CN113143968A - Nerve stem cell exosome nasal spray and preparation method thereof - Google Patents
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Abstract
The present application relates to exosomesThe field of preparation preparations, in particular to a nasal spray of a neural stem cell exosome and a preparation method thereof, wherein the nasal spray of the neural stem cell exosome comprises a buffer solution and the following components suspended in the buffer solution: (1-100). times.108one/mL of neural stem cell exosomes; an osmotic pressure regulator accounting for 1.6-10% of the mass fraction of the buffer solution; the osmotic pressure regulator is one of mannitol, sucrose and sorbitol, or a compound system of any number of mannitol, sucrose and sorbitol. By compounding the neural stem cell exosome and the osmotic pressure regulator, the compound preparation has a good storage effect and good absorption performance.
Description
Technical Field
The application relates to the field of exosome preparations, in particular to a nasal spray of a neural stem cell exosome and a preparation method thereof.
Background
Exosomes are novel therapeutic drug carriers that carry active substances in cells and have wide application in the treatment of disease. In the prior art, there are publications on the use of neural stem cells for the treatment of senile dementia or cerebral thrombosis sequelae.
During the treatment process, the neural stem cell exosome mainly acts on the brain. In the current literature, the method mainly adopted is to directly inject exosome suspension at the striatum part of the brain, and the whole method is complicated and has high risk.
Disclosure of Invention
In order to improve the convenience of the administration of the neural exosomes, the application provides a neural stem cell exosome nasal spray and a preparation method thereof.
Firstly, the neural stem cell exosome nasal spray provided by the application adopts the following technical scheme: the nasal spray for the exosomes of the neural stem cells comprises a buffer solution and the following components suspended in the buffer solution: (1-100). times.108one/mL of neural stem cell exosomes;
an osmotic pressure regulator accounting for 1.6-10% of the mass fraction of the buffer solution;
the osmotic pressure regulator is one of mannitol, sucrose and sorbitol, or a compound system of any number of mannitol, sucrose and sorbitol.
In the present application, the exosomes are administered in the form of a nasal spray. The nasal cavity spray is close to the brain, and in the treatment of cerebral thrombosis sequelae or senile dementia, the main action area of the medicine is in the brain, so that the mode of nasal cavity administration is adopted, and the nasal cavity spray has better administration effect besides convenience. Meanwhile, the treatment effect of the medicine for the exosome on the nervous system is a long-term process, so that the medicine is taken by a nasal spray method, and the patient does not need to often take medicine in medical institutions, so that the medicine is convenient.
The osmotic pressure regulator is one of mannitol, sucrose and sorbitol, has the effects of protecting exosome and improving exosome dispersibility besides regulating osmotic pressure and improving the absorption capacity of a nasal cavity, and is more beneficial to improving the stability of exosome compared with an inorganic salt osmotic pressure regulator by selecting one of mannitol, sucrose and sorbitol.
Optionally, the buffer solution further comprises glycerol accounting for 10-20% of the mass fraction of the buffer solution.
The main purpose of adding glycerol is to protect exosomes and improve the storage performance of exosomes. The essence of the exosome is a vesicle containing a certain amount of protein, the surface of the vesicle contains a phospholipid molecular layer, and the exosome aggregation can be slowed down and the stability and the dispersibility of the exosome can be improved through glycerol. On the other hand, the glycerol is doped, so that the moisturizing performance of the spray is improved, the spray has a certain degree of viscosity performance, exosomes are prevented from being rapidly lost, the exosomes can be well adhered to the nasal cavity, and the absorption performance is improved. In addition, glycerol also has some degree of anti-freeze effect.
Optionally, the buffer solution further comprises sheep placenta polypeptides accounting for 0.05-0.1% of the buffer solution by mass.
The sheep placenta polypeptide has the effect of protecting exosome, and can neutralize impurities such as protease introduced in the preparation process as a sacrificial agent, so that exosome is not easy to deteriorate in the long-term storage process, and the storage time of nasal spray is prolonged.
Optionally, the buffer solution further comprises sodium citrate which accounts for 0.1-0.8% of the mass fraction of the buffer solution.
The sodium citrate has good reducibility, has the effect of an antioxidant, and is beneficial to further improving the preservation time and the preservation effect of the exosome. Meanwhile, citric acid also has the effect of a complexing agent, and can complex part of metal ions (such as calcium ions) mixed in a system and part of metal ions remained in a nasal cavity, so that structural damage of the exosome caused by the exosome is reduced, and the storage performance of the exosome is further improved. Meanwhile, citric acid can stimulate mucous membrane cells in nasal cavity to fully absorb exosomes, and the medication effect is improved.
Optionally, the pH value of the buffer solution is 6.5-7.8.
The buffer solution is a spray agent within the range of 6.5-7.8, and has good preservation time and absorption effect.
Optionally, the concentration of the buffer solution is 1-100 mM, and the buffer solution is phosphoric acid buffer solution.
The phosphoric acid buffer solution has proper buffer range, easy preparation and small influence on human body. And the adoption of 1-100 mM phosphoric acid buffer solution is beneficial to improving the absorption effect of exosome while the stimulation to nasal cavity is small.
Optionally, the buffer solution further comprises chitosan oligosaccharide accounting for 0.6-1.5% of the mass fraction of the buffer solution.
The chitosan oligosaccharide can be combined with a part of glycoprotein structures on a phosphate molecule layer on the surface of exosomes, so that mutual collision among exosomes is further reduced, and the possibility of agglomeration of the exosomes is reduced. In addition, chitosan oligosaccharides also have a positive effect on the absorption properties of exosomes in the nasal cavity.
Optionally, the nasal spray is stored in an environment below 4 ℃.
The nasal cavity spray for the exosome of the neural stem cells prepared in the application has a good storage effect at a temperature below 4 ℃, is good in stability, can still have good dispersibility even after being frozen, is convenient to store at home, and is better in convenience.
In a second aspect, the application provides a preparation method of a nasal spray of a neural stem cell exosome, which adopts the following technical scheme:
a preparation method of a nasal cavity spray of a neural stem cell exosome comprises the following steps:
s1, culturing, amplifying and separating the neural stem cells to obtain exosomes;
s2, adding a buffer solution added with an osmotic pressure regulator in advance into the exosome according to the calculated amount under the condition that the temperature is not higher than 4 ℃, and uniformly blowing.
In the technical scheme, the configuration is carried out in the environment of not more than 4 ℃, which is helpful for protecting exosomes and simultaneously improves the uniformity of dispersion. When the temperature is too high, for example, when the nasal spray is prepared at a temperature close to room temperature, some exosomes are lost, and the content of exosomes in the finally prepared nasal spray is lower than the calculated value.
Optionally, in step S2, glycerol is also added into the buffer solution in advance, and after the mixture is blown and beaten uniformly, the sheep placenta polypeptide and chitosan oligosaccharide are continuously added into the system and stored in an environment below 4 ℃.
Glycerol is added into the buffer solution in advance, the glycerol can slightly improve the viscosity of the buffer solution, and can protect exosomes in the blowing and beating process, so that the loss of the exosomes due to overlarge mechanical stress is reduced. Meanwhile, the freezing point can be lowered, the possibility of icing the buffer solution is reduced, and the configuration process is smoother.
In summary, the present application includes at least one of the following advantages:
1. in the application, the convenience of the medicine for the exosome can be improved by preparing the neural stem cell exosome into a nasal spray.
2. In a further embodiment of the present application, the storage capacity of the absorption capacity of the exosomes is improved by the addition of glycerol.
3. In the further setting of the application, the exosome is not easy to deteriorate in the storage process by adding the sheep placenta polypeptide and the sodium citrate, and the storage performance of the nasal spray is further improved.
4. In the further setting of the application, the chitosan oligosaccharide is used for reducing the agglomeration performance of the exosome, improving the dispersibility of the exosome and ensuring that the nasal spray has better stability in the long-term storage process.
Detailed Description
The present application will be described in further detail with reference to examples.
Preparation example: the neural stem cell exosomes were prepared as follows.
(1) Selecting SD rats of 4 weeks old, extracting thighbone by a marrow cavity flushing method, separating to obtain mesenchymal stem cells, carrying out adherent culture in a culture bottle, and replacing the culture medium every 2 days; cells without iron walls were washed away. And when the adhering wall area in the culture bottle is more than 80%, performing suspension culture by using an ultra-low adhesion culture bottle, and continuing to culture for three days.
(2) Separating the suspended cell bodies, and continuously culturing for two days by using a serum-free DMEM medium;
(3) collecting the culture medium supernatant in the above inoculation culture system, filtering with 0.22 μm filter membrane, centrifuging at 4 deg.C under 300 × g for 10min, and retaining the supernatant;
(4) centrifuging the supernatant obtained in the step (3) for 100min at 2000 Xg, and reserving the supernatant;
(5) centrifuging the supernatant obtained in the step (4) for 30min at 10000 Xg, and reserving the supernatant;
(6) and (4) centrifuging the supernatant obtained in the step (5) for 70min at 100000 Xg, and reserving the precipitate to obtain the exosome. The exosomes are prepared and then prepared as soon as possible.
For the following examples and comparative examples, the following experiments were set up to verify the effectiveness of the technical solution in the present application.
Experiment 1 and stability experiment, the prepared nasal spray is placed under specific stability, and the form of the nasal spray is observed after 10 days, 30 days and 90 days.
Experiment 2, absorption performance experiment, selecting 45d rat, anesthetizing it with ether, nasal spray treating it with nasal spray according to examples and comparative examples below, spraying 1mL, then carefully placing rat to prevent spray from flowing out, after 30min, washing nasal cavity with normal saline, collecting wash solution, filtering out cells and cell debris through 0.22 μm filter membrane, centrifuging 100min at 2000 × g, retaining supernatant and centrifuging 30min at 10000 × g, retaining supernatant and centrifuging 70min at 1000000 × g, resuspending precipitate with 10mM PBS buffer solution with pH value of 7.4, calculating exosome concentration in collected wash solution by NanoSight LM10 particle tracking analysis technique, and reversely deducing exosome absorption rate.
Embodiment 1, a nasal spray of a neural stem cell exosome is prepared by the following steps:
s1, preparing an exosome according to the method of the preparation example 1;
s2, adding 10mM phosphate buffer solution with pH value of 7.4 into the exosome suspension according to calculated amount to prepare 109The cells/mL were aspirated to homogeneity and stored at 4 ℃. In the above process, the exosome concentration can be calculated by NanoSight LM10 particle tracking analysis technique.
In step S2, an osmotic pressure regulator, in this case mannitol, is added to the phosphate buffer solution in advance in an amount of 6% by mass of the phosphate buffer solution.
Examples 2 to 27 and comparative examples 1 to 3, a nasal spray of a neural stem cell exosome, which is different from example 1 in that the types and the amounts of different osmotic pressure regulators and the pH values of buffer solutions are adjusted, as shown in table 1.
Table 1, base recipe adjustment tables in examples 1 to 27 and comparative examples 1 to 3
The stability of examples 1 to 27 and comparative examples 1 to 3 was measured, and is shown in Table 2.
Table 2, examples 1-27 and comparative examples 1-3 stability test data
In the above technical scheme, generally, it can be considered that discoloration and precipitation mean that exosomes are deteriorated, and the fact that sorbitol, mannitol and sucrose are selected as osmotic pressure regulators is proved to have a good exosome protection effect, and have good stability at a standing temperature of 4 ℃ for 10 days. When the time dimension was extended to 30 days, most samples exhibited varying degrees of deterioration, mainly manifested in some degree of precipitation, turbidity and discoloration. Almost all samples had significant discoloration and turbidity on the 90 day dimension. In addition, the applicant tested in the tests that the nasal sprays prepared in examples 1 to 27 could be stored for a long period of time when stored at-80 ℃.
In addition, for the examples with better stability measured in table 2 and comparative example 3, experiment 2 was further performed, and the experimental results are shown in table 3.
Absorption Properties of nasal sprays in Table 3, some examples and comparative examples
As can be seen from the above screening, in the present application, the scheme of preparation example 26, i.e., when the weight ratio of mannitol 25% to sorbitol 75% is set, the pH value is set to 7.4, and the weight fraction of the osmotic adjusting agent is 7%, the storage effect is good, and the nasal absorption capacity is strong.
Further, in example 26, other components were added, and specific components added are shown in table 4.
Table 4, examples 27 to 45
| Numbering | Glycerol | Sheep placenta polypeptide | Citric acid sodium salt | Chitosan oligosaccharide |
| Example 27 | 5 | 0 | 0 | 0.0 |
| Example 28 | 10 | 0 | 0 | 0.0 |
| Example 29 | 20 | 0 | 0 | 0.0 |
| Example 30 | 30 | 0 | 0 | 0.0 |
| Example 31 | 0 | 0.05 | 0 | 0.0 |
| Example 32 | 0 | 0.1 | 0 | 0.0 |
| Example 33 | 0 | 0.2 | 0 | 0.0 |
| Example 34 | 0 | 0 | 0.1 | 0.0 |
| Example 35 | 0 | 0 | 0.2 | 0.0 |
| Example 36 | 0 | 0 | 0.4 | 0.0 |
| Example 37 | 0 | 0 | 0.8 | 0.0 |
| Example 38 | 0 | 0 | 1.5 | 0.0 |
| Example 39 | 0 | 0 | 0 | 0.2 |
| Example 40 | 0 | 0 | 0 | 0.6 |
| EXAMPLE 41 | 0 | 0 | 0 | 1.0 |
| Example 42 | 0 | 0 | 0 | 1.5 |
| Example 43 | 0 | 0 | 0 | 2.0 |
| Example 44 | 15 | 0.1 | 0.2 | 1.0 |
Experiments 1 and 2 were performed on the above examples, and the results are shown in table 5.
Table 5, control of the results of the experiments in examples 27 to 44
In the above examples, the effect of the addition of the respective materials on the examples themselves can be seen. For some of the examples, since it is difficult to determine whether the exosomes are deteriorated by visual judgment with naked eyes, experiment 3 is further set in the present application to determine the storage of the exosomes.
Experiment 3, before the nasal spray is stored for a long time, sampling, centrifuging at 100000r/min for 70min, retaining the precipitate, washing with deionized water, blowing uniformly, centrifuging at 100000r/min for 70min, retaining the precipitate, freeze-drying, weighing, and recording as m1(ii) a After storage for 90 days, an equal volume of sample was removed and centrifuged through a 0.22 μm filter, and the sample was lyophilized and weighed as m2Calculate m2And m1The results obtained are shown in Table 6.
TABLE 6 exosome retention before and after storage
| Numbering | m1/m2(％) | Numbering | m1/m2(％) |
| Example 26 | 72.9 | Example 37 | 78.8 |
| Example 28 | 78.4 | Example 40 | 79.6 |
| Example 32 | 77.2 | EXAMPLE 41 | 83.5 |
| Example 35 | 79.9 | Example 42 | 78.9 |
| Example 36 | 80.1 | Example 44 | 89.7 |
The optimal components of the materials in the present application can be verified by the above further data, and it is proved that the best preservation effect can be obtained when the addition is performed according to the technical scheme in example 44 by using the technical scheme in the present application. In addition, when the addition amount of the sodium citrate is large, the nasal cavity is strongly stimulated, so that the addition amount of the sodium citrate is set to be 0.2 mass percent under the condition of approximate effect, and the applicability is better.
Further, the following examples are provided.
Example 45, a nasal spray of neural stem cell exosomes, was different from example 44 in that the storage temperature was-20 ℃.
Example 46, a nasal spray of neural stem cell exosomes, was different from example 44 in that the storage temperature was-80 ℃.
The storage temperatures of examples 45 and 46 were adjusted, and experiment 1 shows that the storage effect of example 45 is substantially similar to that of example 44, and the crystal is not crystallized, which proves that the solution of the present application can be stored well at-20 ℃. In example 46, the nasal spray is crystallized when stored at-80 ℃, but still has good dispersibility after redissolution, which proves that the prepared exosome nasal spray has good storage effect under the condition of deep cooling at-80 ℃.
Example 47A nasal spray of neural stem cell exosomes, different from example 44 in that the exosomes are present at a concentration of 1010one/mL.
Example 48A nasal spray of neural stem cell exosomes, the difference with example 44 is that of exosomesAt a concentration of 108Per mL
Experiment 3 performed on example 47 and example 48 revealed that the exosome retention amounts were 90.1% and 88.0%, respectively, demonstrating that when the exosome concentration was low, the loss amount was small after long-term storage, and that the exosome nasal spray could be prepared at different concentrations according to actual needs, with good preservation effect within the above range.
The present embodiment is only for explaining the present application and is not limited to the present application, and a person skilled in the art can make modifications of the present embodiment without inventive contribution as required after reading the present specification, but is protected by patent laws within the scope of the claims of the present application.
Claims (10)
1. The nasal spray for the exosomes of the neural stem cells is characterized by comprising a buffer solution and the following components suspended in the buffer solution:
（1～100）×108one/mL of neural stem cell exosomes;
an osmotic pressure regulator accounting for 1.6-10% of the mass fraction of the buffer solution;
the osmotic pressure regulator is one of mannitol, sucrose and sorbitol, or a compound system of any number of mannitol, sucrose and sorbitol.
2. The nasal spray of a neural stem cell exosome according to claim 1, wherein the buffer solution further comprises glycerol accounting for 10-20% of the mass fraction of the buffer solution.
3. The nasal spray of a neural stem cell exosome according to claim 1, wherein the buffer solution further comprises sheep placenta polypeptide accounting for 0.05-0.1% of the mass fraction of the buffer solution.
4. The nasal spray of a neural stem cell exosome according to claim 1, wherein the buffer solution further comprises sodium citrate accounting for 0.1-0.8% of the mass fraction of the buffer solution.
5. The nasal spray of a neural stem cell exosome according to claim 1, wherein the buffer solution has a pH value of 6.5-7.8.
6. The nasal spray of a neural stem cell exosome according to claim 5, wherein the concentration of the buffer solution is 1-100 mM, and the buffer solution is phosphate buffer solution.
7. The nasal spray of a neural stem cell exosome according to claim 1, wherein the buffer solution further comprises chitosan oligosaccharide accounting for 0.6-1.5% of the mass fraction of the buffer solution.
8. The nasal spray of claim 7, wherein the nasal spray is maintained in an environment of less than 4 ℃.
9. A preparation method of a nasal spray of a neural stem cell exosome according to any one of claims 1 to 8, comprising the following steps:
s1, culturing, amplifying and separating the neural stem cells to obtain exosomes;
s2, adding a buffer solution added with an osmotic pressure regulator in advance into the exosome according to the calculated amount under the condition that the temperature is not higher than 4 ℃, and uniformly blowing.
10. The method for preparing a nasal spray of a neural stem cell exosome according to claim 9, wherein in step S2, glycerol is further added into the buffer solution in advance, after the glycerol is uniformly blown, sheep placenta polypeptide and chitosan oligosaccharide are continuously added into the system, and the system is stored in an environment at a temperature below 4 ℃.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115137754A (en) * | 2022-08-04 | 2022-10-04 | 首都医科大学附属北京朝阳医院 | Ophthalmic pharmaceutical composition containing exosomes |
| CN116421561A (en) * | 2023-03-07 | 2023-07-14 | 陕西初源外泌体生物科技有限公司 | Nasal spray for treating exosomes with nerve function deficiency and preparation method thereof |
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