Nasal administration preparations for brain diseases, and methods for preparing and using the same

Abstract

The application provides a nasal administration preparation for brain diseases and a preparation method and application thereof, and relates to the technical field of pharmaceutical preparations.The nasal administration preparation for brain diseases comprises gelatin derivative chitosan microspheres wrapped with stem cell exosomes, and the microspheres are prepared by using a specific structure of gelatin derivative to coat stem cell exosomes, and then cross-linking with chitosan.The gelatin derivative used in the application has good surface activity, can be well compatible with cells and active factors, and forms a stable inner core coating structure;at the same time, the positively charged surface of the chitosan cross-linked with the outer layer of the gelatin derivative can be combined with anions in the nasal mucus, which can effectively prolong the residence time of the microspheres in the nasal cavity.Therefore, the nasal administration preparation of the application can not only slowly release stem cell exosomes, but also improve the absorption efficiency of the nasal cavity for stem cell exosomes, so that the stem cell exosomes can more effectively reach the brain.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical preparations, in particular to a nasal administration preparation for brain diseases and a preparation method and application thereof. BACKGROUND

[0002] Brain neurodegenerative diseases are a class of diseases caused by the progressive loss of neurons or myelin, characterized by cognitive or physical dysfunction. The disease is divided into acute (such as cerebral ischemia) and chronic (such as Alzheimer's disease and Parkinson's disease). In recent years, in the face of the world's problem of brain neurodegenerative diseases, stem cell therapy has provided a new way.

[0003] Mesenchymal stem cells (MSCs) are the most commonly used cell model in clinical experiments. MSCs mainly release their stem cell exosomes through paracrine to play a role. These exosomes contain a large part of active substances such as growth factors, cytokines, mRNAs and miRNAs. They change the activity of target cells by horizontally transferring these bioactive molecules to achieve the purpose of repairing the damaged site.

[0004] Studies have found that after stem cell exosomes injected intravenously enter the body, most of them are cleared by the immune system, and only a small part reaches the site of brain disease. Therefore, in many disease models, stem cell exosomes can only produce short-term efficacy. The nasal administration method can greatly increase the area of epithelial cell absorption of drugs due to the large number of fine villi on the nasal mucosa. At the same time, there is a rich vascular network under the epithelial cells. The venous blood flowing through the nasal cavity can bypass the blood-brain barrier (BBB) and enter the cerebrospinal fluid, and the drug can quickly enter the brain. However, the normal physiological swing of the cilia in the nasal cavity can quickly clear the drug dropped into the nasal cavity, affecting drug absorption.

[0005] Therefore, it is necessary and urgent to develop a nasal administration preparation that can effectively improve the absorption efficiency of stem cell exosomes in the nasal cavity.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The first object of the present application is to provide a nasal administration preparation for brain diseases. The nasal administration preparation can not only slowly release stem cell exosomes, but also improve the absorption efficiency of stem cell exosomes in the nasal cavity, so that stem cell exosomes can more effectively reach the brain.

[0008] The second object of the present application is to provide a preparation method of a nasal administration preparation for brain diseases.

[0009] The third object of the present application is to provide a use of a nasal administration preparation for brain diseases.

[0010] In order to achieve the above-mentioned objects of the present application, the following technical solutions are adopted:

[0011] The present application provides a nasal administration preparation for brain diseases, which comprises: gelatin derivative chitosan microspheres wrapped with stem cell exosomes.

[0012] The gelatin derivative chitosan microspheres wrapped with stem cell exosomes are mainly prepared by cross-linking gelatin derivative coated stem cell exosomes and chitosan.

[0013] The structural formula of the gelatin derivative is as follows:

[0014] .

[0015] Further, the particle size of the gelatin derivative chitosan microspheres wrapped with stem cell exosomes is 30-80 μm.

[0016] The present application provides a preparation method of the above-mentioned nasal administration preparation for brain diseases, which comprises:

[0017] S1: Dissolve gelatin derivative in a solvent to obtain a gelatin derivative solution, mix the gelatin derivative solution with stem cell exosomes, then incubate to obtain solution A, mix solution A with a chitosan solution to obtain solution B;

[0018] S2: Mix solution B as the water phase with an oil phase containing an emulsifier to form a water-in-oil solution; further, the solvent of the gelatin derivative solution is water;

[0019] And / or, the gelatin derivative solution is a gelatin derivative aqueous solution with a concentration of 1-8%;

[0020] And / or, the incubation temperature in step S1 is 25-30℃, and the time is 10-20 min;

[0021] And / or, 1 mL of the gelatin derivative aqueous solution in solution A corresponds to 1x10 8 ~1x10 9 stem cell exosomes after incubation;

[0022] And / or, the stem cell exosomes are mesenchymal stem cell exosomes.

[0023] Further, the chitosan solution is mainly prepared by dissolving chitosan in a 3% acetic acid solution. Preferably, the concentration of the chitosan solution is 1-5%.

[0024] Furthermore, the volume ratio of solution A to chitosan solution is 1:1.

[0025] Furthermore, the preparation method includes:

[0026] (A) Mix the gelatin derivative solution with stem cell exosomes, and then incubate at 25℃~30℃ for 10~20 min to obtain solution A;

[0027] The concentration of gelatin derivative in the gelatin derivative solution is 1-8%;

[0028] The gelatin derivative solution in 1 mL of solution A after incubation corresponds to 1×10 8 ~1×10 9 One stem cell exosome;

[0029] (B) Mix solution A with chitosan solution to obtain solution B;

[0030] (C) Mix 20 mL of liquid paraffin with 0.6 mL of Span-80, and then add solution B dropwise at 35~45℃ and 300~500 r / min to prepare a stable water-in-oil emulsion. After ice bath, cross-linking and curing are carried out sequentially, followed by vacuum drying to obtain gelatin derivative chitosan microspheres.

[0031] Furthermore, the crosslinking and curing method in step (C) is as follows: 0.15~1mL of glutaraldehyde (50%) is added to the water-in-oil emulsion after ice bath for crosslinking and curing for 30~90min, followed by centrifugation at 3000~5000 r / min for 10~20min.

[0032] The present invention provides the application of the above-mentioned nasal administration formulation for brain diseases in the preparation of drugs for neurodegenerative diseases.

[0033] Furthermore, the neurodegenerative diseases include at least one of Alzheimer's disease, Parkinson's syndrome, and leukoencephalopathy.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] This invention provides a nasal delivery formulation for brain diseases, comprising: gelatin derivative chitosan microspheres encapsulating stem cell exosomes. These microspheres are prepared by first encapsulating the stem cell exosomes with a gelatin derivative of a specific structure, followed by cross-linking with chitosan. Because the gelatin derivative of this application incorporates aldehyde groups and miconazole, it possesses excellent surface activity, exhibiting good compatibility with cells and active factors to form a stable core-encapsulation structure. The outer layer of the chitosan microspheres, after cross-linking with chitosan, carries a positive charge, allowing it to bind with anions in nasal mucus. This not only improves the permeability of epithelial cells and promotes drug absorption but also inhibits mucociliary clearance, thereby prolonging the microspheres' residence time in the nasal cavity. Therefore, this nasal delivery formulation not only slowly releases stem cell exosomes but also improves the absorption efficiency of stem cell exosomes in the nasal cavity, enabling them to reach the brain more effectively, laying the foundation for the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's syndrome.

[0036] The present invention provides a method for preparing a nasal drug delivery formulation for brain diseases, comprising the following steps: S1, mixing a solution of the above-mentioned gelatin derivative with a stem cell exosome solution to obtain solution A, and then mixing solution A with a chitosan solution to obtain solution B; S2, mixing solution B as an aqueous phase with an oil phase containing an emulsifier to form an oil-in-water solution; adding a crosslinking agent for crosslinking and curing to obtain gelatin derivative chitosan microspheres encapsulating stem cell exosomes. The above-mentioned preparation method of this application has the technical advantages of simple processing and ease of operation.

[0037] The nasal delivery formulation for brain diseases provided by this invention can be widely used in the preparation of drugs for neurodegenerative diseases. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 The exosome release rate over time of chitosan microspheres with different concentrations of gelatin derivatives provided in Experimental Example 1 of this invention;

[0040] Figure 2 The retention time of chitosan microspheres of different concentrations of gelatin derivative provided in Experimental Example 1 of the present invention in the nasal cavity of mice;

[0041] Figure 3This is a comparison diagram of the fluorescence intensity in the brain of the gelatin derivative chitosan microspheres provided in Example 2 of Experiment 1 of the present invention and the control group. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] According to one aspect of the present invention, a nasal delivery formulation for brain diseases, the nasal delivery formulation comprising: gelatin derivative chitosan microspheres encapsulating stem cell exosomes;

[0044] The gelatin derivative chitosan microspheres containing stem cell exosomes are mainly prepared by coating stem cell exosomes with gelatin derivatives and then cross-linking them with chitosan.

[0045] The structural formula of the gelatin derivative is as follows:

[0046] .

[0047] It should be noted that the gelatin derivative used in the nasal delivery formulation for brain diseases in this application is the gelatin derivative disclosed in the inventor's previous application (CN118161622B). This gelatin derivative has good surface activity due to the introduction of aldehyde groups and miconazole into the gelatin molecule, and can be well compatible with cells and active factors. Compared with other existing gelatin hydrogels / gelatin derivatives, the gelatin derivative hydrogel used in this application is positively charged and has a good three-dimensional network structure, while stem cell exosomes are negatively charged. Therefore, it can effectively encapsulate stem cell exosomes. By combining with chitosan, the surface of the final gelatin derivative chitosan microspheres has a positive charge, while the nasal mucosa is negatively charged. This can increase the retention time of the gelatin derivative chitosan microspheres in the nasal cavity, improve the absorption efficiency of stem cell exosomes in the nasal cavity, and enable stem cell exosomes to reach the brain more effectively.

[0048] This invention provides a nasal delivery formulation for brain diseases, comprising: gelatin derivative chitosan microspheres encapsulating stem cell exosomes. These microspheres are prepared by first encapsulating the stem cell exosomes with a gelatin derivative of a specific structure, followed by cross-linking with chitosan. Because the gelatin derivative of this application incorporates aldehyde groups and miconazole, it possesses excellent surface activity, exhibiting good compatibility with cells and active factors to form a stable core-encapsulation structure. The outer layer of the chitosan microspheres, after cross-linking with chitosan, carries a positive charge, allowing it to bind with anions in nasal mucus. This not only improves the permeability of epithelial cells and promotes drug absorption but also inhibits mucociliary clearance, thereby prolonging the microspheres' residence time in the nasal cavity. Therefore, this nasal delivery formulation not only slowly releases stem cell exosomes but also improves the absorption efficiency of stem cell exosomes in the nasal cavity, enabling them to reach the brain more effectively, laying the foundation for the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's syndrome.

[0049] In a preferred embodiment of the present invention, the stem cell exosomes include one or more of embryonic stem cell exosomes, mesenchymal stem cell exosomes, and induced pluripotent stem cell exosomes, preferably mesenchymal stem cell exosomes.

[0050] In a preferred embodiment of the present invention, the particle size of the gelatin derivative chitosan microspheres encapsulating stem cell exosomes is 30~80μm.

[0051] According to one aspect of the present invention, a method for preparing the above-described nasal administration formulation for brain diseases, the method comprising:

[0052] S1: Dissolve the gelatin derivative in a solvent to obtain a gelatin derivative solution. Mix the gelatin derivative solution with stem cell exosomes and then incubate to obtain solution A. Mix solution A with chitosan solution to obtain solution B.

[0053] S2: Mix solution B as the aqueous phase with the oil phase containing emulsifier to form an oil-in-water solution;

[0054] The present invention provides a method for preparing a nasal drug delivery formulation for brain diseases, comprising the following steps: S1, mixing a solution of the above-mentioned gelatin derivative with a stem cell exosome solution to obtain solution A, and then mixing solution A with a chitosan solution to obtain solution B; S2, mixing solution B as an aqueous phase with an oil phase containing an emulsifier to form an oil-in-water solution; adding a crosslinking agent for crosslinking and curing to obtain gelatin derivative chitosan microspheres encapsulating stem cell exosomes. The above-mentioned preparation method of this application has the technical advantages of simple processing and ease of operation.

[0055] In a preferred embodiment of the present invention, the solvent of the gelatin derivative solution is water;

[0056] And / or, the gelatin derivative solution is an aqueous solution of gelatin derivative with a concentration of 1-8%;

[0057] And / or, the incubation temperature in step S1 is 25℃~30℃, and the incubation time is 10~20min;

[0058] And / or, after incubation, 1 mL of gelatin derivative aqueous solution in solution A encapsulates 1×10 8 ~1×10 9 One stem cell exosome;

[0059] And / or, the stem cell exosomes are mesenchymal stem cell exosomes.

[0060] In a preferred embodiment of the present invention, the chitosan solution is mainly prepared by dissolving chitosan in a 3% acetic acid solution.

[0061] In a preferred embodiment of the present invention, the mixing volume ratio of solution A to chitosan solution is 1:1.

[0062] In a preferred embodiment of the present invention, the preparation method includes:

[0063] (A) Mix the gelatin derivative solution with stem cell exosomes, and then incubate at 25℃~30℃ for 10~20 min to obtain solution A;

[0064] The concentration of gelatin derivative in the gelatin derivative solution is 1-8%;

[0065] The gelatin derivative solution in 1 mL of solution A after incubation corresponds to 1×10 8 ~1×10 9 One stem cell exosome;

[0066] (B) Mix solution A with chitosan solution to obtain solution B;

[0067] (C) Mix 20 mL of liquid paraffin with 0.6 mL of Span-80, and then add solution B dropwise at 35~45℃ and 300~500 r / min to prepare a stable water-in-oil emulsion. After ice bath, cross-linking and curing are carried out sequentially, followed by vacuum drying to obtain gelatin derivative chitosan microspheres.

[0068] In a preferred embodiment of the present invention, the crosslinking and curing method in step (C) is as follows: 0.15~1mL of glutaraldehyde (50%) is added to the water-in-oil emulsion after ice bath for crosslinking and curing for 30~90min, followed by centrifugation at 3000~5000 r / min for 10~20min.

[0069] According to one aspect of the present invention, the use of the above-described nasal administration formulation for brain diseases in the preparation of a drug for neurodegenerative diseases.

[0070] The nasal delivery formulation for brain diseases provided by this invention can be widely used in the preparation of drugs for neurodegenerative diseases.

[0071] Preferably, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's syndrome, and leukoencephalopathy.

[0072] The technical solution of the present invention will be further described below with reference to the embodiments.

[0073] Example 1

[0074] A gelatin derivative, the preparation method comprising:

[0075] (1) Disperse gelatin in deionized water, stir and dissolve at 60°C, cool to 35°C, add potassium periodate, react in the dark for 6 hours, add ethylene glycol, continue stirring for 0.5 hours, dialyze with a dialysis bag with a molecular weight cutoff of 8000-14000D for 72 hours, change the deionized water every 12 hours, collect the solution in the dialysis bag and freeze dry to obtain aldehyde-modified gelatin, wherein the mass ratio of gelatin, deionized water, potassium periodate and ethylene glycol is 1:20:1.2:0.9;

[0076] (2) Miconazole was dissolved in N,N-dimethylformamide to obtain solution 1, and trans-3-chloroacrylic acid was dissolved in deionized water to obtain solution 2. Under a nitrogen atmosphere, solution 2 was added dropwise to solution 1 and stirred. The addition was completed in 1 hour. The temperature was raised to 30°C and stirring was continued for 8 hours to obtain a reaction mixture. Deionized water was added to the reaction mixture so that the volume ratio of N,N-dimethylformamide to deionized water in the reaction mixture was 1:10. N,N-dimethylformamide was removed by rotary evaporation, and then extracted with ethyl acetate. The aqueous layer was concentrated and dried to obtain the miconazole derivative. The mass concentration of miconazole in solution 1 was 0.20 g / mL, the mass concentration of trans-3-chloroacrylic acid in solution 2 was 0.15 g / mL, and the molar ratio of miconazole to trans-3-chloroacrylic acid was 1:0.8.

[0077] (3) Disperse the aldehyde-modified gelatin obtained in step S1 in deionized water, heat to 60°C, stir for 30 min, then add sodium hydroxide, miconazole derivative obtained in step S2 and benzyltriethylammonium chloride in sequence, continue stirring for 4 h, adjust the pH to 6-7 with hydrochloric acid, dialyze for 72 h with a dialysis bag with a molecular weight cutoff of 8000-14000D, change the deionized water every 12 hours, collect the solution in the dialysis bag and freeze dry to obtain the gelatin derivative, wherein the mass ratio of aldehyde-modified gelatin, sodium hydroxide, miconazole derivative and benzyltriethylammonium chloride is 25:1:8:0.9, and the amount of aldehyde-modified gelatin added to the deionized water is 0.15 g / mL.

[0078] The structural formula of the prepared gelatin derivative is as follows:

[0079]

[0080] Example 2

[0081] A method for preparing a nasal delivery formulation for brain diseases, the method comprising:

[0082] (a1) Dissolve the gelatin derivative prepared in Example 1 in water to prepare a gelatin derivative solution with a concentration of 4%, add centrifuged human umbilical cord blood mesenchymal stem cell exosomes and mix, then incubate at 25°C for 15 min to obtain solution A;

[0083] The mixing is performed at a ratio of 1×10 per ml of gelatin derivative solution. 8 One stem cell exosome was used;

[0084] (a2) Chitosan (95% deacetylation degree, food-grade chitosan) was dissolved in a 3% acetic acid solution to prepare a 1% chitosan solution. The chitosan solution was mixed with solution A at a mass ratio of 1:1 to obtain a mixture of gelatin derivative and chitosan.

[0085] (a3) Mix 20 mL of liquid paraffin with 0.6 mL of Span-80. Add the mixture of gelatin derivative and chitosan prepared in step (a2) dropwise at 40 °C and 400 r / min to prepare a stable W / O emulsion.

[0086] The W / O emulsion was placed in an ice bath, and 0.15 mL of glutaraldehyde (50%) was added. The mixture was cross-linked and cured for 1 h, followed by centrifugation at 3000 r / min for 10 min and vacuum drying to obtain gelatin derivative chitosan microspheres.

[0087] Examples 3-6

[0088] Except for the concentration of the chitosan solution in step (a2) of Examples 3-6 of this application, which is different from that in Example 1, the rest are the same as in Example 2, as shown in Table 1.

[0089] Table 1:

[0090]

[0091] That is: (a2) Chitosan is dissolved in a 3% acetic acid solution to prepare 3%, 5%, 7%, and 10% chitosan solutions respectively. The chitosan solutions are mixed with solution A at a mass ratio of 1:1 to obtain a mixture of gelatin derivative and chitosan.

[0092] Comparative Example 1

[0093] This comparative example is the same as Example 2 except that the gelatin derivative prepared in Example 1 in step (a1) is replaced with existing gelatin (Aladdin G108396).

[0094] Comparative Example 2

[0095] A method for preparing a nasal delivery formulation for brain diseases, the method comprising:

[0096] The gelatin derivative prepared in Example 1 was dissolved in water to prepare a 4% gelatin derivative solution. Human umbilical cord blood mesenchymal stem cell exosomes collected by centrifugation were added and mixed. The mixture was then incubated at 25°C for 15 min to obtain solution A, which contains a gelatin derivative encapsulating stem cell exosome structure.

[0097] The mixing is performed at a ratio of 1×10 per ml of gelatin derivative solution. 8 One stem cell exosome was used;

[0098] The difference between this comparative example and Example 2 is that the gelatin derivative of this application did not undergo chitosan cross-linking of the outer layer after coating stem cell exosomes.

[0099] Comparative Example 3

[0100] A method for preparing a nasal delivery formulation for brain diseases, the method comprising:

[0101] (1) The gelatin derivative and carboxymethyl chitosan prepared in Example 1 were dispersed in deionized water, and after high-speed homogenization and vacuum filtration sterilization, human umbilical cord blood mesenchymal stem cell exosomes collected by centrifugation were added and mixed, and the mixture was blown evenly to obtain a pre-prepared solution.

[0102] The amounts of gelatin derivatives and carboxymethyl chitosan added to the pre-prepared solution were 0.035 g / mL and 10.035 g / mL respectively, and the high-speed homogenization process was performed at 1000 r / min for 10 s; the amount of stem cell exosomes added per ml of pre-prepared solution was 1 × 10⁻⁶. 8 indivual;

[0103] (2) The pre-prepared solution was dripped into sterile mineral oil through a syringe, heated to 37°C, and kept warm for 12 hours to obtain hydrogel microspheres loaded with stem cell exosomes. The microspheres were collected by filtration and washed with sterile phosphate buffer to obtain gelatin derivative chitosan microspheres.

[0104] The difference between this comparative example and Examples 2-6 is that this comparative example uses gelatin derivatives and carboxymethyl chitosan to directly mix stem cell exosomes, and then prepares gelatin derivative chitosan microspheres.

[0105] Experimental Example 1

[0106] This experimental example demonstrates the effectiveness of the gelatin derivative chitosan microspheres prepared in Examples 2-6 and Comparative Examples 1-3 as follows:

[0107] (I) Experiment on the exosome release from gelatin-derived chitosan microspheres:

[0108] Different concentrations of gelatin-derived chitosan microspheres were resuspended in 1 mL of PBS buffer and released in a shaker at 37°C and 100 rpm. At fixed time points, 10 μL of the release solution was collected, and 10 μL of the corresponding PBS buffer (pH 7.2-7.4) was added simultaneously. The protein concentration of the collected release solution was measured using Nanodrop One to calculate the exosome release amount, using the following formula:

[0109] Cumulative exosome release rate % = (V0C n + Ve∑n-1) / md × 100%;

[0110] In the formula: V0 is the total volume of the release medium, Cn is the drug concentration of the nth sample, Ve is the volume of each sample, and md is the drug content. Specific results are shown in Table 2.

[0111] Table 2. Release rate of exosomes from stem cells using gelatin-derived chitosan microspheres (%):

[0112]

[0113] Figure 1 The exosome release rate over time of chitosan microspheres with different concentrations of gelatin derivatives.

[0114] Depend on Figure 1It was found that, by simulating the nasal cavity environment (nasal mucus pH around 7.4, at 37℃ and pH around 7.4), the higher the concentration of gelatin derivative chitosan microspheres in the first 30 minutes, the lower the exosome release rate. As time progressed, the release rate of 1%~5% (Examples 2~4) gelatin derivative chitosan microspheres reached over 90%, while the release rate of 10% (Example 6) gelatin derivative chitosan microspheres only reached 80%. Although the exosome release amount over time in Comparative Examples 1~3 was similar to the effect of this application, the effect of the retention time in the nasal cavity in the later stage was poor.

[0115] (ii) Residence time of gelatin derivative chitosan microspheres in mouse nasal cavity.

[0116] 1. Place 10 8 Add 4 mL PBS, 1 mL SnCl2 (5 mg / mL), 1 mL NaAc sodium acetate (100 mg / mL), and an appropriate amount of pertechnetate to the stem cell exosomes. Wash three times with PBS to remove unlabeled pertechnetate. Centrifuge at 10000g to collect the labeled stem cell exosomes. Then prepare gelatin derivative chitosan microspheres according to the methods of Examples 2-6 and Comparative Examples 1-3. Before the operation, detect the radioactivity signal of stem cell exosomes using the Sophy DSX-NXT Spect system with an initial value of I0.

[0117] 2. Twenty-seven healthy mice were selected and randomly divided into nine groups of three mice each.

[0118] After anesthetizing each group of mice, the gelatin derivative chitosan microspheres prepared according to the methods of Examples 2-6 and Comparative Examples 1-3 were diluted to 1 mL with PBS buffer and dripped into the nasal cavity of the anesthetized mice. The mice were placed in a lateral recumbent position, and the values ​​at different time points were detected using the Sophy DSX-NXTSpect system. t The percentage of stay at different times is I t / I0×100% determination of the change in radioactivity in nasal mucus over time.

[0119] Simultaneously, a blank control group was set up. The blank control group consisted of stem cell exosomes labeled in step 1 dissolved in 1 mL of PBS buffer and then dripped into the nasal cavity of mice. Due to the negative charge of the stem cell exosomes and the negative charge of the nasal cavity, as well as the protective and clearing effect of the nasal cilia, the stem cell exosomes in the blank control group had a short residence time in the nasal cavity, as detailed in Table 3.

[0120] Table 3. Retention rate of gelatin derivative chitosan microspheres in mouse nasal cavity over time (%):

[0121]

[0122] Figure 2The retention time of chitosan microspheres with different concentrations of gelatin derivatives in the nasal cavity of mice.

[0123] As shown in Table 2, compared with the embodiments of this application, the number of stem cell exosomes retained in the nasal cavity within 30 minutes in comparative examples 1-3 was significantly lower. Specifically:

[0124] Comparative Example 1 used existing gelatin to encapsulate stem cell exosomes, but due to the lack of a good three-dimensional structure, the encapsulation efficiency was low. Comparative Example 2 did not use chitosan cross-linking, so the entire microsphere was negatively charged and therefore excluded by the nasal cavity's protective mechanism. In Comparative Example 3, because the gelatin derivative (positively charged) and chitosan (positively charged) cross-linked first, the encapsulation efficiency of stem cell exosomes was very low. Therefore, the retention time of stem cell exosomes in the nasal cavity for 30 minutes in the above comparative examples was relatively low. This further demonstrates that our company's gelatin derivative has a good three-dimensional structure and can more efficiently encapsulate stem cell exosomes.

[0125] Depend on Figure 2 It was found that the blank solution was lost by more than 70% after 30 minutes in the nasal cavity of mice. However, the gelatin derivative chitosan microspheres in Examples 2-6 had a longer residence time in the nasal cavity as the concentration increased. Among them, the 5% and 10% gelatin derivative chitosan microspheres could still retain about 80% after 100 minutes. This further illustrates that the 5% and 10% gelatin derivative chitosan microspheres are suitable for encapsulating stem cell exosomes for nasal administration.

[0126] (III) The residence time of gelatin derivative chitosan microspheres in the brain.

[0127] Take 10 mL of PKH26 staining solution (10 μM) and mix with 1×10 9 One stem cell exosome was incubated at 25°C for 1 hour, then centrifuged at 100,000×g for 60 min to remove excess dye. The stem cell exosomes with no precipitate were labeled with PKH26 and divided into two portions.

[0128] One sample of the above-mentioned "PKH26-labeled stem cell exosomes" was prepared into gelatin derivative chitosan microspheres using the preparation method of Example 2, and dissolved in 1 mL of PBS solution;

[0129] Another control group was prepared by directly dissolving the above-mentioned "PKH26-labeled stem cell exosomes" in 1 mL of PBS solution. The two solutions were then dripped into the nasal cavities of two groups of mice (6 mice, divided into two groups of 3 mice each). The fluorescence values ​​of the mouse brain at 1, 12, 24, and 48 h were detected using a mouse in vivo imaging system (PerkinElmer IVIS Lumina LT Series Ⅲ). The fluorescence values ​​were then exported using the ROI tool.

[0130] Figure 3 This is a comparison diagram of the fluorescence intensity in the brain of gelatin derivative chitosan microspheres and the control group in Example 2 of the present invention.

[0131] Depend on Figure 3 It is known that stem cell exosomes encapsulated in gelatin derivative chitosan microspheres can be continuously released into the brain via nasal administration, and the residence time of exosomes in the brain can be greatly extended, thereby achieving the therapeutic effect of stem cell exosomes on brain diseases.

[0132] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nasal delivery formulation for brain diseases, characterized in that, The nasal delivery formulation comprises: gelatin derivative chitosan microspheres encapsulating stem cell exosomes; The preparation of the gelatin derivative chitosan microspheres encapsulating stem cell exosomes includes: coating stem cell exosomes with gelatin derivatives and then cross-linking them with chitosan. The structural formula of the gelatin derivative is as follows: ； The chitosan solution prepared by cross-linking is obtained by dissolving chitosan in a 3% acetic acid solution, and the concentration of the chitosan solution is 1-5%.

2. The nasal delivery formulation for brain diseases according to claim 1, characterized in that, The gelatin derivative chitosan microspheres containing stem cell exosomes have a particle size of 30~80μm.

3. A method for preparing a nasal delivery formulation for brain diseases according to claim 1 or 2, characterized in that, The preparation method includes: S1: Dissolve the gelatin derivative described in claim 1 in a solvent to obtain a gelatin derivative solution, mix the gelatin derivative solution with stem cell exosomes, and then incubate to obtain solution A, and mix solution A with chitosan solution to obtain solution B; S2: Mix solution B as the aqueous phase with the oil phase containing emulsifier to form an oil-in-water solution; add a crosslinking agent to crosslink and cure, and obtain gelatin derivative chitosan microspheres encapsulating stem cell exosomes.

4. The method for preparing the nasal delivery formulation for brain diseases according to claim 3, characterized in that, The solvent for the gelatin derivative solution is water; And / or, the gelatin derivative solution is an aqueous solution of gelatin derivative with a concentration of 1-8%; And / or, the incubation temperature in step S1 is 25℃~30℃, and the incubation time is 10~20min; And / or, after incubation, 1 mL of gelatin derivative aqueous solution in solution A encapsulates 1×10 8 ~1×10 9 One stem cell exosome; And / or, the stem cell exosomes are mesenchymal stem cell exosomes.

5. The method for preparing a nasal delivery formulation for brain diseases according to claim 3, characterized in that, The volume ratio of solution A to chitosan solution is 1:

1.

6. The method for preparing a nasal delivery formulation for brain diseases according to claim 3, characterized in that, The preparation method includes: (A) Mix the gelatin derivative solution with stem cell exosomes, and then incubate at 25℃~30℃ for 10~20 min to obtain solution A; The concentration of gelatin derivative in the gelatin derivative solution is 1-8%; The gelatin derivative solution in 1 mL of solution A after incubation corresponds to 1×10 8 ~1×10 9 One stem cell exosome; (B) Mix solution A with chitosan solution to obtain solution B; (C) Mix 20 mL of liquid paraffin with 0.6 mL of Span-80, and then add solution B dropwise at 35~45℃ and 300~500 r / min to prepare a stable water-in-oil emulsion. After ice bath, cross-linking and curing are carried out sequentially, followed by vacuum drying to obtain gelatin derivative chitosan microspheres.

7. The method for preparing a nasal delivery formulation for brain diseases according to claim 6, characterized in that, The cross-linking and curing method in step (C) is as follows: Add 0.15-1 mL of glutaraldehyde to the water-in-oil emulsion after ice bath for cross-linking and curing for 30-90 min, followed by centrifugation at 3000-5000 r / min for 10-20 min.

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