Stem cell exosome-containing atomized liquid for treating xerophthalmia and preparation method thereof

Abstract

The invention discloses atomized liquid containing stem cell exosomes and used for treating xerophthalmia and a preparation method of the atomized liquid, and relates to the technical field of atomized liquid for eyes, and the atomized liquid is prepared from components as follows: an exosome-loaded nano-carrier, an osmotic pressure regulator, a pH regulator and a solvent; the nano-carrier loaded with the exosome is prepared from a chitosan-carboxymethyl chitosan polymer loaded with the exosome, and the concentration of the nano-carrier loaded with the exosome in the atomized liquid is 30-100 [mu] g / mL; the exosome is a mesenchymal stem cell exosome; the molecular weight of the chitosan is 3 kDa, and the average molecular weight of the carboxymethyl chitosan is 5-50 kDa; according to the present invention, when the atomized liquid relying on hydrogen bond and static slow release is applied to xerophthalmia, the slow release effect failure or obvious burst release cannot be caused even if the tear ion concentration is high, and the pH value is neutral or meta-acid, and the retention time of the carrier on the cornea surface can be prolonged, the tear washing can be resisted, and the retention time at the action site can be prolonged.

Description

Technical Field

[0001] The present invention relates to the technical field of ophthalmic atomization solutions, and particularly to an atomization solution for treating dry eye disease containing stem cell exosomes and a preparation method thereof. Background Art

[0002] Dry Eye Disease (DED) is characterized by decreased tear film stability, ocular surface inflammation, and nerve damage. Common causes include insufficient tear secretion, excessive tear evaporation, and inflammatory reactions. Traditional artificial tears and anti-inflammatory drugs can only relieve symptoms and cannot reverse ocular surface damage or restore lacrimal gland function.

[0003] Exosomes are nanoscale vesicles (30 - 150 nm) secreted by cells, carrying bioactive molecules such as proteins, lipids, mRNA, and miRNA, and participating in intercellular communication and material transport. Epidermal growth factor (EGF), fibroblast growth factor (FGF), etc. carried by exosomes directly stimulate the proliferation of ocular surface cells. miRNA regulates the expression of inflammatory genes, accelerates wound healing, inhibits the release of pro-inflammatory factors, relieves inflammatory reactions, promotes the secretory function of lacrimal duct cells, increases tear secretion, repairs damaged corneal nerve fibers, and improves dry eye-related neuralgia.

[0004] On the premise that exosomes have great potential for treating dry eye disease, there is a need to obtain an atomization solution containing exosomes.

[0005] For example, in Chinese Patent, application number CN202410851614.4, a sustained-release arformoterol tartrate atomization inhalation solution, a preparation method thereof, and applications thereof. The preparation method of the above-mentioned sustained-release arformoterol tartrate atomization inhalation solution includes the following steps: preparing a nanocarrier loaded with arformoterol tartrate, and then dissolving the carrier loaded with arformoterol tartrate and a stabilizer in water for injection to obtain a mixed solution. Adding a cosolvent, an osmotic pressure regulator, and a pH regulator to the mixed solution, and after volume fixation, aliquoting to obtain the sustained-release arformoterol tartrate atomization inhalation solution. The nanocarrier of this invention can form a complex with arformoterol tartrate through electrostatic or hydrogen bond interactions, thereby achieving the purpose of sustained and slow release of the drug. In addition, the sustained-release arformoterol tartrate atomization inhalation solution of this invention also has excellent stability and atomization performance.

[0006] However, when the atomization solution relying on hydrogen bond and electrostatic sustained release is applied to dry eye disease, due to the high ion concentration and neutral pH in the tears of dry eye disease, the electrostatic interaction will quickly fail, and lysozyme in the tears can degrade the chitosan carrier, resulting in sudden drug release and uncontrollable drug concentration. Summary of the Invention

[0007] The embodiments of the present application provide an atomizing solution for treating dry eye containing stem cell exosomes and a preparation method thereof, which solve the problem of burst release caused by electrostatic shielding in the existing tear sustained-release agent and realize a more stable sustained-release atomizing solution.

[0008] The embodiments of the present application provide an atomizing solution for treating dry eye containing stem cell exosomes, and its components include: a nano-carrier loaded with exosomes, an osmotic pressure regulator, a pH regulator, and a solvent;

[0009] The nano-carrier loaded with exosomes is made of a chitosan-carboxymethyl chitosan polymer loaded with exosomes, and its concentration in the atomizing solution is 30-100 μg / mL; the exosomes are mesenchymal stem cell exosomes;

[0010] The molecular weight of chitosan is 3 kDa, and the average molecular weight of the used carboxymethyl chitosan is 5-50 kDa.

[0011] Furthermore, the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, and glucose, and the concentration is 0.85%-1.5%; so that the osmotic pressure of the atomizing solution is close to that of physiological saline or slightly higher.

[0012] Furthermore, the pH regulator is selected from one of phosphate buffer solution and Tris buffer solution, and the pH value of the atomizing solution is adjusted to 7.2-7.4.

[0013] Furthermore, the solvent is physiological saline, deionized water or other suitable biocompatible solvents.

[0014] Furthermore, the specific steps for preparing the nano-carrier loaded with exosomes are as follows:

[0015] S1. Dissolve chitosan in ethanol, add ethylene oxide and react at 55-65 °C for 20-26 h to obtain a chitosan-polyethylene glycol copolymer;

[0016] S2. Dissolve the chitosan-polyethylene glycol copolymer in dimethyl sulfoxide, add hexamethylene diisocyanate and react at 75-85 °C for 5-8 h to obtain a chitosan-polyethylene glycol block copolymer;

[0017] S3. Then dissolve the chitosan-polyethylene glycol block copolymer in ethanol again, add carboxymethyl chitosan and an exosome solution, and sodium tripolyphosphate as a crosslinking agent, stir, and freeze-dry at -20 °C to prepare a nano-carrier loaded with exosomes.

[0018] Furthermore, the mass ratio of the chitosan-polyethylene glycol block copolymer, carboxymethyl chitosan, and the exosome solution is 1:(0.5-2):1, and the exosome concentration in the exosome solution is 1×10 7 -1×10 9 particles / mL.

[0019] Furthermore, the carboxymethyl chitosan includes long-chain carboxymethyl chitosan and short-chain carboxymethyl chitosan, where the molecular weight of the long-chain carboxymethyl chitosan is 5-10 kDa, the molecular weight of the short-chain carboxymethyl chitosan is 20-50 kDa, and the mass ratio of the long-chain carboxymethyl chitosan to the short-chain carboxymethyl chitosan is 7:3.

[0020] Furthermore, the carboxymethyl chitosan is partially alkylated, and the alkylated carboxymethyl chitosan accounts for 15% of the total amount of carboxymethyl chitosan used.

[0021] Furthermore, the alkylation process is as follows: dissolve different molecular weight CMCS combinations in dimethyl sulfoxide, add an excessive amount of dodecyl bromide and the alkaline catalyst sodium hydroxide, and react at 60°C - 80°C for 12 - 24 hours to obtain alkylated CMCS.

[0022] Furthermore, the carrier has a bilayer, including an inner layer and an outer layer, and the bilayer carrier is prepared by freeze-drying and then cross-linking and coating again;

[0023] Among them, the outer layer is a pH-responsive layer, and the CMCS used is low molecular weight CMCS + high proportion alkylation, and the inner layer is a long-acting sustained-release layer, and the CMCS used is high molecular weight CMCS + low proportion alkylation.

[0024] The preparation method of the above atomized liquid for treating dry eye containing stem cell exosomes is as follows: mix the nano-carrier loaded with exosomes, osmotic pressure regulator, pH regulator, and solvent, then subpackage it in a sterile container, seal it, and store it away from light and high temperature.

[0025] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0026] First, exosomes are used as the main drug, and chitosan-carboxymethyl chitosan is used as the carrier, so that exosomes can still play a sustained-release role and be retained for a long time in a high-salt neutral environment. The carboxylic acid groups of carboxymethyl chitosan bind to the glycosyl groups of ocular surface mucin through hydrogen bonds or electrostatic interactions, prolonging the residence time of the carrier on the corneal surface, resisting the flushing of tears, and carboxymethyl chitosan itself has a high viscosity, which can prolong the residence time at the action site.

[0027] Second, use a combination of CMCS with multiple different molecular weights to regulate the structural function differentiation of the multi-level structure of the carrier through molecular weight differences, and achieve the efficient loading and intelligent sustained release of exosomes.

[0028] Third, through the alkylation of the CMCS combination, while increasing the loading amount, the drug release rate increases in patients with severe dry eye inflammation to meet the needs of patients with different conditions.

[0029] Fourthly, through the double-layer carrier design, the outer layer of low-molecular-weight highly alkylated CMCS achieves rapid response in an acidic environment, and the inner layer of high-molecular-weight low-alkylated CMCS ensures stable release in the middle and late stages, significantly optimizing the uniformity of the drug release rate. Brief Description of the Drawings

[0030] Figure 1 It is the graph of the drug release experiment result in the first embodiment of the present invention;

[0031] Figure 2 It is the graph of the ion influence experiment result in the first embodiment of the present invention;

[0032] Figure 3 It is the graph of the drug release experiment result of the combination of two different molecular weight CMCSs in the second embodiment of the present invention. Detailed Embodiments

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this invention belongs; the terms used in the description of the present invention in this specification are only for the purpose of describing specific embodiments, and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0034] Embodiment 1: The present application provides an atomized liquid for treating dry eye containing stem cell exosomes, and its components include: a nano-carrier loaded with exosomes, an osmotic pressure regulator, a pH regulator, and a solvent.

[0035] Among them, the osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, and glucose, and the concentration is 0.85%-1.5%; so that the osmotic pressure of the atomized liquid is close to that of physiological saline or slightly higher;

[0036] The pH regulator is selected from one of phosphate buffer solution and Tris buffer solution, and the pH value of the atomized liquid is adjusted to 7.2-7.4;

[0037] The solvent is physiological saline, deionized water or other suitable biocompatible solvents;

[0038] The nano-carrier loaded with exosomes is made of a chitosan-carboxymethyl chitosan polymer loaded with exosomes, and the concentration in the atomized liquid is 30-100 μg / mL; the exosomes are mesenchymal stem cell exosomes;

[0039] The specific steps for preparing the nano-carrier loaded with exosomes are as follows:

[0040] S1. Dissolve chitosan in ethanol, add ethylene oxide and react at 55-65 °C for 20-26 h to obtain a chitosan-polyethylene glycol copolymer;

[0041] S2. Dissolve the chitosan - polyethylene glycol copolymer in dimethyl sulfoxide, add hexamethylene diisocyanate, and react at 75 - 85 °C for 5 - 8 h to obtain the chitosan - polyethylene glycol block copolymer;

[0042] S3. Then dissolve the chitosan - polyethylene glycol block copolymer in ethanol again, add carboxymethyl chitosan and the exosome solution, as well as sodium tripolyphosphate as a cross - linker, stir, and freeze - dry at - 20 °C to prepare the exosome - loaded nanocarrier.

[0043] Among them, the molar ratio of chitosan, ethylene oxide, and hexamethylene diisocyanate is 1:0.3:0.5;

[0044] The mass ratio of the chitosan - polyethylene glycol block copolymer, carboxymethyl chitosan, and the exosome solution is 1:(0.5 - 2):1;

[0045] The concentration of exosomes in the exosome solution is 1×10 7 -1×10 9 particles / mL;

[0046] The molecular weight of the chitosan used as the raw material is 3 kDa, and the average molecular weight of the carboxymethyl chitosan used is 30 kDa.

[0047] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0048] By using exosomes as the main drug and chitosan - carboxymethyl chitosan as the carrier, the exosomes can still play a sustained - release role and be retained for a long time in a high - salt neutral environment. Thus, this embodiment actually solves the problem of burst release caused by the rapid dissociation of the exosome nebulizer solution in tears due to electrostatic interaction.

[0049] As a natural drug carrier, exosomes have unique structures and functions that can achieve long - distance transportation in the body under physiological and pathological conditions. They have good stability and natural targeting ability and can cross the corneal barrier; carry growth - promoting factors (such as VEGF, bFGF, etc.) and other molecules beneficial to tissue regeneration, promote the proliferation and regeneration of damaged ocular cells, and accelerate the repair of ocular tissues; inhibit inflammatory reactions and relieve ocular redness and pain. For example, miR - 204 in exosomes can inhibit the IL - 6 / IL - 6R / Stat signaling pathway, causing macrophages to transform from the M1 type (pro - inflammatory type) to the M2 type (anti - inflammatory type), thereby reducing inflammatory reactions.

[0050] Carboxymethyl chitosan is a cationic biopolymer containing a large number of highly hydrophilic amino groups, which makes the biopolymer have a net positive charge, thus promoting the interaction with various negatively charged polymers. Carboxymethyl chitosan is partially dissociated into - COO at physiological pH -, it forms a dynamic binding with the amino group (-NH2) in the chitosan - polyethylene glycol block copolymer through hydrogen bonds or weak electrostatic attraction. Even in a high - salt environment, the negative charge of the carboxylic acid group can still partially offset the shielding effect of salt ions, maintaining the binding force between the carrier and exosomes; thus reducing the shielding effect caused by excessive ion concentration and preventing the sudden release of drugs.

[0051] The cross - linker sodium tripolyphosphate (negatively charged) forms an ionic cross - linked network with the amino group (-NH3 + ) of chitosan, further stabilizing the carrier structure and reducing the sudden release of exosomes; preventing the sudden release of drugs caused by the collapse of the carrier due to enzymatic decomposition.

[0052] The carboxylic acid group of carboxymethyl chitosan binds to the glycosyl group of ocular surface mucin through hydrogen bonds or electrostatic interactions, prolonging the residence time of the carrier on the corneal surface and resisting the flushing of tears. Moreover, carboxymethyl chitosan itself has a high viscosity, which can prolong the residence time at the action site.

[0053] First, an experiment was conducted on the ability of exosomes to treat dry eye. An experimental group and a control group were set up. The control group did not adopt any treatment methods. In the experimental group, before treatment, at 2 weeks of treatment, and at 4 weeks of treatment, the ocular symptoms (dryness degree, foreign body sensation) and the amount of tear secretion in the eyes (Schirmer test) of the patients were evaluated. The treatment method was atomization treatment using an atomized solution once a day. The composition of the atomized solution was: exosomes, osmotic pressure regulator, pH regulator, and solvent; except that the exosome concentration was 1×10 8 -1×10 10 particles / mL; the other components were the same as in Example 1, and the results are shown in Table 1.

[0054]

[0055] Table 1

[0056] After knowing that exosomes have a therapeutic effect on dry eye, an experiment on the sustained - release effect of the nano - carrier loaded with exosomes in Example 1 was conducted. Composite carriers of different ratios of C - PEG and CMCS (not loaded with exosomes) were dispersed in simulated tears (0.9% NaCl, pH 7.4 PBS), diluted to 0.1 mg / mL, and the Zeta potential (mV) was measured using a Zeta potential analyzer (Malvern Zetasizer Nano ZS) at a temperature of 25°C. The results are shown in Table 2 (C - PEG is chitosan - polyethylene glycol block copolymer, and CMCS is carboxymethyl chitosan).

[0057] C-PEG:CMCS Zeta potential (mV) Charge state analysis 1:0.5 +8.2±1.5 Amino group (+) dominant, weak electrostatic binding 1:1 -3.5±0.8 Carboxyl group (-) and amino group (+) in dynamic equilibrium 1:1.5 -12.4±1.2 Carboxyl group (-) dominant, but crosslinked network stable 1:2 -18.6±2.1 Carboxyl group (-) overloaded, enhanced electrostatic shielding effect

[0058] Table 2

[0059] After loading Cy5-labeled exosomes onto the carrier, an in vitro sustained-release experiment was carried out. The prepared Cy5-labeled exosome-loaded nanocarrier was put into the release medium, and the release medium was simulated tear fluid (0.9% NaCl, pH 7.4, PBS + 0.1% mucin); the carrier loaded with fluorescently labeled exosomes was dispersed in the medium and oscillated at 37 °C (100 rpm); samples were taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h. After centrifugation (10,000 rpm, 10 min), the supernatant was taken, and the exosome concentration was detected by a fluorescence spectrophotometer to obtain the results of drug release. The results are as Figure 1 ;

[0060] A salt concentration influence experiment was carried out. Gradient salt concentrations were set to simulate high and low ion concentrations. Specifically, the salt gradient was set as follows: 0.5%, 0.9%, 1.5% NaCl (pH 7.4, solvent PBS). The 8-hour exosome release rates of each group of carriers under the salt gradient (the method was the same as the in vitro sustained-release experiment). The results are as Figure 2 ;

[0061] It can be seen that the optimal ratio is C-PEG:CMCS = 1:1.5. At this concentration, the electrostatic effect still partially exists and does not cause sudden drug release. The release rate at 8 hours is 58.6%, the half-life is 5.8 hours, and the loss rate is 18.4 / h. It still has good sustained-release ability under the high ion concentration caused by the patient's pathological state.

[0062] Example 2: In the above example, by using chitosan-carboxymethyl chitosan as the carrier, the exosomes can still play a sustained-release role and be retained for a long time in a high-salt neutral environment. To enhance the adsorption ability and release ability of the carrier, it was further improved on the basis of Example 1.

[0063] Carboxymethyl chitosan also includes long-chain carboxymethyl chitosan and short-chain carboxymethyl chitosan, where the molecular weight of long-chain carboxymethyl chitosan is 5-10 kDa, the molecular weight of short-chain carboxymethyl chitosan is 20-50 kDa, and the mass ratio of long-chain carboxymethyl chitosan to short-chain carboxymethyl chitosan is 7:3.

[0064] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0065] By using a combination of CMCS with multiple different molecular weights and regulating the structural function differentiation of the multi-level structure of the carrier through molecular weight differences, efficient loading and intelligent sustained release of exosomes are achieved. Thus, the problem solved in this embodiment is the problem that the adsorption amount is insufficient and cannot match the sustained-release ability.

[0066] High-molecular-weight CMCS serves as the "structural framework", providing high charge density and strong cross-linking ability. With a high molecular weight and a high density of carboxylic acid groups, it forms a stable electrostatic bond with the amino groups of the chitosan-polyethylene glycol block copolymer, resisting the ion shielding effect in tears. The long-chain CMCS forms a dense network with the cross-linking agent, reducing the porosity of the carrier and preventing the sudden release of exosomes. Low-molecular-weight CMCS, as the "release regulator", forms dynamic pores and promotes the diffusion of exosomes. The short-chain CMCS forms micropores or channels inside the carrier, increasing the specific surface area and enhancing the exosome loading capacity. The low-molecular-weight CMCS chain segments have high flexibility and are easily swollen in the tear environment, expanding the pore size, gradually releasing exosomes, and as the electrostatic and hydrogen bond interactions gradually decrease, the exosomes are gradually released. The overall release is from large pore release in the early stage, electrostatic hydrogen bond release in the middle stage, and accelerated release due to the swelling of CMCS to expand the pores in the later stage;

[0067] The increase in the surface negative charge of high-molecular-weight CMCS not only resists ion interference but also resists tear flushing, prolonging the residence time of the carrier on the ocular surface. The micropores generated by low-molecular-weight CMCS provide physical adsorption space and can also improve the loading efficiency.

[0068] An in vitro sustained-release experiment was carried out using the carrier prepared in Example 2. The sampling times were 0.5 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The results of the drug release are as Figure 3 .

[0069] Example 3: In Example 2, different molecular weight CMCS were used in combination to optimize the release rate of exosomes. However, in actual use, the tears of severely dry eye patients are acidic, and the swelling of the carrier leads to an average 30% increase in the release rate, especially more than 65% increase in the later release speed, resulting in a decrease in the uniformity of the sustained-release speed and easy to cause large fluctuations in the drug concentration in the later stage when the drug concentration is stable. To optimize the drug release rate in an acidic environment, further improvement was made on the basis of Example 2.

[0070] Carboxymethyl chitosan was also partially alkylated, and the alkylated carboxymethyl chitosan accounted for 15% of the total amount of carboxymethyl chitosan used. The alkylation process was as follows: Different molecular weight CMCS were combined and dissolved in dimethyl sulfoxide, an excessive amount of dodecyl bromide and the alkaline catalyst sodium hydroxide were added, and the reaction was carried out at 60 °C - 80 °C for 12 - 24 hours to obtain alkylated CMCS;

[0071] The reaction formula is:

[0072] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0073] By alkylating the CMCS combination, while increasing the loading amount, the drug release rate increases in patients with severe inflammatory dry eye to meet the needs of patients with different conditions. Thus, the problem actually solved in this embodiment is how to achieve sustained and controlled release for patients with severe inflammation and how to adaptively change the release amount according to the condition.

[0074] Through aminoalkylation, long-chain alkyl groups are introduced into carboxymethyl chitosan. The hydrophobic effect of the alkyl chain can promote the entanglement of molecular chains at pH 7.4. At pH 6.5, due to the charge change, the hydrophobic interaction is weakened, reducing the entanglement degree. Moreover, the lower the pH, the greater the degree of weakening of the hydrophobic interaction and the faster the drug release rate, realizing the automatic regulation of the drug release rate according to the patient's condition.

[0075] The alkylation of low-molecular-weight CMCS introduces hydrophobic segments, enhancing the intermolecular hydrophobic interaction. In the state of normal physiological tears, the molecular chains fold or entangle to form a dense structure, so that exosomes are also wrapped due to the mutual entanglement of molecular weights under the action of electrostatic force and hydrogen bond, reducing the release degree in the physiological environment. In an acidic environment, the hydrophobic segments induce local phase separation, and the degree of molecular chain entanglement gradually decreases, gradually releasing the encapsulated exosomes and increasing the release degree in the early and middle stages. The alkylation of high-molecular-weight CMCS improves the mechanical strength of the carrier and the drug encapsulation rate. In an acidic environment, the carboxyl groups are protonated, the degree of swelling decreases, but the hydrophobic cross-linked network is partially dissociated, forming microcracks, and the drug diffuses rapidly through the cracks, and the release degree in the middle and late stages is greatly affected. Due to steric hindrance, the degree of alkylation of low-molecular-weight CMCS is higher than that of high-molecular-weight CMCS. Therefore, during the release process, it actually shows that the release degree is different in the early stage according to the different pathological states of the patients, and the release tends to be stable in the later stage.

[0076] On the basis of the high-molecular-weight CMCS (50 kDa) + low-molecular-weight CMCS (10 kDa) group in Example 2, the carrier prepared in Example 3 was used for in vitro sustained-release experiments. The release media included physiological and pathological environments. The pH of the physiological environment was 7.4, and the pH of the pathological environments were 6.5, 6.8, and 7.0. The differences between alkylation (Example 3) and non-alkylation (Example 2) were compared. The sampling times were 0.5 h, 2 h, 4 h, 8 h, 12 h, and 24 h. The results of drug release are shown in Table 3.

[0077]

[0078] Table 3

[0079] Example 4: Example 3 optimizes the adaptive release in an inflammatory environment through alkylation. To further optimize the release rate, it is further improved on the basis of Example 3.

[0080] The carrier has multiple layers, and a double-layer carrier is prepared by freeze-drying and then cross-linking and coating again; the carrier includes an inner layer and an outer layer;

[0081] The outer layer is a pH-responsive layer, and the CMCS used is low-molecular-weight CMCS (5-10 kDa) + high-proportion alkylation (20% bromododecane modification);

[0082] The inner layer is a long-acting sustained-release layer, and the CMCS used is high-molecular-weight CMCS (20-50 kDa) + low-proportion alkylation (5% bromododecane modification).

[0083] The technical solutions in the embodiments of the present application at least have the following technical effects or advantages:

[0084] Through the double-layer carrier design, the outer low-molecular-weight and high-alkylated CMCS realizes rapid response in an acidic environment, and the inner high-molecular-weight and low-alkylated CMCS ensures stable release in the middle and late stages, significantly optimizing the uniformity of the drug release rate. Thus, this embodiment solves the problem that the drug release amount cannot be sustained-release according to the actual drug absorption or metabolism stage.

[0085] The high alkylation in the outer layer responds rapidly in the inflammatory acidic environment, forms pores through the phase separation of the hydrophobic chain segments, reduces the entanglement degree, accelerates the initial drug release, and alleviates acute inflammation. The inner layer forms microcracks through the dynamic dissociation of the hydrophobic cross-linked network, and the release rate is stable in the middle and late stages. The high-molecular-weight network resists the flushing of tears and prolongs the residence time of the carrier.

[0086] Taking into account patients in pathological states under different etiologies and patients with different conditions, it promotes the uniform release of exosomes.

[0087] Using 5 kDa CMCS + 20% bromododecane modification in the outer layer and 50 kDa CMCS + 20% bromododecane modification in the inner layer, a dissolution test was carried out in a pathological environment with a pH 6.5 dissolution medium, and the results are shown in Table 4;

[0088] Time (h) Release rate of monolayer carrier (alkylated) (%) Release rate of bilayer carrier (%) 0.5 7.8 12.5 2 22.5 29.4 4 45.6 56.2 8 82.0 81.0 12 96.5 92.6 24 99.5 96.1

[0089] Table 4

[0090] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An atomizing solution for treating dry eye containing stem cell exosomes, characterized in that, Its components include: nano - carriers loaded with exosomes, osmotic pressure regulators, pH regulators, and solvents; The nano - carriers loaded with exosomes are made of chitosan - carboxymethyl chitosan polymers loaded with exosomes, and the concentration in the nebulized solution is 30 - 100 μg / mL; the exosomes are mesenchymal stem cell exosomes; The molecular weight of chitosan is 3 kDa, and the average molecular weight of the used carboxymethyl chitosan is 5 - 50 kDa.

2. The atomizing solution for treating dry eye containing stem cell exosomes according to claim 1, characterized in that, The osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, and glucose, and the concentration is 0.85% - 1.5%; making the osmotic pressure of the nebulized solution close to or slightly higher than that of normal saline. The pH regulator is selected from one of phosphate buffer solution and Tris buffer solution, and the pH value of the nebulized solution is adjusted to 7.2 - 7.

4.

3. The atomizing solution for treating dry eye containing stem cell exosomes according to claim 1, wherein, The solvent is normal saline, deionized water or other suitable biocompatible solvents.

4. The atomized liquid for treating dry eye containing stem cell exosomes according to claim 1, characterized in that, The specific steps for preparing the nano - carriers loaded with exosomes are as follows: S1. Dissolve chitosan in ethanol, add ethylene oxide and react at 55 - 65 °C for 20 - 26 h to obtain chitosan - polyethylene glycol copolymer; S2. Dissolve chitosan - polyethylene glycol copolymer in dimethyl sulfoxide, add hexamethylene diisocyanate and react at 75 - 85 °C for 5 - 8 h to obtain chitosan - polyethylene glycol block copolymer; S3. Then dissolve the chitosan - polyethylene glycol block copolymer in ethanol again, add carboxymethyl chitosan and exosome solution, and sodium tripolyphosphate as a cross - linker, stir, and freeze - dry at - 20 °C to obtain the nano - carriers loaded with exosomes.

5. The atomized liquid for treating dry eye containing stem cell exosomes according to claim 1, wherein The mass ratio of chitosan - polyethylene glycol block copolymer, carboxymethyl chitosan, and exosome solution is 1:(0.5 - 2):1, and the exosome concentration in the exosome solution is 1×10 7 -1×10 9 particles / mL.

6. The atomized liquid for treating dry eye containing stem cell exosomes according to claim 5, characterized in that, Carboxymethyl chitosan includes long - chain carboxymethyl chitosan and short - chain carboxymethyl chitosan. Among them, the molecular weight of long - chain carboxymethyl chitosan is 5 - 10 kDa, the molecular weight of short - chain carboxymethyl chitosan is 20 - 50 kDa, and the mass ratio of long - chain carboxymethyl chitosan to short - chain carboxymethyl chitosan is 7:

3.

7. The atomized liquid for treating dry eye containing stem cell exosomes according to claim 6, characterized in that, Carboxymethyl chitosan is partially alkylated, and the alkylated carboxymethyl chitosan accounts for 15% of the total amount of carboxymethyl chitosan used.

8. The atomized liquid for treating dry eye containing stem cell exosomes according to claim 7, characterized in that, The alkylation process is: dissolve a combination of CMCS with different molecular weights in dimethyl sulfoxide, add an excessive amount of dodecyl bromide and the alkaline catalyst sodium hydroxide, and react at 60 °C - 80 °C for 12 - 24 hours to obtain alkylated CMCS.

9. The atomizing solution for treating dry eye containing stem cell exosomes according to claim 8, wherein, The carrier has a bilayer, including an inner layer and an outer layer, and the bilayer carrier is prepared by freeze - drying and then cross - linking and coating again; Among them, the outer layer is a pH - responsive layer, and the used CMCS is low - molecular - weight CMCS + high - proportion alkylation. The inner layer is a long - acting sustained - release layer, and the used CMCS is high - molecular - weight CMCS + low - proportion alkylation.

10. A method for preparing an atomizing solution for treating dry eye containing stem cell exosomes according to any one of claims 1-9, characterized in that, The preparation method is: mix the nano - carriers loaded with exosomes, osmotic pressure regulators, pH regulators, and solvents, then sub - package them in a sterile container, seal and store, avoiding light and high temperature.

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