Preparation method and delivery system of drug for targeting central nervous system in nose

Abstract

The invention discloses a preparation method of an intranasal targeted central nervous system drug, which comprises the following steps: dissolving chitosan in an acetic acid solution, and stirring to obtain a transparent chitosan solution with the concentration of 1.6-2.0% w / v; the preparation method comprises the following steps: adding ultra-pure water into poly (N-isopropylacrylamide) dry powder in a low-temperature state, slowly heating and stirring until the mixture is clarified, cooling to the low-temperature state, and complementing the mixture until the mass-volume ratio is 4-5.5% w / v; mixing the chitosan transparent solution and the poly (N-isopropylacryloyl) solution in a low-temperature state to generate a composite solution, and adding the beta-sodium glycerophosphate solution into the composite solution by using a micro pump to enable the volume ratio of the composite solution to the beta-sodium glycerophosphate solution to be (4: 1)-(5.5: 1); in a low-temperature state, dropwise adding a PEG400 pre-solution into the composite solution, so that the final concentration of the PEG400 reaches 0.05%-0.1% v / v; taking an exosome solution, slowly injecting the exosome solution into the composite solution at the speed of 2mL per minute, and continuously stirring to finally enable the exosome concentration to reach 80-150mu g / mL.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a preparation method and a delivery system for a drug targeting the central nervous system for intranasal use. Background Art

[0002] Diseases of the central nervous system (CNS), especially spinal cord injury (SCI), are highly complex diseases, and their treatment remains a difficult point in global medical research to date. SCI is usually directly triggered by trauma to cause primary injury, and then develops into more destructive secondary injury. The main characteristics of secondary injury include inflammatory response, blood-spinal cord barrier (BSCB) disruption, oxidative stress, and neuronal apoptosis. These pathological processes not only exacerbate the loss of spinal cord function, but also seriously hinder the repair and functional recovery of nerve tissue. Therefore, how to effectively alleviate the inflammatory microenvironment, protect neurons, and promote tissue repair has become the key goal of SCI treatment.

[0003] The blood-spinal cord barrier is a highly selective barrier structure located between the blood and the nerve tissue of the central nervous system, which is composed of a variety of tightly connected cell complexes and has extremely low permeability. This barrier effectively restricts the entry of exogenous substances through the synergistic action of tight junction proteins, transporters, and metabolic enzymes. Due to these characteristics, approximately 98% of small molecule chemical drugs and almost all macromolecular drugs (such as protein, polypeptide drugs, and gene therapy drugs) are difficult to cross the blood-spinal cord barrier, and thus cannot reach an effective therapeutic concentration in the spinal cord. This barrier function plays an important role in maintaining the homeostasis of the central nervous system, but also poses a great challenge to drug treatment at the spinal cord position. How to effectively break through the blood-spinal cord barrier and achieve precise drug delivery in the nervous system has become a major issue in current drug research and development and neurology research. Traditional drug delivery methods such as intravenous injection or local injection have certain effects in SCI treatment, but there are significant limitations. Due to the liver and kidney capture effect and the barrier effect of BSCB in the intravenous injection method, most drugs cannot reach the central nervous system; although local injection can achieve a certain degree of targeted delivery, it is limited by the diffusion range of the injection area and the drug metabolism rate, and the persistence and stability are insufficient. The above limitations seriously affect the therapeutic effect of drugs, and there is an urgent need for an efficient and precise delivery system to meet the treatment needs of SCI and other central nervous system diseases.

[0004] Nasal drug delivery has become a non-invasive delivery strategy because it can bypass the blood-brain barrier (BBB) and the blood-spinal cord barrier (BSCB) and directly deliver drugs to the central nervous system. Through nasal mucosal diffusion or olfactory and trigeminal nerve pathways, drugs can quickly reach the target site. However, the drug absorption efficiency and residence time of nasal drug delivery are limited. In particular, the stability of exosomes in the nasal environment is poor and they are easily affected by mucus flow and clearance mechanisms, resulting in a significant reduction in drug utilization rate. Therefore, how to optimize the stability and delivery efficiency of exosomes in the nasal drug delivery route has become a key problem to be solved.

[0005] It can be seen that there is a need in the prior art for a new preparation method and delivery system for drugs targeting the central nervous system for intranasal use, so as to achieve precise and efficient release of nano-drug loading. Summary of the Invention

[0006] The present invention provides a preparation method and a delivery system for drugs targeting the central nervous system for intranasal use, aiming to overcome many problems existing in traditional drug delivery methods, especially the problems of low drug utilization rate and low delivery efficiency caused by the limitations of the blood-brain barrier (BBB) and the blood-spinal cord barrier (BSCB) during the treatment of central nervous system diseases. The present invention proposes an innovative delivery platform that can be widely applied to various central nervous system diseases.

[0007] Based on the above technical objectives, the present invention provides a preparation method for drugs targeting the central nervous system for intranasal use, and the method includes:

[0008] Take chitosan with a molecular weight of 180 - 220 kDa and dissolve it in an acetic acid solution with a concentration of 1% v / v, and obtain a transparent chitosan solution with a concentration of 1.6% - 2.0% w / v through stirring;

[0009] Add poly(N-isopropylacrylamide) dry powder to ultrapure water at a low temperature, slowly heat it to and stir until it becomes clear, and then cool it back to the low temperature state, and make up the mass-volume ratio to 4% - 5.5% w / v to generate a poly(N-isopropylacrylamide) solution;

[0010] Mix the transparent chitosan solution and the poly(N-isopropylacrylamide) solution at a volume ratio of 2:1 at a low temperature to generate a composite solution, specifically including: dropwise add the poly(N-isopropylacrylamide) solution to the transparent chitosan solution at a predetermined speed and continuously stir;

[0011] Use a micro-pump to add a 50% w / v β-glycerophosphate solution to the composite solution at a predetermined rate so that the volume ratio of the composite solution to the β-glycerophosphate solution reaches 4:1 - 5.5:1, and let it stand to remove bubbles;

[0012] At a low temperature state, a PEG400 pre-solution is added dropwise to the composite solution to make the final PEG 400 concentration reach 0.05% - 0.1% v / v;

[0013] Take the exosome solution and slowly inject it into the composite solution at a rate of 2 mL per minute while continuously stirring, until the final exosome concentration reaches 80 - 150 μg / mL.

[0014] In one embodiment, the low temperature state refers to a temperature less than or equal to 4°C.

[0015] In one embodiment, the molecular weight of the poly(N-isopropylacrylamide) is 40KDa.

[0016] In one embodiment, the stirring speed when generating the chitosan solution is 200 rpm, and continuous stirring is carried out for more than 12 hours.

[0017] In one embodiment, when generating the poly(N-isopropylacrylamide) solution, it is slowly heated to 25°C and stirred for more than 30 minutes until it becomes clear.

[0018] In one embodiment, the poly(N-isopropylacrylamide) solution is added dropwise to the transparent chitosan solution at a rate of 10 - 20 mL / min.

[0019] In one embodiment, the β-glycerophosphate solution is added to the composite solution at a rate of 5 mL per minute until the pH reaches 6.7.

[0020] In one embodiment, when adding the PEG400 pre-solution to the composite solution, the stirring speed does not exceed 100 rpm, and stirring is carried out for 5 - 10 minutes.

[0021] The present invention also provides a drug for intranasal targeting of the central nervous system, the drug comprising: a thermosensitive hydrogel and an exosome nano-biological component; the thermosensitive hydrogel is cross-linked by chitosan, poly(N-isopropylacrylamide), β-glycerophosphate, and PEG400. The preparation of the drug uses the aforementioned preparation method.

[0022] The present invention also provides a drug delivery system for intranasal targeting of the central nervous system, the drug delivery system comprising removing bubbles from the aforementioned drug for intranasal targeting of the central nervous system by low-speed centrifugation, and then aseptically packaging the drug into a sterilized nasal sprayer under aseptic operation conditions.

[0023] Other features and advantages of the present invention will be described in the subsequent specification, and, in part, will be obvious from the specification, or will be understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained by the structures specifically pointed out in the specification, claims, and drawings. Brief Description of the Drawings

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention, but do not constitute a limitation to the present invention. In the accompanying drawings:

[0025] Figure 1 is a schematic diagram of the thermosensitivity curve of the chitosan thermosensitive hydrogel of the first embodiment of the present invention;

[0026] Figure 2 is the exosome release curve of the chitosan thermosensitive hydrogel of the first embodiment of the present invention. Detailed Embodiments

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the following further elaborates on the present invention in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] In the present invention, those not specified with specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer; for reagents or instruments not specified with the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0029] Example 1

[0030] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0031] 1. Raw Materials and Main Parameters

[0032] Chitosan: molecular weight 200 kDa, deacetylation degree ≥ 90%, target preparation concentration 1.75% (w / v).

[0033] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0034] PNIPAAm (Poly-N-isopropylacrylamide): molecular weight 40 kDa, purity ≥ 96%, final concentration 5.0% (w / v).

[0035] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm mixture:β-GP = 5:1 (v / v).

[0036] PEG 400: final concentration 0.1% (v / v).

[0037] Exosomes (reprogrammed macrophage exosomes): final concentration 100 μg / mL, addition rate 2 mL / min.

[0038] Reaction environment: 4°C throughout the process.

[0039] 2. Specific preparation steps

[0040] 2.1 Preparation of chitosan solution:

[0041] Dissolve chitosan in 1% (v / v) acetic acid solution, stir at 200 rpm for 12 hours at 4°C to obtain a transparent solution of 1.75% (w / v).

[0042] 2.2 Preparation of PNIPAAm solution:

[0043] Add PNIPAAm dry powder to ultrapure water at 4°C, slowly warm up to 25°C and stir for 30 minutes until clear; then cool back to 4°C and make up the volume to 5.0% (w / v). Turbidity detection < 10 NTU indicates a homogeneous solution.

[0044] 2.3 Preliminary compounding:

[0045] Mix the chitosan solution and the PNIPAAm solution at a volume ratio of 2:1 in an ice bath, add the PNIPAAm solution dropwise at a rate of 10–20 mL / min, and stir at 150 rpm. Monitor and adjust the pH online to 6.2.

[0046] 2.4 Addition of β-GP:

[0047] Use a micropump to add 50% (w / v) β-GP to the composite solution at a rate of 5 mL per minute until the pH reaches 6.7. Let it stand overnight (12 hours) at 4°C to remove bubbles.

[0048] 2.5 Addition of spray aid:

[0049] Add the PEG 400 pre-solution dropwise to the composite solution at 4°C to make the final PEG 400 concentration 0.1% (v / v), and stir for 5 minutes at 100 rpm.

[0050] 2.6 Addition of exosome loading:

[0051] Take the reprogrammed macrophage exosome solution (concentration 1 mg / mL), slowly inject it at a rate of 2 mL per minute, and stir at 50 rpm. The final exosome concentration is about 100 μg / mL.

[0052] 2.7 Loading of the spray device:

[0053] After removing bubbles by low-speed centrifugation (100 g, 2 minutes), aliquot with a sterile syringe into a nasal sprayer and store at 4°C.

[0054] 3. Results and analysis:

[0055] 3.1 Gelation temperature and rate: Rapid gelation occurs within 1 - 2 minutes at 34°C. Its thermosensitive properties are as followsFigure 1 as shown

[0056] 3.2 Spray performance: At 4°C, the droplet size is about 50 - 100 μm, and the atomization is uniform.

[0057] 3.3 Exosome release detection: The cumulative release exceeds 80% on the 7th day. Considering that some exosomes are inactivated and degraded during the process, the actual active exosomes are also close to this level, which has reached the optimal therapeutic effect range. After 10 days, the cumulative release changes little, indicating that there is not much remaining exosomes. As Figure 2 shown

[0058] Conclusion: The thermosensitive hydrogel spray obtained under this formulation has ideal nasal spray performance and rapid gelation characteristics, and can effectively release 80% of exosomes within 7 days, which is the optimal condition example of the present invention.

[0059] Example 2

[0060] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0061] 1. Raw Materials and Main Parameters

[0062] Chitosan: Molecular weight 200 kDa, deacetylation degree ≥ 90%, target preparation concentration 1.6% (w / v).

[0063] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05, used for preparing chitosan solution.

[0064] PNIPAAm (Poly N-isopropylacrylamide): Molecular weight 40 kDa, purity ≥ 96%, final concentration 5.0% (w / v).

[0065] β-Glycerophosphate (β-GP): 50% (w / v), according to chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0066] PEG 400: Final concentration 0.1% (v / v).

[0067] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min.

[0068] Reaction environment: Keep the operation at 4°C throughout the process to prevent premature gelation.

[0069] 2. Specific Preparation Steps

[0070] 2.1 Preparation of Chitosan Solution

[0071] Weigh the chitosan powder and add it to an acetic acid solution with a concentration of 1.0% (v / v) and a pH of 3.0 ± 0.05 to prepare a chitosan solution with a concentration

[0072] of 1.6% (w / v); under the condition of 4°C, stir at 200 - 300 rpm for 12 hours to completely dissolve the chitosan, obtaining a transparent and uniform low-concentration chitosan solution.

[0073] Preparation of 2.2 PNIPAAm solution

[0074] Take the PNIPAAm dry powder and add it to ultrapure water at 4°C, and initially stir at 100 rpm; slowly raise the temperature to 25°C and change to stirring at 200 rpm for about 30 minutes. After the solution becomes clear, lower the temperature back to 4°C; finally, make up to a concentration of 5.0% (w / v) and confirm that the turbidity < 10 NTU to ensure no obvious microcoagulation.

[0075] 2.3 Preliminary compounding

[0076] Mix the above chitosan solution (1.6%) and PNIPAAm solution (5.0%) at a volume ratio of 2:1, and keep the operating temperature at 4°C or in an ice bath environment; slowly drip the PNIPAAm solution at a rate of 10 - 20 mL / min, and the stirring speed is about 150 rpm; monitor the pH online. If the pH < 6.0, drip 0.1M NaOH; if > 6.2, add 1% acetic acid solution, and finally control it at about pH = 6.2.

[0077] 2.4 Addition of β-GP solution

[0078] Prepare a 50% (w / v) β-GP solution and sterilize it through a 0.22 μm filter membrane; use a micro pump to gradually drip it into the composite solution at a rate of about 5 mL / min while stirring at a low speed (100 - 150 rpm); stop adding the material when the pH value rises to 6.7; if it is still insufficient, a small amount of NaOH can be dripped for adjustment; place the mixed solution at 4°C and let it stand for 12 hours to facilitate the discharge of air bubbles and the stability of the system.

[0079] 2.5 Addition of the spray aid PEG 400

[0080] Prepare a 1.5% (v / v) PEG 400 pre-solution and place it at 4°C; slowly drip it into the composite solution, and the stirring speed does not exceed 100 rpm. Stir for 5 - 10 minutes to make the final concentration of PEG 400 reach 0.1% (v / v); this step can improve the sprayability, but has limited effect on the chitosan system with a lower concentration.

[0081] 2.6 Exosome loading

[0082] Add the exosome (1 mg / mL) solution to the prepared hydrogel solution at a rate of 2 mL / min; reduce the stirring rate to 50 rpm to ensure that the exosomes are evenly dispersed without being damaged. The final exosome concentration is about 100 μg / mL.

[0083] 2.7 Filling of the spray device

[0084] After removing bubbles by low-speed centrifugation (100 g, 2 minutes), under aseptic conditions, the hydrogel solution is dispensed into a sterilized nasal sprayer. Check the seal and store at 4°C.

[0085] 3. Results and analysis

[0086] 3.1 Gelation time and temperature

[0087] Under the condition of 34°C, it takes about 5 minutes to start forming an obvious gel, which is significantly delayed compared with 2 - 3 minutes when the chitosan concentration is 1.75%. The slower gelation speed indicates that the decrease in chitosan concentration weakens the ability of the network to form rapidly.

[0088] 3.2 Mechanical properties

[0089] Compared with Example 1 (chitosan 1.75%), due to the lower chitosan content, the mechanical strength and elastic modulus of the gel in this example have significantly decreased. Rheological tests show that the storage modulus (G′) decreases by about 10 - 20%, and the retention in the application scenario is slightly weaker.

[0090] 3.3 Exosome release

[0091] The exosome release curve was monitored using simulated body fluid (37°C): The cumulative release was about 70% in 7 days, which is lower than 80% of the best example.

[0092] 3.4 Spraying performance

[0093] When spraying at a low temperature of 4°C, it can be atomized normally, but due to a relatively large decrease in the solution viscosity, the droplet size distribution is slightly larger than that of Example 1, and the spraying uniformity decreases slightly.

[0094] Comprehensive evaluation

[0095] Slower gelation speed: It takes 4 - 5 minutes to initially gel, reducing the advantage of rapid gelation; Decreased mechanical properties: The gel stability also weakens, and it may have a shorter maintenance time in the local environment; The 7-day release amount of exosomes can reach 70%, which is relatively close to the target, but it is not ideal for occasions that require higher mechanical support or a faster gelation speed. In this example, after the chitosan concentration was reduced from 1.75% to 1.6%, obvious degradation such as an extended gelation time and reduced mechanical properties occurred, and the overall performance was significantly inferior to the optimal example. Although the cumulative 7-day release amount of exosomes is 70%, close to 80%, due to the influence on both the gelation speed and the gel strength, the clinical practical operability and usage effect are slightly inferior to the best scheme.

[0096] Example 3

[0097] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0098] 1. Raw Materials and Main Parameters

[0099] Chitosan: Molecular weight is about 200 kDa, deacetylation degree ≥ 90%, and the preparation concentration is 2.0% (w / v).

[0100] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0101] PNIPAAm (Poly N-isopropylacrylamide): Molecular weight 40 kDa, purity ≥ 96%. In this example, the preparation concentration is 5.5% (w / v) (higher than 5.0% in Example 1).

[0102] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0103] PEG 400: Final concentration 0.1% (v / v).

[0104] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min.

[0105] Operating temperature: 4°C throughout the process.

[0106] 2. Specific Preparation Steps

[0107] 2.1 Preparation of PNIPAAm Solution (5.5%)

[0108] Weigh the PNIPAAm dry powder and add it to ultrapure water in batches at 4°C. The initial stirring speed is 100 - 150 rpm. Slowly heat up to 25°C and then change to stirring at 200 rpm for about 30 minutes until the solution becomes clear. Cool back to 4°C and make up the volume to 5.5% (w / v). If necessary, measure the turbidity <10 NTU to ensure uniformity.

[0109] 2.2 Preparation of chitosan solution (2.0%)

[0110] Dissolve the chitosan powder in 1.0% (v / v) acetic acid solution (pH = 3.0 ± 0.05) to prepare a 2.0% (w / v) solution. Stir at 200 - 300 rpm at 4°C for 12 hours until a transparent and homogeneous solution is formed.

[0111] 2.3 Preliminary chitosan / PNIPAAm composite (volume ratio 2:1)

[0112] In an ice bath environment, mix the chitosan solution (2.0%) and the PNIPAAm solution (5.5%) at a volume ratio of 2:1. The dropping rate is about 10 - 20 mL / min and the stirring speed is 150 rpm. Monitor online with a pH meter: if pH < 6.0, add 0.1 M NaOH dropwise; if pH > 6.2, add 1% acetic acid, and finally control the pH at 6.2.

[0113] 2.4 Addition of β-GP solution

[0114] Inject the 50% (w / v) β-GP solution (filtered through 0.22 μm) into the above composite solution with a micro pump at a speed of about 5 mL / min; chitosan / PNIPAAm:β-GP = 5:1 (v / v), and the stirring speed is 100 - 150 rpm;

[0115] Stop adding materials when the pH value is about 6.7; place it at 4°C and let it stand for 12 hours to remove bubbles and homogenize the system.

[0116] 2.5 Addition of PEG 400 (0.1%)

[0117] Slowly add the pre-solution of PEG 400 (1.5% v / v) to the above composite solution at 4°C, and the stirring speed does not exceed 100 rpm; finally, the concentration of PEG 400 reaches 0.1%, and stir for about 5 - 10 minutes to reduce the interfacial tension and improve the sprayability.

[0118] 2.6 Exosome loading (100 μg / mL)

[0119] The exosome solution (1 mg / mL) was slowly injected at a rate of 2 mL per minute with stirring at 50 rpm; the temperature was maintained at 4 °C throughout the process to avoid thermal denaturation of exosomes; the final concentration was approximately 100 μg / mL to obtain an exosome-loaded sprayable hydrogel.

[0120] 2.7 Loading and storage

[0121] Low-speed centrifugation (100 g, 2 minutes) was used to remove microbubbles; under a sterile operating environment, the solution was dispensed into a sterilized nasal sprayer using a sterile syringe, ensuring no bubbles and good sealing; it was stored refrigerated at 4 °C to avoid premature gelation caused by a high-temperature environment.

[0122] 3. Results and analysis

[0123] 3.1 Viscosity and spraying operation

[0124] Viscosity increase: After the chitosan content was increased from 1.75% to 2.0%, the solution viscosity also increased significantly at 4 °C, and a greater thrust or an improved spray orifice was required for smooth spraying, and wire drawing or dripping was likely to occur.

[0125] Spraying uniformity: Due to the high viscosity, the droplet size distribution was wider and the atomization effect was slightly worse; PEG 400 (0.1%) was difficult to completely offset the wire-drawing tendency in a high-viscosity system.

[0126] 3.2 Gelation rate and mechanical strength

[0127] Gelation rate: In a 34 °C water bath, initial gelation occurred in about 2 - 3 minutes, which was similar to the standard example; however, due to the high viscosity, local gelation of the sprayer might occur during the operation process.

[0128] Mechanical strength: Increasing the chitosan content usually increased the crosslinking density and mechanical strength of the gel; rheological tests showed that the storage modulus (G′) was higher than that when the chitosan content was 1.75%, and the colloid was relatively tougher.

[0129] 3.3 Exosome release

[0130] Release efficiency: Release measurements were carried out at 37 °C, and the cumulative exosome release amount was 75% at 7 days; although the mechanical strength was higher, the denser network structure would hinder exosome diffusion; some exosomes might be trapped by the network or degraded and inactivated due to long retention in the later stage.

[0131] Release curve: The initial release was faster, and then due to the high chitosan content forming a dense structure, the diffusion rate slowed down significantly in the later stage.

[0132] Comprehensive evaluation

[0133] Increased operation difficulty: High viscosity leads to difficult spraying and cumbersome local operations; if the operation is carried out at room temperature (>25°C) for a slightly longer time, local premature gelation is likely to occur, blocking the nozzle. Suboptimal exosome release: Although the mechanical properties are enhanced after gelation, about 75% is released in 7 days; it is not conducive to the clinical need to maintain the optimal exosome concentration in the first 7 days. The chitosan 2.0% system has higher mechanical strength but sacrifices some release efficiency and ease of operation, and its overall performance is inferior to the optimal example (chitosan 1.75%).

[0134] Example 4

[0135] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0136] 1. Raw Materials and Main Parameters

[0137] Chitosan: Molecular weight is about 200 kDa, deacetylation degree ≥ 90%, and the prepared concentration is 2.5% (w / v).

[0138] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0139] PNIPAAm (Poly-N-isopropylacrylamide): Molecular weight 40 kDa, purity ≥ 96%, and the prepared concentration in this example is 5.0% (w / v).

[0140] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0141] PEG 400: Final concentration 0.1% (v / v).

[0142] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min.

[0143] Operating temperature: 4°C throughout the process.

[0144] 2. Specific Preparation Steps

[0145] 2.1 Preparation of chitosan solution (2.5%)

[0146] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 2.5% (w / v) chitosan solution; stir (200 - 300 rpm) for 12 hours at 4°C to ensure the solution is transparent and uniform.

[0147] 2.2 Preparation of PNIPAAm solution (5.0%)

[0148] Take the PNIPAAm dry powder and slowly add it to ultrapure water at 4°C. The initial stirring speed is 100 - 150 rpm; heat it to 25°C and continue to stir at 200 rpm for 30 minutes until the solution becomes clear; cool it back to 4°C and make up the volume to 5.0% (w / v); measure the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0149] 2.3 Preliminary Composite of Chitosan / PNIPAAm (Volume Ratio 2:1)

[0150] Mix the 2.5% chitosan solution and the 5.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min, and the stirring speed is ~150 rpm; monitor online with a pH meter: if pH < 6.0, add 0.1 M NaOH dropwise; if pH > 6.2, add 1% acetic acid to finally adjust to pH ≈ 6.2.

[0151] 2.4 Addition of β-GP Solution

[0152] Take the 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm, and the pH gradually rises to about 6.7 after addition; stop adding the material and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble-free.

[0153] 2.5 Addition of PEG 400 (0.1%)

[0154] Slowly drip the PEG 400 pre-solution (1.5% v / v) into the composite solution, and control the stirring rate at 100 rpm; stir for 5 - 10 minutes, and the final PEG 400 concentration is 0.1%, stir evenly to improve the sprayability and surface tension.

[0155] 2.6 Exosome Loading (100 μg / mL)

[0156] Slowly add the exosome solution (1 mg / mL) to the composite solution at a rate of 2 mL / min, and the stirring speed is 50 rpm; maintain the operating temperature at 4°C throughout the process to avoid exosome denaturation; the final exosome concentration is 100 μg / mL to ensure uniform dispersion.

[0157] 2.7 Sub-packaging and Storage

[0158] Centrifuge at low speed (100 g, 2 minutes) to remove microbubbles; under a sterile operating environment, sub-package the solution into a sterilized nasal sprayer; after sealing, store it at 4°C to avoid premature gelation caused by a high-temperature environment.

[0159] 3. Results and Analysis

[0160] 3.1 Exosome release (7-day cumulative amount)

[0161] 7-day exosome release: When tested in simulated body fluid (37°C), the cumulative release in 7 days was about 40%;

[0162] Due to the 5.0% PNIPAAm concentration and 2.5% chitosan concentration, the network structure of the hydrogel is very dense, which significantly limits the diffusion of exosomes. The release of exosomes in the hydrogel is inhibited, resulting in a slow release rate. The initial release of exosomes is small, and most of the release is concentrated in the later stage of 7 days. The exosomes are more inactivated, which is also one of the reasons for the low release.

[0163] Release curve:

[0164] The release curve showed a slow release process, with only 50% released within 7 days. This indicates that the dense network structure makes it more difficult to release exosomes and cannot provide a sustained therapeutic effect.

[0165] 3.2 Spray performance

[0166] Spray uniformity: Due to the high concentration of chitosan, the viscosity of the hydrogel solution is extremely high, resulting in poor spray performance. During the spray process, the droplet size is large, unevenly distributed, and the droplets are heavy, which affects the atomization effect of the spray. The spray droplet size is 100-200μm, which is much higher than the uniform range in the standard example. The spray coverage effect is poor, and it is difficult to achieve the ideal therapeutic effect.

[0167] 3.3 Gelation temperature and speed

[0168] Gelation temperature and speed:

[0169] Since the PNIPAAm concentration is 5.0% and the chitosan concentration is as high as 2.5%, the gelation temperature is about 36°C and the gelation time is relatively long, about 8 to 10 minutes. Too high a chitosan concentration results in a denser hydrogel network, a slower gelation rate, and difficulty in completing gelation in a short time.

[0170] Comprehensive evaluation

[0171] Insufficient release of exosomes: The high concentration of chitosan makes the hydrogel structure too dense, which makes the release of exosomes too slow, resulting in only about 50% release within 7 days. The release of exosomes is too slow, and the proportion of exosome activity loss is high, which affects the therapeutic effect.

[0172] Poor spray performance: The high viscosity of the hydrogel leads to poor spray performance, uneven droplet distribution, and poor spray effect, which affects the uniformity and effect of the treatment.

[0173] Slow gelation speed: Due to the too high concentration of chitosan, the hydrogel gels slowly at body temperature, and the gelation temperature is slightly higher than that of the standard example, and the gel formation is relatively slow, which affects the treatment reaction speed.

[0174] High-concentration chitosan (2.5%) results in an overly dense hydrogel network, and the release of exosomes is significantly inhibited. The release amount in 7 days is only 50%. The spraying performance is poor, the droplet size is uneven, and the spraying effect is poor, which affects the treatment effect. The gelation time is slow. Although it gels at body temperature, the formation process of the hydrogel is too slow, which affects the treatment effect and reaction time. The use of high-concentration chitosan leads to slow exosome release and inactivation, and fails to effectively provide a sustained slow-release effect. Therefore, the effect of this example is not ideal and belongs to a failed example.

[0175] Example 5

[0176] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0177] 1. Raw Materials and Main Parameters

[0178] Chitosan: Molecular weight: 30 kDa, degree of deacetylation: ≥90%, target preparation concentration: 1.75% (w / v)

[0179] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05

[0180] PNIPAAm: Molecular weight: 40 kDa, purity ≥96%, final concentration: 5.0% (w / v)

[0181] β-glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm mixture:β-GP = 5:1 (v / v).

[0182] PEG 400: Final concentration: 0.1% (v / v).

[0183] Exosomes (reprogrammed macrophage exosomes): Final concentration: 100 μg / mL, addition rate: 2 mL / min

[0184] Reaction environment: 4°C throughout the process

[0185] 2. Specific Preparation Steps

[0186] 2.1 Preparation of Chitosan Solution

[0187] Take chitosan and dissolve it in 1% (v / v) acetic acid solution, stir at a rotation speed of 200 rpm for 12 hours at 4°C to obtain a transparent solution (1.75% w / v).

[0188] 2.2 Preparation of PNIPAAm Solution

[0189] At 4 °C, PNIPAAm dry powder was added to ultrapure water, and the temperature was slowly raised to 25 °C and stirred for 30 minutes until clear; then it was cooled back to 4 °C, and the volume was made up to 5.0% (w / v). The turbidity was detected to be <10 NTU, indicating that the solution was homogeneous.

[0190] 2.3 Preliminary compounding

[0191] The chitosan solution and the PNIPAAm solution were mixed at a volume ratio of 2:1 in an ice bath. The PNIPAAm solution was added dropwise at a rate of 10–20 mL / min, and the stirring speed was 150 rpm. The pH was monitored online and adjusted to 6.2.

[0192] 2.4 Addition of β-GP

[0193] Using a micro pump, 50% (w / v) β-GP was added to the compound solution at a rate of 5 mL per minute until the pH reached 6.7. It was left standing at 4 °C overnight (12 hours) to remove bubbles.

[0194] 2.5 Addition of co-spraying agent

[0195] At 4 °C, the PEG 400 pre-solution was added dropwise to make the final PEG 400 concentration 0.1% (v / v), and it was stirred for 5 minutes at a speed of 100 rpm.

[0196] 2.6 Nano-drug loading

[0197] Take the reprogrammed macrophage exosome solution (concentration 1 mg / mL), slowly inject it at a rate of 2 mL per minute, and the stirring speed is 50 rpm. The final exosome concentration is about 100 μg / mL.

[0198] 2.7 Loading of the nasal spray device

[0199] After removing bubbles by low-speed centrifugation (100 g, 2 minutes), it was aliquoted into a nasal sprayer with a sterile syringe and stored at 4 °C.

[0200] 3. Results and analysis

[0201] 3.1 Gelation temperature and rate

[0202] Gelation temperature: Since the molecular weight of chitosan was relatively low (30 kDa), the hydrogel network was relatively loose, the gelation temperature increased to about 40 °C, and the gelation rate was slow. It took about 4 hours to form a stable hydrogel.

[0203] 3.2 Spraying performance

[0204] During the spraying process, the droplet size distribution was between 50 and 100 μm, and the atomization effect was uniform. The solution was easily ejected during spraying, and the drug was evenly distributed, which was suitable for nasal spray application.

[0205] 3.3 Exosome Release Detection

[0206] Release amount in 7 days: The release of exosomes is relatively fast in the first few days. However, due to the loose network structure of the hydrogel, the release amount gradually decreases in the later stage. Eventually, the cumulative release amount in 7 days is 50%. After 10 days, the cumulative release amount changes little, indicating that the remaining amount of exosomes is small.

[0207] Comprehensive Evaluation

[0208] The gelation temperature is relatively high, reaching 40 °C. Therefore, the formation of the hydrogel is slow, and the sustained-release effect cannot be fully maintained. The gelation speed is slow, the gelation process is relatively long, and the low cross-linking degree results in a relatively fast release rate of exosomes. Approximately 50% is released within 7 days, and the sustained-release effect is poor. The spraying performance is good, the droplet size is within the ideal range, and the atomization is uniform. The activity of exosomes decreases. Due to the loose structure of the hydrogel, the exosomes are released too fast, resulting in most of the exosomes being released in the early stage and insufficient sustained-release effect in the later stage. In this example, due to the reduction of the chitosan molecular weight to 30 kDa, the gelation temperature increases, the gelation speed is slow, and the hydrogel network is loose, failing to effectively control the release of exosomes. The cumulative release of exosomes within 7 days is only 50%, so the expected sustained-release effect cannot be provided, belonging to a failed example.

[0209] Example 6

[0210] Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes

[0211] 1. Raw Materials and Main Parameters

[0212] Chitosan: Molecular weight: 300 kDa, Degree of deacetylation: ≥90%, Target preparation concentration: 1.75% (w / v)

[0213] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05

[0214] PNIPAAm: Molecular weight: 40 kDa, Purity ≥96%, Final concentration: 5.0% (w / v)

[0215] β-Glycerophosphate (β-GP): 50% (w / v), Chitosan / PNIPAAm:β-GP = 5:1 (v / v)

[0216] PEG 400: Final concentration: 0.1% (v / v)

[0217] Exosomes (or other nano-drugs): Final concentration: 100 μg / mL, Addition rate: 2 mL / min

[0218] Reaction environment: 4 °C throughout the process

[0219] 2. Specific preparation steps

[0220] 2.1 Preparation of chitosan solution

[0221] Dissolve chitosan in 1% (v / v) acetic acid solution, stir at a speed of 200 rpm for 12 hours at 4°C to obtain a transparent solution (1.75% w / v).

[0222] 2.2 Preparation of PNIPAAm solution

[0223] Add PNIPAAm dry powder to ultrapure water at 4°C, slowly warm up to 25°C and stir for 30 minutes until clear; then cool back to 4°C and make up the volume to 5.0% (w / v). The turbidity detection is <10 NTU, indicating that the solution is homogeneous.

[0224] 2.3 Preliminary compounding

[0225] Mix the chitosan solution and the PNIPAAm solution at a volume ratio of 2:1 in an ice bath, add the PNIPAAm solution dropwise at a speed of 10–20 mL / min, and stir at a speed of 150 rpm. Monitor and adjust the pH online to 6.2.

[0226] 2.4 Addition of β-GP

[0227] Use a micropump to add 50% (w / v) β-GP to the composite solution at a rate of 5 mL per minute until the pH reaches 6.7. Let it stand overnight (12 hours) at 4°C to remove bubbles.

[0228] 2.5 Addition of spray aid

[0229] Add the PEG 400 pre-solution dropwise at 4°C to make the final PEG 400 concentration 0.1% (v / v), and stir for 5 minutes at a speed of 100 rpm.

[0230] 2.6 Nanodrug loading

[0231] Take the reprogrammed macrophage exosome solution (concentration 1 mg / mL), slowly inject it at a speed of 2 mL per minute, and stir at a speed of 50 rpm. The final exosome concentration is about 100 μg / mL.

[0232] 2.7 Loading of the spray device

[0233] After removing bubbles by low-speed centrifugation (100 g, 2 minutes), aliquot with a sterile syringe into a nasal sprayer and store at 4°C.

[0234] 3. Results and analysis

[0235] 3.1 Gelation temperature and rate

[0236] Gelation temperature: Due to the high molecular weight of chitosan (300 kDa), the cross-linking degree of the hydrogel is relatively high, and the hydrogel forms at approximately 33 °C.

[0237] Gelation rate: Due to the relatively tight cross-linking network of high molecular weight chitosan, the gelation rate is slow, and it takes about 3 - 4 hours to reach a stable state.

[0238] 3.2 Spray performance

[0239] Poor spray performance: Due to the high molecular weight of chitosan (300 kDa), the viscosity of the hydrogel is relatively high, which results in larger droplet sizes during spraying, usually exceeding 100 μm, and the poor fluidity of the spraying liquid, poor atomization effect, and limited spray performance. It is difficult for a high-viscosity solution to form uniform small droplets in a sprayer. Therefore, the drug distribution may be uneven, with too high a local drug concentration and insufficient concentration in other parts.

[0240] Larger droplet sizes: The droplet sizes of the spray are larger, and most droplet sizes > 100 μm, which leads to an unsatisfactory spray effect and affects the uniform distribution of the drug. Especially during nasal spraying, it may affect the coverage and persistence of the therapeutic effect.

[0241] 3.3 Exosome release detection

[0242] Release amount in 7 days: The release of exosomes is relatively fast in the first few days, but due to the loose network structure of the hydrogel, the release amount gradually decreases in the later stage. Finally, the cumulative release amount in 7 days is 40%. After 10 days, the cumulative release amount changes little, indicating that the remaining amount of exosomes is small.

[0243] Comprehensive evaluation

[0244] Lower gelation temperature: The hydrogel can gel at 34 °C, resulting in a slow gelation rate and a good gelation effect. Slow gelation rate: Due to the relatively high cross-linking degree caused by higher molecular weight chitosan, the gelation rate is slow, and it takes a long time to form a stable hydrogel. Insufficient exosome release: Due to the too high cross-linking degree, the release of exosomes is inhibited, resulting in a cumulative release amount of only 40% within 7 days. Poor spray performance: Due to the high viscosity of the hydrogel, the droplet sizes are larger, and most droplet sizes exceed 100 μm, resulting in a poor spray effect and uneven drug distribution, affecting the therapeutic effect. Decrease in exosome activity: Due to the too fast release of exosomes and the loose structure of the hydrogel, the activity of a part of the exosomes is lost, thus affecting the therapeutic effect and immune regulation function. Summary: High molecular weight chitosan increases the viscosity of the hydrogel, resulting in poor spray performance and affecting the uniform release of exosomes. Therefore, the ideal therapeutic effect cannot be achieved in this experiment.

[0245] As can be seen from the above Examples 1-6, the target preparation concentration and molecular weight of the chitosan solution will directly affect the gelation effect and the sustained release effect of exosomes. In the present invention, the concentration of the chitosan solution meeting the technical requirements of the present invention should be limited to 1.6% to 2.0%, and the molecular weight of chitosan should be limited to 180 to 220.

[0246] Example 7

[0247] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0248] 1. Raw Materials and Main Parameters

[0249] Chitosan: Molecular weight 200 kDa, deacetylation degree ≥ 90%, target preparation concentration 1.75% (w / v)

[0250] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05

[0251] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, final concentration 4.5% (w / v)

[0252] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v)

[0253] PEG 400: Final concentration 0.1% (v / v)

[0254] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min

[0255] Reaction environment: 4°C throughout

[0256] Target release amount: Approximately 80% cumulative release within 7 days

[0257] 2. Specific Preparation Steps

[0258] 2.1 Preparation of Chitosan Solution

[0259] Dissolve chitosan in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05. Prepare a chitosan solution with a concentration of 1.75% (w / v) and stir for 12 hours in a 4°C environment to ensure that the solution is completely transparent and uniform.

[0260] 2.2 Preparation of PNIPAAm Solution

[0261] Accurately weigh the PNIPAAm dry powder and add it to ultrapure water at 4 °C. Slowly warm the mixture to 25 °C and stir at 200 rpm for 30 minutes until the solution becomes clear. After the solution is clear, cool it back to 4 °C and make up to the target volume with a final concentration of 4.5% (w / v). Use a laser scattering particle size analyzer to check the clarity of the solution and ensure that the turbidity is less than 10 NTU.

[0262] 2.3 Mixing of the preliminary composite solution

[0263] Mix the chitosan solution in step 1 and the PNIPAAm solution in step 2 at a volume ratio of 2:1 in an ice bath environment. Slowly add the PNIPAAm solution with a dropping rate controlled at 10–20 mL / min and a stirring speed of 150 rpm. Use a pH meter with an accuracy of 0.01 to monitor the pH value of the mixed solution online. If pH < 6.0, slowly add 0.1 M NaOH to adjust to pH = 6.2; if pH > 6.2, add an appropriate amount of 1% acetic acid solution.

[0264] 2.4 Addition of β-GP solution

[0265] Prepare a 50% (w / v) β-GP solution and sterilize it through a 0.22 μm filter membrane. Use a micro pump to slowly add the β-GP solution to the above composite solution at a rate of 5 mL per minute, and stir (stirring speed 100–150 rpm). Stop adding the β-GP solution when the pH value approaches 6.7. If the pH value is less than 6.7, a small amount of NaOH can be used for fine-tuning. The composite solution is left standing at 4 °C for 12 hours to ensure complete removal of air bubbles and make the solution uniform.

[0266] 2.5 Addition of the co-spraying agent PEG 400

[0267] Take the stock solution of PEG 400 and prepare a 1.5% (v / v) PEG 400 pre-solution using cooled ultrapure water or PBS. Slowly add this pre-solution to the composite solution at 4 °C with a stirring speed not exceeding 100 rpm and stir for 5–10 minutes to ensure uniform mixing and a final concentration of PEG 400 reaching 0.1% (v / v).

[0268] 2.6 Exosome loading

[0269] Take the exosome solution and ensure its concentration is 100 μg / mL. Slowly add the exosome solution to the hydrogel solution at a rate of 2 mL per minute with a stirring speed of 50 rpm. Gently stir to ensure uniform dispersion of exosomes, and finally form an exosome-loaded thermosensitive hydrogel solution.

[0270] 2.7 Loading of the spray device

[0271] Transfer the prepared exosome-loaded hydrogel solution to a sterile room or a laminar flow hood for operation. Use low-speed centrifugation (100 g, 2 minutes) to remove air bubbles and ensure the liquid is completely homogeneous. Use a sterile syringe to fill the solution into a nasal sprayer that has been sterilized by high temperature, and ensure that the liquid is bubble-free and well-sealed. After completion, store the sprayer at 4 °C for future use.

[0272] 3. Results and Analysis

[0273] 3.1 Gelation Rate and Temperature

[0274] The hydrogel solution prepared through the above steps gels rapidly in a 34 °C water bath within approximately 2 - 3 minutes, achieving the expected thermosensitive response characteristics. The gelation rate is moderate, capable of providing the required colloidal support for the treatment area in a short time.

[0275] 3.2 Exosome Release Amount and Rate

[0276] It was determined that approximately 70% of the exosomes were released cumulatively within 7 days. In the initial stage of release (approximately 24 - 48 hours), the exosome release amount was relatively fast, and then the release rate gradually slowed down, finally reaching a cumulative release amount of 70% on the 7th day. This release rate is relatively stable.

[0277] 3.3 Spraying Performance

[0278] Under the condition of 4 °C, the hydrogel solution has a good spraying effect, with droplet diameters of approximately 50 - 100 μm, and uniform atomization, ensuring that the drug can be evenly coated on the inner wall of the nasal cavity.

[0279] In this example, the thermosensitive hydrogel loaded with exosomes has good spraying performance, thermosensitive response, and exosome release characteristics. Approximately 70% of the exosomes were released cumulatively within 7 days. Although slightly lower than the optimal value (80%), it is still within an acceptable range and suitable for general applications. Compared with Example 1, there is a slight decrease in performance, but it can still meet the treatment requirements.

[0280] Example 8

[0281] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0282] 1. Raw Materials and Main Parameters

[0283] Chitosan: Molecular weight is approximately 200 kDa, deacetylation degree ≥ 90%, and the preparation concentration is 1.75% (w / v).

[0284] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0285] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, and the concentration configured in this example is 5.5% (w / v) (higher than 5.0% in the standard example).

[0286] Sodium β - glycerophosphate (β - GP): 50% (w / v), chitosan / PNIPAAm:β - GP = 5:1 (v / v).

[0287] PEG 400: Final concentration 0.1% (v / v).

[0288] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min.

[0289] Operating temperature: 4°C throughout the process.

[0290] 2. Specific preparation steps

[0291] 2.1 Preparation of PNIPAAm solution (5.5%)

[0292] Weigh the PNIPAAm dry powder, add it to ultrapure water in batches under the condition of 4°C, and the initial stirring speed is 100 - 150 rpm. Slowly raise the temperature to 25°C, and then change to stir at 200 rpm for about 30 minutes until the solution becomes clear. Lower the temperature back to 4°C, and make up the volume to 5.5% (w / v). If necessary, measure the turbidity < 10 NTU to ensure uniformity.

[0293] 2.2 Preparation of chitosan solution (1.75%)

[0294] Dissolve the chitosan powder in 1.0% (v / v) acetic acid solution (pH = 3.0 ± 0.05) to prepare a 1.75% (w / v) solution. Stir at 200 - 300 rpm for 12 hours at 4°C until a transparent and uniform solution is formed.

[0295] 2.3 Preliminary compounding of chitosan / PNIPAAm (volume ratio 2:1)

[0296] Under an ice - bath environment, mix the chitosan solution (1.75%) and the PNIPAAm solution (5.5%) at a volume ratio of 2:1. The dropping rate is about 10 - 20 mL / min, and the stirring speed is 150 rpm. Monitor online with a pH meter: If pH < 6.0, add 0.1 M NaOH; if pH > 6.2, add 1% acetic acid, and finally control the pH at 6.2.

[0297] 2.4 Addition of β - GP solution

[0298] Inject a 50% (w / v) β-GP solution (filtered through 0.22 μm) into the above composite solution with a micro pump at a rate of approximately 5 mL / min; chitosan / PNIPAAm:β-GP = 5:1 (v / v), stirring speed 100 - 150 rpm;

[0299] Stop feeding when the pH value is approximately 6.7; let it stand at 4 °C for 12 hours to remove air bubbles and homogenize the system.

[0300] 2.5 Add PEG 400 (0.1%)

[0301] Slowly add the PEG 400 pre-solution (1.5% v / v) to the above composite solution at 4 °C, with a stirring speed not exceeding 100 rpm; the final PEG 400 concentration reaches 0.1%, and stir for about 5 - 10 minutes to reduce the interfacial tension and improve the sprayability.

[0302] 2.6 Exosome loading (100 μg / mL)

[0303] Slowly inject the exosome solution (1 mg / mL) at a rate of 2 mL per minute, stirring at 50 rpm; maintain the temperature at 4 °C throughout the process to avoid thermal denaturation of exosomes; the final concentration is approximately 100 μg / mL to obtain a sprayable hydrogel loaded with exosomes.

[0304] 2.7 Filling and storage

[0305] Centrifuge at low speed (100 g, 2 minutes) to remove microbubbles; under a sterile operating environment, use a sterile syringe to dispense the solution into a sterilized nasal sprayer, ensuring no air bubbles and good sealing; store it at 4 °C in the refrigerator to avoid premature gelation caused by high temperature environment.

[0306] 3. Results and analysis

[0307] 3.1 Gelation temperature and speed

[0308] Since the concentration of PNIPAAm is increased to 5.5%, the phase transition is more easily triggered. Gelation can occur at around ~32 °C, which is lower than ~34 °C of the standard example (5.0% PNIPAAm). It is observed that preliminary gelation can occur in about 3 - 4 minutes. If the ambient temperature is higher, it is easy to gel prematurely during the operation, increasing the process difficulty.

[0309] 3.2 Solution viscosity and spray operation

[0310] The high concentration of PNIPAAm also increases the viscosity of the solution at low temperature (4 °C). Although 0.1% PEG 400 helps to reduce the surface tension, a greater thrust still needs to be applied during the spray operation; there is a certain probability of drawing or uneven droplets, and the atomization quality is slightly inferior to the standard example.

[0311] 3.3 Exosome Release

[0312] Tested in simulated body fluid at 37°C, the cumulative release amount in 7 days was about 63%;

[0313] Lower than the target of 80%. The reason may be that the denser PNIPAAm network restricts the diffusion of exosomes; at the same time, the decrease in the gelation temperature leads to premature gelation locally, which also affects the uniform release of exosomes at body temperature.

[0314] Comprehensive Evaluation

[0315] The decrease in the gelation temperature is beneficial to gelation starting at slightly lower than body temperature (32°C), but it is also easy to cause semi-gelation near room temperature; higher requirements for operation and spraying process, and easy to block the nozzle. The exosome release efficiency decreases, and only 63% is released in 7 days; long-term retention of exosomes may inactivate, reducing the actual effective amount. Although the gelation is faster and firmer, the overall release amount and atomization operability are slightly worse. Lower gelation temperature (∼32°C): Premature gelation may occur during actual use, increasing the operation difficulty; the exosome release amount in 7 days is only 63%: lower than the ideal target of 80%, indicating that increasing the PNIPAAm concentration does not bring better drug release performance; the overall performance is inferior to the best example. Although the network strength is higher, it has adverse effects in terms of easy premature gelation, increased viscosity, and decreased release amount.

[0316] Example 9

[0317] Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes

[0318] 1. Raw Materials and Main Parameters

[0319] Chitosan: Molecular weight about 200 kDa, deacetylation degree ≥90%, preparation concentration 1.75% (w / v).

[0320] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0321] PNIPAAm: Molecular weight 40 kDa, purity ≥96%, preparation concentration 3.0% (w / v) in this example.

[0322] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0323] PEG 400: Final concentration 0.1% (v / v).

[0324] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min.

[0325] Operating temperature: 4°C throughout the process.

[0326] 2. Specific preparation steps

[0327] 2.1 Preparation of chitosan solution (1.75%)

[0328] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 1.75% (w / v) chitosan solution; stir (200 - 300 rpm) for 12 hours at 4°C to ensure the solution is transparent and uniform.

[0329] 2.2 Preparation of PNIPAAm solution (3.0%)

[0330] Take PNIPAAm dry powder and slowly add it to ultrapure water at 4°C with an initial stirring speed of 100 - 150 rpm; heat up to 25°C and continue to stir at 200 rpm for 30 minutes until the solution is clear; cool back to 4°C and make up the volume to 3.0% (w / v); measure the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0331] 2.3 Preliminary composite of chitosan / PNIPAAm (volume ratio 2:1)

[0332] Mix the 1.75% chitosan solution and 3.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min, and the stirring speed is ~150 rpm; monitor online with a pH meter: if pH < 6.0, add 0.1 M NaOH; if pH > 6.2, add 1% acetic acid, and finally adjust to pH ≈ 6.2.

[0333] 2.4 Addition of β-GP solution

[0334] Take a 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro-pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm, and the pH gradually rises to about 6.7 after addition; stop feeding and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble-free.

[0335] 2.5 Addition of PEG 400 (0.1%)

[0336] Slowly drip the PEG 400 pre-solution (1.5% v / v) into the composite solution, and control the stirring rate at 100 rpm; stir for 5 - 10 minutes, and the final PEG 400 concentration is 0.1%, stir evenly to improve the sprayability and surface tension.

[0337] 2.6 Exosome loading (100 μg / mL)

[0338] The exosome solution (1 mg / mL) was slowly added to the composite solution at a rate of 2 mL / min with a stirring speed of 50 rpm; the operating temperature was maintained at 4 °C throughout the process to avoid exosome denaturation; the final exosome concentration was 100 μg / mL to ensure uniform dispersion.

[0339] 2.7 Aliquoting and storage

[0340] Low-speed centrifugation (100 g, 2 minutes) was used to remove microbubbles; under a sterile operating environment, the solution was aliquoted into a sterilized nasal sprayer; after sealing, it was stored at 4 °C to avoid premature gelation caused by high-temperature environment.

[0341] 3. Results and analysis

[0342] 3.1 Exosome release (7-day cumulative amount)

[0343] Exosome release amount in 7 days: Tested in simulated body fluid (37 °C), the 7-day cumulative release amount was approximately 70%;

[0344] Since the PNIPAAm concentration decreased to 3.0%, the hydrogel network became relatively loose, resulting in too fast exosome release in the initial stage. The lower PNIPAAm concentration made the hydrogel more likely to absorb water, causing the diffusion rate of exosomes in the hydrogel to increase, leading to a faster initial release.

[0345] Too much exosome release in the initial stage would consume most of the exosomes, and the remaining exosomes were difficult to provide sufficient sustained-release effect, resulting in insufficient release amount in the later stage.

[0346] Release curve: The release curve showed a rapid release in the initial stage, then entered a relatively flat stage, and the release rate slowed down significantly, resulting in unstable release in the later stage and failing to provide a continuous sustained-release effect.

[0347] 3.2 Spraying performance

[0348] Spraying uniformity: The decrease in PNIPAAm concentration reduced the viscosity of the hydrogel solution accordingly, and the uniformity of the spray droplets was improved. The droplet size distribution was about 50 - 100 μm, and the spraying effect was relatively uniform. Although the spraying performance was improved, due to too low viscosity, the stability and persistence of spraying might be inferior to the standard example, and the droplet size was relatively large, which might lead to inaccurate spraying treatment effect.

[0349] 3.3 Gelation temperature and speed

[0350] Gelation temperature and speed: Since the PNIPAAm concentration decreased to 3.0%, the gelation temperature increased to about 37 °C, and the gelation time was about 10 - 15 minutes. The gelation speed was slow, indicating that the hydrogel formed a relatively loose network structure at a lower concentration, resulting in a slower gelation process.

[0351] 3.4 Conclusions and Potential Problems

[0352] Insufficient exosome release: Due to the decrease in the PNIPAAm concentration, the structure of the hydrogel is relatively loose, resulting in the exosomes being released too quickly in the initial stage and the release rate being insufficient in the later stage. The final release amount in 7 days is 70%. This phenomenon of premature exosome release causes the hydrogel to be unable to provide a sustained slow-release effect, affecting the treatment effect.

[0353] Improved but still unsatisfactory spraying performance: Due to the decrease in the viscosity of the hydrogel solution caused by the lower PNIPAAm concentration, the uniformity of the spray droplets has been improved, but there is still a problem of too large droplets, affecting the stability of the spraying effect.

[0354] Slightly slower gelation speed: The lower concentration of PNIPAAm leads to a slowdown in the gelation speed, making the hydrogel may not be suitable for some occasions that require rapid gelation.

[0355] In this example, the PNIPAAm concentration is reduced to 3.0%, resulting in an increase in the gelation temperature of the hydrogel, a slowdown in the gelation speed, and too fast release of exosomes, ultimately resulting in a 7-day release amount of 70%. The spraying performance has been improved, but the droplet distribution is still relatively large, affecting the uniformity of spraying. The release rate is fast, and the later release is insufficient, resulting in an unsatisfactory long-term slow-release effect.

[0356] Example 10

[0357] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0358] 1. Raw Materials and Main Parameters

[0359] Chitosan: The molecular weight is about 200 kDa, the degree of deacetylation is ≥90%, and the prepared concentration is 1.75% (w / v).

[0360] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0361] PNIPAAm: The molecular weight is 40 kDa, the purity is ≥96%, and the prepared concentration in this example is 7.0% (w / v).

[0362] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0363] PEG 400: The final concentration is 0.1% (v / v).

[0364] Exosomes (derived from reprogrammed M2 macrophages): The final concentration is 100 μg / mL, and the addition rate is 2 mL / min.

[0365] Operating temperature: 4°C throughout the process.

[0366] 2. Specific preparation steps

[0367] 2.1 Preparation of chitosan solution (1.75%)

[0368] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 1.75% (w / v) chitosan solution; stir (200 - 300 rpm) for 12 hours at 4°C to ensure the solution is transparent and uniform.

[0369] 2.2 Preparation of PNIPAAm solution (7.0%)

[0370] Take PNIPAAm dry powder and slowly add it to ultrapure water at 4°C with an initial stirring speed of 100 - 150 rpm; heat up to 25°C and continue to stir at 200 rpm for 30 minutes until the solution is clear; cool back to 4°C and make up the volume to 7.0% (w / v); measure the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0371] 2.3 Preliminary composite of chitosan / PNIPAAm (volume ratio 2:1)

[0372] Mix the 1.75% chitosan solution and 7.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min, and the stirring speed is ~150 rpm; monitor online with a pH meter: if pH < 6.0, add 0.1M NaOH; if pH > 6.2, add 1% acetic acid, and finally adjust to pH ≈ 6.2.

[0373] 2.4 Addition of β-GP solution

[0374] Take a 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm, and the pH gradually rises to about 6.7 after addition; stop feeding and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble-free.

[0375] 2.5 Addition of PEG 400 (0.1%)

[0376] Slowly drip the PEG 400 pre-solution (1.5% v / v) into the composite solution, and control the stirring rate at 100 rpm; stir for 5 - 10 minutes, and the final concentration of PEG 400 is 0.1%, stir evenly to improve the sprayability and surface tension.

[0377] 2.6 Exosome Loading (100 μg / mL)

[0378] The exosome solution (1 mg / mL) was slowly added to the composite solution at a rate of 2 mL / min with a stirring speed of 50 rpm; the operating temperature was maintained at 4 °C throughout the process to avoid exosome denaturation; the final exosome concentration was 100 μg / mL to ensure uniform dispersion.

[0379] 2.7 Subpackaging and Storage

[0380] Low-speed centrifugation (100 g, 2 minutes) was used to remove microbubbles; in a sterile operating environment, the solution was subpackaged into a sterilized nasal sprayer; after sealing, it was stored at 4 °C to avoid premature gelation caused by high-temperature environment.

[0381] 3. Results and Analysis

[0382] 3.1 Exosome Release

[0383] Exosome release amount at 7 days: Tested in simulated body fluid (37 °C), the cumulative release amount in 7 days was approximately 80%; the PNIPAAm concentration was 7.0%, and the hydrogel network was very dense, so the exosome release was relatively concentrated and the release rate was fast. The release amount reached 80% on the 5th day, but the release amount in the later stage was less and the release persistence was insufficient.

[0384] Release curve: The release curve showed rapid release in the initial stage, then reached 80% of the release amount on the 5th day, and the subsequent release rate gradually slowed down, indicating that the early release amount was too high and the slow release in the later stage was insufficient.

[0385] 3.2 Spraying Performance

[0386] Spraying uniformity:

[0387] The PNIPAAm concentration was as high as 7.0%, resulting in extremely high viscosity of the hydrogel solution, and the uniformity of the droplets decreased. The size of the spray droplets was about 100 - 150 μm. Due to the too high viscosity, the spraying effect was not ideal, the droplet distribution was uneven, which might affect the uniformity and coverage effect of the treatment.

[0388] 3.3 Gelation Temperature and Rate

[0389] Gelation temperature and rate:

[0390] After the PNIPAAm concentration increased to 7.0%, the gelation temperature decreased significantly to about 32 °C, which meant that the properties of this hydrogel system might be affected at room temperature.

[0391] The gelation time was about 3 - 4 minutes, indicating that the network structure of the gel was relatively dense and the formation speed was slow.

[0392] 3.4 Conclusions and Potential Problems

[0393] Excessive exosome release: Due to the relatively high concentration of PNIPAAm, the hydrogel network is relatively tight, resulting in excessive initial release of exosomes and insufficient release in the later stage. Although the cumulative release amount within 7 days is 80%, the concentration of release is too strong to provide a uniform long-term sustained release effect. For therapeutic applications that require continuous release, excessive rapid release will affect the long-term therapeutic effect.

[0394] Poor spraying performance: The high concentration of PNIPAAm increases the viscosity of the hydrogel solution, resulting in larger droplets and poor uniformity during spraying, which may lead to poor spraying effects.

[0395] Longer gelation time: Although the gelation temperature is relatively low (32 °C), the relatively high concentration of PNIPAAm prolongs the gelation time and the gel formation is slower, affecting its applicability to therapeutic occasions that require rapid response.

[0396] In this example, the concentration of PNIPAAm is increased to 7.0%, resulting in the hydrogel being able to form a gel at 32 °C. However, the gelation time is slightly longer, and the gel network is denser, resulting in excessive exosome release and difficulty in maintaining the release rate, with a decrease in the release amount in the later stage. The spraying performance is poor, and the droplet size is relatively large, resulting in uneven atomization and affecting the therapeutic effect. The rapidly released exosomes are not ideal for therapeutic applications with high requirements for long-term sustained release effects, and it is difficult to ensure the treatment window.

[0397] From the above Examples 7-10, it can be seen that the concentration of the PNIPAAm solution directly affects the gelation effect and the sustained release effect of exosomes. In the present invention, the concentration of the PNIPAAm solution that meets the technical requirements of the present invention should be limited to 4% - 5.5%.

[0398] Example 11

[0399] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0400] 1. Raw Materials and Main Parameters

[0401] Chitosan: Molecular weight is about 200 kDa, deacetylation degree ≥ 90%, and the target preparation concentration is 1.75% (w / v).

[0402] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0403] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, and the final concentration is 5.0% (w / v).

[0404] Sodium β-glycerophosphate (β-GP): 50% (w / v). Chitosan / PNIPAAm mixture:β-GP = 4:1 (v / v) in this example.

[0405] PEG 400: final concentration 0.1% (v / v).

[0406] Exosomes (derived from reprogrammed M2 macrophages): final concentration 100 μg / mL, addition rate 2 mL / min.

[0407] Operating temperature: 4 °C throughout the process.

[0408] 2. Specific preparation steps

[0409] 2.1 Preparation of chitosan solution

[0410] Take chitosan powder and dissolve it in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05. Prepare a 1.75% (w / v) chitosan solution. Stir at 4 °C (200 - 300 rpm) for 12 hours to ensure complete dissolution of chitosan and formation of a clear solution.

[0411] 2.2 Preparation of PNIPAAm solution

[0412] Take PNIPAAm dry powder and gradually add appropriate amount of ultrapure water at 4 °C with an initial stirring speed of 100 - 150 rpm. Slowly raise the temperature to 25 °C and maintain stirring at 200 rpm for about 30 minutes until the solution becomes clear;

[0413] After cooling to 4 °C, make up the volume to the final concentration of 5.0% (w / v).

[0414] 2.3 Preliminary compounding: In an ice bath environment, mix the chitosan solution (step 2.1) and the PNIPAAm solution (step 2.2) at a volume ratio of 2:1. Control the dropping rate at 10 - 20 mL / min and the stirring speed at about 150 rpm;

[0415] Online monitor the pH of the solution with a pH meter. If pH < 6.0, add 0.1 M NaOH dropwise; if pH > 6.2, slightly add 1% acetic acid to make pH ≈ 6.2.

[0416] 2.4 Addition of β-GP solution

[0417] Sterilize the prepared 50% (w / v) β-GP solution through a 0.22 μm filter membrane; use a micro pump to inject β-GP into the above composite solution at a rate of about 5 mL / min, and maintain stirring at 100 - 150 rpm; when the volume ratio reaches chitosan / PNIPAAm:β-GP = 4:1 (v / v), the pH value can be observed with a pH meter to be about 6.7 - 6.8; after finishing the feeding, let the solution stand at 4 °C for 12 hours to remove air bubbles and homogenize the solution.

[0418] 2.5 Add co-spraying agent (PEG 400)

[0419] Pre-prepare a 1.5% (v / v) PEG 400 solution, place it in an environment at 4 °C, and slowly drop the PEG 400 solution into the composite solution at a stirring speed not exceeding 100 rpm until the final concentration reaches 0.1% (v / v). Continue stirring for 5 - 10 minutes to improve the fluidity and spraying performance of the system.

[0420] 2.6 Exosome loading

[0421] Take the exosome solution (concentration 1 mg / mL), slowly add it at a rate of 2 mL per minute, with a stirring speed of 50 rpm, and maintain an environment at 4 °C throughout the process to avoid damage to exosome activity. The final exosome concentration is about 100 μg / mL to form an exosome-loaded liquid hydrogel.

[0422] 2.7 Loading of the spraying device

[0423] Centrifuge at low speed (100 g, 2 minutes) to remove micro air bubbles. In a sterile environment, use a sterile syringe to fill the solution into a sterilized nasal sprayer, ensure that there are no air bubbles remaining and the seal is intact, and store it at 4 °C for standby.

[0424] 3. Results and analysis

[0425] 3.1 Gelation temperature and speed

[0426] In this example, due to the increase in the amount of β-GP used (4:1 in this example, an increase compared to 5:1 in Example 1), the gelation temperature rises slightly, about around 36 °C. The gelation speed is slightly delayed compared to Example 1, and it takes 5 minutes to form a stable gel structure at 36 °C.

[0427] 3.2 Exosome release detection

[0428] Place the hydrogel sample in simulated body fluid (37 °C) for release measurement. The cumulative release is about 71% at 7 days, lower than the ideal target of 80%; affected by the amount of β-GP, the release is slightly faster in the early stage and the ending is gentle in the later stage, and the overall release curve is still relatively stable.

[0429] 3.3 Spraying performance

[0430] PEG 400 (0.1%) ensures good spray fluidity, and the spray droplet size is about 60-120 μm at 4° C.; compared with the optimal example, the atomization uniformity is slightly worse.

[0431] Comprehensive evaluation

[0432] Due to the increase of β-GP, the gel formation condition is closer to or higher than 36°C; the release in 7 days is 71%, which is a certain gap from the 80% target, but it can still provide a relatively considerable amount of exosome release. In this example, under the condition of a slightly higher β-GP incorporation amount (4:1), the gelation temperature of the system is moved up to about 36°C, and the cumulative release in 7 days is only 71%, which is close to but not the ideal target of 80%; the spraying and gelation performance is also slightly inferior to the better examples, which can still be regarded as a successful case with certain application value, but it is not the best solution.

[0433] Example 12

[0434] <Preparation of chitosan thermosensitive hydrogel loaded with reprogrammed macrophage exosomes>

[0435] 1. Raw materials and main parameters

[0436] Chitosan: molecular weight about 200 kDa, deacetylation degree ≥ 90%, preparation concentration 1.75% (w / v).

[0437] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0438] PNIPAAm: molecular weight 40 kDa, purity ≥ 96%, and the concentration in this embodiment is 5.0% (w / v).

[0439] Sodium β-glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm: β-GP = 3.5:1 (v / v).

[0440] PEG 400: final concentration 0.1% (v / v).

[0441] Exosomes (derived from reprogrammed M2 macrophages): final concentration 100 μg / mL, addition rate 2 mL / min.

[0442] Operating temperature: 4°C throughout the process.

[0443] 2. Specific preparation steps

[0444] 2.1 Preparation of chitosan solution (1.75%)

[0445] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 1.75% (w / v) chitosan solution; stir (200 - 300 rpm) for 12 hours at 4°C to ensure the solution is transparent and uniform.

[0446] 2.2 Preparation of PNIPAAm solution (5.0%)

[0447] Take PNIPAAm dry powder and slowly add it to ultrapure water at 4°C with an initial stirring speed of 100 - 150 rpm; heat up to 25°C and continue to stir at 200 rpm for 30 minutes until the solution is clear; cool back to 4°C and make up the volume to 5.0% (w / v); measure the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0448] 2.3 Preliminary composite of chitosan / PNIPAAm (volume ratio 2:1)

[0449] Mix the 1.75% chitosan solution and the 5.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min and the stirring speed is ~150 rpm; monitor online with a pH meter: if pH < 6.0, add 0.1M NaOH; if pH > 6.2, add 1% acetic acid and finally adjust to pH ≈ 6.2.

[0450] 2.4 Addition of β - GP solution

[0451] Take a 50% (w / v) β - GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro - pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm, and the pH gradually rises to about 6.7 after addition; stop adding the material and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble - free.

[0452] 2.5 Addition of PEG 400 (0.1%)

[0453] Slowly drip the PEG 400 pre - solution (1.5% v / v) into the composite solution with a stirring rate controlled at 100 rpm; stir for 5 - 10 minutes, and the final PEG 400 concentration is 0.1%, stir evenly to improve the sprayability and surface tension.

[0454] 2.6 Exosome loading (100 μg / mL)

[0455] Slowly add the exosome solution (1 mg / mL) to the composite solution at a rate of 2 mL / min with a stirring speed of 50 rpm; maintain the operating temperature at 4°C throughout the process to avoid exosome denaturation; the final exosome concentration is 100 μg / mL to ensure uniform dispersion.

[0456] 2.7 Sub-packaging and storage

[0457] Centrifuge at low speed (100 g, 2 minutes) to remove microbubbles; under aseptic operation environment, sub-package the solution into a sterilized nasal sprayer; after sealing, store at 4°C to avoid premature gelation caused by high temperature environment.

[0458] 3. Results and analysis

[0459] 3.1 Exosome release

[0460] Exosome release amount at 7 days: Tested in simulated body fluid (37°C), the cumulative release amount in 7 days was about 57%, significantly lower than the target value of 80%; due to the increase in the β-GP ratio (3.5:1), the cross-linking degree of the hydrogel increased, resulting in a denser hydrogel network and too fast gelation speed, so exosomes were released too fast in the initial stage and almost no continued release in the later stage, leading to the inability to maintain the therapeutic effect. The too high cross-linking degree of the hydrogel restricted the release of exosomes, and some exosomes could not be fully released, affecting the therapeutic effect.

[0461] Release curve: The release curve showed rapid release, most exosomes were released rapidly in the first few days, and the release amount in the later stage hardly continued to release, unable to achieve the effect of long-term slow release.

[0462] 3.2 Spraying performance

[0463] Spraying uniformity: Due to too high cross-linking degree and too fast gelation speed, local gelation occurred in the hydrogel, and the particle size of the spray droplets was relatively large (>100 μm), resulting in poor atomization effect; the relatively large size of the spray droplets led to unsatisfactory spraying effect and uneven distribution of the drug, affecting the uniformity of treatment.

[0464] 3.3 Gelation temperature and speed

[0465] Gelation temperature and speed: Due to the increase in the β-GP ratio, the cross-linking degree of the hydrogel increased, resulting in an accelerated gelation speed, and the network structure of the hydrogel was relatively dense, and the gelation temperature dropped to about 32°C, resulting in too fast gelation speed.

[0466] Due to the lower gelation temperature and faster gelation speed, the gelation process of the hydrogel was uneven, resulting in local premature gelation and affecting the uniformity of the solution.

[0467] 3.4 Conclusions and potential problems

[0468] Too fast exosome release: Due to the increase in the β-GP concentration, the network structure of the hydrogel was dense, resulting in too fast exosome release speed and unstable release amount. The cumulative release amount within 7 days was only 57%, and almost no release occurred in the later stage. The too high cross-linking degree of the hydrogel affected the continuous release of exosomes, resulting in the inability to maintain the therapeutic effect.

[0469] Poor spray performance: Due to local premature gelation, the droplets during the spraying process are uneven, affecting the spray uniformity of the drug and the therapeutic effect.

[0470] In this example, the concentration of β-GP was increased to 3.5:1, resulting in a higher cross-linking degree of the hydrogel. The hydrogel network was relatively dense, and the release of exosomes was too fast, resulting in a 7-day release amount of only 55%, unable to reach the expected 80%. The spray performance was poor, the droplet size was large, and the atomization effect was poor, affecting the uniformity of drug distribution. This formulation could not provide an ideal sustained-release effect, and the therapeutic window was limited, belonging to a failed example.

[0471] Example 13

[0472] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0473] 1. Raw Materials and Main Parameters

[0474] Chitosan: Molecular weight is about 200 kDa, deacetylation degree ≥ 90%, and the prepared concentration is 1.75% (w / v).

[0475] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0476] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, and the prepared concentration in this example is 5.0% (w / v).

[0477] β-glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0478] PEG 400: Final concentration 0.1% (v / v).

[0479] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min.

[0480] Operating temperature: 4°C throughout the process.

[0481] pH regulation: When adding β-GP and NaOH, the pH increases to 6.8.

[0482] 2. Specific Preparation Steps

[0483] 2.1 Preparation of Chitosan Solution (1.75%)

[0484] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 1.75% (w / v) chitosan solution; stir (200 - 300 rpm) at 4°C for 12 hours to ensure the solution is transparent and uniform.

[0485] 2.2 Preparation of PNIPAAm solution (5.0%)

[0486] Take PNIPAAm dry powder and slowly add it to ultrapure water at 4°C with an initial stirring speed of 100 - 150 rpm; heat up to 25°C and continue to stir at 200 rpm for 30 minutes until the solution is clear; cool back to 4°C and make up the volume to 5.0% (w / v); measure the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0487] 2.3 Preliminary composite of chitosan / PNIPAAm (volume ratio 2:1)

[0488] Mix the 1.75% chitosan solution and the 5.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min and the stirring speed is ~150 rpm;

[0489] 2.4 Addition of β-GP solution

[0490] Take a 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm and monitored online with a pH meter: drop 0.1 M NaOH until pH = 6.8. Stop adding the material and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble-free.

[0491] 2.5 Addition of PEG 400 (0.1%)

[0492] Slowly drop the PEG 400 pre-solution (1.5% v / v) into the composite solution and control the stirring rate at 100 rpm; stir for 5 - 10 minutes until the final PEG 400 concentration is 0.1%, and stir evenly to improve the sprayability and surface tension.

[0493] 2.6 Exosome loading (100 μg / mL)

[0494] Slowly add the exosome solution (1 mg / mL) to the composite solution at a rate of 2 mL / min with a stirring speed of 50 rpm; maintain the operating temperature at 4°C throughout the process to avoid exosome denaturation; the final exosome concentration is 100 μg / mL to ensure uniform dispersion.

[0495] 2.7 Sub-packaging and storage

[0496] Centrifuge at low speed (100g, 2 minutes) to remove microbubbles; under aseptic operation environment, dispense the solution into a sterilized nasal sprayer; after sealing, store at 4°C to avoid premature gelation caused by high temperature environment.

[0497] 3. Results and Analysis

[0498] 3.1 Exosome Release

[0499] Exosome release amount at 7 days: Tested in simulated body fluid (37°C), the cumulative release amount in 7 days was about 50%; due to the failure to control pH in time, local precipitation of chitosan occurred, the structure of the hydrogel changed, and exosomes could not be evenly loaded, resulting in low release amount. High pH value led to premature gelation, some exosomes were prematurely encapsulated, lost their activity, and the release rate was relatively fast, affecting the subsequent sustained release.

[0500] Release curve: The release curve showed rapid release, a large amount of exosomes were rapidly released in the first few days, and almost no further release occurred in the later stage, unable to achieve the effect of long-term sustained release.

[0501] 3.2 Spraying Performance

[0502] Spraying uniformity: Due to local precipitation of chitosan, the network structure of the hydrogel was uneven, resulting in uneven distribution of droplets during spraying, large droplet size (>100μm), affecting the atomization effect; the relatively large size of the spraying droplets led to unsatisfactory spraying effect, uneven distribution of the drug, and affected the uniformity of treatment.

[0503] 3.3 Gelation Temperature and Rate

[0504] Gelation temperature and rate: Since the pH increased to 6.8, the cross-linked network of the hydrogel was relatively loose, resulting in an accelerated gelation rate, but the network structure was unstable, and gelation occurred prematurely in some areas. The gelation temperature was too low, resulting in local gelation of the hydrogel at room temperature, affecting the uniformity of the solution, resulting in uneven gel structure, and unable to ensure the uniform release of exosomes.

[0505] 3.4 Conclusions and Potential Problems

[0506] Too fast exosome release: Due to the increase in pH, local precipitation of chitosan occurred, the cross-linking degree of the hydrogel decreased, resulting in too fast exosome release in the initial stage and less release amount.

[0507] The cumulative release amount within 7 days was only 50%, and almost no further release occurred in the later stage.

[0508] Poor spraying performance: Local gelation of the hydrogel led to uneven distribution of droplets during spraying, affecting the spraying uniformity of the drug and the treatment effect.

[0509] Premature gelation: Local gelation phenomenon results in an uneven gel structure, affecting the distribution of exosomes, leading to unstable and uneven release of exosomes, and affecting the sustained-release effect.

[0510] In this example, the increase in pH to 6.8 caused local precipitation of chitosan, and a good network structure was not formed. The loading of exosomes was uneven, resulting in a release amount of only 50% in 7 days. The spraying performance was poor, the droplet size was large, and the atomization effect was poor, affecting the uniformity of drug distribution. Premature gelation and inactivation of exosomes led to the inability to sustain the therapeutic effect and failed to meet the requirements of long-term treatment, belonging to a failure example.

[0511] Example 14

[0512] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0513] 1. Raw Materials and Main Parameters

[0514] Chitosan: Molecular weight is about 200 kDa, deacetylation degree ≥ 90%, and the preparation concentration is 1.75% (w / v).

[0515] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0516] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, and the preparation concentration in this example is 5.0% (w / v).

[0517] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 6:1 (v / v).

[0518] PEG 400: Final concentration 0.1% (v / v).

[0519] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 100 μg / mL, addition rate 2 mL / min.

[0520] Operating temperature: 4°C throughout the process.

[0521] 2. Specific Preparation Steps

[0522] 2.1 Preparation of chitosan solution (1.75%)

[0523] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 1.75% (w / v) chitosan solution; stir (200 - 300 rpm) for 12 hours at 4°C to ensure the solution is transparent and uniform.

[0524] 2.2 Preparation of PNIPAAm solution (5.0%)

[0525] Take PNIPAAm dry powder and slowly add it to ultrapure water at 4°C. The initial stirring speed is 100 - 150 rpm; heat up to 25°C and continue to stir at 200 rpm for 30 minutes until the solution becomes clear; cool back to 4°C and make up the volume to 5.0% (w / v); measure the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0526] 2.3 Preliminary Composite of Chitosan / PNIPAAm (Volume Ratio 2:1)

[0527] Mix a 1.75% chitosan solution and a 5.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min, and the stirring speed is about 150 rpm; monitor online with a pH meter: if pH < 6.0, add 0.1 M NaOH dropwise; if pH > 6.2, add 1% acetic acid to finally adjust to pH ≈ 6.2.

[0528] 2.4 Addition of β-GP Solution

[0529] Take a 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm, and the pH gradually rises to about 6.8 after addition; stop adding the material and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble-free.

[0530] 2.5 Addition of PEG 400 (0.1%)

[0531] Slowly drip the PEG 400 pre-solution (1.5% v / v) into the composite solution, and control the stirring rate at 100 rpm; stir for 5 - 10 minutes, and the final concentration of PEG 400 is 0.1%, stir evenly to improve the sprayability and surface tension.

[0532] 2.6 Exosome Loading (100 μg / mL)

[0533] Slowly add the exosome solution (1 mg / mL) to the composite solution at a rate of 2 mL / min, and the stirring speed is 50 rpm; maintain the operating temperature at 4°C throughout the process to avoid exosome denaturation; the final exosome concentration is 100 μg / mL to ensure uniform dispersion.

[0534] 2.7 Sub-packaging and Storage

[0535] Centrifuge at low speed (100 g, 2 minutes) to remove microbubbles; under a sterile operating environment, sub-package the solution into a sterilized nasal sprayer; after sealing, store it at 4°C to avoid premature gelation caused by a high-temperature environment.

[0536] 3. Results and Analysis

[0537] 3.1 Exosome release: Exosome release amount at 7 days: Tested in simulated body fluid (37°C), the cumulative release amount in 7 days was approximately 62%; due to the decrease in β-GP concentration (6:1), the cross-linking degree of the hydrogel weakened, resulting in a relatively loose structure of the hydrogel, an accelerated gelation rate, and too rapid exosome release in the initial stage, with almost no continued release in the later stage. The too low cross-linking degree of the hydrogel led to uneven exosome release, affecting its sustained-release effect.

[0538] Release curve: The release curve showed rapid release, with a large amount of exosomes released rapidly in the first few days, and almost no continued release in the later stage, failing to achieve the effect of long-term sustained release.

[0539] 3.2 Spray performance

[0540] Spray uniformity:

[0541] Due to the low cross-linking degree, the structure of the hydrogel was relatively loose, and local gelation might cause uneven droplet distribution during the spraying process. The droplet size was relatively large (>100μm), affecting the atomization effect; the relatively large size of the spray droplets led to an unsatisfactory spray effect and uneven distribution of the drug, affecting the uniformity of treatment.

[0542] 3.3 Gelation temperature and rate

[0543] Gelation temperature and rate: Due to the increase in the β-GP ratio (6:1), the cross-linking degree of the hydrogel weakened, resulting in a slowdown in the gelation rate to 15 - 20 minutes and an increase in the gelation temperature to approximately 37°C.

[0544] 3.4 Conclusions and potential problems

[0545] Gelation problem: Due to the low β-GP concentration, the gelation rate was slow, and the gelation temperature increased to 37°C. Although there was no premature gelation, the gelation process was relatively slow, which might lead to too rapid exosome release.

[0546] Insufficient release effect: The cumulative exosome release within 7 days was 62%. The release curve of exosomes was steep, with a relatively fast release in the initial stage and a small release amount in the later stage, resulting in the failure to achieve the sustained-release effect.

[0547] Spray performance: The atomization effect was good, the spraying was uniform, and the droplet size was within the ideal range, which was conducive to the uniform distribution of the drug and ensured the stability of the treatment effect.

[0548] Treatment window problem: Due to the rapid release of exosomes, the treatment window could not be effectively extended, and a continuous treatment effect could not be provided.

[0549] In this example, the ratio of β-GP was reduced to 6:1, resulting in an increase in the gelation temperature to 37 °C, a slowdown in the gelation rate, and no premature gelation. The 7-day release amount was 68%, and the release rate was relatively fast, failing to achieve an ideal sustained-release effect. The spraying performance was good, and the droplet size was within the ideal range, ensuring uniform drug distribution. This example failed to meet the long-term sustained-release treatment requirements and thus belongs to a failure case.

[0550] From the above Examples 11-14, it can be seen that the volume ratio of the mixed solution of chitosan and PNIPAAm to the sodium β-glycerophosphate solution during mixing will directly affect the gelation effect and the sustained-release effect of exosomes. In the present invention, the volume ratio of the mixed solution of chitosan and PNIPAAm to the sodium β-glycerophosphate solution that meets the technical requirements of the present invention should be limited to 4:1 to 5.5:1.

[0551] Example 15

[0552] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0553] 1. Raw Materials and Main Parameters

[0554] Chitosan: Molecular weight is about 200 kDa, deacetylation degree ≥ 90%, and the target preparation concentration is 1.75% (w / v).

[0555] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0556] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, and the final concentration is 5.0% (w / v).

[0557] Sodium β-glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0558] PEG 400: Final concentration 0.1% (v / v).

[0559] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 80 μg / mL, addition rate 2 mL / min.

[0560] Operating temperature: 4 °C throughout the process.

[0561] 2. Specific Preparation Steps

[0562] 2.1 Preparation of Chitosan Solution

[0563] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 1.75% (w / v) chitosan solution; stir (200 - 300 rpm) at 4°C for 12 hours to ensure a transparent and homogeneous solution.

[0564] 2.2 Preparation of PNIPAAm solution

[0565] Take PNIPAAm dry powder and slowly add it to ultrapure water at 4°C with an initial stirring speed of 100 - 150 rpm; heat up to 25°C and continue to stir at 200 rpm for 30 minutes until the solution is clear; cool back to 4°C and make up the volume to 5.0% (w / v); detect the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0566] 2.3 Preliminary composite of chitosan / PNIPAAm (volume ratio 2:1)

[0567] Mix the 1.75% chitosan solution and the 5.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min and the stirring speed is 150 rpm; monitor the pH value online. If pH < 6.0, add 0.1 M NaOH; if pH > 6.2, add 1% acetic acid, and finally adjust to pH ≈ 6.2.

[0568] 2.4 Addition of β-GP solution

[0569] Use a 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm, and the pH gradually rises to about 6.7 after addition; stop adding the material and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble-free.

[0570] 2.5 Addition of PEG 400 (0.1%)

[0571] Slowly drop the PEG 400 pre-solution (1.5% v / v) into the composite solution and control the stirring rate at 100 rpm; stir for 5 - 10 minutes, and the final PEG 400 concentration is 0.1% to moderately control the surface tension of the solution.

[0572] 2.6 Exosome loading (80 μg / mL)

[0573] Slowly add the exosome solution (1 mg / mL) to the composite solution at a rate of 2 mL / min with a stirring speed of 50 rpm; maintain the operating temperature at 4°C throughout the process to avoid exosome denaturation; the final exosome concentration is 80 μg / mL to ensure uniform dispersion.

[0574] 2.7 Sub-packaging and storage

[0575] Centrifuge at low speed (100 g, 2 minutes) to remove air bubbles; under aseptic conditions, dispense the solution into a sterilized nasal sprayer; after sealing, store at 4°C to avoid premature gelation caused by local warming.

[0576] 3. Results and Analysis

[0577] 3.1 Exosome Release

[0578] Exosome release after 7 days: Tested in simulated body fluid (37°C), the cumulative exosome release after 7 days was approximately 60%. The exosome release was relatively low. The main reason was that the exosome concentration decreased, resulting in a lower exosome loading in the colloid and insufficient release. Higher exosome concentrations could improve their cumulative release and enhance the sustained-release effect. Therefore, reducing the concentration to 80 μg / mL directly affected the release performance.

[0579] 3.2 Spraying Performance

[0580] Spraying uniformity: Compared with the standard example, the spraying performance remained consistent, and the droplet size distribution was approximately 50 - 100 μm, indicating that the addition of PEG 400 effectively assisted in the spraying uniformity. However, due to the low exosome concentration, the overall loading decreased, affecting its atomization distribution and effect.

[0581] 3.3 Gelation Temperature and Rate

[0582] Gelation temperature and rate: Since the chitosan concentration in the system was 1.75% and the PNIPAAm concentration was 5.0%, the gelation temperature of this system was approximately 34°C, which was consistent with the standard example; the gelation time was about 2 - 3 minutes, with no significant change compared to the standard example, indicating that the gelation rate was basically stable.

[0583] 3.4 Conclusions and Potential Problems

[0584] Decrease in exosome release: After the exosome concentration was reduced to 80 μg / mL, the cumulative exosome release only reached 60%, failing to achieve the ideal 80%. Insufficient release may lead to a weakened therapeutic effect, especially during treatment processes that require continuous exosome sustained release, where the effect cannot be maintained.

[0585] Spraying performance not significantly affected: The spraying uniformity and atomization effect were good, but the low exosome concentration reduced the overall treatment amount, resulting in poor release performance.

[0586] Applicability issues: In early treatment, the reduction in exosome concentration and release may not meet the required therapeutic effect, especially when dealing with the clinical needs of slow exosome release, the effect may be affected.

[0587] In this example, reducing the exosome concentration to 80 μg / mL results in a 7-day release amount of exosomes being reduced to 60%, with a poor release effect; the spraying performance and gelation speed are similar to those of the standard example, but the release efficiency is insufficient, making it suitable for applications with low requirements for exosome release or short-term treatments.

[0588] Example 16

[0589] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0590] 1. Raw Materials and Main Parameters

[0591] Chitosan: Molecular weight is about 200 kDa, deacetylation degree ≥ 90%, and the prepared concentration is 1.75% (w / v).

[0592] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0593] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, and the configured concentration in this example is 5.0% (w / v).

[0594] β-Glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0595] PEG 400: Final concentration 0.1% (v / v).

[0596] Exosomes (derived from reprogrammed M2 macrophages): Final concentration 150 μg / mL, addition rate 2 mL / min.

[0597] Operating temperature: 4°C throughout the process.

[0598] 2. Specific Preparation Steps

[0599] 2.1 Preparation of chitosan solution (1.75%)

[0600] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05; prepare a 1.75% (w / v) chitosan solution; stir (200 - 300 rpm) for 12 hours at 4°C to ensure the solution is transparent and uniform.

[0601] 2.2 Preparation of PNIPAAm solution (5.0%)

[0602] Take the PNIPAAm dry powder and slowly add it to ultrapure water at 4°C with an initial stirring speed of 100 - 150 rpm; heat it to 25°C and continue to stir at 200 rpm for 30 minutes until the solution becomes clear; cool it back to 4°C and make up the volume to 5.0% (w / v); measure the turbidity of the solution to ensure it is below 10 NTU and maintain uniformity.

[0603] 2.3 Preliminary chitosan / PNIPAAm composite (volume ratio 2:1)

[0604] Mix a 1.75% chitosan solution and a 5.0% PNIPAAm solution in a volume ratio of 2:1 in an ice bath; the dropping rate of the PNIPAAm solution is 10 - 20 mL / min and the stirring speed is ~150 rpm; monitor online with a pH meter: if pH < 6.0, add 0.1M NaOH dropwise; if pH > 6.2, add 1% acetic acid and finally adjust to pH ≈ 6.2.

[0605] 2.4 Addition of β-GP solution

[0606] Take a 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it with a micro pump at a speed of 5 mL / min; the stirring speed is 100 - 150 rpm, and the pH gradually rises to about 6.7 after addition; stop feeding and let it stand at 4°C for 12 hours to ensure the solution is uniform and bubble-free.

[0607] 2.5 Addition of PEG 400 (0.1%)

[0608] Slowly drip the PEG 400 pre-solution (1.5% v / v) into the composite solution and control the stirring rate at 100 rpm; stir for 5 - 10 minutes, and the final PEG 400 concentration is 0.1%. Stir evenly to improve the sprayability and surface tension.

[0609] 2.6 Exosome loading (150 μg / mL)

[0610] Slowly add the exosome solution (1 mg / mL) to the composite solution at a rate of 2 mL / min with a stirring speed of 50 rpm; maintain the operating temperature at 4°C throughout the process to avoid exosome denaturation; the final exosome concentration is 150 μg / mL to ensure uniform dispersion.

[0611] 2.7 Sub-packaging and storage

[0612] Centrifuge at low speed (100 g, 2 minutes) to remove microbubbles; under a sterile operating environment, sub-package the solution into a sterilized nasal sprayer; after sealing, store it at 4°C to avoid premature gelation caused by a high-temperature environment.

[0613] 3. Results and analysis

[0614] 3.1 Exosome Release (7-day Cumulative Amount)

[0615] Exosome release amount in 7 days: Tested in simulated body fluid (37°C), the cumulative release amount in 7 days is 65%. After the exosome concentration is increased to 150 μg / mL, the overall viscosity of the hydrogel increases significantly, resulting in a limited diffusion rate of exosomes.

[0616] Too rapid release: High concentration of exosomes leads to rapid initial release of exosomes, with a large proportion of exosomes released in the first few hours. Subsequently, the network structure of the hydrogel becomes denser, restricting the continuous release in the later stage and resulting in an overall release amount of only 65%.

[0617] The release curve shows rapid initial release, then enters a relatively flat stage with a significantly slowed release rate, failing to provide a continuous sustained release effect. The high concentration of exosomes causes aggregation of exosomes within the hydrogel, further exacerbating this uneven release phenomenon.

[0618] 3.2 Spray Performance

[0619] Spray uniformity: High concentration of exosomes increases the viscosity of the hydrogel solution, resulting in a less uniform spray than the standard example, with a larger droplet size distribution (about 70 - 120 μm). Spraying requires more thrust, which may lead to a spray effect not as expected and spray blockage or non-uniformity during operation.

[0620] 3.3 Gelation Temperature and Rate

[0621] Gelation temperature and rate: The gelation temperature remains at about 34°C with no significant change.

[0622] The gelation time is 3 - 4 minutes, slightly longer than 1 - 2 minutes in the standard example because the increased viscosity of the hydrogel causes the gelation process to be slightly slower.

[0623] 3.4 Conclusions and Potential Problems

[0624] Insufficient release due to excessive exosome concentration: When the exosome concentration is increased to 150 μg / mL, the exosome release amount in 7 days is 65%. The excessive initial release of exosomes consumes most of the exosomes, and the remaining exosomes cannot be effectively released, resulting in insufficient total release. High concentration of exosomes may also cause aggregation or retention of exosomes, further restricting their release.

[0625] Spray performance is affected: High concentration of exosomes increases the viscosity of the hydrogel solution, resulting in a decline in spray performance, unsatisfactory atomization effect, non-uniform droplets, and difficult operation.

[0626] Reduced sustained-release effect: Although high-concentration exosomes increase the loading capacity, due to the too-fast release, it is impossible to maintain the ideal sustained-release effect. Especially for treatment scenarios that require long-term sustained release of exosomes, the effect is inferior to that of the standard example.

[0627] In this example, when the exosome concentration is increased to 150 μg / mL, the exosome release amount within 7 days drops to 65%, failing to reach the ideal 80%. The spraying performance deteriorates, and the increased viscosity results in uneven spraying, larger droplets, and increased difficulty in spraying operations. The premature release of exosomes leads to a reduction in the total release amount and poor sustained-release effect in the later stage, and the overall performance is inferior to that of the best example. It is suitable for short-term treatment needs, but may not be ideal for treatment scenarios with high requirements for the continuous release of exosomes.

[0628] From the above Examples 15-16, it can be seen that the concentration of exosomes also directly affects the gelation effect and the sustained-release effect of exosomes. In the present invention, the concentration of exosomes that meets the technical requirements of the present invention should be limited to 80-150 μg / mL.

[0629] Example 17

[0630] <Preparation of Chitosan Thermosensitive Hydrogel Loaded with Reprogrammed Macrophage Exosomes>

[0631] 1. Raw Materials and Main Parameters

[0632] Chitosan: Molecular weight is about 200 kDa, degree of deacetylation ≥ 90%, and the prepared concentration is 1.75% (w / v).

[0633] Acetic acid solution: 1.0% (v / v), pH = 3.0 ± 0.05.

[0634] PNIPAAm: Molecular weight 40 kDa, purity ≥ 96%, and the final concentration is 5.0% (w / v).

[0635] β-glycerophosphate (β-GP): 50% (w / v), chitosan / PNIPAAm:β-GP = 5:1 (v / v).

[0636] PEG 400: The final concentration is only 0.05% (v / v) (lower than 0.1% in Example 1).

[0637] Exosomes (derived from reprogrammed M2 macrophages): The final concentration is 100 μg / mL, and the addition rate is 2 mL / min.

[0638] Operating temperature: 4°C throughout the process.

[0639] 2. Specific Preparation Steps

[0640] 2.1 Preparation of Chitosan Solution

[0641] Dissolve chitosan powder in 1.0% (v / v) acetic acid solution with pH = 3.0 ± 0.05 to prepare a 1.75% (w / v) chitosan solution. At 4°C, stir continuously at a speed of 200 - 300 rpm for 12 hours until the chitosan is completely dissolved to form a transparent solution.

[0642] 2.2 Preparation of PNIPAAm solution

[0643] Take PNIPAAm dry powder and slowly add it to ultrapure water at 4°C. Initially stir at 100 - 150 rpm, heat up to 25°C and then change to stir at 200 rpm for about 30 minutes until the solution becomes clear. Cool back to 4°C and make up the volume to 5.0% (w / v).

[0644] 2.3 Mixing of chitosan / PNIPAAm

[0645] Under an ice bath, mix the chitosan solution (1.75%) and the PNIPAAm solution (5.0%) at a volume ratio of 2:1. Control the dropping rate of the PNIPAAm solution at 10 - 20 mL / min and the stirring speed at about 150 rpm. Monitor the pH value online. If pH < 6.0, dropwise add 0.1 M NaOH; if pH > 6.2, add a small amount of 1% acetic acid; finally maintain pH ≈ 6.2.

[0646] 2.4 Addition of β-GP solution

[0647] Take a 50% (w / v) β-GP solution (sterilized by a 0.22 μm filter membrane) and slowly add it to the above composite solution with a volume ratio of 5:1 (v / v) using a micro pump. The stirring speed is 100 - 150 rpm and the injection rate is about 5 mL / min. Stop adding the material when the pH approaches 6.7. Let it stand at 4°C for 12 hours to complete defoaming and ensure uniform dispersion of the solution.

[0648] 2.5 Adjust the concentration of PEG 400 to 0.08%

[0649] Compared with Example 1, in this example, the final concentration of PEG 400 is reduced from 0.1% to 0.05%. First, prepare a 1.5% (v / v) PEG 400 pre-solution and dropwise add it to the composite solution at a stirring speed not exceeding 100 rpm at 4°C. Stir for about 5 - 10 minutes to confirm the uniform dispersion of PEG 400 and basically control the viscosity and surface tension.

[0650] 2.6 Exosome loading

[0651] Take the exosome solution (concentration 1 mg / mL) and slowly add it to the composite solution at a rate of 2 mL per minute with a stirring speed of 50 rpm. Keep the whole process at 4°C to avoid exosome denaturation. Finally, the exosome concentration is about 100 μg / mL, forming an exosome-loaded liquid thermosensitive hydrogel.

[0652] 2.7 Sub-packaging and storage

[0653] Centrifuge at low speed (100 g, 2 minutes) to remove bubbles. In a sterile environment, fill the solution into a nasal sprayer that has been sterilized by high temperature, ensure there are no bubbles and it is well-sealed, and store it at 4°C for later use to avoid premature gelation caused by local heating.

[0654] 3. Results and analysis

[0655] 3.1 Spraying performance

[0656] Compared with 0.1% PEG 400, the concentration of PEG 400 in this example is reduced to 0.05%, and it can be seen that the viscosity increases slightly;

[0657] The droplet size during spraying is relatively large (up to 100 - 150 μm), slightly worse than that in Example 1 and when the concentration of PEG 400 is higher, and it is easy to have uneven droplets.

[0658] 3.2 Gelation time and thermosensitive response

[0659] Place it in a 34°C water bath, and phase change starts in about 2 - 3 minutes. After gelation, the overall stability of the colloid is good and there is no obvious collapse.

[0660] 3.3 Exosome release detection

[0661] Place it in simulated body fluid (37°C) for 7 days, and measure that the cumulative exosome release amount is about 78%, close to the ideal target of 80%, but there is still a slight gap, indicating that the low concentration of PEG 400 has a slight impact on the release rate.

[0662] In this example, the concentration of PEG 400 is reduced from 0.1% to 0.05%, resulting in a decrease in both the spraying uniformity and the gelation speed. The cumulative exosome release amount in 7 days is about 78%, similar to the 80% standard, and it can still meet certain clinical needs, but the overall performance is slightly inferior to the optimal example. It is applicable to occasions where the requirements for spray atomization are not extremely high but a stable exosome release is still required. Therefore, the concentration of PEG 400 in this invention should be limited to 0.05% - 0.1%.

[0663] Example 18

[0664] <In vivo efficacy evaluation>

[0665] 1. Evaluation of drug delivery efficiency: Hydrogels loaded with exosomes were labeled with DiD fluorescent dye and administered to mice by nasal spray. Mice were sacrificed at 3 hours, 24 hours, and 3 days after administration, and spinal cord tissue and other major organs were taken to detect the distribution of tissue fluorescence signals using a small animal imaging system. The targeted delivery efficiency of the drug was quantitatively analyzed by fluorescence intensity.

[0666] The results showed that at 3 hours, the radioactivity intensity in the nasal region of mice in the Exosomes@Hydrogel group was significantly higher than that in the Exosomes group alone, indicating that the hydrogel could effectively prolong the retention time of exosomes in the nasal cavity. At the same time, significant radioactivity intensity was shown in the spinal cord injury area, with significant targeting compared to the CTRL group (P<0.001). At 24 hours and 3 days, the radioactivity intensity in the spinal cord injury area of both the Exosomes@Hydrogel group and the Exosomes group continued to increase, and the radioactivity intensity in the Exosomes@Hydrogel group was significantly higher than that in the Exosomes group alone (P<0.001), indicating that hydrogel loading not only improved the targeting of exosomes but also significantly enhanced their accumulation and utilization in the target tissue. These results suggest that the hydrogel for nasal-targeted delivery of exosomes to the spinal cord is an effective delivery system with significant targeting and can show good effects in the treatment of spinal cord injury.

[0667] 2. Analysis of functional recovery:

[0668] (1) Behavioral tests: The recovery of motor function in mice was evaluated by the Basso Mouse Scale (BMS); gait coordination and motor ability were detected by the open field test. The BMS score results showed that the motor function of the treatment group mice was significantly improved compared to the control group (P<0.001).

[0669] (2) Histological analysis: HE staining and Nissl body staining were performed on the spinal cord injury area tissue to evaluate the degree of tissue repair.

[0670] (3) Detection of inflammatory factors: The expression levels of anti-inflammatory factors IL-10 and TGF-β in the injured tissue were detected by ELISA. The hydrogel spray loaded with reprogrammed macrophage exosomes significantly reduced the inflammatory response in the spinal cord injury area and enhanced the expression levels of anti-inflammatory factors (IL-10 and TGF-β increased significantly, P<0.001).

[0671] (4) Neuronal survival rate: The number and morphological changes of neurons were detected by βIII-tubulin immunofluorescence labeling. The hydrogel spray loaded with reprogrammed macrophage exosomes significantly enhanced the survival of neurons in the spinal cord injury area (P<0.001).

[0672] The results showed that the thermosensitive delivery system targeting the spinal cord via the nose loaded with macrophage exosomes not only significantly improved the delivery efficiency of exosomes in the spinal cord injury area, but also effectively improved the motor function of mice by inhibiting inflammation, promoting neuron survival and regeneration, providing a new and efficient treatment method for the treatment of central nervous system diseases.

[0673] The above are only some preferred embodiments or comparative examples of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a drug targeting the central nervous system for intranasal use, characterized in that, The method includes: Dissolve chitosan with a molecular weight of 180 - 220 KDa in an acetic acid solution with a concentration of 1% v / v, and stir to obtain a transparent chitosan solution with a concentration of 1.6% - 2.0% w / v; Add poly(N-isopropylacrylamide) dry powder to ultrapure water at a low temperature, slowly heat up to and stir until it becomes clear, and then cool back to the low temperature state, and make up the mass-volume ratio to 4% - 5.5% w / v to generate a poly(N-isopropylacrylamide) solution; Mix the transparent chitosan solution and the poly(N-isopropylacrylamide) solution at a volume ratio of 2:1 at a low temperature to generate a composite solution, specifically including: dropwise add the poly(N-isopropylacrylamide) solution to the transparent chitosan solution at a predetermined speed and continuously stir; Use a micro pump to add a 50% w / v β-glycerophosphate solution to the composite solution at a predetermined rate so that the volume ratio of the composite solution to the β-glycerophosphate solution reaches 4:1 - 5.5:1, and let it stand to remove bubbles; At a low temperature, add a PEG400 pre-solution dropwise to the composite solution so that the final PEG 400 concentration reaches 0.05% - 0.1% v / v; Take an exosome solution and slowly inject it into the composite solution at a speed of 2 mL per minute and continuously stir until the final exosome concentration reaches 80 - 150 μg / mL.

2. The preparation method according to claim 1, wherein The low temperature state refers to a temperature less than or equal to 4°C.

3. The preparation method according to claim 1, characterized in that, The molecular weight of the poly(N-isopropylacrylamide) is 40 KDa.

4. The preparation method according to claim 1, characterized in that, The stirring speed when generating the chitosan solution is 200 rpm, and stir continuously for more than 12 hours.

5. The preparation method according to claim 1, characterized in that, When generating the poly(N-isopropylacrylamide) solution, slowly heat up to 25°C and stir for more than 30 minutes until it becomes clear.

6. The preparation method according to claim 1, characterized in that, Dropwise add the poly(N-isopropylacrylamide) solution to the transparent chitosan solution at a speed of 10 - 20 mL / min.

7. The preparation method according to claim 1, characterized in that, Add the β-glycerophosphate solution to the composite solution at a rate of 5 mL per minute until the pH reaches 6.

7.

8. The preparation method according to claim 1, wherein When adding the PEG400 pre-solution dropwise to the composite solution, the stirring speed does not exceed 100 rpm, and stir for 5 - 10 minutes.

9. A drug for intranasal administration targeting the central nervous system, the drug comprising: Thermosensitive hydrogel and exosome nano-biological components; the thermosensitive hydrogel is cross-linked by chitosan, poly(N-isopropylacrylamide), β-glycerophosphate, and PEG400; the preparation of the drug is carried out using the preparation method described in any one of the preceding claims 1 - 8.

10. A drug delivery system for intranasal targeting of the central nervous system, the drug delivery system includes centrifuging the drug for intranasal targeting of the central nervous system described in the preceding claim 9 at a low speed to remove bubbles, and then aseptically packaging the drug into a sterilized nasal sprayer under aseptic operating conditions.

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