Multifunctional fusion peptide with both targeting and penetration effects and its application

Abstract

The present invention provides a multifunctional fusion peptide having both targeting and penetration effects and its application, wherein the multifunctional fusion peptide comprises in sequence: a functional domain targeting outer hair cells as shown in SEQ ID NO.1, a first flexible connector, a cell-penetrating peptide as shown in SEQ ID NO.2, a second flexible connector and an inner terminal region; the inner terminal region is a lysine connected with maleimide. The multifunctional fusion peptide can achieve surface modification of the carrier through the addition reaction of maleimide on the terminal lysine, and the hair cell targeting region at the free end can target and transport the carrier to the inner ear hair cells. The cell-penetrating peptide with a positive charge in the middle part penetrates through nonspecific electrostatic aggregation with the negatively charged round window membrane on the side. The fusion peptide gives the carrier active targeting and high penetration characteristics, which can better protect hearing and hair cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a multifunctional fusion peptide having both targeting and penetration effects and application thereof. Background Art

[0002] Noise-induced hearing loss is a public health disease with an increasing prevalence worldwide, causing serious damage to individuals' quality of life. The sensory cells of the mammalian cochlea do not have the ability to regenerate, which makes hair cell loss irreversible. However, delivering drugs to the inner ear for treatment after noise exposure before permanent sensorineural damage occurs is an important means.

[0003] Due to the special anatomical position of the cochlea in the inner ear and the presence of the blood maze barrier, drug absorption is greatly limited. Therefore, drug delivery efficiency is particularly critical in the treatment of inner ear diseases. Intratympanic administration is often the first choice because of the minimal side effects and the highest drug concentration. It is currently the mainstream route of administration in preclinical animal studies, and its application is being expanded to clinical trials. The round window membrane and the oval window are the only two permeable barriers between the middle ear and the inner ear. Since the oval window is partially blocked by the stapes baseplate, the round window membrane becomes the main route for drug diffusion into the inner ear.

[0004] It is known that nanoparticles can enter the inner ear from the round window membrane through endocytosis and exocytosis, but the efficiency is limited. This situation can be improved by surface modification of nanoparticles, but the current modification strategy only uses hair cell targeting peptides or cell penetrating peptides alone. Although these strategies can increase the delivery efficiency to a certain extent, they still have limitations, such as single function.

[0005] In order to further improve the efficiency of inner ear drug delivery, multifunctional nanoparticles are urgently needed to deliver drugs to the inner ear. Therefore, it is necessary to develop a multifunctional product that has both the ability to target inner ear hair cells and permeate the round window membrane. Summary of the invention

[0006] The purpose of the present invention is to provide a multifunctional fusion peptide with both targeting and penetration effects and its application. The fusion peptide can enable the drug carrier to penetrate more from the round window membrane into the inner ear and deliver the drug to the hair cells in a targeted manner, thereby increasing the drug delivery efficiency, further amplifying the biological activity of the drug, and reducing the hearing loss caused by noise and the loss of outer hair cells of the inner ear basilar membrane to a greater extent, thereby achieving a satisfactory inner ear treatment auxiliary effect.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In the first aspect of the present invention, a multifunctional fusion peptide having both targeting and penetration effects is provided, wherein the multifunctional fusion peptide comprises:

[0009] a functional domain targeting outer hair cells, a first flexible linker, a cell penetrating peptide, a second flexible linker, and a medial terminal region;

[0010] The amino acid sequence of the functional domain targeting outer hair cells is shown in SEQ ID NO.1 (LSTHTTESRSMV), the amino acid sequence of the cell penetrating peptide is shown in SEQ ID NO.2 (RRRRRRRR), the inner terminal region is lysine connected to maleimide, and the first flexible linker and the second flexible linker are selected from one of G, GG, GGS, GGGS, (GGS)n, (GGGS)n, where n is an integer of 1-10.

[0011] Furthermore, the amino acid sequence of the first flexible linker and the second flexible linker is GGS.

[0012] As a specific embodiment, the multifunctional fusion peptide having both targeting and penetration effects is LR27, and the amino acid sequence is shown in SEQ ID NO.3:

[0013] (LSTHTTESRSMVGGSRRRRRRRRGGS[K(maleimide)], wherein the terminal lysine is linked to maleimide.

[0014] In the second aspect of the present invention, a multifunctional fusion peptide-nanoparticle with both targeting and penetration effects is provided, wherein the multifunctional fusion peptide-nanoparticle is formed by covalently bonding the multifunctional fusion peptide with both targeting and penetration effects to the surface of the nanoparticle loaded with a drug.

[0015] Furthermore, the nanoparticles are selected from one of polymer nanoparticles, liposomes, exosomes, silicon nanoparticles, and metal nanoparticles.

[0016] In the third aspect of the present invention, a method for preparing the multifunctional fusion peptide-nanoparticles having both targeting and penetration effects is provided, the method comprising:

[0017] The multifunctional fusion peptide with both targeting and penetration effects is subjected to addition reaction with nanoparticles loaded with drugs under pH 6.5-7.5 to obtain multifunctional fusion peptide-nanoparticles with both targeting and penetration effects.

[0018] Furthermore, the multifunctional fusion peptide having both targeting and permeation effects is subjected to an addition reaction with the nanoparticles at pH 6.5-7.5, specifically comprising:

[0019] The multifunctional fusion peptide with both targeting and permeation effects is dissolved in phosphate buffered saline at pH 7.4 (the mass fraction of the obtained solution containing the multifunctional fusion peptide is preferably 1%-10%wt), 50-70 mg of nanoparticles are resuspended therein, and combined by addition reaction under stirring at room temperature, and then the nanoparticles are washed with ddH2O to remove excess unbound multifunctional fusion peptide.

[0020] Furthermore, the drug-loaded nanoparticles are prepared by a double emulsion method.

[0021] In the fourth aspect of the present invention, an inner ear drug delivery system is provided, wherein the nanogel delivery system is obtained by dispersing the multifunctional fusion peptide-nanoparticles having both targeting and penetration effects in a hydrogel or other aqueous solvent.

[0022] In the above technical solution, when the multifunctional fusion peptide-nanoparticles are dispersed in the hydrogel, the inner ear drug delivery system is called a nanogel delivery system.

[0023] In a fifth aspect of the present invention, a method for preparing the inner ear drug delivery system is provided, the method comprising:

[0024] The multifunctional fusion peptide-nanoparticles having both targeting and penetration effects are dispersed in a hydrogel or other aqueous solvent and mixed uniformly.

[0025] Furthermore, the ratio of the volume of the hydrogel to the mass of the multifunctional fusion peptide-nanoparticles having both targeting and penetration effects is 10 mL: (0.1-1) g.

[0026] In the sixth aspect of the present invention, provided is the use of the multifunctional fusion peptide having both targeting and penetration effects, or the multifunctional fusion peptide-nanoparticle having both targeting and penetration effects, or the inner ear drug delivery system in the preparation of inner ear drug delivery materials.

[0027] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0028] 1. The multifunctional fusion peptide with both targeting and penetration effects of the present invention comprises, in sequence: a functional domain targeting outer hair cells, a first flexible connector, a cell penetrating peptide, a second flexible connector and an inner terminal region;

[0029] The functional domain targeting outer hair cells is actively targeted at the free end, and the cell-penetrating peptide penetrates in the middle part through nonspecific electrostatic aggregation on both sides. The functional domain targeting outer hair cells and the cell-penetrating peptide are assembled through the first flexible connector, which has both hair cell targeting and round window membrane permeability. In addition, the inner terminal region is lysine connected to maleimide, and the covalent binding with the nanoparticle is through the addition reaction of maleimide and thiol.

[0030] 2. After the multifunctional fusion peptide with both targeting and penetration effects of the present invention is covalently bound to nanoparticles or carriers, the hair cell targeting region at the free end can target and transport the carrier to the inner ear hair cells. The cell-penetrating peptide with a positive charge in the middle part penetrates through nonspecific electrostatic aggregation with the negatively charged round window membrane on the side. The design and synthesis of the LR27 fusion peptide endows the carrier with active targeting and high penetration properties. In the in vivo mouse noise-induced hearing loss model, the drug delivery system modified by LR27 injected into the middle ear can better protect hearing and hair cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0032] Figure 1 This is the principle diagram of the technical solution of this application; Figure 1 Number description: 1-LS16, 2-R8, 3-lysine linked to maleimide, 4-GGS, 5-LR27, 6-PTH1-34, 7-thiol-modified nanoparticles, 8-PTH1-34-loaded nanoparticle surface covalently bound to LR27, 9-maleimide group, 10-thiol group, 11-pH 6.5-7.5, 12-thioether bond, 13-hydrogel, 14-C57BL / 6J mice, 15-middle ear injection, 16-noise model, 17-inner ear, 18-oval window, 19-round window membrane;

[0033] Figure 2 This is a schematic diagram of the structure of a multifunctional fusion peptide with both targeting and penetration effects, referred to as LR27, according to an embodiment of the present invention;

[0034] Figure 3 This is the result of LR27 high performance liquid chromatography (HPLC) test;

[0035] Figure 4 Detailed data of each peak of LR27 by high performance liquid chromatography (HPLC);

[0036] Figure 5 This is the result of LR27 mass spectrometry (MS);

[0037] Figure 6 This is the reaction process of LR27 covalently binding to nanoparticles;

[0038] Figure 7 Scanning electron microscopy image of LR27 modified nanoparticles;

[0039] Figure 8 The biocompatibility of LR27 modified nanoparticles to cells; Figure 8 A is the flow cytometry image of different nanoparticles co-cultured with cells for 3 days. Figure 8 B is the percentage quantification of cell viability;

[0040] Fig. 9 Scanning electron microscopy image of the LR27 modified nanogel delivery system;

[0041] Fig.10 The biocompatibility of the LR27-modified nanogel delivery system to cells; Fig.10 A is the flow cytometry image of different nanoparticles co-cultured with cells for 3 days. Fig.10 B is the percentage quantification of cell viability;

[0042] Fig.11 The hearing protection effect of the LR27-modified nanogel delivery system on the noise-induced hearing loss model; Fig.11 A is the animal experiment flow chart. Fig.11 B is the baseline value of hearing threshold of mice in each group. Fig.11 C is the hearing threshold of mice in each group on the third day after noise exposure. Fig.11 D is the hearing threshold of mice in each group on the 7th day after noise exposure. Fig.11 E is the hearing threshold of mice in each group on the 14th day after noise exposure;

[0043] Fig.12 The protective effect of the LR27-modified nanogel delivery system on the outer hair cells of the inner ear in a noise-induced hearing loss model, Fig.12 A is the immunofluorescence staining image of the cochlear basilar membrane of mice in each group. Fig.12 B is the quantification of the survival rate of hair cells turning to the basement membrane. Fig.12 C is the quantification of the survival rate of hair cells in the basement membrane. Fig.12 D is the quantification of the survival rate of hair cells transfected with the basement membrane. DETAILED DESCRIPTION

[0044] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0045] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0046] The general idea of ​​the present invention is as follows:

[0047] In previous studies, simple hair cell targeting peptides usually form amide bonds with the surface of nanoparticles under the catalysis of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Nanoparticles loaded with antioxidant drugs PTH1-34 can also be covalently bound to the surface of nanoparticles through EDC and NHS, which affects the release and activity of PTH1-34.

[0048] The present invention provides a multifunctional fusion peptide having both targeting and penetration effects, wherein the multifunctional fusion peptide comprises, in sequence: a functional domain targeting outer hair cells, a first flexible connector, a cell penetrating peptide, a second flexible connector and an inner terminal region; the amino acid sequence of the functional domain targeting outer hair cells is shown in SEQ ID NO.1 (LSTHTTESRSMV), the amino acid sequence of the cell penetrating peptide is shown in SEQ ID NO.2 (RRRRRRRR), and the inner terminal region is lysine connected to maleimide.

[0049] The terminal band of the multifunctional fusion peptide with both targeting and penetration effects of the present invention is covalently bonded to the nanoparticles through the addition reaction of maleimide and thiol, thereby protecting the activity of the loaded drug of the present application.

[0050] Since the PTH1-34 loaded on the nanoparticles does not participate in the reaction between maleimide and thiol, the surface modification of the nanoparticles by the multifunctional fusion peptide LR27 with both targeting and penetration of the present invention will not affect the release and activity of the PTH1-34 drug. PTH1-34 does not participate in the chemical reaction between maleimide and thiol.

[0051] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0052] The present application will be described in detail below with reference to embodiments and experimental data.

[0053] Example 1. Preparation and characterization of a multifunctional fusion peptide with both targeting and penetration effects

[0054] 1. Preparation of multifunctional fusion peptides with both targeting and penetration effects.

[0055] (1) Design of multifunctional fusion peptides with targeting and penetration effects

[0056] Prestin is a highly conserved membrane protein that is specifically expressed on the membrane of outer hair cells in the inner ear, making it an ideal target for inner ear drug delivery. There is already a targeting peptide PrTP1 (LSTHTTESRSMVGGSCGGS[K(N3)]) for Prestin. We simplified and redesigned its structure, retaining the functional domain for targeting outer hair cells, connecting maleimide to the terminal lysine to obtain LS16 (LSTHTTESRSMVGGS[K(maleimide)]), and adding the cell-penetrating peptide R8 (RRRRRRRR) to the middle part through two flexible connectors GGS to obtain the fusion peptide LR27 (LSTHTTESRSMVGGSRRRRRRRRGGS[K(maleimide)]). The functional domain of LS16 is exposed at the free end to play a targeting role, and the positively charged R8 penetrates the negatively charged round window membrane through nonspecific electrostatic aggregation at the exposed sites on both sides. This shows that the structural design of LR27 is reasonable and it is feasible to have both targeting and penetration functions.

[0057] In other embodiments, the flexible linker may also be replaced by other linkers, such as G, GG, GGGS, (GGS)n, (GGGS)n, etc., wherein n is an integer of 1-10.

[0058] (2) Preparation of multifunctional fusion peptides with targeting and penetration effects

[0059] The preparation of LR27 is completed by solid phase synthesis. The basic principle of solid phase synthesis is to connect the hydroxyl group of the hydroxyl terminal amino acid of the peptide chain to be synthesized to an insoluble polymer resin through a covalent bond structure, and then use the amino acid bound to the solid phase carrier as the amino component, remove the amino protecting group and react with excess activated carboxyl components to extend the peptide chain. Repeat the operation (condensation, washing, deprotection, neutralization and washing, next round of condensation) to achieve the desired length of the peptide chain, and then cleave the peptide chain from the resin, and after purification and other treatments, the desired polypeptide is obtained.

[0060] (3) Characterization of the synthesis of fusion peptide LR27 using high performance liquid chromatography (HPLC) and mass spectrometry (MS)

[0061] HPLC analysis results are as follows Figure 3 , Figure 4 , Figure 5 , the purity of LR27 is 96.42%. The MS results show that the actual molecular weight of LR27 is 3279.75 g mol-1, which is calculated from the m / z value of the molecular [M + nH] nH ion it produces. The theoretical molecular weight of LR27 is 3279.61 g mol-1, and its actual molecular weight is very close to the theoretical molecular weight. Based on the above results, it can be proved that LR27 was successfully synthesized.

[0062] Example 2: Multifunctional fusion peptide-nanoparticles (PTH / LRNPs) with both targeting and penetration effects and preparation method thereof

[0063] 1. Preparation method

[0064] (1) Obtaining drug-loaded nanoparticles

[0065] The drug of the embodiment of the present invention is specifically selected from PTH1-34 (the first 34 amino acids of the N-terminus of the parathyroid hormone-related peptide PTH) with an antioxidant effect. In other embodiments, other drugs may also be loaded.

[0066] The nanoparticles of the embodiment of the present invention can be selected from polymer nanoparticles, liposomes, exosomes, silicon nanoparticles, metal nanoparticles and other unlisted nanoparticles. The embodiment of the present invention specifically uses thiol-modified polylactic-co-glycolic acid (PLGA-SH) nanoparticles, which are purchased from Xi'an Ruixi Biotechnology Co., Ltd., item number R-PL9654.

[0067] As a specific embodiment, PLGA-SH is fully dissolved in dichloromethane, PTH1-34 is added thereto as the inner aqueous phase, and ultrasonicated on ice at 100W for 2 minutes to obtain an emulsion. The emulsion is added dropwise to a polyvinyl alcohol aqueous solution as the outer aqueous phase, and stirred overnight at room temperature to completely evaporate the dichloromethane. The precipitate is collected by high-speed centrifugation to obtain PTH / NPs.

[0068] (2) The multifunctional fusion peptide with both targeting and penetration effects prepared in Example 1 is subjected to an addition reaction with the drug-loaded nanoparticles (PTH / NPs) at room temperature at pH 6.5-7.5 to obtain multifunctional fusion peptide-nanoparticles with both targeting and penetration effects.

[0069] Figure 6 LR27 is modified by covalently binding to the surface of the nanoparticles. The maleimide connected to the terminal lysine of LR27 reacts with the thiol group on the surface of the nanoparticles under pH 6.5-7.5 to form a stable thioether bond.

[0070] Example 3: Verification of the biocompatibility of PTH / LRNPs on mouse hair cell line (HEI-OC1)

[0071] 1. Operation method

[0072] After PTH / LRNPs were co-cultured with HEI-OC1 for 3 days, the cell pellets were digested and collected, the cells were treated with a cell apoptosis detection kit, and the results were detected by flow cytometry.

[0073] 2. Experimental results

[0074] Flow cytometry results Figure 8 As shown, PTH / LRNPs did not induce obvious cell apoptosis when co-cultured with HEI-OC1 cells for 3 days. The viability of HEI-OC1 cells after treatment with NPs, PTH / NPs, PTH / LNPs, and PTH / LRNPs was 95.86±0.72%, 96.56±1.26%, 95.92±0.25%, and 95.68±0.47%, respectively.

[0075] Example 4. Nanogel delivery system (Gel-PTH / LRNPs) and preparation method thereof

[0076] 1. Preparation method

[0077] PTH / LRNPs were dispersed in hydrogel to obtain Gel-PTH / LRNPs.

[0078] The embodiment of the present invention specifically selects polyether F127 hydrogel, which is purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd. with the item number EFL-F127-001.

[0079] 2. Characterization

[0080] After being dried in a freeze dryer for 24 hours, the surface structure was characterized by scanning electron microscopy.

[0081] The results are as follows Fig. 9 As shown, the images show that each group of nanogel delivery systems has a porous structure with similar pore size, and no obvious nanoparticles are observed. This may be due to the good interfacial compatibility between the nanoparticles and the gel. The scale bar of the picture is 50μm.

[0082] Example 5: Verification of the biocompatibility of Gel-PTH / LRNPs to HEI-OC1

[0083] Flow cytometry results Fig.10As shown, Gel-PTH / LRNPs did not induce obvious cell apoptosis when co-cultured with HEI-OC1 cells for 3 days. After treatment with Gel, Gel-NPs, Gel-PTH / NPs, Gel-PTH / LNPs and Gel-PTH / LRNPs, the viability of HEI-OC1 cells was 95.91±0.77%, 95.97±0.27%, 96.30±0.26%, 96.09±0.36% and 95.04±0.67%, respectively.

[0084] Example 6. In vivo experiments

[0085] 1. C57BL / 6J mice with normal hearing were screened by brainstem auditory evoked potential (ABR) at 7 weeks of age for in vivo experiments. Three days before exposure to noise, Gel, Gel-NPs, Gel-PTH / NPs, Gel-PTH / LNPs and Gel-PTH / LRNPs (the nanogel delivery system of Example 4 of the present invention) were injected into the middle ear cavity through the auditory bulla.

[0086] Wherein, Gel: is a hydrogel;

[0087] Gel-NPs are prepared by dissolving nanoparticles in hydrogel.

[0088] The preparation method of Gel-PTH / NPs is to dissolve nanoparticles loaded with drug PTH1-34 in hydrogel.

[0089] Preparation of Gel-PTH / LNPs by Single Targeting Peptide Modification

[0090] LS16 (LSTHTTESRSMVGGS [K (maleimide)]) reacts with drug-loaded nanoparticles at pH 6.5-7.5 to obtain multifunctional fusion peptide-nanoparticles with both targeting and penetration effects dissolved in hydrogel.

[0091] When C57BL / 6J mice were 8 weeks old, they were exposed to 110 dB broadband white noise for 2 hours to establish a noise-induced hearing loss model. The control group was only injected with Gel in advance without noise exposure. ABR tests were performed 3, 7, and 14 days after noise exposure to observe changes in hearing thresholds.

[0092] The results are as follows Fig.11As shown, the powerful function of LR27 is revealed as time goes by and the degree of damage increases. The ABR results on the 14th day showed that the hearing thresholds of mice treated with Gel-PTH / NPs, Gel-PTH / LNPs and Gel-PTH / LRNPs at 24kHz were 46.25 ± 4.79, 40 ± 4.08 and 28.75 ± 2.5 dB, respectively; and the hearing thresholds at 32 kHz were 52.5 ± 6.45, 42.5 ± 2.89 and 31.25 ± 4.79 dB, respectively. The results showed that compared with the unmodified group and the group modified with only a single targeting peptide LS16 (Gel-PTH / LNPs), the drug delivery efficiency was the highest after being modified with LR27, and the mice treated with it had the lowest hearing threshold and the most obvious hearing protection effect.

[0093] 2. C57BL / 6J mice were killed and samples were collected after completing ABR on the 14th day after noise exposure. The cochlear basilar membrane was immunofluorescently stained, the hair cell-specific protein Myosin Ⅶa was stained red, and the cytoskeleton was labeled with green phalloidin. The results showed that Gel-PTH / LRNPs treatment successfully rescued the loss of outer hair cells in noise-damaged mice. Before noise exposure, mice were injected with Gel, Gel-NPs, Gel-PTH / NPs, Gel-PTH / LNPs and Gel-PTH / LRNPs. The survival rates of outer hair cells transferred in the basilar membrane were 80.80±3.22%, 80.10±2.99%, 93.48±1.87%, 98.17±1.38% and 98.52±1.18%, respectively, while the survival rates of outer hair cells transferred at the base of the basilar membrane were 51.81±6.31%, 54.05±8.37%, 72.83±3.62%, 83.70±2.99% and 94.6±1.39%, respectively. Consistent with the hearing test results, LR27 amplified the protective effect of the drug on hair cells to the greatest extent. The image scale is 20 μm.

[0094] In summary, the presence of the multifunctional fusion peptide LR27 of the present invention allows the nanoparticles to penetrate into the inner ear from the round window membrane and release protective drugs in a targeted manner to the hair cells, thereby improving the drug delivery efficiency, further exerting and amplifying the biological activity of the drug, such as combating noise-induced oxidative stress, reducing hair cell damage, and lowering the hearing threshold, ultimately achieving a satisfactory therapeutic effect.

[0095] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0097] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A multifunctional fusion peptide having both targeting and penetration effects, characterized in that: The multifunctional fusion peptide comprises in sequence: a functional domain targeting outer hair cells, a first flexible linker, a cell penetrating peptide, a second flexible linker, and a medial terminal region; The amino acid sequence of the functional domain targeting outer hair cells is shown in SEQ ID NO.1, the amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.2, the inner terminal region is lysine connected to maleimide, and the first flexible linker and the second flexible linker are selected from one of G, GG, GGS, GGGS, (GGS)n, (GGGS)n, where n is an integer of 1-10.

2. The multifunctional fusion peptide having both targeting and penetration effects according to claim 1, characterized in that: The amino acid sequence of the first flexible linker and the second flexible linker is GGS.

3. A multifunctional fusion peptide-nanoparticle with both targeting and penetration effects, characterized in that: The multifunctional fusion peptide-nanoparticle is formed by covalently bonding the multifunctional fusion peptide with both targeting and penetration effects as described in any one of claims 1 to 2 to the surface of the nanoparticle loaded with the drug.

4. The multifunctional fusion peptide-nanoparticle with both targeting and penetration effects according to claim 3, characterized in that: The nanoparticles are selected from polymer nanoparticles, liposomes, exosomes, silicon nanoparticles, and metal nanoparticles.

5. A method for preparing multifunctional fusion peptide-nanoparticles having both targeting and penetration effects as claimed in any one of claims 3 to 4, characterized in that: The method comprises: The multifunctional fusion peptide with both targeting and penetration effects is subjected to addition reaction with nanoparticles loaded with drugs under pH 6.5-7.5 to obtain multifunctional fusion peptide-nanoparticles with both targeting and penetration effects.

6. An inner ear drug delivery system, characterized in that: The inner ear drug delivery system is obtained by dispersing the multifunctional fusion peptide-nanoparticles with both targeting and penetration effects as described in any one of claims 3 to 4 in a hydrogel or other solvent.

7. A method for preparing the inner ear drug delivery system according to claim 6, characterized in that: The method comprises: The multifunctional fusion peptide-nanoparticles with both targeting and penetration effects as described in any one of claims 3 to 4 are dispersed in a hydrogel or other solvents by ultrasonic dispersion and mixing.

8. Use of the multifunctional fusion peptide with both targeting and penetration effects as described in any one of claims 1-2, or the multifunctional fusion peptide-nanoparticle with both targeting and penetration effects as described in any one of claims 3-4, or the inner ear drug delivery system as described in claim 6 in the preparation of inner ear drug delivery materials.

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