A dry eye treatment aerosol liquid containing stem cell exosomes and a preparation method thereof

Abstract

The application discloses an atomized liquid containing stem cell exosomes for treating dry eye and a preparation method thereof, and relates to the technical field of eye atomized liquids, which comprises the following components: exosome-loaded nanocarriers, osmotic pressure regulators, pH regulators and solvents; the exosome-loaded nanocarriers are made of exosome-loaded chitosan-carboxymethyl chitosan polymers, and the concentration in the atomized liquid is 30-100 mu g / mL; the exosomes are mesenchymal stem cell exosomes; the chitosan has a molecular weight of 3 kDa, and the carboxymethyl chitosan used has an average molecular weight of 5-50 kDa; the atomized liquid can be applied to dry eye by relying on hydrogen bond and electrostatic slow release, even if the ion concentration of the tear liquid is high, the ph is neutral or slightly acidic, the slow release effect will not fail or obvious burst release will not be caused, and the retention time of the carrier on the corneal surface can be prolonged, the tear liquid can be resisted, and the residence time at the action site can be prolonged.

Description

Technical Field

[0001] This invention relates to the field of ophthalmic nebulizer technology, and more particularly to a nebulizer containing stem cell exosomes for treating dry eye syndrome and its preparation method. Background Technology

[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 response. 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 substance transport. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) carried by exosomes directly stimulate the proliferation of ocular surface cells; miRNAs regulate the expression of inflammatory genes, accelerate wound healing, inhibit the release of pro-inflammatory factors, alleviate inflammatory responses, promote the secretory function of lacrimal gland duct cells, increase tear secretion, repair damaged corneal nerve fibers, and improve dry eye-related neuralgia.

[0004] Given the great potential of exosomes in treating dry eye, there is a demand for nebulized solutions containing exosomes.

[0005] For example, Chinese Patent Application No. CN202410851614.4 discloses a sustained-release arbutorium tartrate nebulized inhalation solution, its preparation method, and its application. The preparation method of the aforementioned sustained-release arbutorium tartrate nebulized inhalation solution includes the following steps: preparing a nanocarrier loaded with arbutorium tartrate; then dissolving the arbutorium tartrate-loaded carrier and a stabilizer in water for injection to obtain a mixed solution; adding a co-solvent, an osmotic pressure regulator, and a pH adjuster to the mixed solution; and finally, dispensing the solution after adjusting the volume to obtain the sustained-release arbutorium tartrate nebulized inhalation solution. The nanocarrier of this invention can form a complex with arbutorium tartrate through electrostatic or hydrogen bonding interactions, thereby achieving the purpose of sustained and slow drug release. Furthermore, the sustained-release arbutorium tartrate nebulized inhalation solution of this invention also exhibits excellent stability and nebulization performance.

[0006] However, when nebulizers that rely on hydrogen bonds and electrostatic slow release are applied to dry eye syndrome, the high ion concentration and neutral pH in dry eye tears can cause the electrostatic effect to fail rapidly. Lysozyme in the tears can degrade the chitosan carrier, leading to a burst release of the drug and making the drug concentration uncontrollable. Summary of the Invention

[0007] This application provides an atomizing solution containing stem cell exosomes for treating dry eye and its preparation method, which solves the problem of sudden release caused by electrostatic shielding in tear sustained-release agents in the prior art, and achieves a more stable sustained-release atomizing solution.

[0008] This application provides an atomizing solution for treating dry eye syndrome containing stem cell exosomes, the components of which include: a nanocarrier loaded with exosomes, an osmotic pressure regulator, a pH regulator, and a solvent;

[0009] The nanocarriers loaded with exosomes are made of chitosan-carboxymethyl chitosan polymer loaded with exosomes, and the 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 carboxymethyl chitosan used is 5-50 kDa.

[0011] Furthermore, one or more of sodium chloride, mannitol, and glucose are selected as the osmotic pressure regulator, with a concentration of 0.85%-1.5%; so that the osmotic pressure of the nebulizer is close to or slightly higher than that of physiological saline.

[0012] Furthermore, the pH adjuster is selected from phosphate buffer and Tris buffer to adjust the pH of the nebulizer to 7.2-7.4.

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

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

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

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

[0017] S3. Then, the chitosan-polyethylene glycol block copolymer was dissolved again in ethanol, and carboxymethyl chitosan and exosome solution were added, along with sodium tripolyphosphate as a crosslinking agent. The mixture was stirred and freeze-dried at -20°C to obtain the nanocarrier loaded with exosomes.

[0018] Furthermore, the mass ratio of chitosan-polyethylene glycol block copolymer, carboxymethyl chitosan, and exosome solution was 1:(0.5-2):1, and the exosome concentration in the exosome solution was 1×10⁻⁶. 7 -1×10 9 particles / mL.

[0019] Furthermore, carboxymethyl chitosan includes long-chain carboxymethyl chitosan and short-chain carboxymethyl chitosan, wherein 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.

[0020] Furthermore, the carboxymethyl chitosan is partially alkylated, with alkylated carboxymethyl chitosan accounting for 15% of the total carboxymethyl chitosan used.

[0021] Furthermore, the alkylation process is as follows: CMCS combinations of different molecular weights are dissolved in dimethyl sulfoxide, excess dodecane bromo and alkaline catalyst sodium hydroxide are added, and the mixture is reacted at 60℃-80℃ for 12-24 hours to obtain alkylated CMCS.

[0022] Furthermore, the carrier has a bilayer, consisting of an inner layer and an outer layer, and the bilayer carrier is prepared by cross-linking and coating again after freeze-drying.

[0023] The outer layer is a pH-responsive layer, using low-molecular-weight CMCS with a high proportion of alkylation, while the inner layer is a long-acting sustained-release layer, using high-molecular-weight CMCS with a low proportion of alkylation.

[0024] The preparation method of the above-mentioned nebulized solution for treating dry eye containing stem cell exosomes is as follows: after mixing the nanocarrier loaded with exosomes, osmotic pressure regulator, pH regulator and solvent, it is dispensed into sterile containers, sealed and stored, and protected from light and high temperature.

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

[0026] Firstly, exosomes are used as the main drug, and chitosan-carboxymethyl chitosan serves as the carrier, enabling the exosomes to exert a sustained-release effect and remain for a long time even in a high-salt, neutral environment. The carboxylic acid groups of carboxymethyl chitosan bind to the glycosyl groups of ocular surface mucins through hydrogen bonds or electrostatic interactions, prolonging the carrier's residence time on the corneal surface and resisting tear washout. Furthermore, carboxymethyl chitosan itself has high viscosity, which can extend its residence time at the site of action.

[0027] Secondly, by using a combination of CMCSs with different molecular weights, the structural and functional differences of the multi-level structure of the carrier can be regulated through molecular weight differences, thereby achieving efficient loading and intelligent sustained release of exosomes.

[0028] Thirdly, by alkylating the CMCS combination, the drug loading capacity is increased, while the drug release rate is increased in patients with severe dry eye syndrome, so as to meet the needs of patients with different conditions.

[0029] Fourth, through the dual-layer carrier design, the outer layer of low molecular weight, high alkylated CMCS enables rapid response in acidic environments, while the inner layer of high molecular weight, low alkylated CMCS ensures stable release in the mid-to-late stages, significantly optimizing the uniformity of drug release rate. Attached Figure Description

[0030] Figure 1 This is a diagram showing the drug release experiment results in Example 1 of the present invention;

[0031] Figure 2 This is a diagram showing the experimental results of ion influence in Example 1 of the present invention;

[0032] Figure 3 The figure shows the drug release experimental results of two different molecular weight CMCS combinations in the embodiments of the present invention. Detailed Implementation

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] Example 1: This application provides an atomizing solution for treating dry eye syndrome containing stem cell exosomes, the components of which include: a nanocarrier loaded with exosomes, an osmotic pressure regulator, a pH regulator, and a solvent.

[0035] The osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, and glucose, with a concentration of 0.85%-1.5%; so that the osmotic pressure of the nebulizer is close to or slightly higher than that of physiological saline.

[0036] The pH adjuster is selected from phosphate buffer or Tris buffer to adjust the pH of the nebulizer to 7.2-7.4;

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

[0038] The nanocarriers loaded with exosomes are made of chitosan-carboxymethyl chitosan polymer loaded with exosomes, and the concentration in the atomizing solution is 30-100 μg / mL; the exosomes are mesenchymal stem cell exosomes;

[0039] The specific steps for preparing nanocarriers loaded with exosomes are as follows:

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

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

[0042] S3. Then, the chitosan-polyethylene glycol block copolymer was dissolved again in ethanol, and carboxymethyl chitosan and exosome solution were added, along with sodium tripolyphosphate as a crosslinking agent. The mixture was stirred and freeze-dried at -20°C to obtain the nanocarrier loaded with exosomes.

[0043] The molar ratio of chitosan, ethylene oxide, and hexamethylene diisocyanate is 1:0.3:0.5.

[0044] The mass ratio of chitosan-polyethylene glycol block copolymer, carboxymethyl chitosan, and 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 is 3kDa, and the average molecular weight of the carboxymethyl chitosan used is 30kDa.

[0047] The technical solutions described in the embodiments of this application have at least 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 exert a sustained-release effect and be retained for a long time in a high-salt neutral environment. Thus, this embodiment 1 actually solves the problem of sudden release caused by rapid dissociation of exosome nebulized liquid in tears due to electrostatic effect.

[0049] Exosomes, as natural drug carriers, possess unique structures and functions that enable long-distance transport within the body under physiological and pathological conditions. They exhibit good stability, natural targeting, and the ability to cross the corneal barrier. They carry growth factors (such as VEGF and bFGF) and other molecules beneficial to tissue regeneration, promoting the proliferation and regeneration of damaged ocular cells and accelerating the repair of ocular tissues. They also inhibit inflammatory responses, reducing ocular redness and pain. For example, miR-204 in exosomes can inhibit the IL-6 / IL-6R / Stat signaling pathway, causing macrophages to shift from M1 (pro-inflammatory) to M2 (anti-inflammatory), thereby reducing the inflammatory response.

[0050] Carboxymethyl chitosan is a cationic biopolymer containing a large number of highly hydrophilic amino groups, giving it a net positive charge. This promotes its interaction with various negatively charged polymers. Carboxymethyl chitosan partially dissociates into -COO- at physiological pH. -It forms a dynamic bond with the amino group (-NH2) in the chitosan-polyethylene glycol block copolymer through hydrogen bonding 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 and maintain the binding force between the carrier and the exosome. This reduces the shielding effect caused by excessively high ion concentration, thus preventing drug burst release.

[0051] The cross-linking agent sodium tripolyphosphate (negatively charged) reacts with the amino group (-NH3) of chitosan. + This forms an ion cross-linking network, further stabilizing the carrier structure and reducing exosome burst release; it also prevents drug burst release caused by carrier collapse due to enzymatic degradation.

[0052] The carboxylic acid groups of carboxymethyl chitosan bind to the glycosyl groups of ocular surface mucins through hydrogen bonds or electrostatic interactions, prolonging the retention time of the carrier on the corneal surface and resisting tear washout. Furthermore, carboxymethyl chitosan itself has high viscosity, which can prolong its residence time at the site of action.

[0053] First, an experiment was conducted to investigate the therapeutic effect of exosomes on dry eye syndrome. An experimental group and a control group were set up. The control group received no treatment. In the experimental group, ocular symptoms (degree of dryness, foreign body sensation) and tear secretion (Schirmer test) were assessed before treatment, at 2 weeks, and at 4 weeks. The treatment method involved nebulization therapy once daily using a solution composed of exosomes, an osmotic pressure regulator, a pH regulator, and a solvent; the exosome concentration was 1×10⁻⁶. 8 -1×10 10 particles / mL; the remaining components are the same as in Example 1, and the results are shown in Table 1;

[0054]

[0055] Table 1

[0056] After learning that exosomes have a therapeutic effect on dry eye, an experiment was conducted to test the sustained-release effect of the nanocarrier loaded with exosomes in Example 1. Different proportions of C-PEG and CMCS composite carrier (without exosomes) were dispersed in simulated tear fluid (0.9% NaCl, pH 7.4 PBS) and diluted to 0.1 mg / mL. The zeta potential (mV) was detected using a zeta potential analyzer (Malvern Zetasizer Nano ZS) at 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(+) dominant, with weak electrostatic binding. 1:1 -3.5±0.8 Dynamic equilibrium between carboxylic acid group (-) and amino group (+) 1:1.5 -12.4±1.2 The carboxylic acid group (-) is dominant, but the cross-linked network is stable. 1:2 -18.6±2.1 Carboxylic acid group (-) overload enhances electrostatic shielding effect.

[0058] Table 2

[0059] Cy5-labeled exosomes were loaded onto a carrier for in vitro sustained-release experiments. The prepared Cy5-labeled exosome-loaded nanocarriers were placed in a release medium, which was a simulated tear solution (0.9% NaCl, pH 7.4, PBS + 0.1% mucin). The carriers loaded with fluorescently labeled exosomes were dispersed in the medium and shaken at 37°C (100 rpm). Samples were taken at 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h, centrifuged (10,000 rpm, 10 min), and the supernatant was collected. The exosome concentration was detected using a fluorescence spectrophotometer to obtain the drug release results. The results are as follows: Figure 1 ;

[0060] An experiment was conducted to investigate the effect of salt concentration. A gradient salt concentration was set up to simulate varying ion concentrations. Specifically, the salt gradient was set to 0.5%, 0.9%, and 1.5% NaCl (pH 7.4, solvent PBS). The exosome release rate of each carrier group under the salt gradient for 8 hours was measured (using the same method as the in vitro sustained-release experiment). The results are as follows: Figure 2 ;

[0061] The optimal ratio is C-PEG:CMCS = 1:1.5. At this concentration, the electrostatic effect still exists to some extent and will not lead to a burst release of the drug. 58.6% is released after 8 hours, the half-life is 5.8 hours, and the loss rate is 18.4 / h. It still has good sustained-release ability even under high ion concentrations caused by the patient's pathological state.

[0062] Example 2: The above example uses chitosan-carboxymethyl chitosan as a carrier, which enables exosomes to exert a sustained-release effect and be retained for a long time in a high-salt neutral environment. To enhance the adsorption and release capacity of the carrier, further improvements were made based on Example 1.

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

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

[0065] By using a combination of CMCSs with different molecular weights, the structural and functional differences of the multi-level structure of the carrier are regulated by molecular weight differences, thereby achieving efficient loading and intelligent sustained release of exosomes. Thus, the problem solved by this embodiment is the insufficient adsorption capacity and the mismatch between adsorption capacity and sustained release capacity.

[0066] High molecular weight CMCSs, acting as a "structural framework," provide high charge density and strong cross-linking ability. Their high molecular weight and high density of carboxylic acid groups form stable electrostatic bonds with the amino groups of the chitosan-polyethylene glycol block copolymer, resisting the ion shielding effect in tears. Long-chain CMCSs form a dense network with the cross-linking agent, reducing carrier porosity and preventing exosome burst release. Low molecular weight CMCSs, acting as a "release regulator," form dynamic pores and promote exosome diffusion. Short-chain CMCSs form micropores or channels within the carrier, increasing specific surface area and enhancing exosome loading. The high flexibility of low molecular weight CMCS segments allows them to easily swell in the tear environment, expanding pore size and gradually releasing exosomes. This release gradually decreases with electrostatic and hydrogen bonding effects, resulting in a progressive release pattern: initial release through large pores, mid-stage release through electrostatic hydrogen bonding, and final release accelerated by CMCS swelling and pore expansion.

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

[0068] In vitro sustained-release experiments were conducted using the carrier prepared in Example 2, with sampling times of 0.5h, 2h, 4h, 8h, 12h, and 24h; the drug release results are as follows. Figure 3 .

[0069] Example 3: Example 2 optimized the release rate of exosomes by using a combination of CMCS with different molecular weights. However, in actual use, the tears of patients with severe dry eye are acidic, and the release rate is increased by an average of 30% due to carrier swelling, especially in the later stage where the release rate increases by more than 65%. This leads to a decrease in the uniformity of the sustained release rate and easily causes large fluctuations in drug concentration in the later stage when the drug concentration is stable. In order to optimize the drug release rate in an acidic environment, further improvements were made based on Example 2.

[0070] Carboxymethyl chitosan is also partially alkylated, with alkylated carboxymethyl chitosan accounting for 15% of the total carboxymethyl chitosan used. The alkylation process is as follows: CMCS combinations of different molecular weights are dissolved in dimethyl sulfoxide, excess dodecane bromo and alkaline catalyst sodium hydroxide are added, and the reaction is carried out at 60℃-80℃ for 12-24 hours to obtain alkylated CMCS.

[0071] The reaction formula is:

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

[0073] By alkylating the CMCS combination, the drug release rate is increased in patients with severe dry eye syndrome and inflammation, thereby adapting to the needs of patients with different conditions. Thus, the actual problem solved by this embodiment is how to achieve sustained and controlled release for patients with severe inflammation and how to automatically change the release amount according to the condition.

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

[0075] Alkylation of low molecular weight CMCS introduces hydrophobic segments, enhancing intermolecular hydrophobic interactions. Under normal physiological tear conditions, the molecular chains fold or entangle, forming a dense structure. This causes exosomes to be encapsulated by molecular entanglement under electrostatic and hydrogen bonding, reducing release rates in physiological environments. In acidic environments, hydrophobic segments induce local phase separation, gradually reducing the degree of molecular chain entanglement and releasing encapsulated exosomes, increasing release rates in the early and mid-stages. Alkylation of high molecular weight CMCS improves the mechanical strength of the carrier and drug encapsulation efficiency. In acidic environments, carboxyl groups are protonated, reducing swelling, but the hydrophobic cross-linked network partially dissociates, forming microcracks. Drugs diffuse rapidly through these cracks, significantly affecting release rates in the mid and late stages. Due to steric hindrance, the degree of alkylation of low molecular weight CMCS is higher than that of high molecular weight CMCS. Therefore, during release, the actual release rate varies depending on the patient's pathological state in the early stage, and tends to stabilize in the later stage.

[0076] Based on the high molecular weight CMCS (50kDa) + low molecular weight CMCS (10kDa) group in Example 2, an in vitro sustained-release experiment was conducted using the carrier prepared in Example 3. 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 alkylated (Example 3) and non-alkylated (Example 2) were compared. The sampling times were 0.5h, 2h, 4h, 8h, 12h, and 24h. The drug release results are shown in Table 3.

[0077]

[0078] Table 3

[0079] Example 4: Example 3 optimized adaptive release under inflammatory conditions through alkylation. To further optimize the release rate, further improvements were made based on Example 3.

[0080] The carrier has multiple layers and is prepared by cross-linking and coating again after freeze-drying; 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 (5-10kDa) + high proportion alkylation (20% bromododecane modification);

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

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

[0084] Through a dual-layer carrier design, the outer layer of low molecular weight, highly alkylated CMCS enables rapid response in acidic environments, while the inner layer of high molecular weight, low alkylated CMCS ensures stable release in the mid-to-late stages, significantly optimizing the uniformity of drug release rate. Thus, this embodiment solves the problem that the drug release amount cannot be sustained according to the actual drug absorption or metabolism stage.

[0085] The outer layer, with its high alkylation, responds rapidly in inflammatory acidic environments. It forms pores through hydrophobic segment phase separation, reducing entanglement and accelerating initial drug release to alleviate acute inflammation. The inner layer dynamically dissociates through a hydrophobic crosslinking network, forming microcracks. The release rate is stable in the middle and later stages, and the high molecular weight network resists tear erosion, prolonging the carrier's retention time.

[0086] It caters to patients with different pathological states due to different causes, as well as patients with different conditions, and promotes the uniform release of exosomes.

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

[0088] Time (h) Monolayer carrier (alkylation) release rate (%) Release rate of the dual-layer 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 description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nebulizing solution containing stem cell exosomes for treating dry eye syndrome, characterized in that, Its components include: nanocarriers loaded with exosomes, osmotic pressure regulators, pH regulators, and solvents; The nanocarriers loaded with exosomes are made of chitosan-carboxymethyl chitosan polymer loaded with exosomes, with a concentration of 30-100 μg / mL in the atomizing solution; the exosomes are mesenchymal stem cell exosomes; The molecular weight of chitosan is 3 kDa, while the average molecular weight of carboxymethyl chitosan used is 5-50 kDa. The specific steps for preparing nanocarriers loaded with exosomes are as follows: S1. Dissolve chitosan in ethanol, add ethylene oxide and react at 55-65℃ for 20-26h to obtain chitosan-polyethylene glycol copolymer. S2. Dissolve the chitosan-polyethylene glycol copolymer in dimethyl sulfoxide, add hexamethylene diisocyanate and react at 75-85℃ for 5-8 hours to obtain the chitosan-polyethylene glycol block copolymer. S3. Then, the chitosan-polyethylene glycol block copolymer was dissolved in ethanol again, and carboxymethyl chitosan and exosome solution were added, along with sodium tripolyphosphate as a crosslinking agent. The mixture was stirred and freeze-dried at -20°C to obtain the nanocarrier loaded with exosomes. The mass ratio of chitosan-polyethylene glycol block copolymer, carboxymethyl chitosan, and exosome solution was 1:(0.5-2):1, and the exosome concentration in the exosome solution was 1×10⁻⁶. 7 -1×10 9 particles / mL; Carboxymethyl chitosan includes long-chain carboxymethyl chitosan and short-chain carboxymethyl chitosan. The molecular weight of long-chain carboxymethyl chitosan is 5-10 kDa, and the molecular weight of short-chain carboxymethyl chitosan is 20-50 kDa. The mass ratio of long-chain carboxymethyl chitosan to short-chain carboxymethyl chitosan is 7:

3. Carboxymethyl chitosan undergoes partial alkylation, with alkylated carboxymethyl chitosan accounting for 15% of the total carboxymethyl chitosan used.

2. The nebulizing solution for treating dry eye syndrome containing stem cell exosomes as described in claim 1, characterized in that, The osmotic pressure regulator is selected from one or more of sodium chloride, mannitol, and glucose, with a concentration of 0.85%-1.5%; so that the osmotic pressure of the nebulizer is close to that of physiological saline. The pH regulator is selected from one of phosphate buffer and Tris buffer, and the pH value of the nebulizer is adjusted to 7.2-7.

4.

3. The nebulizing solution for treating dry eye syndrome containing stem cell exosomes as described in claim 1, characterized in that, The solvent is a biocompatible solvent.

4. The nebulizing solution for treating dry eye syndrome containing stem cell exosomes as described in claim 3, characterized in that, The biocompatible solvents include physiological saline and deionized water.

5. The nebulizing solution for treating dry eye syndrome containing stem cell exosomes as described in claim 1, characterized in that, The alkylation process is as follows: carboxymethyl chitosan combinations of different molecular weights are dissolved in dimethyl sulfoxide, excess dodecane bromo and alkaline catalyst sodium hydroxide are added, and the reaction is carried out at 60℃-80℃ for 12-24 hours to obtain alkylated carboxymethyl chitosan.

6. The nebulizing solution for treating dry eye syndrome containing stem cell exosomes as described in claim 5, characterized in that, The carrier has two layers, including an inner layer and an outer layer, and the two-layer carrier is prepared by freeze-drying and then cross-linking and coating again. The outer layer is a pH-responsive layer, using low molecular weight carboxymethyl chitosan with a high proportion of alkylation, while the inner layer is a long-lasting sustained-release layer, using high molecular weight carboxymethyl chitosan with a low proportion of alkylation.

7. A method for preparing a nebulizing solution containing stem cell exosomes for treating dry eye as described in any one of claims 1-6, characterized in that, The preparation method is as follows: after mixing the nanocarrier loaded with exosomes, osmotic pressure regulator, pH regulator and solvent, the mixture is dispensed into sterile containers, sealed and stored, and protected from light and high temperature.

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