CN109381447B - Astaxanthin-loaded phospholipid nanoparticle and preparation method and application thereof - Google Patents

Abstract

The invention discloses an Astaxanthin (AST) -loaded phospholipid nanoparticle and a preparation method thereof, wherein the phospholipid nanoparticle is obtained by mixing an organic phase in which a high-molecular polymer and astaxanthin are dissolved and a water phase in which phospholipid is dissolved and performing an emulsion solvent volatilization method, wherein the high-molecular polymer is monomethoxypolyethylene glycol-polylactic acid (MPEG-PLA), and the phospholipid is dimyristoyl phosphatidylcholine (DMPC). According to the invention, the astaxanthin is modified by carrying out phospholipid nanoparticle entrapment, the solubility and size of the astaxanthin are changed, the water solubility of the fat-soluble medicine astaxanthin is greatly improved, and meanwhile, the phospholipid nanoparticles endow the astaxanthin with a slow release characteristic, namely the medicine is continuously and slowly released after entering cells of a pathological change part, so that the administration frequency can be reduced, and the treatment effect can be enhanced.

Description

Astaxanthin-loaded phospholipid nanoparticle and preparation method and application thereof

Technical Field

The invention relates to a phospholipid nanoparticle carrying a high antioxidant activity medicament, in particular to a preparation method and application of the phospholipid nanoparticle carrying astaxanthin.

Background

The common pathogenic mechanism of inner ear diseases such as noise-induced and drug-induced deafness relates to the increase of active oxygen (ROS) in the microenvironment of the inner ear and the reduction of antioxidant stress. A large number of small molecular compounds with antioxidant activity are proved to have the effects of preventing and antagonizing ototoxicity caused by the increase of ROS in vivo and in vitro experimental researches, and part of medicaments enter a clinical test stage. However, due to the special structure of the inner ear, small molecule compounds, which are often administered systemically, cannot enter the lesion and clinical trials fail.

Astaxanthin has strong antioxidant activity, and can be used for treating liver injury, brain injury and multiple organ injury caused by anoxia. However, there has been no report on the use of astaxanthin for ototoxicity such as hearing loss due to oxidative stress in the inner ear. For this reason, there may be the following two points: 1) astaxanthin is liposoluble, is slightly soluble in water, and is not beneficial to pharmacy; 2) the inner ear is a semi-closed structure and has double barrier protection of blood-brain and blood-labyrinth, and systemic medication is difficult to reach the lesion part. The local inner ear medicine is usually lost through the eustachian tube, and needs to be repeatedly used for many times, so that the probability of artificial trauma and infection is easily increased.

Therefore, those skilled in the art have focused on improving the physicochemical properties of astaxanthin so that it can be used for the prevention or treatment of inner ear diseases.

Disclosure of Invention

The invention aims to improve the physicochemical properties of astaxanthin (ASTxanthin, AST) through a pharmacy approach, namely, prepare and construct astaxanthin-Loaded Phospholipid Nanoparticles (LPN) so as to improve the water solubility of the astaxanthin and endow the astaxanthin with slow release properties, thereby realizing the purpose of preventing or treating inner ear diseases by local intratympanic administration of the inner ear.

In order to achieve the above purpose, the invention firstly provides a preparation method of astaxanthin-loaded phospholipid nanoparticles (AST-LPN), wherein the phospholipid nanoparticles are obtained by adopting dichloromethane as an organic phase solvent of astaxanthin and volatilizing the organic phase by an emulsion solvent volatilization method; specifically, the organic phase in which a high molecular polymer and astaxanthin are dissolved is mixed with an aqueous phase in which a phospholipid is dissolved, and the mixture is ultrasonically emulsified, and then the organic phase is volatilized to obtain the astaxanthin-containing water-based ink.

Wherein the high molecular polymer is preferably methoxypolyethylene glycol-polylactic acid (MPEG-PLA), and the phospholipid is preferably dimyristoyl phosphatidylcholine (DMPC).

Preferably, the aqueous phase solvent is aqueous methanol.

Preferably, the mass ratio of DMPC to MPEG-PLA used to prepare the astaxanthin-loaded phospholipid nanoparticles is 1:1 to 20, more preferably 1:9 to 10.

Further, the preparation method of the astaxanthin-loaded phospholipid nanoparticles comprises the following steps:

1) dissolving MPEG-PLA and AST in dichloromethane to obtain an organic phase;

2) dissolving DMPC in methanol water solution to serve as water phase;

3) dropping the organic phase into the water phase, and emulsifying by ultrasonic;

4) after emulsification, pouring the emulsion into a sodium cholate aqueous solution;

5) stirring for a certain time to completely volatilize the organic solvent.

Preferably, the dissolution concentration of MPEG-PLA in dichloromethane in step 1) is 20-40g/L, most preferably 30 g/L; the solubility concentration of AST in methylene chloride is 1-10g/L, more preferably 2-5 g/L.

Preferably, the concentration of the methanol aqueous solution in the step 2) is 1 to 8 percent, and optimally 4 percent based on the volume ratio of the methanol to the water; the solubility concentration of DMPC in the water phase is 0.5-1.5g/L, preferably 1 g/L.

Preferably, the mass ratio of the DMPC to the MPEG-PLA in the mixed solution for emulsification in the step 3) is 1:8-12, more preferably 1: 9-10.

Further, the time for dripping the organic phase in the step 3) is preferably not more than 5 minutes, more preferably not more than 3 minutes; preferably, the sonication is carried out in an ice bath environment, and the sonication time period is preferably 50 to 70 seconds, and most preferably 60 seconds.

Preferably, the concentration of the aqueous solution of sodium cholate in step 4) is 0.3% to 0.7%, and most preferably 0.5%, in grams of sodium cholate contained in each 100mL of water.

Preferably, the stirring time in step 5) is 6 to 12 hours.

Further, the method also comprises the following steps after the organic solvent in the step 5) is completely volatilized: centrifuging and collecting precipitate; wherein the centrifugation rotating speed is preferably 11000-13000rpm, the centrifugation temperature is preferably 0-5 ℃, and the centrifugation time is preferably 25-40 minutes.

Further, the invention provides astaxanthin-loaded phospholipid nanoparticles prepared by the method; the encapsulation rate of the phospholipid nanoparticles is 20% -80%, preferably 40% -60%, and more preferably 50% -55%; the drug loading rate of the phospholipid nanoparticles is 1% -5%, preferably 2% -4%, and more preferably 2% -3%; the particle size of the phospholipid nanoparticle is less than 200nm, preferably 100-180nm, and more preferably 140-160 nm; the surface of the phospholipid nanoparticle is negatively charged.

Furthermore, the solubility of the astaxanthin-loaded phospholipid nanoparticles in water is at least more than 1 g/L.

Furthermore, the sustained release time of the astaxanthin-loaded phospholipid nanoparticles in the in-vitro artificial lymph fluid is more than 5 days, preferably more than 7 days, and more preferably more than 10 days; the sustained release time of the astaxanthin-loaded phospholipid nanoparticles in vivo is more than 6 hours, preferably more than 10 hours.

Furthermore, the invention also provides application of the astaxanthin-loaded phospholipid nanoparticles in preparation of medicines for preventing and treating inner ear diseases, in particular application in diseases caused by increase of active oxygen in microenvironment of inner ears, such as noise-induced deafness and drug-induced deafness.

Further, the prevention and treatment drug comprises the astaxanthin-loaded phospholipid nanoparticles provided by the invention, and can also comprise a pharmaceutically acceptable carrier or excipient, and the drug can also be a pharmaceutical composition.

Preferably, the administration mode of the astaxanthin-loaded phospholipid nanoparticles provided by the invention is local intratympanic administration of the inner ear.

Experiments prove that the astaxanthin-loaded phospholipid nanoparticles (AST-LPN) can smoothly enter lymph fluid of an inner ear after being administrated through a round window membrane, and the AST-LPN in the lymph fluid of the inner ear can slowly release a medicament, so that the concentration of the medicament can be maintained to 24 hours, and necessary conditions are created for the AST to resist hearing loss caused by ROS increase. Finally, AST-LPN shows a remarkable inhibiting effect on the hearing threshold rise caused by cisplatin in the range of 2.8-22kHz after being administered through a round window membrane, and the average inhibiting rate is about 20 dB.

The invention adopts an emulsifying solvent volatilization method, selects phospholipid DMPC and a high molecular polymer material MPEG-PLA to carry out lipid nanoparticle entrapment modification on AST, optimizes the preparation method, obtains astaxanthin-loaded phospholipid nanoparticles, changes the dissolution characteristic and the particle size of the medicine, and greatly improves the water solubility of the fat-soluble medicine astaxanthin (by about 24 times). The phospholipid nanoparticles have a phospholipid bilayer similar to a cell membrane, so that the biocompatibility of a carrier system is improved, and meanwhile, the phospholipid nanoparticles endow the astaxanthin slow release characteristic, namely, the medicine is continuously and slowly released after entering cells at a pathological change part, so that the administration frequency can be reduced, and the treatment effect is enhanced.

Drawings

FIG. 1 shows the particle size variation of lipid nanoparticles prepared by the nanoprecipitation method using acetonitrile as an organic solvent in different mass ratios of phospholipid-high molecular polymer in example 1;

FIG. 2 is the potential change of lipid nanoparticles prepared by the nanoprecipitation method using acetonitrile as an organic solvent in different mass ratios of phospholipid-high molecular polymer in example 1;

FIG. 3 shows the variation of the dispersion coefficients of lipid nanoparticles prepared by the nanoprecipitation method using acetonitrile as an organic solvent in different mass ratios of phospholipid-high molecular polymer in example 1;

FIG. 4 is a comparison of the effect of two different preparation methods on astaxanthin encapsulation efficiency in example 1 and example 2;

FIG. 5 is a comparison of the effect of two different preparation methods on astaxanthin loading in example 1 and example 2;

FIG. 6 is a comparison of particle sizes of astaxanthin-loaded and astaxanthin-free lipid nanoparticles prepared by emulsion solvent evaporation;

FIG. 7 is a comparison of surface potentials of astaxanthin-loaded and astaxanthin-free lipid nanoparticles prepared by emulsion solvent evaporation;

FIG. 8 is a transmission electron microscope image of astaxanthin-loaded lipid nanoparticles prepared by an emulsion solvent evaporation method, Bar: 100 nm;

FIG. 9 is a release profile of AST-LPN in vitro artificial lymph;

FIG. 10 is a lymph fluid release profile in AST-LPN guinea pigs;

fig. 11 is a graph of the results of the toxic effect of blank unloaded phospholipid nanoparticles on HEI-OC1 cells;

FIG. 12 is a graph showing the results of the toxic effect of various concentrations of Cisplatin (CDDP) on HEI-OC1 cells;

FIG. 13 is a result of the protective effect of AST-LPN and AST on CDDP induced cytotoxicity;

FIG. 14 is a statistical analysis of ROS activity fluorescence intensity in HEI-OC1 cells after treatment with different doses;

FIG. 15 is a graph of the change in ROS activity in HEI-OC1 cells following treatment with different doses;

FIG. 16 shows the results of low dose multiple (4 mg/kg/day, 3 consecutive days) intraperitoneal administration of CDDP, with ABR measurements taken 1 day before and 3 days after intraperitoneal administration;

FIG. 17 is a graph of the results of a high dose single (12mg/kg, 1) intraperitoneal injection of cisplatin with ABR detection 1 day before and 3 days after intraperitoneal injection, respectively;

fig. 18 is a graph of AST-LPN administered via retroauricular approach to expose round window membrane 1h prior to intraperitoneal cisplatin administration, ABR was recorded before and 3 days post administration, and measured ABR values before and after administration of p <0.05, > p <0.01, > p < 0.001; the value is the mean ± sd, N is 4;

fig. 19 is AST-LPN administered via retroauricular approach exposing round window membrane 1h before and 3 days after intraperitoneal cisplatin administration, ABR domain shift before and after administration, p <0.05, p <0.01, p <0.001 compared to cisplatin group; the values mean ± sd, and N4.

Detailed Description

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention, but the present invention is not limited thereto.

The first embodiment is as follows: preparation of astaxanthin phospholipid nanoparticles (AST-LPN) by nano precipitation method

Dissolving 30mg of MPEG-PLA (high molecular polymer) and 2-5mg of AST in 1mL of acetonitrile solution to obtain an organic phase; 3mg of DMPC (phospholipid) was dissolved in 10mL of 4% ethanol solution as an aqueous phase. The aqueous phase was preheated to 65 ℃ and stirred at 900rpm until the DMPC was completely dissolved. Dropwise adding the organic phase containing the drug, stirring for 2min after finishing dropping within 3min, adjusting the stirring speed to 300rpm, and stirring gently overnight until the organic phase is completely volatilized. Centrifuging at 4000g and 4 deg.C for 40min with Millipore 10kDa centrifugal concentration tube, removing unencapsulated drug and organic solvent, and continuously centrifuging for 2 times to obtain about 500 μ L nanoparticle concentrate. The mass ratio of DMPC to MPEG-PLA is 1:0, 1:1, 1:5, 1:10, 1:20, 0:1 respectively, and the AST-LPN prepared in the range has a particle size of 90.2-138.4nm (figure 1) and a potential of-9.8-15.3 mV (figure 2).

The results in FIG. 1 show that the phospholipid nanoparticles have a minimum particle size of about 80nm at a phospholipid/polymer ratio of 1: 20. When the content of phospholipid is increased or decreased, the particle size of the phospholipid nano-particles is increased. The particle size of nanoparticles prepared from DMPC to MPEG-PLA ratio of 1:0 is about 120nm, while the particle size of liposomes prepared from DMPC to MPEG-PLA ratio of 0:1 is up to 1750 nm.

The results in FIG. 2 show that the phospholipid nanoparticle potentials increased with increasing DMPC content, but were all negatively charged. The maximum is about-3 mV and the minimum is about-15 mV. In subsequent experiments, the ratio of DMPC to MPEG-PLA is 1:10 to prepare the astaxanthin-loaded phospholipid nanoparticles.

The results in FIG. 3 show that the particle size dispersion coefficients of the prepared phospholipid nanoparticles are all between 0.1 and 0.2 and the dispersity is better when the ratio of DMPC to MPEG-PLA is 1:0, 1:1, 1:5 and 1: 10.

The AST-LPN prepared by the method has low encapsulation efficiency (EE%) which is only 3.2-8%; drug Loading (DL%) was also low, only about 0.38-0.5%, and both encapsulation efficiency and Drug Loading were reduced with increasing Drug Loading, which may be associated with Drug overload (fig. 4 and 5).

EXAMPLE 2 preparation of astaxanthin phospholipid nanoparticles (AST-LPN) by emulsion solvent volatilization method

The main reasons for the low encapsulation efficiency and the low drug-loading rate of the astaxanthin lipid nanoparticles prepared by the nano-precipitation method are analyzed, and the low solubility of AST in acetonitrile is probably caused. The same mass of AST was dissolved in acetonitrile and dichloromethane, and after vortex dissolution the dichloromethane solution was more clear and transparent, presumably with AST more soluble in dichloromethane than acetonitrile. Thus, AST-LPN was prepared by dissolving AST and MPEG-PLA in methylene chloride instead of acetonitrile and DMPC in 4% (volume ratio of methanol to water) methanol in water. The detailed steps are as follows: MPEG-PLA 30mg and AST 2-5mg were dissolved in 1mL of methylene chloride as an organic phase; 3mg of DMPC was dissolved in 3mL of 4% aqueous methanol as an aqueous phase. Dropwise adding the organic phase containing the medicine into the water phase, completing the dropwise adding within 3min, emulsifying in an ultrasonic crushing instrument with power of about 480w, and continuously performing ultrasonic treatment for 60 s. In order to avoid the influence of heat generated in the ultrasonic process on the activity of astaxanthin, the ultrasonic process was carried out on ice. After the sonication, the emulsion was poured into 26mL of 0.5% sodium cholate solution and placed on a rotary stirring apparatus for overnight stirring (300rpm) to completely evaporate the residual organic solvent. And centrifuging the final solution at 12,000rpm and 4 ℃ for 30min to obtain a precipitate, namely AST-LPN.

As shown in FIGS. 4 and 5, EE% and DL% of AST-LPN were about 51.8% and 2.65%, respectively, prepared by substituting methylene chloride for acetonitrile and by an emulsion solvent evaporation method instead of a nano-precipitation method. The EE% and DL% of AST-LPN prepared by the method are improved by about 6.48-16.19% and 5.3-7.5% compared with those of the nano precipitation method, the water solubility of AST medicine is greatly improved, and the method has strong clinical application value.

The AST-LPN and the unloaded lipid Nanoparticles (NP) prepared by the emulsion solvent evaporation method have the particle size of about 138.97nm, and have no significant difference (figure 6). Both the AST-LPN and NP were negatively charged at about-9.3 and-22.8 mV (FIG. 7), respectively, and the AST-LPN surface charge was significantly higher than that of NP, probably due to the lipid-soluble high molecular weight AST coating the nanoparticle surface, shielding some of the charge. The results of a projection electron microscope show that the AST-LPN has a round and smooth surface and uniform particle size of about 150nm, and are basically consistent with the detection results of a particle size analyzer (figure 8).

EXAMPLE 3 examination of Release characteristics in AST-LPN inner ear lymph fluid

As shown in FIG. 9, AST-LPN was slowly released in the artificial lymph fluid in vitro for 15 days. In addition, as shown in fig. 10, the test results of drug concentration detection in lymph of inner ear demonstrated that, after AST solution (6 μ g) was administered through round window membrane, lymph was taken 30min after administration for mass spectrometry analysis, and AST content was not detected. This result suggests that lipid-soluble AST cannot enter the inner ear through the round window membrane. Based on this, we determined that AST does not exert the protective effect of cisplatin ototoxicity after being administered through the round window membrane. Referring to FIG. 10, after AST-LPN administration through Round Window Membrane (RWM) to guinea pigs (AST concentration: 1.0mg/mL, 6. mu.L), the concentration in lymph fluid reached the maximum (600.67 + -247.95 ng/mL) after 1 hour, and the drug was continuously released slowly for 24 hours; however, after administration of AST through the abdominal cavity (IP) and Round Window Membrane (RWM), the AST content was not detected in the inner ear lymph.

The research result shows that AST is modified by phospholipid nanoparticles, so that the water solubility is greatly improved, the particle size and the surface potential endow AST with the characteristic of penetrating through a round window membrane, the AST can smoothly enter lymph fluid of inner ear, and the drug is slowly released in the lymph fluid (24 h). The lipid nanoparticles widen the application range of AST disease treatment, improve the solubility of AST to enable the AST to be applied to the prevention or treatment of inner ear diseases, and provide theoretical basis and experimental basis for AST clinical transformation.

Example 4 protective Effect of AST-LPN on cisplatin cytotoxicity

Cisplatin ototoxicity is a common drug-induced deafness. Cisplatin easily enters inner ear lymph fluid through a blood labyrinth barrier, ROS (reactive oxygen species) of auditory epithelial cells are increased to induce a mitochondrion-dependent apoptosis pathway, and hair cells are subjected to apoptosis to further cause hearing loss and even total deafness. An auditory cell line HEI-OC1 is taken as an in-vitro drug toxicity prevention cell model, and the protective effect of AST-LPN on cisplatin-induced HEI-OC1 cytotoxicity is investigated. At the same time, the mechanism of the cytotoxicity protection effect is further discussed. The experimental results show that: the blank unloaded phospholipid nanoparticles had no cytotoxic effect on HEI-OC1 (FIG. 11), and the toxic effect of Cisplatin (CDDP) on HEI-OC1 cells increased with increasing concentration of cisplatin (FIG. 12); AST-LPN significantly inhibited cisplatin cytotoxicity (P <0.001) at concentrations of 1, 5, 10. mu.g/mL, whereas AST did not have cisplatin cytotoxicity inhibition (P >0.05) at the same concentrations (FIG. 13).

Example 5 AST-LPN antagonism of cis-platin induced intracellular ROS elevation

As shown in figures 14 and 15, AST remarkably inhibits the increase of intracellular ROS caused by CDDP (60 mu M, 24h) under the concentration of 1, 5, 10 and 15 mu g/mL, and shows strong antioxidant property. Particularly, the intracellular ROS increasing effect of AST-LPN under the concentration of 5, 10 and 15 mug/mL is obviously stronger than the intracellular ROS increasing effect of AST under the same concentration, and the intracellular ROS level can be reduced to the normal level.

Example 6 AST-LPN animal experiments on the protective Effect of cisplatin ototoxicity

1. Cisplatin ototoxicity guinea pig model construction

Different administration modes are selected to establish a guinea pig cisplatin ototoxicity model. The results are shown in FIGS. 16 and 17, and the Auditory Brainstem Response (ABR) was measured on the third day after low dose multiple (4 mg/kg/day, 3 consecutive days) intraperitoneal injection of Cisplatin (CDDP), with a small amplitude of threshold shift, averaging about 15 dB. However, ABR was measured on the third day after a single (12mg/kg, 1) intraperitoneal injection of cisplatin at high doses, with a sufficiently large amplitude of threshold shift, especially in the frequency range of 4-16kHz, with an average threshold shift of about 52 dB. Based on this, the test subsequently selects a guinea pig ototoxicity model constructed by a high dose single (12mg/kg, 1) administration mode.

Protection of AST-LPN against Hearing-loss in cisplatin ototoxic guinea pigs

The AST-LPN is administered by round window membrane, i.e. the round window membrane is exposed through retroauricular approach after anesthesia of guinea pig, gelatin sponge absorbing appropriate amount of AST-LPN solution is placed in round window niche, the auditory bulb is sealed, and suture is performed. After holding the position for 1 hour, cisplatin (12mg/kg) was intraperitoneally injected, and the guinea pigs were waited for awakening, and were returned to cages for rearing after the state was ensured. Hearing thresholds were measured 1 day before dosing and 3 days after dosing, respectively.

The hearing protection results are shown in fig. 18 and 19. Figure 18 shows that there was no significant difference in the pre-dose cisplatin left and right ear hearing thresholds. After AST-LPN pre-protection is given to the round window membrane, the hearing of the dosed lateral ear is remarkably different from the hearing threshold of the non-dosed lateral ear. The opposite ear to which the AST-LPN solution was not administered was used as a cisplatin control group, and the cochlear hearing of the AST-LPN administration side was significantly reduced within the range of 2.8 to 22kHz with an average reduction of about 20dB when the AST-LPN was previously placed for 1 hour (FIG. 19).

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (7)

1. The preparation method of the astaxanthin-loaded phospholipid nanoparticles is characterized in that dichloromethane is used as an organic phase solvent of astaxanthin, an organic phase is volatilized by an emulsion solvent volatilization method to obtain the astaxanthin-loaded phospholipid nanoparticles, the phospholipid nanoparticles are obtained by mixing and ultrasonically emulsifying an organic phase in which a high-molecular polymer and astaxanthin are dissolved and an aqueous phase in which phospholipid is dissolved, and volatilizing the organic phase, the high-molecular polymer is MPEG-PLA, the phospholipid is DMPC, and the mass ratio of the DMPC to the MPEG-PLA is 1: 1-20.

2. The method for preparing astaxanthin-loaded phospholipid nanoparticles according to claim 1, which comprises the following steps:

1) dissolving MPEG-PLA and AST in dichloromethane to obtain an organic phase;

2) dissolving DMPC in methanol water solution to serve as water phase;

3) dropping the organic phase into the water phase, and emulsifying by ultrasonic;

4) after emulsification, pouring the emulsion into a sodium cholate aqueous solution;

5) stirring to completely volatilize the organic solvent to obtain the astaxanthin-loaded phospholipid nanoparticles.

3. The method for preparing astaxanthin-loaded phospholipid nanoparticles as claimed in claim 2, wherein the dissolution concentration of MPEG-PLA in methylene chloride in step 1) is 20-40g/L, the dissolution concentration of AST in methylene chloride is 1-10g/L, and the dissolution concentration of DMPC in methanol aqueous solution in step 2) is 0.5-1.5 g/L.

4. The method for preparing astaxanthin-loaded phospholipid nanoparticles as claimed in claim 2, wherein the concentration of the aqueous methanol solution in step 2) is 1% to 8% by volume of methanol to water, and the concentration of the aqueous sodium cholate solution in step 4) is 0.3% to 0.7% by weight of sodium cholate contained in each 100mL of water.

5. The astaxanthin-loaded phospholipid nanoparticles prepared by the method for preparing astaxanthin-loaded phospholipid nanoparticles according to claim 1 or 2, wherein the encapsulation efficiency is 20% to 80% and the drug loading is 1% to 5%.

6. Use of the astaxanthin-carrying phospholipid nanoparticles prepared by the method for preparing astaxanthin-carrying phospholipid nanoparticles as defined in claim 1 or 2 for the preparation of a medicament for the prevention and treatment of inner ear diseases.

7. A medicament for preventing or treating inner ear diseases comprising astaxanthin-loaded phospholipid nanoparticles prepared by the method of claim 1 or 2.

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