A liposome dispersion and ear drops for penetrating the eardrum and the round window membrane of the inner ear

Abstract

The present application relates to the technical field of biological medicine, and more particularly to a liposome dispersion liquid and ear drops for penetrating the tympanic membrane and the inner ear round window membrane. The present application designs a liposome dispersion liquid which can effectively penetrate the complete path of the tympanic membrane and the inner ear round window membrane after non-invasive ear drops through the external auditory canal, so as to avoid the tympanic membrane perforation caused by intratympanic injection and reduce the pain and discomfort of patients. The liposome dispersion liquid of the present application has good stability, the prepared ear drops can realize efficient delivery of targeted inner ear drugs, has the characteristics of non-invasive and convenience, and has a wide application prospect in the medical field.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a liposome dispersion and ear drops that can penetrate the tympanic membrane and the round window membrane of the inner ear. Background Technology

[0002] Inner ear diseases, including hearing loss, tinnitus, vertigo, and balance disorders, severely impact patients' quality of life, social participation, and work capacity. Due to the inner ear's deep location, complex structure, and the presence of the blood-labyrinthine barrier, systemic drug administration is difficult to achieve sufficiently effective drug concentrations in the inner ear and is prone to causing significant side effects, resulting in limited therapeutic efficacy.

[0003] Currently, the most common method of local drug administration to the inner ear in clinical practice is tympanic membrane puncture and injection. Although this can increase the local drug concentration, it is an invasive procedure that can cause pain, dizziness, tympanic membrane damage, and even infection. Patient compliance is low, and stable drug delivery cannot be achieved. In contrast, external auditory canal ear drops have advantages such as being non-invasive, convenient, and safe, making them one of the most promising methods of inner ear drug administration for clinical application. Theoretically, ear drops need to pass through the external auditory canal → tympanic membrane → tympanic cavity → round window membrane to enter the inner ear, achieving truly non-invasive local drug administration. However, the components and formulations of traditional ear drops do not have the ability to penetrate deep tissues and cannot cross the two key barriers of the tympanic membrane and round window membrane, often only used for external auditory canal anti-infection purposes.

[0004] The tympanic membrane, composed of an outer layer of keratinized epithelium, a middle layer of fibrous tissue, and an inner layer of mucosa, acts as a significant physical barrier, allowing only trace amounts of small molecules to pass through. The round window membrane, on the other hand, is a three-layered structure consisting of an outer epithelial layer, a middle matrix collagen layer, and an inner epithelial layer, providing strong barrier properties against most drugs, proteins, and conventional nanoparticles. Furthermore, the tympanic cavity between the tympanic membrane and the round window membrane is connected to the nasopharynx via the Eustachian tube, allowing drugs to easily escape through the Eustachian tube or diffuse and dilute within the tympanic cavity, further reducing the effective amount of drug reaching the round window membrane area. Simultaneously, ear drops have a short residence time on the external auditory canal and the surface of the tympanic membrane, easily being drained, evaporated, or cleared by mucus, making it difficult to create a sufficient drug concentration gradient locally, and even more difficult to sustain action on the tympanic membrane and round window membrane surfaces, thus hindering effective inner ear drug delivery.

[0005] Therefore, there is an urgent need for a non-invasive drug delivery system that can actively cross the barriers of the tympanic membrane and round window membrane and efficiently deliver drugs to the inner ear, in order to solve the problems of high invasiveness, insufficient efficiency and limited safety of existing treatments for inner ear diseases.

[0006] In recent years, various nanodelivery systems, such as liposomes, polymer nanoparticles (e.g., PLGA nanoparticles), nanogels, dendritic polymers, and exosome mimics, have been used to explore inner ear drug delivery. However, existing nanodelivery technologies still have significant limitations in the field of otology. For example, current nanodelivery systems lack active transmembrane mechanisms such as induced endocytosis, endocytic transport, or transcellular transport. After entering the ear canal, they have difficulty passing through the tympanic membrane and cannot accumulate in the round window membrane to enter the inner ear, resulting in low overall transmembrane efficiency. In addition, although traditional liposomes or polymer nanoparticles can improve drug loading, they usually cannot simultaneously possess both long-term residence and transmembrane functions. For example, while ordinary liposomes can fuse with biological membranes, their retention is weak; PLGA nanoparticles have good stability, but insufficient transmembrane and adhesion abilities; nanogels have strong adhesion, but poor penetration. On the other hand, long-term static storage of liposome dispersions can lead to the gradual aggregation of liposome particles and significant stratification due to density differences and gravity, resulting in irreversible enlargement of liposome particle size. When the stability of the liposome membrane deteriorates, the overall encapsulation efficiency decreases, the effective drug loading decreases, and the efficacy of liposomes is affected.

[0007] Therefore, there is still a need to design liposome dispersions that can effectively penetrate the complete path of the tympanic membrane and the round window membrane of the inner ear and have good stability in order to improve the drug delivery efficiency targeting the inner ear. Summary of the Invention

[0008] To address the aforementioned technical challenges, this invention provides a liposome dispersion comprising: a drug-loaded liposome suspension and a stabilizer; the drug-loaded liposomes in the drug-loaded liposome suspension are phospholipid-containing drug-loaded liposomes with a particle size of 80-150 nm; the stabilizer is at least one selected from trehalose, glycerol, lecithin, sorbitol, propylene glycol, glucose, and polyethylene glycol (PEG); and the polydispersity index (PDI) of the liposome dispersion is below 0.25.

[0009] This invention significantly improves the stability of drug-loaded liposomes in dispersions by controlling the particle size of drug-loaded liposomes to 80-150 nm and adding stabilizers to keep the PDI value of the liposome dispersion below 0.25.

[0010] Meanwhile, by using drug-loaded liposomes containing phospholipids, the phospholipids can fuse with the lipid bilayer between keratinocytes, promoting membrane fusion and transcellular transport, and facilitating penetration of interstitial spaces, thereby achieving effective delivery of targeted drugs to the inner ear.

[0011] By using drug-loaded liposomes containing phospholipids of appropriate particle size and by using stabilizers to improve the stability of the drug-loaded liposomes, the drug-loaded liposomes can effectively penetrate the tympanic membrane and the complete path of the inner ear round window membrane, significantly improving the drug delivery efficiency targeting the inner ear.

[0012] Preferably, the particle size of the drug-loaded liposomes is 90~100nm.

[0013] Preferably, in the liposome dispersion, the concentration of trehalose is 2-6 w / v%, and / or the concentration of glycerol is 1-4 w / v%, and / or the concentration of glucose is 4-10 w / v%, and / or the concentration of PEG is 2-5 w / v%, and / or the concentration of sorbitol is 2-6 w / v%, and / or the concentration of propylene glycol is 2-8 w / v%, and / or the concentration of lecithin is 3-5 w / v.

[0014] Preferably, the stabilizer is a combination of trehalose and glycerol; and / or, the PDI value of the liposome dispersion is 0.100~0.170.

[0015] This invention has found that when the stabilizer is a combination of trehalose and glycerol, the stability of drug-loaded liposomes in the liposome dispersion can be further improved, thereby significantly improving the drug delivery efficiency targeting the inner ear.

[0016] Preferably, in the liposome dispersion, the concentration of trehalose is 4-5 w / v and the concentration of glycerol is 1-2 w / v.

[0017] Preferably, the trehalose is present inside the drug-loaded liposome or dispersed in the drug-loaded liposome suspension; and / or, the glycerol is dispersed in the drug-loaded liposome suspension.

[0018] Preferably, the drug-loaded liposome is prepared from soybean lecithin, cholesterol, and the drug.

[0019] Preferably, the surface of the drug-loaded liposome is coated with hyaluronic acid; the molecular weight of the hyaluronic acid is 800kDa~1500kDa.

[0020] This invention further reveals that hyaluronic acid with a concentration of 800kDa to 1500kDa exhibits significant adhesiveness, firmly adhering to the skin of the external auditory canal and the epithelial layer of the round window membrane of the tympanic membrane, thereby further increasing retention time. Simultaneously, the hydrogel-like shell formed by the hyaluronic acid prevents nanoparticles from being rapidly washed away within the external auditory canal. Therefore, the hyaluronic acid layer on the surface of the drug-loaded liposomes simultaneously serves as a biological lubricant and protector, further enhancing the stability of the drug-loaded liposomes, thus maintaining the local drug concentration gradient and promoting continuous drug penetration into the tympanic membrane.

[0021] Furthermore, hyaluronic acid within the aforementioned molecular weight range can specifically recognize and bind to CD44 receptors on the surface of membrane epithelial cells and fibroblasts. This not only induces drug-loaded liposomes to further aggregate toward the target site, but also activates myosin to cause a reversible temporary rearrangement and opening of tight junctions on the tympanic membrane. This further promotes the more effective entry of drug-loaded liposomes into the tympanic membrane and into the middle ear, and successfully recognizes the corresponding receptors on the round window membrane to achieve targeted drug delivery to the inner ear.

[0022] Preferably, the molecular weight of the hyaluronic acid is 1000kDa~1200kDa.

[0023] Preferably, the surface of the hyaluronic acid is modified with a cell-penetrating peptide (CPP), which is at least one of low molecular weight protamine (LMWP), transactivator of transcription (TAT), YGRKKRRQRRR (SEQ ID No. 1), octaarginine (R8), nonaarginine (R9), a protein transduction domain (Penetratin), a viral transmission peptide (VP22), a cell-penetrating carrier peptide (Pep-1), and a fluorescein-labeled membrane-penetrating peptide (MPG).

[0024] The amino acid sequence of LMWP is as follows: VSRRRRRRGGRRRR (SEQ ID No. 2).

[0025] The amino acid sequence of TAT is as follows: GRKKRRQRRR (SEQ ID No. 3).

[0026] The amino acid sequence of R8 is as follows: RRRRRRRRR (SEQ ID No. 4).

[0027] The amino acid sequence of R9 is as follows: RRRRRRRRR (SEQ ID No. 5).

[0028] The amino acid sequence of Penetratin is as follows: RQIKIWFQNRRMKWKK (SEQ ID No. 6).

[0029] The amino acid sequence of VP22 is as follows: NAKTRRHERRRKLAIER (SEQ ID No. 7).

[0030] The amino acid sequence of Pep-1 is as follows: KETWWETWWTEWSQPKKKRKV (SEQ ID No. 8).

[0031] The amino acid sequence of MPG is as follows:

[0032] ACGALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID No. 9).

[0033] Preferably, the carboxyl terminus of the MPG is amidated:

[0034] ACGALFLGFLGAAGSTMGAWSQPKKKRKV-NH2.

[0035] Cell-penetrating peptides can form electrostatic adsorption with negatively charged glycoproteins on the surface of the round window membrane, eliminating the repulsive force between the drug-loaded liposomes (hyaluronic acid is negatively charged) and the membrane. At the same time, cell-penetrating peptides can enhance the uptake capacity of the round window membrane epithelial cells, accelerate the endocytosis of the drug-loaded liposomes by the cells, further promote the penetration of the drug-loaded liposomes through the cell layer and matrix layer of the round window membrane to reach the inner ear, thereby improving the transmembrane efficiency of the drug-loaded liposomes.

[0036] More preferably, the cell-penetrating peptide is LMWP.

[0037] Furthermore, the present invention provides a method for preparing the liposome dispersion, comprising: dispersing the raw material of drug-loaded liposomes in an organic solvent to obtain solution A; mixing solution A with the stabilizer to obtain a drug-loaded organic phase of liposomes; removing the organic solvent by rotary evaporation to obtain a film; and hydrating and breaking the film to obtain the liposome dispersion.

[0038] Preferably, the raw materials for the drug-loaded liposomes include soybean lecithin, cholesterol, and drugs; and / or, the organic solvent is a mixture of ethanol and chloroform.

[0039] Preferably, the volume ratio of ethanol to chloroform is 1:(0.8~1.2).

[0040] Preferably, in the liposome-loaded drug organic phase, the final concentration of soybean lecithin is 3-5 w / v, the final concentration of cholesterol is 3-5 w / v, and the final concentration of the drug is 1-3 w / v.

[0041] Preferably, based on the volume of solution A, the concentration of trehalose is 2-8 w / v and the concentration of glycerol is 3-5 w / v.

[0042] Preferably, based on the volume of solution A, the concentration of trehalose is 4-6 w / v and the concentration of glycerol is 3-5 w / v.

[0043] Preferably, the preparation method further includes: adding hyaluronic acid solution to the liposome drug-loaded organic phase to obtain a lipid-polysaccharide complex solution, removing the organic solvent by rotary evaporation to obtain a film; and hydrating and breaking the film to obtain the liposome dispersion.

[0044] Preferably, the concentration of the hyaluronic acid solution is 0.03%~0.05w / v.

[0045] Preferably, the rotary evaporation temperature is 60~75℃, the vacuum degree is 80~90kPa, and the rotation speed is 95-105r / min.

[0046] Preferably, the hydration temperature is 26°C to 37°C.

[0047] Preferably, the hydration can be achieved by gentle agitation or magnetic stirring at 26°C to 37°C for 15 to 30 minutes.

[0048] Preferably, the hydrated material is broken up using ultrasonic treatment or microfluidic high-pressure homogenization.

[0049] Preferably, the conditions for the ultrasonic treatment include: power of 200~400W, 5~10s per session under ice bath conditions, 5~10s interval, and 5~10min of cycle.

[0050] Preferably, ultrasonic processing is performed using a probe.

[0051] Preferably, the conditions for the microjet high-pressure homogenization method include: pressure of 500~1000 bar, and 3~5 cycles.

[0052] Preferably, the preparation method further includes: modifying the surface of the drug-loaded liposomes with cell-penetrating peptides using a chemical coupling method, wherein the amount of cell-penetrating peptides added is 1~50 μg / mL, based on the volume of the liposome dispersion obtained after disruption.

[0053] The present invention further discovers that controlling the amount of cell-penetrating peptide added within the above range is beneficial for drug-loaded liposomes to obtain the best transmembrane efficiency.

[0054] Preferably, the modification of cell-penetrating peptides on the surface of drug-loaded liposomes using chemical coupling specifically involves modifying cell-penetrating peptides on the surface of drug-loaded liposomes using carbodiimide / succinimide ester (EDC / NHS) coupling.

[0055] Preferably, after modification by EDC / NHS coupling method, the N-terminus or C-terminus of the cell-penetrating peptide is covalently linked to the hydrophobic phospholipid DSPE-PEG, and / or, after the carboxyl group of the outer layer of hyaluronic acid is activated, it contacts the cell-penetrating peptide containing the N-terminal amino group, so that the carboxyl group and the amino group are covalently linked.

[0056] Preferably, in the EDC / NHS reaction system, the molar ratio of EDC to carbonate is (2~5):1.

[0057] Preferably, excess cell-penetrating peptides are removed by centrifugation, dialysis, ultrafiltration, or gel filtration.

[0058] In some implementations, the drug includes, but is not limited to, small molecule drugs, hormones, antioxidants, neurotrophic factors, etc.

[0059] Furthermore, the present invention provides an ear drop solution containing the liposome dispersion described above or the liposome dispersion prepared by the preparation method described above.

[0060] In some embodiments, the ear drops also include pharmaceutically acceptable excipients.

[0061] In some implementations, the pharmaceutically acceptable excipients include buffer salts, isotonic regulators (such as glycerol or sodium chloride), or preservatives.

[0062] Preferably, the pharmaceutically acceptable excipients include phosphate buffer solution and isotonicity regulator sodium chloride.

[0063] Furthermore, the present invention provides the application of the liposome dispersion, the preparation method, or the ear drops in the preparation of a drug having the ability to penetrate the tympanic membrane and the round window membrane.

[0064] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0065] This invention presents a non-invasive liposome dispersion that effectively penetrates the tympanic membrane and the complete path of the round window membrane of the inner ear after being administered via the external auditory canal. This avoids tympanic membrane perforation caused by intratympanic injection and reduces patient pain and discomfort. The liposome dispersion of this invention exhibits good stability, and the prepared ear drops can achieve highly efficient targeted delivery of drugs to the inner ear. It is non-invasive and convenient, and has broad application prospects in the medical field. Attached Figure Description

[0066] Figure 1 This is a comparison chart of ear drops and intratympanic injection.

[0067] Figure 2 This is fluorescence imaging of liposome dispersions modified with different CPPs.

[0068] Figure 3 This is a fluorescence imaging of liposome dispersions modified with different CPP concentrations. Detailed Implementation

[0069] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels. The drug in the drug-loaded liposomes in the following embodiments is dexamethasone sodium phosphate, used as an example to demonstrate the advantages of this invention, and should be understood as not constituting a limitation on the implementation of this invention. In the following embodiments, w / v represents the mass-to-volume ratio in g / mL. The molecular weight of soybean lecithin in the following embodiments is 700-1000 Da. The molecular weight of hyaluronic acid in the following embodiments is 1000-1200 Da. The molecular weight of PEG in the following embodiments is 6000-10000 Da. The molecular weight of cholesterol in the following embodiments is 387 Da.

[0070] Example 1

[0071] This embodiment provides a liposome dispersion, comprising: a drug-loaded liposome suspension and a stabilizer; the drug-loaded liposomes in the drug-loaded liposome suspension are phospholipid-containing drug-loaded liposomes, and the particle size of the drug-loaded liposomes is 90-100 nm; the stabilizer is trehalose and glycerol, and in the liposome dispersion, the concentration of trehalose is 4 w / v and the concentration of glycerol is 2 w / v. The drug-loaded liposomes are prepared from soybean lecithin, cholesterol, and dexamethasone sodium phosphate; the surface of the drug-loaded liposomes is coated with hyaluronic acid, and the surface of the hyaluronic acid is modified with the cell-penetrating peptide LMWP.

[0072] The method for preparing the liposome dispersion includes:

[0073] Step 1: Preparation of the organic phase for liposome drug delivery

[0074] Soy lecithin, cholesterol, and dexamethasone sodium phosphate were added to 500 mL of ethanol / chloroform solution (1:1, v / v). The final concentrations of soybean lecithin, cholesterol, and dexamethasone sodium phosphate were 4 w / v, 0.04 w / v, and 1 w / v, respectively. The mixture was stirred at room temperature for 10 min to obtain a clear or homogeneous solution A.

[0075] Step 2: Add stabilizer

[0076] Add trehalose powder and glycerol evenly to solution A obtained in step 1, so that the final concentration of trehalose is 5 w / v and the final concentration of glycerol is 4 w / v. Stir at room temperature for 30 min to obtain a homogeneous solution B.

[0077] Step 3: Add hyaluronic acid to form a lipid-polysaccharide complex solution.

[0078] A 0.04 w / v hyaluronic acid aqueous solution was prepared and slowly added dropwise to solution B at a rate of 4 mL / min under stirring to obtain a mixture. The mixture was then treated with a high-shear dispersing emulsifier (300 W, 8000 rpm) for 5 min each time to ensure thorough dispersion of the organic phase and the aqueous phase containing hyaluronic acid, resulting in solution C (lipid-polysaccharide complex solution).

[0079] Step 4: Rotary evaporation to form a thin film

[0080] Transfer the mixed solution C into a rotary evaporator flask and evaporate it at 100 r / min under a vacuum of 80 kPa and 60 °C, alternating between clockwise and counterclockwise for 10 min in each direction, for a total time of approximately 40 min. When the volume of the mixed solution remains at 5%, a small amount of hyaluronic acid aqueous solution can be added again (to make the mass-to-volume ratio of hyaluronic acid to the remaining mixed solution 1.0 mg / mL), and rotary evaporation can be continued until the organic solvent is basically removed, resulting in a lipid-hyaluronic acid film uniformly adhered to the flask wall.

[0081] Step 5: Preparation of hyaluronic acid-modified liposome dispersion

[0082] Add an appropriate amount of pure water to the above lipid-hyaluronic acid film, and gently shake at 30°C to allow the film to fully detach and form a crude liposome dispersion. Use a microfluidic high-pressure homogenization method to break the crude liposome dispersion into a uniform hyaluronic acid-modified liposome dispersion with a particle size of 90~100nm.

[0083] Step 6: Surface finishing with CPP (LMWP is used in this embodiment)

[0084] The hyaluronic acid-modified liposome dispersion obtained in the above steps was used to activate the carboxyl groups of the outer layer of hyaluronic acid with EDC / NHS at pH 6.0. The molar ratio of EDC to carbonate was 2:1. Subsequently, CPP aqueous solution was added to the system at a final CPP concentration of 10 μg / mL to covalently link the carboxyl groups to the N-terminal amino groups of CPP. After reacting for 3 h, the liposome dispersion was obtained by dialysis and gel filtration to remove free excess cell-penetrating peptides and byproducts. The final concentration of trehalose in the liposome dispersion was 4 w / v, and the final concentration of glycerol was 2 w / v.

[0085] Furthermore, this embodiment provides an ear drop solution containing the liposome dispersion, including the liposome dispersion, a phosphate buffer solution, an isotonic adjuster sodium chloride, and adjusting the pH to 6.5.

[0086] Example 2

[0087] This embodiment provides a liposome dispersion and ear drops, the only difference from Example 1 is that the CPP used is VP22.

[0088] Example 3

[0089] This embodiment provides a liposome dispersion and ear drops, the only difference from Example 1 is that the CPP uses R8.

[0090] Example 4

[0091] This embodiment provides a liposome dispersion and ear drops, the only difference from Example 1 is that the CPP uses TAT.

[0092] Example 5

[0093] This embodiment provides a liposome dispersion and ear drops, which differ from Example 1 in that:

[0094] In the liposome dispersion, the concentration of trehalose was 2 w / v and the concentration of glycerol was 3 w / v.

[0095] Example 6

[0096] This embodiment provides a liposome dispersion and ear drops, which differ from Example 1 in that:

[0097] In the liposome dispersion, the concentration of trehalose was 6 w / v and the concentration of glycerol was 3 w / v.

[0098] Example 7

[0099] This embodiment provides a liposome dispersion and ear drops, the only difference from Example 1 being that the stabilizer in the liposome dispersion is trehalose, and the concentration of trehalose is 6w / v.

[0100] Example 8

[0101] This embodiment provides a liposome dispersion and ear drops, the only difference from Example 1 being that the stabilizer in the liposome dispersion is glycerol, and the concentration of glycerol is 6 w / v.

[0102] Example 9

[0103] This embodiment provides a liposome dispersion and ear drops, which differ from Example 1 in that:

[0104] In the liposome dispersion, the stabilizers were trehalose and PEG, with the concentration of trehalose being 4 w / v and the concentration of PEG being 2 w / v.

[0105] Example 10

[0106] This embodiment provides a liposome dispersion and ear drops, which differ from Example 1 in that:

[0107] The stabilizers in the liposome dispersion were sorbitol and PEG, with sorbitol at a concentration of 4 w / v and PEG at a concentration of 2 w / v.

[0108] Example 11

[0109] This embodiment provides a liposome dispersion and ear drops, which differ from Example 1 in that:

[0110] In the liposome dispersion, the stabilizers were propylene glycol and sorbitol, with propylene glycol at a concentration of 4 w / v and sorbitol at a concentration of 2 w / v.

[0111] Example 12

[0112] This embodiment provides a liposome dispersion and ear drops, which differ from Example 1 in that:

[0113] In the liposome dispersion, the stabilizers are lecithin and glucose, with the concentration of lecithin being 3 w / v and the concentration of glucose being 6 w / v.

[0114] Example 13

[0115] This embodiment provides a liposome dispersion and ear drops, which differ from Example 1 in that:

[0116] The particle size of the drug-loaded liposomes is 140~150nm.

[0117] Comparative Example

[0118] This comparative example provides a liposome dispersion and ear drops, differing only from Example 1 in that:

[0119] The particle size of the drug-loaded liposomes is 160~170nm.

[0120] Experimental Example 1

[0121] This experimental example tested the PDI value of the liposome dispersions prepared in the above examples and comparative examples. The steps were as follows: the liposome dispersions were thoroughly mixed under ultrasonication, and large particles and insoluble matter were removed by passing the mixture through a 0.22 μm filter membrane. The samples were then added to the sample cell of a DLS instrument, and the scattered light signal was analyzed to obtain the standard deviation of the particle size distribution and the average particle size of the liposome dispersions. The formula for calculating PDI is as follows:

[0122] PDI = Standard Deviation 2 / Average particle size 2

[0123] The test results are shown in Table 1.

[0124] Table 1

[0125]

[0126] Experimental Example 2

[0127] In this experimental example, fluorescently labeled liposome dispersions and ear drops containing them were prepared according to the preparation method of Example 1 above. Based on the preparation steps of Example 1, only the hyaluronic acid was replaced with an equal amount of fluorescently labeled hyaluronic acid. The preparation method of fluorescently labeled hyaluronic acid is as follows:

[0128] Hyaluronic acid was dissolved in phosphate buffer to obtain solution A. At room temperature, the fluorescent label 1,1'-dioctyl-3,3,3',3'-tetramethylindole carbapenem (Dil) was dissolved in organic solvent B (anhydrous dimethyl sulfoxide) to obtain solution C. The mass-to-volume ratio of Dil to anhydrous dimethyl sulfoxide was 1:1, that is, 1 mg of Dil solid powder was dissolved in 1 mL of anhydrous dimethyl sulfoxide. Solution A and solution C were stirred thoroughly to obtain solution D. The reaction was carried out at room temperature for 4 h, followed by dialysis for 48 h. After lyophilization, solid E was obtained, which is the fluorescently labeled hyaluronic acid.

[0129] Small animal live imaging experiment:

[0130] Mice were anesthetized with 1% sodium pentobarbital, and the left external auditory canal was exposed. Under a microscope, 15 μL of the fluorescently labeled liposome dispersion from Example 1 (the concentration of the fluorescently labeled liposome dispersion was uniformly adjusted to 20 mg / mL) was evenly dropped onto the tympanic membrane of the mice using a microsyringe. The fluorescence intensity of the left ear of the mice was photographed using small animal in vivo imaging on days 1, 2, 3, 5, and 7 after drug administration, and the results were analyzed. Simultaneously, mice treated with the same drug regimen by puncturing the tympanic membrane with a microsyringe served as a control.

[0131] The results are as follows Figure 1 As shown, the fluorescence intensity in the left ear of both groups gradually decreased over time, until the fluorescence signal gradually disappeared on day 7. Compared with intratympanic injection, ear drops have a stronger sustained-release effect and a longer duration of action in the middle and inner ear. Therefore, compared with intratympanic injection, ear drops can also achieve liposome delivery to the inner ear, proving that the liposome dispersion of the present invention can effectively penetrate the complete pathway of the tympanic membrane and the round window membrane of the inner ear, achieving efficient delivery of targeted drugs to the inner ear.

[0132] Experimental Example 3

[0133] This experimental example follows the preparation method of Experiment 2 to prepare the fluorescently labeled liposome dispersions and ear drops containing them corresponding to Examples 2-4. The fluorescence intensity of the liposome dispersions modified with different CPPs was compared at the same time point. The results are as follows: Figure 2 As shown, the delivery efficiency of LMWP is significantly better than that of TAT, R8 and VP22.

[0134] Test Example 4

[0135] Based on Example 1, this experiment adjusted the final CPP concentrations to 1 μg / mL, 10 μg / mL, and 100 μg / mL, with no CPP added serving as a control group. The fluorescence intensity of the liposome dispersions modified with different CPP concentrations was compared according to the method in Example 2. The results are as follows: Figure 3 As shown, delivery efficiency decreases when CPP concentration is too high.

[0136] Experimental Example 5

[0137] This experiment tested the therapeutic effects of ear drops prepared in different embodiments and comparative examples using a hearing test. The hearing test procedure was as follows: C57 mice with normal hearing were screened using auditory brainstem response (ABR) testing. After noise exposure, the mice were given ear drops in their left external auditory canal, while the right ear was not treated. On day 7 after administration, ABR testing was performed again, and the changes in hearing of the mice were statistically analyzed. The test results are shown in Table 2.

[0138] Table 2

[0139]

[0140] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A liposome dispersion, characterized in that, include: The drug-loaded liposome suspension and stabilizer; the drug-loaded liposomes in the drug-loaded liposome suspension are phospholipid-containing drug-loaded liposomes, and the particle size of the drug-loaded liposomes is 90~100nm; the PDI value of the liposome dispersion is 0.100~0.170; the stabilizer is a combination of trehalose and glycerol; in the liposome dispersion, the concentration of trehalose is 4~5 w / v%, and the concentration of glycerol is 1~2 w / v%; the drug-loaded liposomes are prepared from soybean lecithin, cholesterol, and drugs as raw materials; the surface of the drug-loaded liposomes is coated with hyaluronic acid; the surface of the hyaluronic acid is modified with cell-penetrating peptides; the cell-penetrating peptides are LMWP, VP22, R8, or TAT.

2. The liposome dispersion according to claim 1, characterized in that, The trehalose is present inside the drug-loaded liposome or dispersed in the drug-loaded liposome suspension; and / or, the glycerol is dispersed in the drug-loaded liposome suspension.

3. The liposome dispersion according to claim 1 or 2, characterized in that, The molecular weight of the hyaluronic acid is 800 kDa to 1500 kDa.

4. A method for preparing the liposome dispersion according to any one of claims 1 to 3, characterized in that, include: The raw materials for drug-loaded liposomes are dispersed in an organic solvent to obtain solution A; Solution A is mixed with the stabilizer to prepare a liposome-loaded organic phase. Hyaluronic acid solution is added to the liposome-loaded organic phase to obtain a lipid-polysaccharide complex solution. The organic solvent is removed by rotary evaporation to obtain a film. The film is hydrated and then broken to obtain a hyaluronic acid-modified liposome dispersion. Cell-penetrating peptides are modified on the surface of the drug-loaded liposomes in the hyaluronic acid-modified liposome dispersion using a chemical coupling method. The amount of cell-penetrating peptides added is 1~50 μg / mL, based on the volume of the hyaluronic acid-modified liposome dispersion, to obtain the liposome dispersion.

5. The preparation method according to claim 4, characterized in that, The organic solvent is a mixture of ethanol and chloroform.

6. The preparation method according to claim 4, characterized in that, Based on the volume of solution A, the concentration of trehalose is 2-8 w / v and the concentration of glycerol is 3-5 w / v.

7. An ear drop solution, characterized in that, It contains the liposome dispersion of any one of claims 1 to 3 or the liposome dispersion prepared by any one of claims 4 to 6.

8. The use of the liposome dispersion according to any one of claims 1 to 3, the liposome dispersion prepared by the preparation method according to any one of claims 4 to 6, or the ear drops according to claim 7 in the preparation of a drug having penetration of the tympanic membrane and the round window membrane.

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