Preparation and application of aggregate delivery system for treating hearing impairment

Abstract

The invention provides a preparation method of drug-entrapped aggregate micro-droplets. The method comprises the following steps: preparing a polypeptide-nucleic acid conjugate, and entrapping a drug by using the prepared polypeptide-nucleic acid conjugate. The delivery efficiency of the drug-entrapped aggregate micro-droplet is remarkably improved, so that the drug-entrapped aggregate micro-droplet can be applied to treatment of diseases, especially NIHL.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to the preparation of a condensate microdroplet delivery system based on a polypeptide-nucleic acid conjugate and the application of the prepared condensate microdroplets in the treatment of noise-induced hearing loss. Background Art

[0002] Noise-induced hearing loss (NIHL) is mainly caused by excessive reactive oxygen species (ROS). Macromolecular antioxidant drugs can reduce the damage caused by noise-induced oxidative stress overload. These drugs can be directly delivered to the inner ear through inner ear administration to exert their antioxidant effects.

[0003] However, the high molecular weight and complex structure of macromolecular antioxidant drugs hinder their ability to penetrate cell membranes. Currently, various carrier systems have been developed to improve their biological activity and delivery efficiency. However, the delivery efficiency of current carriers is still limited, and the application of macromolecular antioxidant drugs in the treatment of NIHL is severely restricted. In particular, when using various carrier systems in the prior art to deliver macromolecular antioxidant drugs, such as protein drugs, there are significant deficiencies in the release of protein drugs.

[0004] Therefore, there is an urgent need in the art to develop a novel macromolecular antioxidant drug delivery system for the treatment of NIHL. Summary of the Invention

[0005] The present invention provides a preparation method of a condensate microdroplet delivery system and the condensate microdroplet delivery system prepared thereby. The condensate microdroplet delivery system of the present invention can effectively encapsulate macromolecular drugs, especially catalase.

[0006] In a first aspect, the present invention provides a method for preparing a condensate microdroplet encapsulating a drug, the method comprising the following steps:

[0007] S1: Prepare a polypeptide-nucleic acid conjugate

[0008] S2: Use the polypeptide-nucleic acid conjugate prepared in step S1 to encapsulate the drug, thereby obtaining the condensate microdroplet encapsulating the drug;

[0009] The polypeptide sequence is a polypeptide containing a free thiol group;

[0010] The nucleic acid is a single-stranded DNA modified with a disulfide bond.

[0011] In a preferred embodiment, the drug is a macromolecular drug.

[0012] In a preferred embodiment, the macromolecular drug is a protein drug.

[0013] In a specific embodiment, the protein drug is catalase.

[0014] In a preferred embodiment, the free sulfhydryl group in the polypeptide is provided by cysteine.

[0015] In a preferred embodiment, the length of the polypeptide is 25 - 30 amino acid residues; preferably 30 amino acid residues.

[0016] In a specific embodiment, the sequence of the polypeptide is as shown in (VPGXG)6-Cys-NH2 (SEQ ID NO:3), where X is any amino acid other than proline.

[0017] In a preferred embodiment, the length of the single-stranded DNA is 18 - 45 nt; preferably 18 nt.

[0018] In a specific embodiment, the position of the disulfide bond modification in the disulfide bond-modified single-stranded DNA is 5'-terminal modification.

[0019] In a specific embodiment, in the polypeptide-nucleic acid conjugate, the polypeptide is connected to the 5'-end of the nucleic acid through the C-terminus.

[0020] In a specific embodiment, the sequence of the polypeptide is as shown in SEQ ID NO:1.

[0021] In a specific embodiment, the sequence of the nucleic acid is as shown in SEQ ID NO:2.

[0022] In a preferred embodiment, the phase transition temperature T of the condensate microdroplets t is 27 - 30 °C, preferably 28 °C; the particle size of the condensate microdroplets is about 2 - 5 μM, preferably 2 μM; the condensate microdroplets can undergo photobleaching recovery, and the recovery time is about 70 - 90 s, preferably 80 s.

[0023] In a preferred embodiment, step S2 includes co-incubating the drug with the polypeptide-nucleic acid conjugate prepared in step S1.

[0024] In a preferred embodiment, the method optionally further includes step S3: verifying the function or activity of the condensate microdroplets encapsulating the drug in vitro.

[0025] In a second aspect, the present invention provides a condensate microdroplet encapsulating a drug, which is prepared by the preparation method described in the first aspect.

[0026] In a third aspect, the present invention provides the use of the condensate microdroplet encapsulating a drug prepared by the method described in the first aspect, or the condensate microdroplet encapsulating a drug described in the second aspect, in the preparation of a drug.

[0027] In a preferred embodiment, the drug is used for treating hearing impairment.

[0028] In a preferred embodiment, the hearing impairment is noise-induced hearing impairment.

[0029] In a preferred embodiment, the hearing impairment is caused by excessive reactive oxygen species (ROS).

[0030] In a third aspect, the present invention provides a method for treating hearing impairment, the method comprising administering to a subject in need of treatment for hearing impairment a therapeutically effective amount of the condensed microdroplets encapsulating the drug prepared by the method of the first aspect, or the condensed microdroplets encapsulating the drug of the second aspect.

[0031] In a preferred embodiment, the hearing impairment is noise-induced hearing impairment.

[0032] In a preferred embodiment, the hearing impairment is caused by excessive reactive oxygen species (ROS).

[0033] In a preferred embodiment, the subject is a mammal.

[0034] In a preferred embodiment, the mammal is a human.

[0035] In a preferred embodiment, 1 - 2 μL of the condensed microdroplets encapsulating the drug is applied to the round window membrane of the cochlea.

[0036] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shows the self-assembly and characterization of the sELP-ON microdroplets of the present invention. Among them, Figure 1 a is the turbidity curve absorbance of sELP-ON at different concentrations at 350 nm. Figure 1 b is the confocal image of TAMRA-labeled sELP-ON microdroplets (1 mM). Figure 1 c is the dynamic light scattering (DLS) analysis and transmission electron microscopy (TEM) image of sELP-ON microdroplets (1 mM). Figure 1 d is the time-lapse image of the fusion process of sELP-ON microdroplets (1 mM) stained with fluorescein isothiocyanate (FITC). Figure 1 e is the time-lapse image of the wetting process of sELP-ON microdroplets (1 mM) on a glass substrate. Figure 1f is a fluorescence recovery delay image after the photobleaching (FRAP) measurement of a single sELP-ON droplet labeled with FITC. Scale bar: 2 μm.

[0038] Figure 2 The encapsulation of catalase by the sELP-ON microdroplets of the present invention and the scavenging of reactive oxygen species in vitro are shown. Among them, Figure 2 a is a confocal microscope image of TAMRA-labeled sELP-ON microdroplets encapsulating FITC-labeled catalase. Figure 2 b is a time-lapse image of the GOx / HRP cascade reaction to scavenge H2O2 by CAT-encapsulated sELP-ON and sELP-ON microdroplets without CAT. H2O2 was detected using the H2O2 fluorescent probe Amplex red. Concentration of sELP-ON microdroplets: 1 mM. Scale bar: 5 μm.

[0039] Figure 3 The in vivo efficacy evaluation of the CAT@sELP-ON microdroplets of the present invention for the treatment of hearing impairment is shown. Among them, Figure 3 a is a schematic diagram of the animal experiment; Figure 3 b shows the changes in the hearing thresholds of each group: (i) in the left ear (sELP-ON@CAT injection ear, experimental group) before noise exposure (blue line, PRE) and on the third day (red line, D3), seventh day (green line, D7), and fourteenth day (purple line, D14) after exposure; (ii) in the right ear (PBS injection ear, control group); (iii) the hearing levels of both ears before noise exposure, L (left ear, blue line), R (right ear, red line); (iv) the hearing levels of both ears on the 3rd day after noise exposure; (v) the hearing levels of both ears on the 7th day after noise exposure; (vi) the hearing levels of both ears on the 14th day after noise exposure.

[0040] Figure 4 The in vivo tissue damage evaluation of the sELP-ON microdroplets of the present invention for the treatment of hearing impairment is shown. Among them, Figure 4 a shows that the CAT@sELP-ON microdroplets reduce the loss of cochlear OHCs and synapses. Hair cells were immunolabeled with phalloidin (red), ribbon synapses were immunolabeled with CtBP2 (green), and cell nuclei were counterstained with DAPI (blue). Left ear (sELP-ON@CAT injection ear, experimental group), right ear (PBS injection ear, control group), *lost hair cells, scale bar: 50 μm; Figure 4 b shows the survival rate (%) of IHCs; Figure 4 c shows the survival rate (%) of OHCs; Figure 4 d shows the survival rate of IHCs ribbon synapses; Figure 4 e shows the observation of cochlear SGNs and ANFs by TEM, scale bar: 5 μm; Figure 4f shows SVs observed by toluidine blue staining, scale bar: 200 μm; Figure 4 g, h, and i are statistical analyses of SGNs survival rate, ANFs density, and SVs thickness; *P < 0.05, **P < 0.01, and ***P < 0.001.

[0041] Figure 5 Shows the preparation of the sELP-ON microdroplets of the present invention and a schematic diagram of administering to mice with noise-induced hearing loss for treating hearing loss. Detailed implementation manners

[0042] After extensive and in-depth research, the inventors unexpectedly discovered a condensate microdroplet delivery system based on a polypeptide-nucleic acid conjugate. The condensate microdroplet delivery system can effectively encapsulate catalase, and when it is dropped into the round window membrane of the cochlea, it can reduce oxidative stress damage in the ear, thereby effectively treating NIHL. The present invention was completed on this basis.

[0043] The condensate microdroplet delivery system of the present invention and its preparation method

[0044] In this article, "the condensate microdroplet delivery system of the present invention", "the condensate microdroplet delivery system", or "the condensate microdroplets" have the same or similar meanings. These terms all refer to micron-sized droplets formed by the liquid-liquid phase separation of the polypeptide-nucleic acid conjugate.

[0045] The condensate microdroplet delivery system of the present invention is based on a polypeptide-nucleic acid conjugate, so that it can encapsulate drugs and then achieve efficient drug delivery. Therefore, the drug-encapsulated condensate microdroplets of the present invention can be prepared as follows: First, prepare the polypeptide-nucleic acid conjugate; secondly, use the prepared polypeptide-nucleic acid conjugate to encapsulate the drug, thereby obtaining the drug-encapsulated condensate microdroplets.

[0046] The condensate microdroplets of the present invention are particularly suitable for encapsulating macromolecular drugs. In a specific implementation manner, the macromolecular drug is a protein drug.

[0047] Based on the specific application field, those skilled in the art can determine the specific polypeptide and nucleic acid in the condensate microdroplets of the present invention according to the drug to be encapsulated. Generally, in the condensate microdroplets of the present invention, the sequence of the polypeptide is (VPGXG)6-Cys-NH2, where X is any amino acid other than proline; the length of the polypeptide is 25-30 amino acid residues; preferably 30 amino acid residues. The nucleic acid is a single-stranded DNA modified with a disulfide bond. In a specific embodiment, the length of the single-stranded DNA is 18-45 nt; preferably 18 nt; the position of the disulfide bond modification in the single-stranded DNA modified with a disulfide bond is the 5' end. The condensate microdroplets of the present invention are particularly suitable for encapsulating catalase. Correspondingly, the sequence of the polypeptide in the polypeptide-nucleic acid conjugate is as shown in SEQ ID NO:1; the sequence of the nucleic acid is as shown in SEQ ID NO:2.

[0048] In the polypeptide-nucleic acid conjugate of the condensate microdroplets of the present invention, the polypeptide and the nucleic acid can be connected in various ways. For example, the polypeptide is connected to the 5' end of the nucleic acid through the C-terminus.

[0049] The condensate microdroplets of the present invention can efficiently deliver macromolecular drugs. Correspondingly, the phase transition temperature T of the condensate microdroplets of the present invention t is 27-30 °C, preferably 28 °C; the particle size is about 2-5 μM, preferably 2 μM; the condensate microdroplets can be restored after photobleaching, and the restoration time is about 70-90, 80 s.

[0050] Based on the teachings of the present invention, those skilled in the art know how to use the polypeptide-nucleic acid conjugate to encapsulate drugs. For example, the drug is co-incubated with the polypeptide-nucleic acid conjugate. After obtaining the condensate microdroplets encapsulating the drug, the function or activity of the condensate microdroplets encapsulating the drug can also be verified in vitro.

[0051] Application

[0052] Based on the teachings of the present invention and the characteristics of the encapsulated drug, those skilled in the art can know that the condensate microdroplets encapsulating the drug of the present invention can be used to prepare drugs, thereby treating diseases corresponding to the drug. For example, the drug can be used to treat hearing impairment. In a specific embodiment, the hearing impairment is noise-induced hearing impairment. In a preferred embodiment, the hearing impairment is caused by excessive reactive oxygen species (ROS).

[0053] Treatment method

[0054] Based on the drug-loaded coacervate microdroplets of the present invention, the present invention also provides a method for treating diseases using the drug-loaded coacervate microdroplets. The method includes administering a therapeutically effective amount of the drug-loaded coacervate microdroplets to a subject in need of treating a disease. The subject can be a mammal; preferably a human.

[0055] In a specific embodiment, the treatment method is a method for treating hearing impairment. Accordingly, the treatment method includes administering a therapeutically effective amount of the drug-loaded coacervate microdroplets of the present invention to a subject in need of treating hearing impairment. The hearing impairment is noise-induced hearing impairment; preferably, the hearing impairment is caused by excessive reactive oxygen species (ROS).

[0056] During the treatment, the dosage of the drug-loaded coacervate microdroplets of the present invention can be determined by a clinician according to specific circumstances, such as the age, gender, weight of the subject, the severity of the disease, and the presence of other co-administered drugs, etc. In a specific embodiment, 1 - 2 μL of the drug-loaded coacervate microdroplets is applied to the round window membrane of the cochlea of the subject.

[0057] Advantages or beneficial technical effects of the present invention:

[0058] 1. Using the coacervate microdroplet delivery system of the present invention can significantly improve the delivery efficiency of macromolecular drugs;

[0059] 2. The present invention uses the polypeptide-nucleic acid conjugate self-assembly technology to prepare a coacervate microdroplet delivery system loaded with catalase. The microdroplets are formed by self-assembly, and the construction steps are simple;

[0060] 3. The coacervate microdroplet delivery system loaded with catalase of the present invention can effectively restore noise-induced hearing loss, and the treatment effect is remarkable.

[0061] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The reagents used in the embodiments are all commercially available reagents. Unless otherwise stated, percentages and parts are calculated by weight.

[0062] Examples

[0063] Experimental materials

[0064] Tetramethylethylenediamine (TCEP) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; 2,2'-Dithiopyridine (Py2S2) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; Fluorescein isothiocyanate (FITC) and Rhodamine B isothiocyanate (RBITC) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Polypeptides (HPLC purified) were purchased from Shanghai Qiangyao Biotechnology Co., Ltd.; Disulfide bond-modified single-stranded DNA was purchased from Shengong Bioengineering (Shanghai) Co., Ltd.; Catalase (CAT) was purchased from Adamas Reagents (Shanghai) Co., Ltd.; 10-Acetyl-3,7-dihydroxyphenazine (Amplex Red) was purchased from Beyotime Biotechnology Co., Ltd.; Ultrafiltration tubes (3 kDa, 10 kDa and 30 kDa) were purchased from Millipore Corporation, USA.

[0065] Example 1. Synthesis of polypeptide-nucleic acid conjugate and preparation of condensate microdroplets

[0066] The TCEP solution (50 mM, 20 μL) was added to the disulfide bond-modified single-stranded DNA solution (100 mM, 100 μL, 0.05 M triethylamine acetate buffer (TEEA buffer)). The mixture was incubated at 37 °C for 1 hour. The oligonucleotide with free thiol groups was concentrated using a 3 kDa ultrafiltration tube. Then 2,2'-Dithiopyridine (Py2S2) (20 mM 10 μL) was added, and the mixture was incubated at 37 °C for 2 hours. The product was purified by centrifugation using a 3 kDa filter tube. The activated DNA was added to the ELP solution containing cysteine thiol groups (10 mg / mL, 120 μL, dissolved in dimethyl sulfoxide, DMSO), and stirred overnight at 37 °C. After the reaction, the obtained conjugate was purified and concentrated using a 3 kDa ultrafiltration tube.

[0067] The reaction route is as follows:

[0068]

[0069] sELP-ON conjugate structure:

[0070]

[0071] The polypeptide and nucleic acid sequences used in the sELP-ON conjugate are shown in Table 1.

[0072] Table 1A. Polypeptide sequence of sELP-ON conjugate

[0073]

[0074] Table 1B. Nucleic acid sequence of sELP-ON conjugate

[0075]

[0076] Example 2. Encapsulation of Catalase

[0077] The sELP-ON microdroplets (20 μM) obtained in Example 1 were pre-incubated with FITC-CAT (1 μM) at 37 °C for 2 h. The mixture was centrifuged for 10 min, and the supernatant was discarded. The fluorescence intensity was monitored by a microplate reader, and the encapsulation efficiency of FITC-CAT was calculated.

[0078] Finally, the encapsulation efficiency of FITC-CAT was 76%.

[0079] Example 3. Characterization of Condensate Microdroplets Encapsulating Catalase

[0080] Confocal microscopy characterization:

[0081] Fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC) (10 mM, 20 μL, dissolved in DMSO) was added to the CAT solution (30 μM, 1000 μL, dissolved in 0.1 M NaHCO3-Na2CO3, pH = 9.5). The mixed solution was stirred at 37 °C for 4 h and incubated in the dark at 4 °C for 8 h. Then, an ultrafiltration tube filter was used to remove the excess FITC or RBITC. The sELP-ON microdroplets (20 μM) were pre-incubated with FITC-CAT (1 μM) at 37 °C for 2 h. A confocal laser scanning microscope (CLSM) equipped with a 63× oil immersion objective and a 37 °C stage heater was used to visualize the samples in the observation chamber. Blue channel, excitation: 405 nm, collection: 425 - 475 nm; green channel, excitation: 488 nm, collection: 500 - 550 nm; red channel, excitation: 561 nm, collection: 570 - 620 nm.

[0082] The results of the construction and self-assembly characterization of sELP-ON condensate microdroplets are as Figure 1 shown.

[0083] Bright-field and fluorescence images observed using a confocal laser scanning microscope (CLSM) showed that spherical particles with a micron size (about 2 μm) were formed ( Figure 1 b), which was further confirmed by dynamic light scattering and transmission electron microscopy (TEM; Figure 1 c). Time-lapse CLSM images showed the dynamic fusion behavior ( Figure 1 d) and wetting behavior ( Figure 1 e) of the microdroplets, indicating their existence in the droplet state. The fluorescence recovery experiment after photobleaching (FRAP) showed that the fluorescence in the droplets recovered rapidly, confirming that the sELP-ON co-gel microdroplets had liquid-like properties ( Figure 1 f).

[0084] Example 4. In vitro scavenging of hydrogen peroxide by coacervate microdroplets encapsulating catalase

[0085] In this example, the in vitro scavenging of hydrogen peroxide by the coacervate microdroplets encapsulating catalase prepared in Example 2 was tested.

[0086] To evaluate the ability of the coacervate microdroplets encapsulating catalase to scavenge hydrogen peroxide, a cascade reaction of glucose oxidase / horseradish peroxidase (GOx / HRP) was added to the system. Glucose can freely pass through the microdroplets and is oxidized by glucose oxidase (GOx) to provide H2O2. In the presence of horseradish peroxidase, the reactive oxygen probe Amplex Red reacts with H2O2 to generate the red fluorescent oxidation product resorufin. The GOx / HRP catalytic reaction was carried out in sELP-ON microdroplets. The sELP-ON droplets were incubated with 1 nM GOx and 1 nM HRP at 37 °C for 2 hours, and then 1 mM HRP and 10 mM glucose were added to the microdroplet solution. For confocal imaging, 10 μM Amplex Red and 5 mM glucose were added, and fluorescence images were captured at 1-minute intervals for 10 minutes.

[0087] The results of the in vitro scavenging of hydrogen peroxide by the microdroplets are as Figure 2 shown. Among them, Figure 2 b shows that after adding glucose to the microdroplet control group pre-encapsulated with GOx, HRP, and the reactive oxygen probe Amplex Red, the red fluorescence in the microdroplets gradually increased, indicating the successful GOx / HRP cascade reaction and resorufin generation. In contrast, the fluorescence in the microdroplets of the CAT pre-encapsulated group was significantly lower than that of the control group, which indicates that CAT effectively consumed H2O2, blocked the HRP-mediated reaction, and reduced the generation of fluorescent molecules.

[0088] Example 5. Evaluation of the treatment of hearing impairment by coacervate microdroplets

[0089] In this example, the therapeutic effect of the coacervate microdroplets encapsulating catalase prepared in Example 2 on hearing-impaired mice was evaluated.

[0090] The animal model used for evaluation was SPF-grade 1-month-old C57BL6 mice, purchased from Vital River Laboratories. The modeling conditions for the mouse noise injury model were as follows: SPF-grade 1-month-old C57BL / 6 mice were exposed to noise with a frequency range of 2–20 kHz and an intensity of 120 dB SPL for 2 hours. The experiment was carried out in a ventilated soundproof exposure chamber. The noise generator was driven by the RZ6 system and calibrated using an AWA5688 type sound level meter.

[0091] During the evaluation process, the method of administering the droplets to the round window membrane of the cochlea was as described below:

[0092] Under a surgical microscope, the skin behind the mouse ear was incised, and the muscle and connective tissue were bluntly separated in sequence to expose the auditory bulla and round window. A 2 μL dose of the catalase-loaded coacervate microdroplets prepared in Example 2 was injected into the round window membrane using a specially made extremely fine glass needle. The left ear was the experimental ear, and the right ear was the control ear.

[0093] The auditory brainstem response (ABR) and noise exposure methods are as described below:

[0094] The workstation was used to record ABR to evaluate the auditory function of mice in response to sound stimuli. Before measurement, the mice were anesthetized and their body temperature was maintained at 37 °C outside the anechoic chamber. The ABR stimuli were emitted by an MF1-M speaker in the free field, including clicks with a duration of 100 μs and tone bursts with frequencies of 4, 8, 16, 24, and 32 kHz, with a rise-fall time of 1 ms and a plateau of 3 ms. ABR recordings were made on 1-month-old mice, 3 days before noise exposure and 1, 7, and 14 days after exposure. More than 512 presentations of the same stimulus were made, and the responses were amplified, filtered (0.3 - 3 kHz), and averaged. The hearing threshold level was determined offline using SPL, which could visually distinguish the first peak (0.1 V) of wave I above the noise floor. The animals were exposed to 120 dB SPL in a ventilated noise exposure chamber at a frequency range of 2 kHz to 20 kHz for 2 hours. The noise generator was driven by an RZ6 system, with a maximum stimulus intensity variation of 3 dB, and was calibrated using a sound level meter before exposure to ensure the accuracy and precision of noise exposure.

[0095] In vivo efficacy evaluation of the catalase-loaded coacervate microdroplets prepared in Example 2 for treating hearing impairment. Auditory brainstem response (ABR) tests were performed 3 days before (-D3) and on days 3 (D3), D7, and D14 after noise exposure, and then cochlear pathology tests were performed on D14. The results are as Figure 3 shown in

[0096] At D3, the ABR thresholds of the treated left ear at clicks, 4, 8, 16, 24, and 32 kHz were 61.00 ± 6.24, 68.00 ± 13.12, 48.00 ± 8.50, 48.00 ± 8.49, 68.00 ± 18.40, and 66.00 ± 22.48 dB SPL ( Figure 3b). In addition, from D7 to D14, the hearing of the treated ear continued to improve compared with the untreated ear. By D14, the ABR thresholds at 4, 8, and 11 kHz frequencies in the treated ear had fully recovered to the levels before noise exposure. In contrast, the untreated right ear showed a permanent ABR threshold shift. These auditory results indicate that sELP-ON@CAT can effectively promote the hearing recovery of NIHL patients.

[0097] In vivo tissue damage assessment of the microdroplets encapsulating catalase prepared in Example 2 for treating hearing injury. Excessive noise exposure can damage hair cells (HCs), ribbon synapses, spiral ganglion neurons (SGNs), and nerve fibers in the cochlea. After administration, the morphology of HCs was examined using lipoid-like staining and confocal microscopy. The CtBP2 level in IHCs was analyzed using immunofluorescence. The morphology of spiral neurons (SNGs) and related innervating nerve fibers was analyzed using transmission electron microscopy (TEM).

[0098] The results are as Figure 4 shown, where, at the D14 stage, there was no significant difference in the number of inner hair cells (IHCs) between the treated ear and the untreated ear ( Figure 4 a-b). However, at the apex, middle, and basal turns of the treated ear, a significant increase in the number of outer hair cells (OHCs) was found, indicating an increased survival rate of OHCs after treatment with microdroplets encapsulating catalase ( Figure 4 a and c). In addition, the treated ear showed significantly more ribbon synapses at all cochlear turns at the D14 stage than the untreated ear ( Figure 4 a and d). In addition, TEM showed that by D14, the spiral neurons (SNGs) and related innervating nerve fibers in the untreated group were significantly reduced, while the treated group had a significant protective effect ( Figure 4 e, g, and h). In summary, these results indicate that treatment with microdroplets encapsulating catalase can significantly reduce the damage caused by excessive noise exposure to ribbon synapses, SGNs, and nerve fibers.

[0099] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for preparing drug-loaded aggregate microdroplets, the method comprising the following steps: S1: Preparation of peptide-nucleic acid conjugates S2: using the polypeptide-nucleic acid conjugate prepared in step S1 to encapsulate the drug, thereby obtaining the drug-encapsulated aggregate microdroplets; The polypeptide sequence is a polypeptide containing a free thiol group; The nucleic acid is a disulfide bond-modified single-stranded DNA.

2. The preparation method according to claim 1, characterized in that The protein drug is catalase.

3. The preparation method according to claim 1 or 2, characterized in that: The sequence of the polypeptide is shown as (VPGXG)6-Cys-NH2, where X is any amino acid except proline.

4. The preparation method according to any one of claims 1 to 3, characterized in that The position of the disulfide bond modification in the disulfide bond-modified single-stranded DNA is the 5' terminal modification.

5. The preparation method according to any one of claims 1 to 4, characterized in that In the polypeptide-nucleic acid conjugate, the polypeptide is connected to the 5' end of the nucleic acid via the C-terminus.

6. The preparation method according to any one of claims 1 to 5, characterized in that The sequence of the polypeptide is shown in SEQ ID NO:

1.

7. The preparation method according to any one of claims 1 to 6, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO:

2.

8. A drug-loaded agglomerate microdroplet, wherein the agglomerate microdroplet is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the drug-loaded agglomerate microdroplets prepared by the method according to any one of claims 1 to 7, or the drug-loaded agglomerate microdroplets according to claim 8 in preparing drugs.

10. A method for treating hearing loss, comprising administering a therapeutically effective amount of drug-loaded agglomerate microdroplets prepared by the method of any one of claims 1 to 7, or the drug-loaded agglomerate microdroplets of claim 8 to a subject in need of treatment for hearing loss.

Images