A composite nanoparticle for treating otitis media and a preparation method and application thereof

Abstract

The present application discloses a kind of composite nanoparticles, including inorganic nanoparticles and polypeptide attached to the surface of the inorganic nanoparticles.The composite nanoparticles of the present application can overcome bacterial drug resistance and better treat otitis media.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of nanotechnology, in particular to a kind of composite nanoparticles for treating otitis media and preparation method and application thereof. BACKGROUND

[0002] Otitis media is an inflammatory disease involving all or part of the structures of the middle ear (including Eustachian tube, tympanic cavity, tympanic sinus and mastoid air cell), and is prone to occur in children. It can be divided into non-suppurative and suppurative two categories, non-suppurative otitis media, barotrauma otitis media, etc., suppurative otitis media has acute and chronic. The current treatment of otitis media is as follows: (1) external ear canal flushing, flushing and then dropping antibiotics; (2) systemic use of antibiotics and corticosteroids.

[0003] The main cause of otitis media is the invasion of pathogens into the external ear canal, which causes external otitis secondary, with the gradual prevalence of bacterial resistance to antibiotics, the existing antibiotic treatment of otitis media is not easy to achieve the desired effect.

[0004] Therefore, it is necessary to develop an effective drug for treating otitis media to overcome bacterial resistance. SUMMARY

[0005] In order to solve one of the above technical problems in the prior art, the present application provides a kind of composite nanoparticles for treating otitis media, which can overcome bacterial resistance and better treat otitis media.

[0006] In the first aspect, the present application provides a kind of composite nanoparticles, comprising

[0007] Inorganic nanoparticles, including titanium oxide, zinc oxide; and

[0008] Polypeptide attached to the surface of the inorganic nanoparticles.

[0009] In some embodiments, the mass ratio of polypeptide, titanium oxide and zinc oxide in the composite nanoparticles is (0.4-0.5):1:(0.2-0.3), preferably 0.5:1:0.3.

[0010] In some embodiments, in the composite nanoparticles, zinc oxide is distributed inside and on the surface of titanium oxide to form inorganic nanoparticles, the inorganic nanoparticles are negatively charged, and the polypeptide is attached to the outer layer of the inorganic nanoparticles by electrostatic adsorption.

[0011] In some embodiments, the polypeptide of the present application is an antibacterial peptide (AMP), which is positively charged and can be used for the preparation of composite nanoparticles, and the molecular weight range can be 3.0-3.5 KDa, preferably 3.3 KDa.

[0012] In some embodiments, the antibacterial peptide can be obtained by commercial purchase.

[0013] In some embodiments, the titanium oxide is TiO2.

[0014] In a second aspect, the present application provides a method for preparing the composite nanoparticle, the method comprising the steps of: heating the titanium oxide and zinc acetate under stirring, adding dropwise a strong base alcohol solution to react, obtaining inorganic nanoparticles, and then reacting with the polypeptide on a shaker to obtain the composite nanoparticle.

[0015] In some embodiments, the heating is at a rate of 175-185°C / min to rapidly heat to 59-61°C, then at a rate of 145-155°C / min to slowly heat to 64-66°C, and then constant temperature for 0.75-1.25h, which are necessary conditions for preparing ZnO nanoparticles of ideal size, with a particle size of about 8-12nm.

[0016] In some embodiments, the heating is at a rate of 180°C / min to rapidly heat to 60°C, then at a rate of 150°C / min to slowly heat to 65°C, and then constant temperature for 1h.

[0017] In some embodiments, the stirring is at a rate of 300-500r / min, for example, it can be 300r / min, 400r / min, 500r / min, to prepare ZnO nanoparticles of ideal size, with a particle size of about 8-12nm.

[0018] In some embodiments, the zinc acetate reacts with the strong base in the presence of dimethyl sulfone to generate zinc oxide nanoparticles.

[0019] In some embodiments, the zinc acetate and dimethyl sulfone are dissolved in an alcohol solvent, the titanium oxide is dispersed in the alcohol solvent, mixed, heated under stirring, and then reacted by adding dropwise a strong base alcohol solution, and then the polypeptide is reacted by a shaker to form a composite nanoparticle.

[0020] In some embodiments, the mass ratio of zinc acetate to dimethyl sulfone is (2-3.5):1. For example, it can be 2:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, 2.5:1, 2.6:1, 2.7:1, 2.75:1, 2.8:1, 2.9:1, 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1.

[0021] In some embodiments, the zinc acetate and dimethyl sulfone are dissolved in 15-25 times the volume of the alcohol solvent, for example, it can be 15 times, 17.5 times, 20 times, 22.5 times, 25 times.

[0022] In some implementations, the mass ratio of zinc acetate to the strong base is (1.5-2.5):1. For example, the mass ratio can be 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.87:1, 1.9:1, 2.0:1, 2.1:1, 2.2:1, 2.3:1, 2.4:1, or 2.5:1.

[0023] In some embodiments, the strong base is dissolved in 15-20 times its volume of alcohol solvent, for example, 15, 16, 17, 18, 19, or 20 times its volume.

[0024] In some implementations, a strong base is dissolved in an alcohol solvent by means of ultrasound.

[0025] In some embodiments, the dropping rate of the strong base alcohol solution is 0.5-1 drop / s, for example, 0.5 drops / s, 1 drop / s.

[0026] In some implementations, after the strong base alcohol solution is completely added, the reaction continues for 2-3 hours, for example, 2 hours, 2.5 hours, or 3 hours.

[0027] In some embodiments, titanium dioxide can be dispersed in an alcohol solvent by: taking a titanium dioxide solution, centrifuging it at 7000-8000 r / min for 4-8 minutes, washing it 2-3 times with an alcohol solvent, and dispersing the precipitate in the alcohol solvent.

[0028] In some embodiments, the concentration of the titanium dioxide solution is 8-12 mg / mL, for example 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL.

[0029] In some embodiments, the volume of the titanium dioxide solution is 2 to 4 times that of the alcohol solvent, for example, it can be 2 times, 2.5 times, 3 times, 3.5 times, or 4 times.

[0030] In some embodiments, titanium dioxide can be dispersed in an alcohol solvent by centrifuging 225 mL of 10 mg / mL TiO2 solution at 8000 r / min for 5 minutes, washing three times with methanol, and dispersing the precipitate in 75 mL of methanol.

[0031] In some embodiments, the mass ratio of titanium oxide to zinc acetate is 1:(1.2-1.5), for example, it can be 1:1.2, 1:1.22, or 1:1.5.

[0032] In some embodiments, the mass ratio of titanium dioxide to the antimicrobial peptide is 1:(0.4-0.5). For example, it can be 1:0.4, 1:0.45, or 1:0.5. Within this range, the encapsulation efficiency of the antimicrobial peptide is effectively improved, avoiding waste of raw materials.

[0033] In some embodiments, the reaction temperature of the shaker is 4-8℃, the rotation speed of the shaker is 100-150r / min, and the reaction time is 3.5-5h. For example, the reaction is carried out at 4℃ with a rotation speed of 120r / min for 4h in a shaker.

[0034] In some embodiments, the alcohol is selected from C1-6alcohols, for example, methanol, ethanol, propanol, n-butanol, isobutyl alcohol, n-pentanol, isopentyl alcohol, neopentyl alcohol, n-hexanol.

[0035] In some embodiments, the strong base is selected from potassium hydroxide, sodium hydroxide.

[0036] In some embodiments, the method for preparing the composite nanoparticles of the present application comprises the following steps:

[0037] Zinc acetate and dimethyl sulfone are dissolved in an alcohol solvent, titanium oxide is dispersed in the alcohol solvent, mixed, heated with stirring, and reacted by dropwise adding an alcohol solution of a strong base to obtain inorganic nanoparticles; the inorganic nanoparticles are subjected to a shaker reaction with a polypeptide to obtain composite nanoparticles.

[0038] In a third aspect, the present application provides a composite nanoparticle, which is prepared by the method described above.

[0039] In a fourth aspect, the present application provides the use of the composite nanoparticle or the composite nanoparticle prepared by the method of the present application in the preparation of a medicament for treating otitis media.

[0040] Effects of the present application

[0041] The present inventors found in the research process that the preparation of nanoparticles from polypeptides and zinc oxide has poor antibacterial effect and unstable combination. However, the present inventors unexpectedly found that when titanium oxide is added, the polypeptide, titanium oxide and zinc oxide form composite nanoparticles, which can overcome bacterial drug resistance and better treat otitis media. In the composite nanoparticles of the present application, titanium oxide has a hollow structure with a particle size range of 180-220nm, and zinc oxide has a smaller particle size of about 8-12nm, which can be uniformly embedded in the interior and surface of the titanium oxide nanoparticles to form inorganic nanoparticles, and the polypeptide is attached to the outer layer of the inorganic nanoparticles by electrostatic adsorption; when used as a drug, the polypeptide penetrates the bacterial cell membrane and the zinc oxide generates hydroxyl radicals to damage the bacteria, achieving effective antibacterial effect.

[0042] The present application successfully prepared antibacterial peptide-TiO2-ZnO (ATZ) composite nanoparticles. In the composite nanoparticles of the present application, the ZnO nanoparticles are crystalline, the TiO2-ZnO (TZ) nanoparticles are spherical, and the ATZ composite nanoparticles have a spherical shape with scattered drugs distributed on the edges.

[0043] The application adopts protein quantification method and ICP mass spectrometry to determine the encapsulation rate and drug loading of ZnO nanoparticles and AMP in the nanoparticles. In the ATZ composite nanoparticles, the encapsulation rate of ZnO nanoparticles is more than 80%, and the drug loading is more than 20%; the encapsulation rate of AMP is more than 80%, and the drug loading is more than 40%. The monodisperse index is less than 0.1, indicating good dispersibility.

[0044] The ATZ composite nanoparticles of the application can overcome bacterial drug resistance and better treat otitis media. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 The preparation process of the ATZ composite nanoparticles is shown.

[0046] Figure 2 The transmission electron microscopy images of ZnO, TZ and ATZ composite nanoparticles are shown.

[0047] Figure 3 The transmission electron microscopy element distribution images of the ATZ composite nanoparticles are shown.

[0048] Figure 4 The antibacterial effects of ZnO nanoparticles, AMP and ATZ composite nanoparticles are shown.

[0049] Figure 5 The bactericidal activity of ZnO nanoparticles, AMP and ATZ composite nanoparticles on Staphylococcus aureus is shown.

[0050] Figure 6 The bactericidal activity of ZnO nanoparticles, AMP and ATZ composite nanoparticles on Streptococcus pneumoniae is shown.

[0051] Figure 7 The histopathological changes (H.E. 4x) in the middle ear tissues of mice are shown, wherein A. healthy group, B. control group, C. model group, D. ZnO nanoparticle treatment group, E. AMP treatment group, D. ATZ composite nanoparticle treatment group. DETAILED DESCRIPTION

[0052] In order to make the objects, technical solutions and advantages of the application clearer, the following further describes the application in detail with examples. The specific examples described herein are only used to explain the application and do not constitute any limitation on the application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0053] Definitions

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied, for the purposes of interpretation of this specification, and shall have the meanings set forth below, and such meanings should be applied, mutatis mutandis, to terms where they appear in the singular or in the plural.

[0055] As used herein, the terms "a" and "one" include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0056] As used herein, the term "about" means ±20% of the number that it precedes. In some embodiments, the term "about" means ±10% of the number that it precedes. In some embodiments, the term "about" means ±5% of the number that it precedes.

[0057] NPs: nano-particles.

[0058] TZ: TiO2-ZnO.

[0059] ATZ: AMP-TiO2-ZnO.

[0060] AMP: Antimicrobial peptides.

[0061] MSM: Methyl sulfonyl methane.

[0062] Examples and figures are provided below to assist in the understanding of the present application. It is understood that the examples and figures are used only for illustrative purposes and should not be used to limit the present application in any manner. The actual scope of the present application is set forth in the claims. It is understood that any modifications and changes can be made without departing from the spirit of the present application.

[0063] Example 1: Preparation of ATZ composite nanoparticles

[0064] This example provides the preparation of ATZ composite nanoparticles.

[0065] (1) 225 mL of 10 mg / mL TiO2 solution was centrifuged at 8000 r / min for 5 minutes, washed with methanol for 3 times, and the precipitate was dispersed in 75 mL of methanol for standby;

[0066] (2) 2.75 g of zinc acetate and 1 g of dimethyl sulfone were weighed and dissolved in 75 mL of methanol, and mixed with the methanol solution of TiO2 obtained in step (1);

[0067] (3) The mixed solution of step (2) is placed on a magnetic stirrer, first heated rapidly to 60℃ at a rate of 180℃ / min, then slowly heated to 65℃ at a rate of 150℃ / min, and kept constant for 1h, and the rotation speed of the magnetic stirrer is ensured to be 400rmp during the whole process;

[0068] (4) 1.47g of potassium hydroxide is weighed, and is fully dissolved in 25mL of methanol by means of ultrasonic, and is added to the mixed solution of step (3) at a rate of 1 drop / 2s; after the dropwise addition is completed, the reaction is continued for 2.5h, and TZ nanoparticles are obtained;

[0069] (5) The TZ nanoparticles are taken, and AMP (purchased from Nanjing Kingsway Biotech Co., Ltd., molecular weight 3.3KDa) is added at a mass ratio of TiO2 to AMP of 2:1, and is allowed to act on a 4℃ 120r / min shaking table for 4h, and is washed with distilled water for 3 times, and ATZ composite nanoparticles are obtained.

[0070] The preparation process of the ATZ composite nanoparticles is shown in Figure 1 .

[0071] The prepared ATZ composite nanoparticles are observed by transmission electron microscopy (TEM). The transmission electron microscopy (TEM) images of the ZnO nanoparticles, the TZ nanoparticles and the ATZ composite nanoparticles observed are shown in Figure 2 . Among them, the A, B and C diagrams are respectively the ZnO nanoparticles, the TZ nanoparticles and the ATZ composite nanoparticles. It can be seen from the TEM images that the ZnO nanoparticles are in a crystal shape (average particle size 10nm), the TZ nanoparticles are in a spherical shape (average particle size 200nm), and the ATZ composite nanoparticles have a spherical shape edge (arrow position) and can see the scattered distribution of the drug.

[0072] The transmission electron microscopy element distribution chart result of the ATZ composite nanoparticles is shown in Figure 3 . The A diagram is the morphology of the ATZ composite nanoparticles under the dark field of the transmission electron microscope; the B diagram indicates the position of the O element, which is the common element of the ZnO nanoparticles and the TiO2 nanoparticles; the C diagram indicates the position of the Ti element, which can indicate the position of the TiO2 nanoparticles; the D diagram indicates the position of the N element, which is one of the main elements of the AMP and can indicate the position of the AMP; the E diagram indicates the position of the Zn element, which can indicate the position of the ZnO nanoparticles; and the F diagram is the superimposed picture of the B, C, D and E diagrams. Through this group of pictures, it can be proved that the ATZ composite nanoparticles are successfully prepared.

[0073] The mass of Zn element and Ti element in the ATZ composite nanoparticles is determined by inductively coupled plasma mass spectrometer (ICP), and the mass of ZnO is calculated; the content of AMP in the ATZ composite nanoparticles is determined by protein quantification (BCA) method, and then the encapsulation rate and drug loading of ZnO nanoparticles and AMP in the ATZ composite nanoparticles are obtained. In the ATZ composite nanoparticles, the encapsulation rate of ZnO nanoparticles is 82.67%, the drug loading is 20.73%, the encapsulation rate of AMP is 83.75%, and the drug loading is 41.86%. The monodisperse index is 0.074, indicating good dispersity.

[0074] Example 2: Preparation of ATZ composite nanoparticles

[0075] In this example, the operation is the same as that in Example 1, except that 3g of zinc acetate and 1g of dimethyl sulfone are weighed and dissolved in 75mL of ethanol.

[0076] The alcohol solvent is replaced by ethanol.

[0077] The prepared ATZ composite nanoparticles have similar properties to those in Example 1.

[0078] Example 3: Preparation of ATZ composite nanoparticles

[0079] In this example, the operation is the same as that in Example 1, except that the temperature is rapidly increased to 61℃ at a rate of 185℃ / min, then slowly increased to 66℃ at a rate of 155℃ / min, and then kept constant for 1h.

[0080] The prepared ATZ composite nanoparticles have similar properties to those in Example 1.

[0081] Example 4: Preparation of ATZ composite nanoparticles

[0082] In this example, the operation is the same as that in Example 1, except that AMP is added in a mass ratio of 3:1 of TiO2 to AMP.

[0083] The prepared ATZ composite nanoparticles have similar properties to those in Example 1.

[0084] Comparative Example 1

[0085] In this comparative example, the operation is the same as that in Example 1, except that the constant temperature time in step (3) is 2h.

[0086] The drug loading of ZnO nanoparticles in the prepared ATZ composite nanoparticles is less than 10%. It can be seen that a longer constant temperature time is not conducive to the loading of ZnO nanoparticles.

[0087] Comparative Example 2

[0088] The comparative example was operated in the same manner as example 1, except that the rotation speed of the magnetic stirrer was 100 rpm during the whole process in step (3).

[0089] The prepared ATZ composite nanoparticles had poor dispersibility and formed agglomerated state. It can be seen that the low rotation speed affected the dispersion effect of the composite nanoparticles.

[0090] Comparative example 3

[0091] The comparative example was operated in the same manner as example 1, except that the AMP was added in a mass ratio of 1:1 of TiO2 to AMP.

[0092] The prepared ATZ composite nanoparticles had an AMP encapsulation rate of less than 60% and a drug loading of less than 15%. It can be seen that the mass ratio of TiO2 to AMP is one of the key factors affecting the encapsulation rate of the antibacterial peptide. A large mass ratio of AMP reduces the encapsulation rate of the antibacterial peptide, resulting in waste of raw materials.

[0093] Comparative example 4. Preparation of AMP-ZnO nanoparticles

[0094] The comparative example provides the preparation of AMP-ZnO nanoparticles, including the following steps:

[0095] 1. Preparation of ZnO nanoparticles

[0096] (1) 2.75 g of zinc acetate and 1 g of dimethyl sulfone were weighed and dissolved in 75 mL of methanol;

[0097] (2) The above dissolution product was placed on a magnetic stirrer, first heated to 60°C at a power of 180°C, then slowly heated to 65°C at a power of 150°C, and kept at a constant temperature for 1 h, ensuring that the rotation speed of the magnetic stirrer was 400 rpm during the whole process;

[0098] (3) 1.47 g of potassium hydroxide was weighed and dissolved in 25 mL of methanol by ultrasonic, and was added to the above solution at a rate of 1 drop / 2 s;

[0099] (4) After the potassium hydroxide dispersed in methanol was completely added to the above solution, the reaction was continued for 2.5 h to obtain ZnO nanoparticles;

[0100] (5) The prepared ZnO nanoparticles were centrifuged at 6000 r / min for 5 minutes, washed with methanol three times, and then dispersed in methanol for standby.

[0101] 2. The ZnO nanoparticles were redispersed in distilled water and oscillated with AMP at a ratio of 1:1, 1:2, 1:3, 1:4 and 1:5, respectively. The stability of the prepared product was poor, because the size of the ZnO nanoparticles was small and the AMP could not be effectively loaded.

[0102] Experimental Example 1. Antibacterial effect of composite nanoparticles

[0103] The bacterial concentration was adjusted to 1 x 10 5 CFU / mL according to the method of Clinical and Laboratory Standards Institute (CLSI). 90 μL of bacteria of different strains (1 x 10 5 CFU / mL) was added to the wells of a 96-well plate. ZnO nanoparticles (prepared according to Step 1 of Reference Comparative Example 4) and AMP (Nanjing Kingsway Biotech Co., Ltd.) were gradient diluted to 5000, 2000, 1000, 500, 100, 10 ug / ml, respectively, and ATZ nanoparticles were gradient diluted to 2000, 1000, 500, 100, 50, 10 ug / ml. Different concentrations of materials were added to different wells. Wells without materials, 100 μL of sterile broth, only 100 μL of bacteria of different strains (1 x 10 5 CFU / mL), 90 μL of bacteria of different strains (1 x 10 5 CFL / mL) and 10 μL of antibiotics were added as blank, negative control and positive control, respectively. After incubation in a 37 °C incubator for 16-18 h, resazurin solution (0.0625%) was added, 20 μL per well, and incubation in a 37 °C incubator was continued for 4 h, and color changes were observed.

[0104] The minimum inhibitory concentration (MIC) values of the ATZ composite nanoparticles prepared in Example 1 for Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae were detected by microdilution method, and the results are shown in Table 1 and Table 2. Figure 4

[0105] Table 1. MIC results of ZnO nanoparticles, AMP and ATZ composite nanoparticles

[0106]

[0107] The above results show that the concentration required for the antibacterial effect of ATZ composite nanoparticles is lower than that of ZnO and AMP alone, proving that the antibacterial effect of ATZ composite nanoparticles is stronger than that of ZnO and AMP alone.

[0108] Experimental Example 2. Antibacterial effect of composite nanoparticles ​

[0109] Further, the minimum bactericidal concentration (MBC) values were determined in 96-well plates in a similar way to the MIC method. The difference is that the MBC value is determined by measuring the number of bacteria in the liquid in the well without visible turbidity, and the concentration of the antibacterial peptide in the well in which the number of bacteria is reduced by three orders of magnitude is taken as the MBC value of the material against the bacteria. The results are shown in Table 2. Figure 5 、 Figure 6 and Table 2.

[0110] Table 2. MBC results of ZnO nanoparticles, AMP and ATZ composite nanoparticles

[0111]

[0112] The above results show that the concentration required for the bactericidal effect of the ATZ composite nanoparticles is lower than that of ZnO and AMP alone, proving that the bactericidal effect of the ATZ composite nanoparticles is stronger than the antibacterial ability of ZnO nanoparticles and AMP alone.

[0113] Experimental Example 3. Effect of composite nanoparticles on treatment of otitis media

[0114] ICR female mice purchased from Vantianlihua Company were selected, which were healthy, had sensitive pinna reflexes, and were adaptively fed in an animal house for 1 week. The mice with uniform body weight were randomly divided into 6 groups, 6 mice in each group. Five groups of mice were anesthetized by intraperitoneal injection of 50 mg / kg of Cetamin, and the bilateral external auditory canals were cleaned with 75% alcohol. Four groups were inoculated into the tympanic cavity by puncturing the tympanic membrane with a micro-syringe to take 5 μL of the bacterial suspension, and the remaining one group was injected with the same volume of PBS in the same way. The healthy group of mice was not treated.

[0115] On the first day of modeling, three groups of the modeling group were taken as the treatment group, 5 uL of 50 ug / mL ZnONPs, AMP and ATZ NPs were taken by a micro-syringe, and the drug was administered by the method of external ear canal dripping, and the same treatment was continued at the same time on the second day. The mice were weighed and recorded every day.

[0116] On the third day of modeling, the mice were sacrificed by eyeball blood sampling, and the bilateral middle ear tissues were fixed with 4% formaldehyde. The middle ear tissues were soaked in EDTA decalcification solution for 2 h, washed with tap water for 1 h, and subjected to histological sectioning and HE staining, and observed under an optical microscope.

[0117] As Figure 7 Definitions Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 DefinitionsAs shown, A shows that there is no inflammatory exudate in the middle ear cavity of the healthy group of mice, and there is only a layer of mucosa on the surface of the cavity bone wall. B is the PBS control group, and the tissue morphology has no obvious difference with the healthy group. C is the modeling group of tympanic membrane puncture injection of S. pneumoniae liquid, and a large amount of exudate and inflammatory cell infiltration appears in the middle ear cavity. D, E and F are the ZnO, AMP and ATZ composite nanoparticle treatment groups, respectively. Compared with the modeling group, the inflammatory cells and exudate are significantly reduced. The effect of F is the most significant, indicating that the ATZ composite nanoparticle has a good therapeutic effect on otitis media.

[0118] The composite nanoparticles of Examples 2-4 are used to operate in the same way according to the method of Experimental Example 1, and the bacteriostatic and bactericidal effects of the composite nanoparticles and the treatment of otitis media have similar effects to the ATZ composite nanoparticles of Example 1.

[0119] The technical solutions of the present application are not limited to the above specific examples, and any technical modification made according to the technical solutions of the present application falls within the protection scope of the present application.

Claims

1. Use of composite nanoparticles in the preparation of a medicament for treating otitis media, said composite nanoparticles comprising: Inorganic nanoparticles, including titanium dioxide and zinc oxide; and Peptides attached to the surface of the inorganic nanoparticles; The preparation method of the composite nanoparticles includes the following steps: heating titanium oxide and zinc acetate under stirring, reacting with an alcoholic solution of strong alkali dropwise to obtain inorganic nanoparticles, and then reacting them with peptides in a shaker to obtain the composite nanoparticles; The heating rate is 175-185℃ / min to raise the temperature to 59-61℃, then 145-155℃ / min to raise the temperature to 64-66℃, and hold the temperature for 0.75-1.25h. The stirring speed is 300-500 r / min; The polypeptide is an antimicrobial peptide, and the mass ratio of titanium dioxide to the antimicrobial peptide is 1:(0.4-0.5). In the composite nanoparticles, zinc oxide is distributed inside and on the surface of titanium oxide to form inorganic nanoparticles, and polypeptides are attached to the outer layer of the inorganic nanoparticles.

2. The use according to claim 1, characterized in that, In the composite nanoparticles, the mass ratio of polypeptide, titanium dioxide, and zinc oxide is (0.4-0.5): 1:(0.2-0.3).

3. The use according to claim 1, characterized in that, The step of heating titanium oxide and zinc acetate under stirring includes dissolving zinc acetate and dimethyl sulfone in an alcohol solvent, dispersing titanium oxide in the alcohol solvent, mixing, and heating under stirring.

4. The use according to claim 1, characterized in that, The reaction temperature of the shaker is 4-8℃, the shaking speed is 100-150 r / min, and the reaction time is 3.5-5 h.

5. The use according to any one of claims 1-4, characterized in that, The alcohol is selected from C1-6 alcohols; and / or the strong base is selected from potassium hydroxide and sodium hydroxide.

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