A kind of external use ear drop for promoting auditory nerve growth and its preparation method

Abstract

The application belongs to the technical field of biological medicine preparation, and particularly relates to an external use ear drop for promoting auditory nerve growth and a preparation method thereof. The composition raw materials comprise the following components: active small molecules, glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, Tween-80, chitosan quaternary ammonium salt, diethylene glycol monoethyl ether, penetration aid, penetrant, sodium cholate, N-acetyl cysteine, F127 surfactant, F68 surfactant and sodium hyaluronate. Through the synergistic effect of the crystalline lipid core, the positive chitosan quaternary ammonium salt shell, the heat-sensitive in-situ gel outer layer and the mild penetration system, the drug is long-acting retained, smoothly released and efficiently penetrated into the inner ear target area under the condition of not puncturing the eardrum, and the synergistic system significantly improves the stability, local exposure and treatment efficiency of the drug.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical preparation technology, specifically relating to a topical ear drop that promotes auditory nerve growth and its preparation method. Background Technology

[0002] Topical ear drops, as a traditional localized medication, have a long history and wide application in treating ear infections, reducing inflammation and pain, and removing earwax. Their conventional formulations are mostly aqueous solutions, suspensions, or alcoholic solutions, relying on the fluidity of the liquid to cover the ear canal mucosa. However, the unique physiological structure of the ear canal presents significant challenges for local medication, especially in addressing the complex condition of sensorineural hearing loss. This type of hearing loss, along with related synaptic damage and spiral ganglion degeneration, is one of the main causes of communication impairment and decreased quality of life in clinical practice. The pathological changes are often related to noise exposure, drug ototoxicity, and aging, with the core pathological feature being the weakening or even detachment of the synaptic connection between hair cells and auditory nerve endings. Currently, targeted drug treatment mainly relies on systemic administration or intratympanic injection. However, the former is difficult to achieve effective drug exposure in the inner ear and easily causes systemic adverse reactions, while the latter, although it can increase local concentration, is an invasive procedure with limitations such as poor patient compliance due to tympanic membrane puncture, difficulty in repeated administration, and strong dependence on medical settings. From a delivery perspective, achieving this goal faces multiple physiological barriers: after being instilled through the external auditory canal, the drug must first cross the tympanic membrane, then pass through the middle ear mucosa and round window membrane into the perilymphatic cavity, and finally achieve sufficient quantity and duration of exposure in the cochlea and auditory nerve endings. The keratinized epithelium and fibrous layer of the tympanic membrane, along with the dense structure of the round window membrane, constitute multiple barriers, significantly blocking both hydrophobic small and large molecules. Simultaneously, the external auditory canal's own clearance and drainage mechanisms further shorten drug residence time, leading to insufficient flux and unstable exposure. Traditional ear drops are mostly simple aqueous solutions or solvent systems, lacking effective retention and sustained-release designs, making it difficult to balance transmembrane flux with formulation safety boundaries. Furthermore, the inner ear, under pathological conditions, is often accompanied by increased oxidative stress and microenvironmental imbalance, which not only accelerates the degradation of certain active small molecules (such as neurotrophic factors) but also weakens their neurotrophic signaling effects, further increasing the complexity of formulation development. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this invention is to provide a topical ear drop solution that promotes auditory nerve growth and its preparation method.

[0004] The technical effects described in this invention are achieved through the following technical solution: a topical ear drop solution that promotes auditory nerve growth, the raw materials of which include the following components: active small molecules, glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, Tween-80, chitosan quaternary ammonium salt, diethylene glycol monoethyl ether, penetration enhancer, penetrant, sodium cholate, N-acetylcysteine, F127 surfactant, F68 surfactant and sodium hyaluronate;

[0005] Preferably, the active small molecule is either 7,8-dihydroxyflavone or neurotrophic factor 3; more preferably, it is 7,8-dihydroxyflavone.

[0006] Preferably, the penetration enhancer is either menthol or d-limonene; more preferably, it is menthol.

[0007] Preferably, the penetrant is either glycerol or propylene glycol; more preferably, it is glycerol.

[0008] Preferably, another aspect of the present invention provides a method for preparing a topical ear drop solution that promotes auditory nerve growth, specifically comprising the following steps:

[0009] S1: After melting glyceryl stearate, glyceryl monostearate, soybean lecithin and vitamin E succinate sequentially in a 75℃ water bath, add an active small molecule solution and stir at 300 rpm for 10-15 min to obtain the oil phase.

[0010] S2: In a water bath at 70–72°C, Tween-80, diethylene glycol monoethyl ether, and the penetrant are added sequentially to deionized water, stirred until dissolved, and then brought to a final volume to obtain the hot water phase; N-acetylcysteine ​​is added to deionized water at 35–45°C, stirred until dissolved, and then brought to a final volume to obtain a 10 wt% cold water phase A; the penetration enhancer is dissolved in diethylene glycol monoethyl ether, and then added to deionized water at 40°C to a final volume to obtain the cold water phase B;

[0011] S3: Slowly pour the 75°C oil phase from step S1 into 5 times the volume of the 70-72°C hot water phase from step S2, and treat with high-speed shearing at 10000rpm for 5-8 minutes. Then transfer to an ice bath and sonicate with a probe at 400W, 30% amplitude, for 30s on / 30s off, for a total of 5-8 cycles, controlling the material temperature to <25°C. After treatment, quickly cool to 4-8°C to obtain the dispersion.

[0012] S4: Prepare a 0.8% chitosan quaternary ammonium salt aqueous solution at 4°C, filter it through a 0.22µm filter for sterilization, and slowly add it to the dispersion in step S3 at 4-8°C. Stir gently at 100rpm for 15-20min, slowly add 0.2-0.3% sodium cholate in three batches, and continue to stir gently for 5-10min. Then add the cold water phase A and cold water phase B from step S2 in sequence, and continue to stir gently during the addition process. After stirring evenly, a nano-dispersion is obtained.

[0013] S5: Cool deionized water at 4°C overnight, then add surfactants F127 and F68 sequentially, stir at 250 rpm for 12-16 h, then add sodium hyaluronate and stir until completely dissolved, filter with 0.22 μm for sterilization, adjust pH to 6-6.4 with citrate buffer to obtain a thermosensitive in situ gel matrix;

[0014] S6: Slowly add the nano-dispersion from step S4 to the thermosensitive in-situ gel matrix from step S5, stir at 200 rpm for 10-15 min, mix evenly, and make up to volume with citrate buffer to obtain the external ear drops.

[0015] Preferably, in step S1, the active small molecule solution is prepared by pre-dissolving the active small molecules in diethylene glycol monoethyl ether at a ratio of 1g:5mL.

[0016] Preferably, in step S1, the ratio of the amounts of glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, and active small molecule solution is 18-22g:4-6g:5-7g:0.4-0.6g:5mL;

[0017] Preferably, in step S2, the ratio of Tween-80, diethylene glycol monoethyl ether, and penetrant is 8-12 g: 10-12 mL: 50 mL.

[0018] Preferably, in step S2, the ratio of the penetration enhancer to diethylene glycol monoethyl ether is 1g:18-22mL;

[0019] Preferably, in step S4, the volume ratio of the chitosan quaternary ammonium salt aqueous solution to the dispersion is 1:6.

[0020] Preferably, in step S4, the volume ratio of the cold water phase A, the cold water phase B, and the dispersion is 0.3–0.35:0.25–0.3:1.

[0021] Preferably, in step S5, the ratio of the amounts of surfactant F127, surfactant F68, sodium hyaluronate, and deionized water is 28-32g:4-4.5g:0.3g:100mL.

[0022] Preferably, in step S6, the volume ratio of the nano-dispersion to the thermosensitive in-situ gel matrix is ​​44-48:50-54.

[0023] The beneficial effects of this invention are as follows:

[0024] This invention utilizes a synergistic structure of a lipid nanocore, a positively charged adhesive shell, a thermosensitive in-situ gel outer layer, and a mild, permeation-enhancing microenvironment to achieve accessible, stable, and gradual release of topical ear drops into the inner ear target area without tympanic membrane puncture. The inner layer, composed of a crystalline lipid core made of glyceryl stearate and glyceryl monostearate, encapsulates active small molecules (7,8-dihydroxyflavone). Surrounded by a stable amphiphilic interface of soybean lecithin and Tween-80, it improves the dispersion and encapsulation of hydrophobic drugs and achieves continuous transmembrane drug delivery through the gradual rearrangement of the crystalline phase. The vitamin E succinate added to the lipid core provides a lipid-phase antioxidant barrier, forming a mutually reinforcing reducing protection with N-acetylcysteine ​​in the aqueous phase, ensuring activity stability during both formulation and administration, and providing a favorable microenvironment for the expression of neurotrophic signals under injury-related oxidative stress. The intermediate adhesion layer uses chitosan quaternary ammonium salt (HACC) to positively modify the surface of nanoparticles at low temperatures. This moderate electrostatic interaction with the negatively charged groups of the tympanic membrane and middle ear mucosa prolongs the local retention time, reducing the risk of early loss, while maintaining interfacial permeability to facilitate material exchange with the outer gel layer. The outer layer is a thermosensitive in-situ gel composed of F127 / F68, combined with sodium hyaluronate to form a hydrophilic and elastic three-dimensional network. It exhibits a flow-like dynamic upon droplet insertion, rapidly transforming into a gel upon reaching body surface temperature and forming a thin layer on the tympanic membrane surface, stably embedding the positively charged nanoparticles. Its own swelling and diffusion behavior provides a smooth release background. After passing through the tympanic membrane and the circular window, the nanolipid cores continue to be released via carrier rearrangement. The two layers work in succession in time and space, forming a stable rather than instantaneous target area exposure. The permeation-enhancing system utilizes diethylene glycol monoethyl ether (Transcutol) to improve drug distribution in the tympanic membrane lipid phase, supplemented by low-dose combinations of menthol or d-limonene with sodium cholate. This allows for reversible fine-tuning of lipid order and tight junctions, reducing the diffusion barrier while maintaining barrier structural integrity. The permeabilizer regulates system osmotic pressure and maintains stratum corneum hydration, minimizing irritation caused by osmotic imbalance. These layers are physicochemically coupled: the crystalline lipid core provides a stable drug reservoir; the positively charged adhesion shell immobilizes nanoparticles and maintains interfacial stability without causing sealing; and the thermosensitive gel anchors nanoparticles to the membrane surface through weak interactions such as hydrogen bonding and electrostatics, while simultaneously regulating the local activity of the permeation-enhancing components to prevent excessive one-time release and maintain a safe flux window. The overall process adopts a series of steps: forming a stable primary emulsion through hot emulsification at the same temperature, promoting lipid core crystallization through rapid cooling and ultrasound, incorporating surface modification and penetration-enhancing systems under low temperature conditions, and then preparing a thermosensitive gel through cold swelling, followed by low-temperature compounding and aseptic packaging. This approach minimizes the adverse effects on heat-sensitive and volatile components and improves batch-to-batch consistency and interlayer compatibility. Attached Figure Description

[0025] Figure 1 The graph shows the biosafety test results of the external ear drops prepared in Examples 1-3 and Comparative Examples 1-4 of this invention;

[0026] Figure 2 The graph shows the results of the in vitro tympanic membrane permeation test of the external ear drops prepared in Example 1 and Comparative Examples 1-4 of this invention;

[0027] Figure 3 The figures show the results of the in vitro tympanic membrane retention test of the external ear drops prepared in Example 1 and Comparative Examples 1-4 of this invention;

[0028] Figure 4 The figures show the results of the small animal inner ear exposure test for the preparation of external ear drops in Example 1 and Comparative Examples 1-4 of the present invention. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0030] Example 1: A topical ear drop solution that promotes auditory nerve growth, comprising the following components: active small molecules, glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, Tween-80, chitosan quaternary ammonium salt, diethylene glycol monoethyl ether, penetration enhancer, penetrant, sodium cholate, N-acetylcysteine, F127 surfactant, F68 surfactant, and sodium hyaluronate;

[0031] The preparation of the topical ear drops that promote auditory nerve growth specifically includes the following steps:

[0032] S1: After melting 20g of glyceryl stearate, 5g of glyceryl monostearate, 6g of soybean lecithin and 0.5g of vitamin E succinate in a 75℃ water bath, add 5mL of 7,8-dihydroxyflavone solution prepared by pre-dissolving active small molecules in diethylene glycol monoethyl ether, and stir at 300rpm for 13min to obtain the oil phase;

[0033] S2: In a 71℃ water bath, 10g Tween-80, 10mL diethylene glycol monoethyl ether, and 50mL glycerol were added sequentially to deionized water, stirred until dissolved, and then brought to a final volume of 200mL to obtain the hot water phase; 10g N-acetylcysteine ​​was added to 40℃ deionized water, stirred until dissolved, and then brought to a final volume of 100mL to obtain 10wt% cold water phase A; 1g menthol was dissolved in 20mL diethylene glycol monoethyl ether, and then added to 40℃ deionized water and brought to a final volume of 80mL to obtain cold water phase B;

[0034] S3: Slowly pour 40 mL of the 75°C oil phase from step S1 into 200 mL of the 71°C hot water phase from step S2, perform high-speed shearing at 10000 rpm for 6 min, then transfer to an ice bath, and sonicate with a probe at 400W, 30% amplitude, for 30 s on / 30 s off, for a total of 6 cycles, controlling the material temperature to <25°C; after treatment, rapidly cool to 6°C to obtain the dispersion;

[0035] S4: Prepare 40 mL of 0.8% chitosan quaternary ammonium salt aqueous solution at 4°C, filter it through 0.22 µm for sterilization, and slowly add it to 240 mL of dispersion in step S3 at 6°C. Stir gently at 100 rpm for 18 min, slowly add 0.75 g of sodium cholate in three batches, and continue to stir gently for 8 min. Then add 100 mL of cold water phase A and 80 mL of cold water phase B from step S2 in sequence, and continue to stir gently during the addition process. After stirring evenly, obtain the nano-dispersion.

[0036] S5: Cool 600 mL of deionized water at 4 °C overnight, then add 180 g of F127 surfactant and 26 g of F68 surfactant in sequence, stir at 250 rpm for 15 h, then add 1.8 g of sodium hyaluronate, stir until completely dissolved, filter through 0.22 μm for sterilization, adjust the pH to 6.2 with citrate buffer to obtain the thermosensitive in situ gel matrix;

[0037] S6: Slowly add 460 mL of nano-dispersion from step S4 to 530 mL of thermosensitive in-situ gel matrix from step S5, stir at 200 rpm for 13 min, mix evenly, and bring the volume to 1000 mL with citrate buffer to obtain external ear drops.

[0038] Example 2: A topical ear drop solution that promotes auditory nerve growth, comprising the following components: active small molecules, glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, Tween-80, chitosan quaternary ammonium salt, diethylene glycol monoethyl ether, penetration enhancer, penetrant, sodium cholate, N-acetylcysteine, F127 surfactant, F68 surfactant, and sodium hyaluronate;

[0039] The preparation of the topical ear drops that promote auditory nerve growth specifically includes the following steps:

[0040] S1: After melting 22g of glyceryl stearate, 6g of glyceryl monostearate, 7g of soybean lecithin and 0.6g of vitamin E succinate in a 75℃ water bath, add 5mL of neurotrophic factor 3 solution prepared by pre-dissolving active small molecules in diethylene glycol monoethyl ether, and stir at 300rpm for 15min to obtain the oil phase.

[0041] S2: In a 72℃ water bath, 12g Tween-80, 11mL diethylene glycol monoethyl ether, and 50mL glycerol were added sequentially to deionized water, stirred until dissolved, and then brought to a final volume of 200mL to obtain the hot water phase; 15g N-acetylcysteine ​​was added to deionized water at 45℃, stirred until dissolved, and then brought to a final volume of 150mL to obtain a 10wt% cold water phase A; 2g menthol was dissolved in 44mL diethylene glycol monoethyl ether, and then added to deionized water at 40℃ and brought to a final volume of 160mL to obtain the cold water phase B;

[0042] S3: Slowly pour 40 mL of the 75°C oil phase from step S1 into 200 mL of the 72°C hot water phase from step S2, perform high-speed shearing at 10000 rpm for 8 min, transfer to an ice bath, and sonicate with a probe at 400W, 30% amplitude, for 30 s on / 30 s off, for a total of 8 cycles, controlling the material temperature to <25°C; after treatment, rapidly cool to 4°C to obtain the dispersion;

[0043] S4: Prepare 40 mL of 0.8% chitosan quaternary ammonium salt aqueous solution at 4°C, filter it through 0.22 µm for sterilization, and slowly add it to 240 mL of dispersion in step S3 at 4°C. Stir gently at 100 rpm for 20 min, slowly add 0.84 g of sodium cholate in three batches, and continue to stir gently for 10 min. Then add 105 mL of cold water phase A and 90 mL of cold water phase B from step S2 in sequence, and continue to stir gently during the addition process. After stirring evenly, obtain the nano-dispersion.

[0044] S5: Cool 600 mL of deionized water at 4 °C overnight, then add 192 g of F127 surfactant and 27 g of F68 surfactant in sequence, stir at 250 rpm for 16 h, then add 1.8 g of sodium hyaluronate, stir until completely dissolved, filter through 0.22 μm for sterilization, adjust the pH to 6 with citrate buffer to obtain the thermosensitive in situ gel matrix.

[0045] S6: Slowly add 480 mL of nano-dispersion from step S4 to 500 mL of thermosensitive in-situ gel matrix from step S5, stir at 200 rpm for 15 min, mix evenly, and bring the volume to 1000 mL with citrate buffer to obtain external ear drops.

[0046] Example 3: A topical ear drop solution that promotes auditory nerve growth, comprising the following components: active small molecules, glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, Tween-80, chitosan quaternary ammonium salt, diethylene glycol monoethyl ether, penetration enhancer, penetrant, sodium cholate, N-acetylcysteine, F127 surfactant, F68 surfactant, and sodium hyaluronate;

[0047] The preparation of the topical ear drops that promote auditory nerve growth specifically includes the following steps:

[0048] S1: After melting 36g of glyceryl stearate, 8g of glyceryl monostearate, 10g of soybean lecithin and 0.8g of vitamin E succinate in a 75℃ water bath, add 10mL of neurotrophic factor 3 solution prepared by pre-dissolving active small molecules in diethylene glycol monoethyl ether, and stir at 300rpm for 10min to obtain the oil phase;

[0049] S2: In a 70℃ water bath, 8g Tween-80, 12mL diethylene glycol monoethyl ether, and 50mL propylene glycol were added sequentially to deionized water, stirred until dissolved, and then brought to a final volume of 200mL to obtain the hot water phase; 10g N-acetylcysteine ​​was added to deionized water at 35℃, stirred until dissolved, and then brought to a final volume of 100mL to obtain a 10wt% cold water phase A; 1g d-limonene was dissolved in 18mL diethylene glycol monoethyl ether, and then added to deionized water at 40℃ and brought to a final volume of 80mL to obtain the cold water phase B;

[0050] S3: Slowly pour 40 mL of the 75°C oil phase from step S1 into 200 mL of the 70°C hot water phase from step S2, perform high-speed shearing at 10000 rpm for 5 min, transfer to an ice bath, and sonicate with a probe at 400W, 30% amplitude, for 30 s on / 30 s off, for a total of 5 cycles, controlling the material temperature to <25°C; after treatment, rapidly cool to 8°C to obtain the dispersion;

[0051] S4: Prepare 40 mL of 0.8% chitosan quaternary ammonium salt aqueous solution at 4°C, filter it through 0.22 µm for sterilization, and slowly add it to 240 mL of dispersion in step S3 at 8°C. Stir gently at 100 rpm for 15 min, slowly add 0.56 g of sodium cholate in three batches, and continue to stir gently for 5 min. Then add 90 mL of cold water phase A and 75 mL of cold water phase B from step S2 in sequence, and continue to stir gently during the addition process. After stirring evenly, obtain the nano-dispersion.

[0052] S5: Cool 600 mL of deionized water at 4 °C overnight, then add 168 g of F127 surfactant and 24 g of F68 surfactant in sequence, stir at 250 rpm for 12 h, then add 1.8 g of sodium hyaluronate, stir until completely dissolved, filter through 0.22 μm for sterilization, adjust the pH to 6.4 with citrate buffer to obtain the thermosensitive in situ gel matrix.

[0053] S6: Slowly add 440 mL of nano-dispersion from step S4 to 540 mL of thermosensitive in-situ gel matrix from step S5, stir at 200 rpm for 10 min, mix evenly, and bring the volume to 1000 mL with citrate buffer to obtain external ear drops.

[0054] Comparative Example 1: The raw materials and processes of Comparative Example 1 are basically the same as those of Example 1. The main difference is that the thermosensitive in-situ gel matrix is ​​not prepared in Comparative Example 1, but is directly adjusted to the same volume with isotonic citrate buffer; the remaining steps and parameters are consistent with those of Example 1.

[0055] Comparative Example 2: The raw materials and processes of Comparative Example 2 are basically the same as those of Example 1. The main difference is that the surface positive charge modification of chitosan quaternary ammonium salt is not carried out in Comparative Example 2, and an equal volume of sterile water is used to replace the chitosan quaternary ammonium salt solution. The amount of sodium cholate remains unchanged. The remaining steps and parameters are consistent with those of Example 1.

[0056] Comparative Example 3: The raw materials and processes of Comparative Example 3 are basically the same as those of Example 1. The main difference is that the mild penetration-enhancing combination is removed in Comparative Example 3, that is, menthol and sodium cholate are not added, and the total amount of diethylene glycol monoethyl ether remains unchanged; the remaining steps and parameters are consistent with those of Example 1.

[0057] Comparative Example 4: The raw materials and processes of Comparative Example 4 are basically the same as those of Example 1. The main difference is that the crystalline lipid core system is replaced with a liquid oil core system in Comparative Example 4, and the medium-chain triglyceride inert oil phase is used to replace glyceryl stearate and glyceryl monostearate in equal mass. The total lipid phase, surfactant dosage and HLB gradation remain unchanged. The remaining steps and parameters are consistent with those of Example 1.

[0058] Performance testing:

[0059] Biosafety testing: The ear drop samples from Examples 1-3 and Comparative Examples 1-4 were tested on human keratinocytes (HaCaT) using the undiluted solution. Cells were seeded in 96-well plates and cultured to 70-80% confluence. The culture medium was discarded, and 100 µL / well of the test solution was added to each well. The cells were incubated at 37°C and 5% CO2 for 24 h and 48 h, respectively. Control groups used unextracted fresh culture medium (negative control) and culture medium containing 10% dimethyl sulfoxide (positive control). After incubation, OD was measured using the CCK-8 assay according to the reagent instructions. 450 Survival rate (%) was calculated as follows: (OD sample - OD positive control) / (OD negative control - OD positive control) × 100%; each batch of samples was tested three times independently, and the average result was taken. The survival rate results are shown below. Figure 1 As shown.

[0060] based on Figure 1The results analysis showed that the external ear drops prepared in the embodiments of the present invention have excellent biocompatibility and can be used safely. Based on the results of Comparative Example 1 and Example 1, Comparative Example 1, which removed the heat-sensitive gel, retained the same surfactants and penetration enhancers in its formulation, but lost the gel network's dilution / blocking / slow-release function for free components. When the original solution was directly exposed, the cell membrane was exposed to higher instantaneous surface tension and membrane fluidity disturbances, resulting in an early decline in metabolic activity. The cumulative effect after 48 hours led to an accelerated decrease in cell viability. Comparative Example 2 removed the chitosan quaternary ammonium salt. Although it retained the gel and penetration enhancer system, the removal of the positive surface modification of the cationic polysaccharide weakened the electrostatic adsorption and reversible disturbance of the positively charged polysaccharide on the cell membrane and glycoproteins. Its compatibility in the in vitro direct contact scenario was slightly higher than that of Example 1. Correspondingly, this also confirms that the positive adhesion in the examples mainly serves the purpose of retention and transmembrane transport in vivo, rather than improving in vitro cell compatibility. Comparative Example 3 removed menthol and sodium cholate, while maintaining the total amount of diethylene glycol monoethyl ether. This eliminated the two types of permeation enhancers that most directly affect membrane lipid order and tight junctions. The release was primarily driven by diethylene glycol monoethyl ether and the carrier, significantly reducing disturbances to cell membrane fluidity and permeability. Therefore, the survival rates at 24h and 48h were typically the highest among all groups. Comparative Example 4 replaced the crystalline lipid core with a liquid oil core, maintaining the outer interface and permeation-enhancing components. However, because the oil core is non-crystalline, the adsorption / redistribution and subsequent sustained-release capacity of hydrophobic small molecules and monoterpenes within the carrier are weaker than with the crystalline lipid core. Upon direct contact with the stock solution, the effective activity of free components in the culture medium was slightly higher, exhibiting slightly worse compatibility than Example 1.

[0061] Ex vivo tympanic membrane permeation and retention test: Fresh porcine tympanic membranes were taken, and the attached soft tissue was removed. The test samples were the topical ear drops prepared in Example 1 and Comparative Examples 1-4; an effective diffusion area of ​​0.64 cm² was selected. 2 The Franz diffusion cell was used, with a acceptor chamber volume of 5 mL. The water bath was maintained at 37°C, and the magnetic spinner speed was 300 rpm. The acceptor phase formulation was PBS (pH 7.4) + 0.5% Tween-80 (to maintain the confluence conditions). Before use, the membrane was preheated to 37°C and degassed. It was clamped with the tympanic membrane facing outwards from the external auditory canal towards the donor side to ensure no leakage. After loading, the membrane was allowed to stand for 60 min for pre-equilibration. 100 µL of sample was added to the donor side of each diffusion cell. If a thermosensitive gel formulation was used (except for Comparative Example 1), it was allowed to stand for 5 min after addition to complete in-situ gelation before being placed in the donor cell. 0.5 mL samples of the acceptor phase were taken at 0.5, 1, 2, 4, 6, 8, 12, and 24 h, with an equal volume of fresh acceptor phase added immediately after each sample. The sample solution was stored at -20°C, and 7,8-DHF was determined by HPLC. The cumulative permeate Q at each time point was calculated. (t) (µg / cm 2 The result is as follows Figure 2As shown; after 24 hours, the tympanic membrane was removed, gently rinsed once with PBS, and lightly blotted dry with filter paper; the membrane was cut according to the effective area of ​​the donor, and the wet weight (mg) was recorded. Extraction was performed using 70wt% methanol solution (containing 0.1% formic acid) (tissue homogenization + sonication for 10 min), centrifuged at 8000g for 10 min, and the supernatant was used for quantitative analysis of 7,8-DHF. The retention volume (ng / mg tissue) was calculated, and the results are as follows. Figure 3 As shown.

[0062] based on Figure 2 and Figure 3 Results analysis showed that Example 1 exhibited a stable flux increase from 0.5 to 6 hours, a relatively high slope in the linear region from 4 to 12 hours, and reached a significant cumulative permeate volume at 24 hours, while also exhibiting the highest intramembrane retention at 24 hours. This may be due to the formation of a stable hydration layer on the donor surface by the thermosensitive gel, reducing loss and maintaining constant donor activity; the positively charged adhesion brought by chitosan quaternary ammonium salts improved the short-term pausing and distribution of drugs on negatively charged membrane surfaces / mucosa; the temperature-dependent reduction of lipid order / fine-tuning of tight connections reduced diffusion resistance; and the temporary retention-continuous release of hydrophobic small molecules and permeation enhancers by the crystalline lipid core, inhibiting early peaks and lengthening the effective donor window. The combined effect of these three factors resulted in simultaneous benefits to both transmembrane steady-state flux and intramembrane available drug volume. Comparative Example 1 showed a lower cumulative permeation rate than Example 1 throughout the entire process, with the difference widening over longer periods, indicating a significant decrease in retention. This may be because, after the gel was removed, although the transient activity of the free surfactant / permeation enhancer on the donor surface was high, the residence time was shortened, and the sample was easily diluted by lateral flow / evaporation, leading to a faster decline in the effective activity of the donor over time. Furthermore, the lack of film-forming support on the membrane surface reduced the drug's residence and redistribution within the membrane. Comparative Example 2 removed the chitosan quaternary ammonium salt while retaining the gel and permeation enhancer system. Its cumulative permeation rate was slightly lower than Example 1, and its retention was lower. This may be due to the lack of positive charge and electrostatic interaction with the biomembrane surface, which weakened the brief residence-redistribution process of the drug on the membrane surface, resulting in a decrease in its membrane phase partition coefficient. Consequently, the amount entering and remaining within the membrane per unit time decreased, manifested as a simultaneous slight decline in both cumulative permeation rate and retention. Comparative Example 3, with menthol and sodium cholate removed while maintaining the total amount of diethylene glycol monoethyl ether, exhibited the lowest cumulative permeate, slowest ramp-up, and lowest retention. This may be because, while the gel and positive charge remained, the donor activity and membrane contact were not significantly impaired, but the barrier was not fine-tuned. The lipid orderliness / tight junctions remained unchanged, reducing the transmembrane diffusion coefficient and the number of effective membrane channels, resulting in insufficient transmembrane driving force. The intramembrane concentration gradient was difficult to establish, thus reducing retention. Comparative Example 4, replacing the crystalline lipid core with a liquid oil core, maintained the outer interface and permeation-enhancing composition. The early cumulative permeate was slightly higher than in the example, but the later cumulative permeate rate and retention were lower. This may be because the liquid core reduced the diffusion resistance within the carrier, resulting in faster initial release and facilitating early transmembrane crossing. However, the lack of the intraphase barrier of the crystalline core led to insufficient subsequent release, and the donor-side effective activity decayed earlier, resulting in lower late-stage flux and intramembrane redistribution capacity compared to the example.

[0063] Small animal inner ear exposure test: Thirty healthy adult SD rats were randomly divided into two groups: Example 1 and Comparative Examples 1-4, with n=6 in each group. Animals underwent unilateral ear drop administration under mild isoflurane inhalation anesthesia. The ear was cleaned and dried before slowly instilling 0.30 mg / L of the drug into the ear. mL of sample was taken. After administration, the animal's head was tilted to the healthy side for 5 min to promote uniform spreading of the formulation on the tympanic membrane and complete in-situ gelation. The contralateral ear was left untreated for in vivo background correction. Samples were taken at 0.5, 2, 6, and 24 h after administration, with only one time point for each animal. 5 µL of perilymph was sampled through the circular window area under direct microscopic observation. All samples were immediately placed in pre-cooled tubes, and an equal volume of 70 wt% methanol (containing 0.1% formic acid) was added for protein precipitation / extraction. The samples were centrifuged at 8000 g for 10 min at 4°C, and the supernatant was collected and stored at -20°C for analysis. 7,8-DHF perilymph was quantified using a method-validated HPLC, with results expressed as ng / mL. The average concentration was calculated at each time point, and a concentration-time curve was plotted. The AUC was calculated using the linear trapezoidal method based on the average curve. 0-24 Read C max With T max The concentration-time curve results are as follows: Figure 4 As shown; AUC 0-24h C max and T max The results are shown in Table 1.

[0064] Table 1. Results of rat inner ear exposure tests in the examples and comparative examples.

[0065]

[0066] based on Figure 4 Analysis of the results in Table 1 shows that the in vivo drug concentration-time curve of Example 1 indicates that the drug concentration increases steadily within the range of 0.5 to 2 hours, with a peak time (T0). max The effect is delayed to 6 hours, and a high blood concentration is maintained even after 24 hours. This characteristic results in a lower area under the curve (AUC) of the drug. 0-24 The highest value among all groups fully demonstrates that this topical ear drop has a synergistic effect of long-lasting retention, efficient penetration, and stable release. In Comparative Example 1, after gel removal, the donor surface was prone to loss and dilution: the slightly moist environment of the external auditory canal and gravity / evaporation caused the effective activity to decay more quickly, resulting in weak early rise and poor maintenance in the later stages due to unstable supply, manifested as C. maxAlong with the simultaneous decrease in AUC and a significantly lower 24-hour concentration, although the permeation-enhancing and positively charged components remained, the lack of film formation and a viscous layer made contact time and stable supply bottlenecks. Comparative Example 2 retained the gel matrix and permeation-enhancing components, but lacked the positively charged interface constructed by chitosan quaternary ammonium salt. The absence of this interface led to a decrease in the drug's partition coefficient and transient retention time on the biomembrane surface, effectively weakening its first-pass interface opportunity during transmembrane transport, resulting in a decrease in the effective flux entering the membrane per unit time. Therefore, despite its T... max The release time remained around 6 hours, with no significant shift in the release rhythm, but overall absorption efficiency was affected, resulting in a slight decrease in Cmax and a slight decline in AUC due to persistently insufficient flux. Comparative Example 3, while retaining the gel and positively charged interface and effectively maintaining donor drug activity, suffered from a lack of a penetration enhancer to gently loosen the barrier, leading to a minimum transmembrane diffusion coefficient and effective channel number, resulting in significantly insufficient driving force; ultimately, the overall plasma drug concentration curve was difficult to improve: Cmax... max Both AUC and T were the lowest in their respective groups; max The release time remained stable at 6 hours, confirming stable drug release on the donor side, but the drug permeation efficiency was extremely poor. Comparative Example 4 retained the same outer interface, permeation enhancer, and gel system as Example 1, thus possessing considerable donor retention and barrier regulation capabilities; however, its liquid core, due to lower intraphase diffusion resistance and the lack of a release barrier provided by a crystalline lipid core, resulted in excessively rapid early drug release, manifested as a concentration surge within 0.5-2 hours and a T0... max Moved to 2 hours earlier, C max Although similar to Example 1, in the later stages, due to insufficient system release capacity and earlier decay of donor drug activity, its blood drug concentration was caught up with and surpassed by Example 1, ultimately leading to AUC. 0-24 Slightly lower; this result directly verifies the key role of "crystallization nuclei" in regulating release behavior - effectively improving the stability and maintenance capacity of overall drug exposure through peak suppression and tailing effect.

[0067] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A topical ear drop solution that promotes auditory nerve growth, characterized in that, Its raw materials include the following components: active small molecules, glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, Tween-80, chitosan quaternary ammonium salt, diethylene glycol monoethyl ether, penetration enhancer, penetrant, sodium cholate, N-acetylcysteine, F127 surfactant, F68 surfactant and sodium hyaluronate. The active small molecule is 7,8-dihydroxyflavone; The penetration enhancer is either menthol or d-limonene; The penetrant is either glycerol or propylene glycol; The aforementioned topical ear drops that promote auditory nerve growth are prepared by the following steps: S1: After melting glyceryl stearate, glyceryl monostearate, soybean lecithin and vitamin E succinate sequentially in a water bath, add an active small molecule solution and stir to obtain the oil phase; S2: In a water bath, Tween-80, diethylene glycol monoethyl ether, and penetrant are added sequentially to deionized water, stirred until dissolved, and then brought to a final volume to obtain the hot water phase; N-acetylcysteine ​​is added to deionized water, stirred until dissolved, and then brought to a final volume to obtain the cold water phase A; the penetration enhancer is dissolved in diethylene glycol monoethyl ether, and then deionized water is added to a final volume to obtain the cold water phase B; S3: Slowly pour the oil phase from step S1 into the hot water phase from step S2, perform high-speed shearing, transfer to an ice bath, perform ultrasonic treatment, and control the material temperature; after the treatment is completed, quickly cool down to obtain a dispersion. S4: Prepare a chitosan quaternary ammonium salt aqueous solution, filter to remove bacteria, and then slowly add it to the dispersion in step S3. Stir gently, add sodium cholate slowly in three batches, continue to stir gently, and then add the cold water phase A and cold water phase B from step S2 in sequence. Continue to stir gently during the addition process. After stirring evenly, obtain the nano dispersion. S5: Cool the deionized water overnight, then add surfactants F127 and F68 in sequence, stir, then add sodium hyaluronate, stir until completely dissolved, filter to remove bacteria, adjust the pH with citrate buffer, and obtain the thermosensitive in-situ gel matrix. S6: Slowly add the nano-dispersion from step S4 to the thermosensitive in-situ gel matrix from step S5, stir, mix evenly, and adjust the volume with citrate buffer to obtain the external ear drops. In step S1, the active small molecule solution is prepared by pre-dissolving the active small molecules in diethylene glycol monoethyl ether at a ratio of 1g:5mL; the ratio of the amounts of glyceryl stearate, glyceryl monostearate, soybean lecithin, vitamin E succinate, and the active small molecule solution is 18-22g:4-6g:5-7g:0.4-0.6g:5mL. In step S2, the ratio of Tween-80, diethylene glycol monoethyl ether, and penetrant is 8-12 g: 10-12 mL: 50 mL; the ratio of penetration enhancer to diethylene glycol monoethyl ether is 1 g: 18-22 mL. In step S4, the volume ratio of the chitosan quaternary ammonium salt aqueous solution to the dispersion is 1:6; the volume ratio of the cold water phase A, the cold water phase B, and the dispersion is 0.3-0.35:0.25-0.3:

1. In step S5, the ratio of the amounts of surfactant F127, surfactant F68, sodium hyaluronate, and deionized water is 28-32g:4-4.5g:0.3g:100mL. In step S6, the volume ratio of the nano-dispersion to the thermosensitive in-situ gel matrix is ​​44-48:50-54.

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