivery. In addition, after entering the brain through the nose, the drug is distributed non-selectively, making it difficult to accumulate in the substantia nigra dopaminergic neurons and easily damaging normal brain tissue.

[0003] Celastrol (CLT) is a pentacyclic triterpenoid monomer compound isolated from Tripterygium wilfordii plants. Studies have found that CLT has significant neuroprotective function in addition to its anti-inflammatory and antioxidant activities. However, CLT has poor water solubility, low bioavailability, and is prone to systemic toxic side effects, which greatly limits its clinical application.

[0004] The present invention uses CLT as a model drug, and loads the transfersome (RVG29-TFs) obtained by modifying tripterygium wilfordii with the polypeptide RVG29 into deacetylgellan gum (DGG) to obtain a gel preparation. The gel preparation is administered through the nasal cavity, and adheres to the surface of the nasal mucosa, which can increase the solubility of poorly soluble drugs, improve the bioavailability of drugs, and slow down the clearance of the nasal cavity. At the same time, it can efficiently achieve active targeted entry into cells and improve the "nose-to-brain" transport efficiency. Summary of the invention

[0005] The purpose of the present invention is to provide an RVG29 peptide-modified tripterygium wilfordii transfersome in-situ gel for nasal use, and a preparation method and application thereof. The prepared RVG29 peptide-modified tripterygium wilfordii transfersome in-situ gel for nasal use can increase the solubility of poorly soluble drugs, improve the bioavailability of drugs, and can respond to the cationic environment of nasal mucus to undergo phase change, adhere to the surface of the nasal mucosa, and prolong the nasal retention time of the transfersome. Subsequently, the transfersome diffuses from the gel and enters the brain across the membrane with the help of its own membrane fusion and deformation penetration, and then, under the mediation of the RVG29 targeting peptide, is efficiently taken up by dopaminergic neurons to exert its drug effect.

[0006] The technical solution of the present invention:

[0007] An RVG29 peptide-modified celastrol transfersome in-situ gel for nasal use, wherein the nasal in-situ gel is prepared from RVG29 peptide-modified celastrol transfersome and deacetyl gellan gum solution;

[0008] The RVG29 peptide modified tripterygium wilfordii transfersome is prepared from 2.5-4.5 parts of tripterygium wilfordii, 30-70 parts of soybean lecithin, 6-20 parts of cholesterol, 5-15 parts of mPEG2000-DSPE, 10-28 parts of mPEG2000-DSPE-RVG29 and 4-12 parts of sodium deoxycholate;

[0009] The concentration of the deacetylated gellan gum solution is 0.6-1.4%, and the solvent is distilled water;

[0010] The volume ratio of the RVG29 peptide-modified tripterygium wilfordii transfersome to the deacetylated gellan gum solution is 0.8-1.2:0.8-1.2.

[0011] The aforementioned nasal in situ gel is prepared from RVG29 peptide-modified tripterygium wilfordii transfersomes and deacetylated gellan gum solution;

[0012] The aforementioned RVG29 peptide modified tripterygium wilfordii transfersome is prepared from 3-4 parts of tripterygium wilfordii, 40-60 parts of soybean lecithin, 10-15 parts of cholesterol, 8-12 parts of mPEG2000-DSPE, 15-20 parts of mPEG2000-DSPE-RVG29 and 6-10 parts of sodium deoxycholate;

[0013] The concentration of the deacetylated gellan gum solution is 0.8-1.2%, and the solvent is distilled water;

[0014] The volume ratio of the aforementioned RVG29 peptide-modified tripterygium wilfordii transfersome and deacetylated gellan gum solution is 0.9-1.1:0.9-1.1.

[0015] Specifically, the aforementioned nasal in situ gel is prepared from RVG29 peptide-modified tripterygium wilfordii transfersome and deacetylated gellan gum solution;

[0016] Specifically, the aforementioned RVG29 peptide modified tripterygium wilfordii transfersome is prepared from 3.5 parts of tripterygium wilfordii, 50 parts of soybean lecithin, 13 parts of cholesterol, 10 parts of mPEG2000-DSPE, 18 parts of mPEG2000-DSPE-RVG29, and 8 parts of sodium deoxycholate;

[0017] Specifically, the concentration of the deacetylated gellan gum solution is 1.0%, and the solvent is distilled water;

[0018] Specifically, the volume ratio of the aforementioned RVG29 peptide-modified tripterygium wilfordii transfersome and the deacetylated gellan gum solution is 1:1.

[0019] The preparation method of the aforementioned RVG29 peptide-modified tripterygium wilfordii transfersome in situ gel for nasal use is carried out according to the following specific steps:

[0020] (1) Preparation of lipid film: 2.5-4.5 mg of celastrol, 30-70 mg of soybean lecithin, 6-20 mg of cholesterol, 5-15 mg of mPEG2000-DSPE, 10-28 mg of mPEG2000-DSPE-RVG29 and 4-12 mg of sodium deoxycholate were weighed in proportion and dissolved in 3-7 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a water bath at 35-39° C. to obtain a uniform lipid film;

[0021] (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: add 5-15 mL of distilled water to the lipid film, hydrate it at 50-70°C in a constant temperature oscillator at 100 rpm for 20-40 min, and perform ice bath sonication for 2-4 min at a rate of 3-7 s per sonication and 3-7 s pause, with an ultrasonic power of 300 W, to obtain RVG29-CLT-TFs;

[0022] (3) Preparation of deacetylated gellan gum solution: 0.6-1.4 g of deacetylated gellan gum powder is placed in a beaker, 100 ml of distilled water is added, and the mixture is weighed. The mixture is stirred at a constant temperature of 85-95° C. for 1.5-2.5 h to completely dissolve the mixture. The mixture is cooled to room temperature, and after distillation, the mixture is sealed and placed in a refrigerator at 3-5° C. to fully swell for 22-26 h to obtain a deacetylated gellan gum solution.

[0023] (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and deacetylated gellan gum solution were mixed at a volume ratio of 0.8-1.2:0.8-1.2, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

[0024] The above-mentioned preparation method is carried out according to the following specific steps:

[0025] (1) Preparation of lipid film: 3-4 mg of celastrol, 40-60 mg of soybean lecithin, 10-15 mg of cholesterol, 8-12 mg of mPEG2000-DSPE, 15-20 mg of mPEG2000-DSPE-RVG29 and 6-10 mg of sodium deoxycholate were weighed and dissolved in 4-6 mL of dichloromethane according to the proportion. The dichloromethane was removed by rotary evaporation at 30 rpm in a water bath at 36-38° C. to obtain a uniform lipid film;

[0026] (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: Take the lipid film and add 8-12 mL of distilled water, hydrate it at 55-65°C constant temperature oscillator at 100 rpm for 25-35 min, perform ice bath sonication for 2.5-3.5 min with a method of sonication for 4-6 s and pause for 4-6 s, and the ultrasonic power is 300 W to obtain RVG29-CLT-TFs;

[0027] (3) Preparation of deacetylated gellan gum solution: 0.8-1.2 g of deacetylated gellan gum powder is placed in a beaker, 100 ml of distilled water is added, and the mixture is weighed. The mixture is stirred at 90° C. for 2 h to completely dissolve the mixture. The mixture is cooled to room temperature, distilled, sealed, and placed in a refrigerator at 4° C. to fully swell for 24 h to obtain a deacetylated gellan gum solution.

[0028] (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and deacetylated gellan gum solution were mixed at a volume ratio of 0.9-1.1:0.9-1.1, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

[0029] Specifically, the above preparation method is carried out according to the following specific steps:

[0030] (1) Preparation of lipid film: 3.5 mg of celastrol, 50 mg of soybean lecithin, 13 mg of cholesterol, 10 mg of mPEG2000-DSPE, 18 mg of mPEG2000-DSPE-RVG29 and 8 mg of sodium deoxycholate were weighed in proportion and dissolved in 5 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a 37°C water bath to obtain a uniform lipid film;

[0031] (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: Take the lipid film and add 10 mL of distilled water, hydrate it at 60°C constant temperature oscillator at 100 rpm for 30 min, and perform ice bath sonication for 3 min with a method of sonication for 5 s and pause for 5 s, with an ultrasonic power of 300 W, to obtain RVG29-CLT-TFs;

[0032] (3) Preparation of deacetylated gellan gum solution: 1.0 g of deacetylated gellan gum powder was placed in a beaker, 100 ml of distilled water was added, and the mixture was weighed. The mixture was stirred at 90° C. for 2 h to completely dissolve the mixture. The mixture was cooled to room temperature, distilled, sealed, and placed in a refrigerator at 4° C. to fully swell for 24 h to obtain a deacetylated gellan gum solution.

[0033] (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and deacetylated gellan gum solution were mixed at a volume ratio of 1:1 and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

[0034] The use of the aforementioned RVG29 peptide-modified tripterygium wilfordii transfersome nasal in situ gel in the preparation of drugs for treating Parkinson's disease.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1.RVG29 peptide modified tripterygium wilfordii transfersomes can increase the solubility of poorly soluble drugs and improve the bioavailability of drugs. The encapsulation efficiency and drug loading of the transfersomes are 94.22±0.21% and 2.31±0.25%, respectively.

[0037] 2. The viscosity of RVG29 peptide-modified tripterygium wilfordii transfersome nasal in situ gel is low, which can flow into the nasal cavity better. After entering the nasal cavity, RVG29-TFs is an aqueous preparation and is easily lost in the nasal cavity. Deacetylated gellangum (DGG) is a typical ion-sensitive in situ gel matrix that can respond to cationic Na in the nasal environment. + ,Ka + , Ca 2+ A phase change occurs, from a solution to a gel, allowing the gel to be effectively adsorbed on the nasal mucosa, thereby prolonging the absorption time of the drug into the brain through the nose and slowing down nasal clearance.

[0038] 3. The 29-amino acid peptide RVG29 derived from rabies virus glycoprotein can specifically recognize and bind to AchR. The peptide RVG29-modified transfersomes (RVG29-TFs) enter the brain through the nose with their own high deformability and permeability, and under the mediation of the RVG29 peptide, the active nasal-brain targeting of dopaminergic neurons is achieved.

[0039] 4. The gel strength is between 25-50s, which can resist the clearance of nasal cilia and protect the integrity of the gel while achieving sustained release of drugs in specific absorption sites in the nasal cavity.

[0040] 5. The water retention capacity is higher than 85%. This excellent water retention capacity can maintain the stability of the gel when it is stored or subjected to external forces.

[0041] 6. The pH value of the gel is between 5.5-7.0, while the pH value of human nasal mucus is usually between 5.5-7.0, which can reduce irritation to the nasal mucosa and is suitable for nasal medication.

[0042] In summary, the RVG29 peptide-modified tripterygium wilfordii transfersome nasal in-situ gel developed by the present invention can increase the solubility of poorly soluble drugs, improve the bioavailability of drugs, and slow down the clearance of the nasal cavity. The RVG29 peptide that can specifically recognize the acetylcholine receptor (AchR) highly expressed by dopaminergic neurons is modified on the surface of the transfersome, which can effectively achieve efficient active targeted entry into the cell, increase the retention time of the carrier in the nasal cavity, increase the absorption time and absorption amount, and improve the "nose-brain" transport efficiency, which is targeted at the treatment of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 :Comparison of RVG29-CLT-TFs / DGG before and after phase transition;

[0044] Figure 2 : Particle size distribution of RVG29 peptide modified tripterygium wilfordii transfersomes;

[0045] Figure 3 : TEM image of RVG29 peptide modified tripterygium wilfordii transfersome (scale bar: 100 nm);

[0046] Figure 4 : Specific HPLC graph (A: tripterygium wilfordii solution; B: blank transfersome gel solution; C: tripterygium wilfordii transfersome gel solution);

[0047] Figure 5 : Viscosity measurement results of transfersome in situ gels with different DGG concentrations;

[0048] Figure 6 : Results of determination of gel strength of RVG29-CLT-TFs / DGG at different DGG concentrations ( n=3);

[0049] Figure 7 :Determination results of water holding capacity of RVG29-CLT-TFs / DGG at different DGG concentrations ( n=3);

[0050] Figure 8 :Determination results of volume expansion coefficient of RVG29-CLT-TFs / DGG at different DGG concentrations ( n=3);

[0051] Fig. 9 :Determination results of pH value of RVG29-CLT-TFs / DGG at different DGG concentrations ( n=3);

[0052] Fig.10 :Release curve of tripterygium wilfordii n=3). DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0054] Embodiment 1:

[0055] Preparation method of nasal in-situ gel of RVG29 peptide-modified tripterygium wilfordii transfersome

[0056] (1) Preparation of lipid film: 3.5 mg of celastrol, 50 mg of soybean lecithin, 13 mg of cholesterol, 10 mg of mPEG2000-DSPE, 18 mg of mPEG2000-DSPE-RVG29 and 8 mg of sodium deoxycholate were weighed in proportion and dissolved in 5 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a 37°C water bath to obtain a uniform lipid film;

[0057] (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: Take the lipid film and add 10 mL of distilled water, hydrate it at 60°C constant temperature oscillator at 100 rpm for 30 min, and perform ice bath sonication for 3 min with a method of sonication for 5 s and pause for 5 s, with an ultrasonic power of 300 W, to obtain RVG29-CLT-TFs;

[0058] (3) Preparation of deacetylated gellan gum solution: 1.0 g of deacetylated gellan gum powder was placed in a beaker, 100 ml of distilled water was added, and the mixture was weighed. The mixture was stirred at 90° C. for 2 h to completely dissolve the mixture. The mixture was cooled to room temperature, distilled, sealed, and placed in a refrigerator at 4° C. to fully swell for 24 h to obtain a 1.0% deacetylated gellan gum solution.

[0059] (4) Preparation of RVG29 peptide-modified tripterygium wilfordii transfersome in situ gel for nasal use: RVG29-CLT-TFs and 1.0% deacetylated gellan gum solution were mixed at a volume ratio of 1:1 and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG.

[0060] Dosage: 2-4 drops each time, 3 times a day.

[0061] Main indications: Parkinson's disease.

[0062] Embodiment 2:

[0063] Preparation method of nasal in-situ gel of RVG29 peptide-modified tripterygium wilfordii transfersome

[0064] (1) Preparation of lipid film: 2.5 mg of celastrol, 30 mg of soybean lecithin, 6 mg of cholesterol, 15 mg of mPEG2000-DSPE, 28 mg of mPEG2000-DSPE-RVG29 and 12 mg of sodium deoxycholate were weighed in proportion and dissolved in 7 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a 39°C water bath to obtain a uniform lipid film;

[0065] (2) Preparation of RVG29 peptide-modified tripterygium wilfordii transfersomes: Take the lipid film and add 5 mL of distilled water, hydrate it at 70°C in a constant temperature oscillator at 100 rpm for 20 min, and perform ice bath sonication for 2 min with a 7 s ultrasonication and a 7 s pause method at an ultrasonic power of 300 W to obtain RVG29-CLT-TFs;

[0066] (3) Preparation of deacetylated gellan gum solution: 1.4 g of deacetylated gellan gum powder was placed in a beaker, 100 ml of distilled water was added, and the mixture was weighed. The mixture was stirred at 95° C. for 2.5 h to completely dissolve the mixture. The mixture was cooled to room temperature, distilled, sealed, and placed in a refrigerator at 3° C. to fully swell for 26 h to obtain a 1.4% deacetylated gellan gum solution.

[0067] (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and 1.4% deacetylated gellan gum solution were mixed at a volume ratio of 1.2:0.8, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

[0068] Dosage: 2-4 drops each time, 3 times a day.

[0069] Main indications: Parkinson's disease.

[0070] Embodiment 3:

[0071] Preparation method of nasal in-situ gel of RVG29 peptide-modified tripterygium wilfordii transfersome

[0072] (1) Preparation of lipid film: 4.5 mg of celastrol, 70 mg of soybean lecithin, 20 mg of cholesterol, 5 mg of mPEG2000-DSPE, 10 mg of mPEG2000-DSPE-RVG29 and 4 mg of sodium deoxycholate were weighed in proportion and dissolved in 3 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a 35°C water bath to obtain a uniform lipid film;

[0073] (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: 15 mL of distilled water was added to the lipid film, and the film was hydrated at 50°C in a constant temperature oscillator at 100 rpm for 40 min. The film was ultrasonicated for 3 s and paused for 3 s for 4 min in an ice bath at an ultrasonic power of 300 W to obtain RVG29-CLT-TFs.

[0074] (3) Preparation of deacetylated gellan gum solution: 0.6 g of deacetylated gellan gum powder was placed in a beaker, 100 ml of distilled water was added, and the mixture was weighed. The mixture was stirred at a constant temperature of 85-95° C. for 1.5 h to completely dissolve the mixture. The mixture was cooled to room temperature, distilled, sealed, and placed in a refrigerator at 5° C. to fully swell for 22 h to obtain a 0.6% deacetylated gellan gum solution.

[0075] (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and 0.6% deacetylated gellan gum solution were mixed at a volume ratio of 0.8:1.2, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

[0076] Dosage: 2-4 drops each time, 3 times a day.

[0077] Main indications: Parkinson's disease.

[0078] Embodiment 4:

[0079] Preparation method of nasal in-situ gel of RVG29 peptide-modified tripterygium wilfordii transfersome

[0080] (1) Preparation of lipid film: 4 mg of celastrol, 60 mg of soybean lecithin, 10 mg of cholesterol, 7.5 mg of mPEG2000-DSPE, 18 mg of mPEG2000-DSPE-RVG29 and 10 mg of sodium deoxycholate were weighed in proportion and dissolved in 4 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a 36°C water bath to obtain a uniform lipid film;

[0081] (2) Preparation of RVG29 peptide-modified tripterygium wilfordii transfersomes: 7 mL of distilled water was added to the lipid film, and the film was hydrated at 65 °C in a constant temperature oscillator at 100 rpm for 25 min. The film was ultrasonicated for 6 s and paused for 6 s for 3 min in an ice bath at an ultrasonic power of 300 W to obtain RVG29-CLT-TFs.

[0082] (3) Preparation of deacetylated gellan gum solution: 0.8 g of deacetylated gellan gum powder was placed in a beaker, 100 ml of distilled water was added, and the mixture was weighed. The mixture was stirred at 90° C. for 2 h to completely dissolve the mixture. The mixture was cooled to room temperature, distilled, sealed, and placed in a refrigerator at 4° C. to fully swell for 25 h to obtain a 0.8% deacetylated gellan gum solution.

[0083] (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and 0.8% deacetylated gellan gum solution were mixed at a volume ratio of 1.1:0.9, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

[0084] Dosage: 2-4 drops each time, 3 times a day.

[0085] Main indications: Parkinson's disease.

[0086] Embodiment 5:

[0087] Preparation method of nasal in-situ gel of RVG29 peptide-modified tripterygium wilfordii transfersome

[0088] (1) Preparation of lipid film: 3 mg of celastrol, 40 mg of soybean lecithin, 18 mg of cholesterol, 12 mg of mPEG2000-DSPE, 15 mg of mPEG2000-DSPE-RVG29 and 6 mg of sodium deoxycholate were weighed in proportion and dissolved in 6 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a 38°C water bath to obtain a uniform lipid film;

[0089] (2) Preparation of RVG29 peptide-modified tripterygium wilfordii transfersomes: 13 mL of distilled water was added to the lipid film, and the film was hydrated at 55°C in a constant temperature oscillator at 100 rpm for 35 min. The film was ultrasonicated for 4 s each and paused for 4 s for 3 min in an ice bath at an ultrasonic power of 300 W to obtain RVG29-CLT-TFs.

[0090] (3) Preparation of deacetylated gellan gum solution: 1.2 g of deacetylated gellan gum powder was placed in a beaker, 100 ml of distilled water was added, and the mixture was weighed. The mixture was stirred at 90° C. for 2 h to completely dissolve the mixture. The mixture was cooled to room temperature, distilled, sealed, and placed in a refrigerator at 4° C. to fully swell for 23 h to obtain a 1.2% deacetylated gellan gum solution.

[0091] (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and 1.2% deacetylated gellan gum solution were mixed at a volume ratio of 0.9:1.1, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

[0092] Dosage: 2-4 drops each time, 3 times a day.

[0093] Main indications: Parkinson's disease.

[0094] The present invention has carried out a lot of experimental studies, and the following are the results of the experimental studies of the present invention:

[0095] 1. Research on the preparation process of RVG29-CLT-TFs / DGG

[0096] 1 Materials

[0097] 1.1 Instrument

[0098] The main instruments include Nano ZS90 laser particle size analyzer (Malvern, UK), H-600 transmission electron microscope (Hitachi, Japan), LC2030 3D high performance liquid chromatograph (Shimadzu, Japan), SB-4200D ultrasonic cell crusher (Ningbo Xinzhi Ultrasonic Equipment Co., Ltd.), RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory), LQ-C2002 electronic balance (Shanghai Yaoxin Electronic Technology Co., Ltd.), H2050R high-speed refrigerated centrifuge (Hunan Xiangyi Centrifuge Instrument Co., Ltd.), XMTD-8222 constant temperature oscillator (Shanghai Jinghong Experimental Equipment Co., Ltd.), HJ-6A digital display constant temperature magnetic stirrer (Changzhou Guohua Electric Co., Ltd.), NDJ-5S digital viscometer (Shanghai Sunny Hengping Scientific Instrument Co., Ltd.), PHS-3C pH meter (Shanghai Hongyi Instrument Co., Ltd.)

[0099] 1.2 Drug testing

[0100] Triptolide (Chengdu Pufeide Biotechnology Co., Ltd., purity>99%, batch number 19041101); distearoylphosphatidylethanolamine-polyethylene glycol 2000 (mPEG2000-DSPE) (Xi'an Ruixi Biotechnology Co., Ltd.); distearoylphosphatidylethanolamine-polyethylene glycol 2000-rabies virus targeting peptide RVG29 (mPEG2000-DSPE-RVG29) (Xi'an Ruixi Biotechnology Co., Ltd.); chromatographic grade glacial acetic acid (Shanghai MacLean Biochemical Technology Co., Ltd., batch number 19041101); C14357167); chromatographic grade methanol (Shanghai MacLean Biochemical Technology Co., Ltd.); cholesterol (Solabo Biotechnology Co., Ltd.); sodium deoxycholate (Solabo Biotechnology Co., Ltd.); soybean lecithin (Shanghai MacLean Biochemical Technology Co., Ltd.); deacetylated gellan gum (DGG) (Zhejiang Tianwei Biotechnology Co., Ltd., batch number: TW-J500); Tween80 (Solabo Biotechnology Co., Ltd., batch number 420G011); other reagents were of analytical grade, and water was pure water or Watsons distilled water.

[0101] 2 Methods and Results

[0102] 2.1 Preparation of RVG29-CLT-TFs

[0103] RVG29-CLT-TFs were prepared by thin film hydration method. The specific operation is as follows: 3.50 mg of tripterygium wilfordii (CLT), 50.00 mg of soybean lecithin, 13.00 mg of cholesterol, 10.00 mg of mPEG2000-DSPE, 18.00 mg of mPEG2000-DSPE-RVG29 and 8.00 mg of sodium deoxycholate were accurately weighed and dissolved in 5 mL of dichloromethane. After removing dichloromethane by rotary evaporation at 30 rpm in a 37 ° C water bath, a uniform lipid film was obtained. 10 mL of distilled water was added, and the mixture was hydrated for 30 min at 60 ° C constant temperature oscillator at 100 rpm, and ultrasonicated for 3 min in an ice bath (supersonic for 5 s, stop for 5 s) at a power of 300 W to obtain RVG29-CLT-TFs.

[0104] 2.1.1 Determination of particle size, zeta potential, polydispersity index and morphology

[0105] The diluted RVG29-CLT-TFs were placed in a NanoZS90 laser particle size analyzer for measurement. The particle size distribution is shown in Figure 2 The average particle size was (102.33±0.26)nm, the polydispersity index was 0.203±0.018, and the Zeta potential was (-41.13±1.7)mV, indicating that the particle size and polydispersity index were small and the particle size distribution was uniform. RVG29-CLT-TFs were observed under transmission electron microscopy. The results are shown in Figure 3RVG29-CLT-TFs are spherical, relatively regular and round, and the particle size is about 100 nm, which is consistent with the particle size measurement result of the laser particle size analyzer.

[0106] 2.1.2 Establishment of CLT content determination method

[0107] 2.1.2.1 The content of CLT in the transfersomes was determined by HPLC.

[0108] Chromatographic conditions of HPLC method: Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 mm); mobile phase methanol: 10% acetic acid water = 95:5; flow rate 1.0 mL min -1 ; Detection wavelength was 425nm; column temperature was 35℃; injection volume was 10μL.

[0109] 2.1.2.2 Exclusivity

[0110] Tripterine-methanol solution: Take 3.5 mg of tripterine and add 10 mL of methanol to dissolve it.

[0111] Blank transfersome gel solution: without adding tripterygium wilfordii, the preparation process is in accordance with the preparation method in item "2.1" to obtain a blank transfersome gel solution.

[0112] Celastrol transfersome gel solution: prepared according to the method in item "2.1", i.e. RVG29-CLT-TFs.

[0113] Accurately pipette 1mL of freshly prepared blank transfersome gel solution and tripterygium wilfordii transfersome gel solution into a 5mL brown volumetric flask, add 3mL of methanol and vortex for 5min to break the emulsion, dilute to 5mL with methanol, mix the solution ultrasonically and centrifuge at 13000rpm / min for 10min, and filter the supernatant with a 0.22μm filter membrane. Pipet 10μL of tripterygium wilfordii-methanol solution, blank transfersome gel solution and tripterygium wilfordii transfersome gel solution into a high performance liquid chromatograph, analyze under the chromatographic conditions under "2.1.2.1", and record the chromatogram.

[0114] like Figure 4 The blank transfersome gel solution has no peak at the peak position of CLT, indicating that the material does not interfere with the determination of CLT and the specificity of the method meets the requirements.

[0115] 2.1.2.3 Drawing of standard curve

[0116] Accurately weigh 10.00 mg of CLT standard, dissolve it in methanol and make up to 10 mL to prepare 1.00 mg mL -1Accurately pipette different volumes of mother solution into a 5 mL volumetric flask, dilute to 5 mL with methanol, and obtain 10.0, 20.0, 40.0, 60.0, 80.0, 100.0, 120 mg·mL -1 The CLT series solutions of different concentrations were analyzed under the chromatographic conditions in "2.1.2.1". The peak area (As) was linearly regressed against the concentration (C). The regression equation was: As = 18866C + 80348 (R 2 =0.999), linear range: 10-120 mg·mL -1 .

[0117] 2.1.2.4 Precision investigation

[0118] Intra-day precision: take the lower value (10.00 mg·mL -1 ), medium (60.00 mg·mL -1 ), high (120.00 mg·mL -1 ) Three concentrations of CLT solutions were analyzed according to the established HPLC. Each concentration was measured three times on the same day, once every 2 hours for 6 hours. The peak areas were recorded and substituted into the standard curve to examine the intra-day precision of the method.

[0119] Inter-day precision: Each concentration was measured once a day, and the samples were injected and measured at the same time for 5 consecutive days to examine the inter-day precision of the method.

[0120] The intra-day RSDs of low, medium and high concentrations of CLT solution were measured to be 0.44%, 0.19% and 0.60%, respectively, and the inter-day RSDs were 1.21%, 1.48% and 1.75%, respectively. Both intra-day and inter-day precisions met the requirements.

[0121] 2.1.2.5 Recovery rate determination

[0122] Prepare low (10.00mg·mL -1 ), medium (60.00 mg·mL -1 ), high (120.00 mg·mL -1) Three concentrations of CLT solution, take appropriate amount and add it to the blank transfersome gel solution, add 3 times amount of methanol to mix, vortex shake for 5 minutes to completely break the emulsion, dilute to 5mL with methanol, centrifuge at 10000rpm for 20 minutes, take the supernatant and filter it with 0.22mm filter membrane. Inject into high performance liquid chromatograph, analyze under the chromatographic conditions under "2.1.2.1", and calculate its RSD value. Through the standard curve, calculate the measured concentration, and the ratio of the measured concentration to the prepared concentration is the recovery rate. The average recovery rates of low, medium and high concentrations are 102.24%, 95.85%, and 103.05%, respectively, and the RSDs are 0.18%, 0.13%, and 0.11%, respectively, indicating that the recovery rate of the method meets the requirements.

[0123] 2.1.3 Determination of encapsulation efficiency and drug loading

[0124] The encapsulation efficiency and drug loading of RVG29-CLT-TFs were measured by HPLC combined with ultrafiltration, and the results were (94.22±0.21)% and (2.31±0.25)%, respectively.

[0125] 2.2RVG29-CLT-TFs / DGG prescription screening

[0126] 2.2.1 Preparation of RVG29-CLT-TFs / DGG with different DGG (deacetylated gellan gum) concentrations

[0127] Weigh different amounts of DGG powder into a beaker, add an appropriate amount of distilled water, stir at 90°C for 2 hours to completely dissolve it, cool to room temperature, add water to the required amount, seal, and place in a 4°C refrigerator to fully swell for 24 hours to obtain 0.6%, 0.8%, 1.0%, 1.2%, and 1.4% DGG solutions. Mix equal volumes of RVG29 peptide-modified RVG29-CLT-TFs and DGG solution, stir evenly at 100rpm, and obtain concentrations of 0.3%, 0.4%, 0.5%, 0.6%, and 0.7% RVG29-CLT-TFs / DGG.

[0128] 2.2.2 Determination of viscosity of RVG29-CLT-TFs / DGG at different DGG concentrations

[0129] The viscosity of the transfersomes with different DGG concentrations before and after the in-situ gel phase transition was measured using a rotational viscometer at 32-34°C. Figure 5 It can be seen that with the increase of DGG concentration, the viscosity of the transfersome gel solution before and after the phase change also increases, while the viscosity of the transfersome gel solution before the phase change is much smaller than that after the phase change, which meets the requirements that the in-situ gel solution has low viscosity and the gel formed after gelation should have an appropriate viscosity.

[0130] 2.2.3 Determination of critical ion concentration of RVG29-CLT-TFs / DGG at different DGG concentrations

[0131] Artificial nasal solution: Accurately weigh 7.91g NaCl, 3.68g KCl, 0.51g CaCl2, and 2.56g NaHCO3 and dissolve them in appropriate amount of distilled water, and make up to 1000mL to obtain artificial nasal solution for use.

[0132] 50, 100, 150, 200, 250, 300, and 350 mL of artificial nasal mimic solutions were mixed with 1 mL of transfersome in situ gel solution of each DGG concentration in centrifuge tubes, placed in a 32-34°C water bath, and inverted after 30 seconds to evaluate the gel formation. If the gel adheres to the bottom and does not flow or slide along the side, it is considered to have formed a gel, indicated by '+'. Gels that flow or slide are indicated by '-', indicating that no gel is formed, and preparations with intermediate behavior are indicated by '±'. Each sample was measured three times in parallel. The results are shown in Table 1. When the matrix dosage is 0.4% or less, a large amount of artificial nasal mimic solution is required to trigger the phase change of the transfersome gel solution; when the matrix dosage is 0.7% or more, a gel can be formed with very little or no artificial nasal mimic solution; and when the matrix dosage is 0.5% or 0.6%, an appropriate amount of artificial nasal mimic solution is required for phase change.

[0133] Table 1 Determination results of critical ion concentration (n=3)

[0134]

[0135] 2.2.4 Determination of RVG29-CLT-TFs / DGG gel strength at different DGG concentrations

[0136] After taking an appropriate amount of transfersome gel solution and an appropriate amount of artificial nasal fluid to react to form a gel, accurately transfer 50g of the transfersome gel after phase change into a 100mL measuring cylinder, place a 35g weight on the top of the gel, and record the time it takes for the weight to sink 5cm. All measurements were performed at 32-34℃ and three replicates were measured. The results are shown in Figure 6 When the matrix dosage is 0.4% or less, the gel strength is insufficient (<20s); when the matrix dosage is 0.7% or more, the gel strength is too strong (>50s); and when the matrix dosage is 0.4%-0.6%, the gel strength meets the requirements and is between 25-50s.

[0137] 2.2.5 Determination of water holding capacity of RVG29-CLT-TFs / DGG at different DGG concentrations

[0138] Accurately transfer 1 mL of the transfersome gel solution and place it in a precisely weighed centrifuge tube, then add 400 mL of artificial nasal fluid and mix with it. Use filter paper to absorb the moisture, accurately weigh the mass of the gel, and record it as W0. Centrifuge at 8000 rpm for 10 min, and use filter paper to absorb the moisture. Accurately weigh the mass of the gel, and record it as W1. Calculate the water retention capacity according to the formula: Water retention capacity (%) = W1 / W0 × 100%. All measurements were carried out at 32-34°C, and each sample was measured in triplicate. See the results. Figure 7 The water retention capacity of the transfersome in situ gel solution at each DGG concentration was higher than 85%, which met the requirements.

[0139] 2.2.6 Determination of volume expansion coefficient of RVG29-CLT-TFs / DGG at different DGG concentrations

[0140] Take 1 mL of transfersome gel solution and mix it with 0.4 mL of artificial nasal solution in a 10 mL measuring cylinder and place it in a 32-34 ° C water bath. At this time, the total volume of the liquid (V M ) is 1.4mL. Continue to add 2.0mL of artificial nasal solution and record the total volume of liquid as V1. The volume of gel after phase transition (V G )=(V1-2.0)mL. Calculate the expansion coefficient S% of the transfersome in situ gel=(V G -V M ) / V M × 100%, all measurements were performed at 32-34°C, and the results were measured in triplicate. Figure 8 The S% of the transfersome in situ gel solutions at various DGG concentrations were all lower than 5%, meeting the requirements.

[0141] 2.2.7 Determination of pH of RVG29-CLT-TFs / DGG at different DGG concentrations

[0142] The pH of the transfersome in situ gel solution was measured using a pH meter. All measurements were performed at 32-34°C and were repeated three times. Fig. 9 The pH values ​​of the transfersome in situ gel solutions at various DGG concentrations were measured to be between 5.5 and 7.0, which all met the requirements.

[0143] Based on the above investigation results, DGG with a concentration of 1.0% was finally selected as the gel matrix.

[0144] 2.3 Determination of the optimal formulation process

[0145] RVG29-CLT-TFs were prepared by thin film hydration method. RVG29-CLT-TFs and 1.0% DGG solution were mixed at a ratio of 1:1 (v / v) and stirred at 100 rpm to obtain orange-yellow 0.5% RVG29-CLT-TFs / DGG.

[0146] 2.4 In vitro drug release

[0147] The in vitro drug release behavior of the drug-loaded transfersome gel was evaluated using the dialysis bag method. The release medium was 0.01 mol·L -1 PBS buffer (pH=7.4, containing 1% Tween80). RVG29-CLT-TFs / DGG was prepared according to the method under "2.3", and after mixing with an appropriate amount of artificial pseudo-nasal solution, 2 mL (1.18 mg CLT) was precisely drawn into a dialysis bag with a molecular cutoff of 8000-14000 kDa, and then the tightly tied dialysis bag was placed in 40 mL of dissolution medium, and slowly shaken and incubated in a constant temperature shaker at 37°C. The in vitro drug release behavior of RVG29-CLT-TFs was studied as above. Another 2 mL of free CLT solution with the same concentration as RVG29-CLT-TFs / DGG was taken and operated in the same manner as above as a control. 1 mL was sampled from the release medium at predetermined time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 h), and an equal volume of blank release medium was added at the same time. All the samples were mixed with 3 times the amount of methanol, vortexed and shaken for 5 minutes to completely break the emulsion, filtered through a 0.22 μm filter membrane, and the CLT content was detected by the established HPLC method. The release curve was drawn and the cumulative release rate was calculated.

[0148] Release curve see Fig.10 Compared with the Free CLT and RVG29-CLT-TFs groups, the release time of RVG29-CLT-TFs / DGG was significantly increased, and the cumulative release within 72 h reached 59.44%, while that of RVG29-CLT-TFs and free CLT were 74.79% and 94.55%, respectively.

[0149] Results: The 0.01 mol·L -1 PBS buffer (pH=7.4) was used as the release medium to simulate the in vivo environment, and the dialysis bag method was used to investigate the release behavior of tripterygium wilfordii transfersomes after they passed through the nasal mucosa and entered the body. The experimental results showed that the release time of the transfersome in situ gel was significantly increased, indicating that it achieved a sustained release effect.

[0150] 3 Discussions

[0151] The nasal transfersome gel solution should have a low viscosity and flow better into the nasal cavity during administration. After entering the nasal cavity, the gel formed by complexation with cations in the nasal cavity should have an appropriate viscosity and be effectively adsorbed on the nasal mucosa, thereby prolonging the absorption time of the drug into the brain through the nose. The critical ion concentration is an important parameter of ionic in situ gelation, an important indicator for activating in situ gelation, and the minimum ion concentration that promotes the phase change process. Gel strength is defined as a measure of the ability of polymers to form and maintain gel morphology. Under normal physiological conditions, nasal cilia oscillate at a rate of 1000 times per minute. The gel strength is generally in the range of 25-50s, which can resist the removal of nasal cilia, protect the integrity of the gel, and achieve the continuous release of drugs in specific absorption sites in the nasal cavity. Excellent water retention ability can maintain the stability of the gel during storage or when subjected to external forces. Due to the limited volume of the human nasal cavity, an excessively large gel volume expansion coefficient causes nasal discomfort, and S% is required to be less than 5%. The pH value of nasal mucus is usually between 5.5 and 7.0. The pH of the in situ gel should be close to that of nasal mucus to reduce irritation to the nasal mucosa.

Claims

1. An RVG29 peptide-modified tripterygium wilfordii transfersome in-situ gel for nasal use, characterized in that: The nasal in-situ gel is prepared from RVG29 peptide-modified tripterygium wilfordii transfersomes and deacetylated gellan gum solution; The RVG29 peptide modified tripterygium wilfordii transfersome is prepared from 2.5-4.5 parts of tripterygium wilfordii, 30-70 parts of soybean lecithin, 6-20 parts of cholesterol, 5-15 parts of mPEG2000-DSPE, 10-28 parts of mPEG2000-DSPE-RVG29 and 4-12 parts of sodium deoxycholate; The concentration of the deacetylated gellan gum solution is 0.6-1.4%, and the solvent is distilled water; The volume ratio of the RVG29 peptide-modified tripterygium wilfordii transfersome to the deacetylated gellan gum solution is 0.8-1.2:0.8-1.

2.

2. The RVG29 peptide-modified tripterygium wilfordii transfersome in situ gel for nasal use according to claim 2, characterized in that: The nasal in-situ gel is prepared from RVG29 peptide-modified tripterygium wilfordii transfersomes and deacetylated gellan gum solution; The RVG29 peptide modified tripterygium wilfordii transfersome is prepared from 3-4 parts of tripterygium wilfordii, 40-60 parts of soybean lecithin, 10-15 parts of cholesterol, 8-12 parts of mPEG2000-DSPE, 15-20 parts of mPEG2000-DSPE-RVG29 and 6-10 parts of sodium deoxycholate; The concentration of the deacetylated gellan gum solution is 0.8-1.2%, and the solvent is distilled water; The volume ratio of the RVG29 peptide-modified tripterygium wilfordii transfersome to the deacetylated gellan gum solution is 0.9-1.1:0.9-1.

1.

3. The RVG29 peptide-modified tripterygium wilfordii transfersome in situ gel for nasal use according to claim 1 or 2, characterized in that: The nasal in-situ gel is prepared from RVG29 peptide-modified tripterygium wilfordii transfersomes and deacetylated gellan gum solution; The RVG29 peptide modified tripterygium wilfordii transfersome is prepared from 3.5 parts of tripterygium wilfordii, 50 parts of soybean lecithin, 13 parts of cholesterol, 10 parts of mPEG2000-DSPE, 18 parts of mPEG2000-DSPE-RVG29 and 8 parts of sodium deoxycholate; The concentration of the deacetylated gellan gum solution is 1.0%, and the solvent is distilled water; The volume ratio of the RVG29 peptide-modified tripterygium wilfordii transfersome and the deacetylated gellan gum solution is 1:

1.

4. The method for preparing the RVG29 peptide-modified tripterygium wilfordii transfersome in-situ gel for nasal use according to any one of claims 1 to 3, characterized in that: The preparation method is carried out according to the following specific steps: (1) Preparation of lipid film: 2.5-4.5 mg of celastrol, 30-70 mg of soybean lecithin, 6-20 mg of cholesterol, 5-15 mg of mPEG2000-DSPE, 10-28 mg of mPEG2000-DSPE-RVG29 and 4-12 mg of sodium deoxycholate were weighed in proportion and dissolved in 3-7 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a water bath at 35-39° C. to obtain a uniform lipid film; (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: add 5-15 mL of distilled water to the lipid film, hydrate it at 50-70°C in a constant temperature oscillator at 100 rpm for 20-40 min, and perform ice bath sonication for 2-4 min at a rate of 3-7 s per sonication and 3-7 s pause, with an ultrasonic power of 300 W, to obtain RVG29-CLT-TFs; (3) Preparation of deacetylated gellan gum solution: 0.6-1.4 g of deacetylated gellan gum powder is placed in a beaker, 100 ml of distilled water is added, and the mixture is weighed. The mixture is stirred at a constant temperature of 85-95° C. for 1.5-2.5 h to completely dissolve the mixture. The mixture is cooled to room temperature, and after distillation, the mixture is sealed and placed in a refrigerator at 3-5° C. to fully swell for 22-26 h to obtain a deacetylated gellan gum solution. (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and deacetylated gellan gum solution were mixed at a volume ratio of 0.8-1.2:0.8-1.2, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

5. The preparation method according to claim 4, characterized in that: The preparation method is carried out according to the following specific steps: (1) Preparation of lipid film: 3-4 mg of celastrol, 40-60 mg of soybean lecithin, 10-15 mg of cholesterol, 8-12 mg of mPEG2000-DSPE, 15-20 mg of mPEG2000-DSPE-RVG29 and 6-10 mg of sodium deoxycholate were weighed and dissolved in 4-6 mL of dichloromethane according to the proportion. The dichloromethane was removed by rotary evaporation at 30 rpm in a water bath at 36-38° C. to obtain a uniform lipid film; (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: Take the lipid film and add 8-12 mL of distilled water, hydrate it at 55-65°C constant temperature oscillator at 100 rpm for 25-35 min, perform ice bath sonication for 2.5-3.5 min with a method of sonication for 4-6 s and pause for 4-6 s, and the ultrasonic power is 300 W to obtain RVG29-CLT-TFs; (3) Preparation of deacetylated gellan gum solution: 0.8-1.2 g of deacetylated gellan gum powder is placed in a beaker, 100 ml of distilled water is added, and the mixture is weighed. The mixture is stirred at 90° C. for 2 h to completely dissolve the mixture. The mixture is cooled to room temperature, distilled, sealed, and placed in a refrigerator at 4° C. to fully swell for 24 h to obtain a deacetylated gellan gum solution. (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and deacetylated gellan gum solution were mixed at a volume ratio of 0.9-1.1:0.9-1.1, and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

6. The preparation method according to claim 5, characterized in that: The preparation method is carried out according to the following specific steps: (1) Preparation of lipid film: 3.5 mg of celastrol, 50 mg of soybean lecithin, 13 mg of cholesterol, 10 mg of mPEG2000-DSPE, 18 mg of mPEG2000-DSPE-RVG29 and 8 mg of sodium deoxycholate were weighed in proportion and dissolved in 5 mL of dichloromethane. The dichloromethane was removed by rotary evaporation at 30 rpm in a 37°C water bath to obtain a uniform lipid film; (2) Preparation of RVG29 peptide modified tripterygium wilfordii transfersomes: Take the lipid film and add 10 mL of distilled water, hydrate it at 60°C constant temperature oscillator at 100 rpm for 30 min, and perform ice bath sonication for 3 min with a method of sonication for 5 s and pause for 5 s, with an ultrasonic power of 300 W, to obtain RVG29-CLT-TFs; (3) Preparation of deacetylated gellan gum solution: 1.0 g of deacetylated gellan gum powder was placed in a beaker, 100 ml of distilled water was added, and the mixture was weighed. The mixture was stirred at 90° C. for 2 h to completely dissolve the mixture. The mixture was cooled to room temperature, distilled, sealed, and placed in a refrigerator at 4° C. to fully swell for 24 h to obtain a deacetylated gellan gum solution. (4) Preparation of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use: RVG29-CLT-TFs and deacetylated gellan gum solution were mixed at a volume ratio of 1:1 and stirred evenly at 100 rpm to obtain orange-yellow RVG29-CLT-TFs / DGG, i.e., the finished product of RVG29 peptide-modified celastrol transfersome in situ gel for nasal use.

7. Use of the RVG29 peptide-modified tripterygium wilfordii transfersome nasal in situ gel according to any one of claims 1 to 6 in the preparation of drugs for treating Parkinson's disease.

Images