Hair cell protective agent based on ferritin nano platform and application of hair cell protective agent in treatment of hearing loss

Abstract

The invention discloses a hair cell protective agent based on a ferritin nano platform and application of the hair cell protective agent to treatment of hearing loss, and belongs to the field of biomedicine.The ferritin nanoparticle loaded with cimicifugin comprises ferritin (Fn) serving as a carrier and cimicifugin (Ci-Fn) loaded in the ferritin. According to the present invention, the cimicifugin (Cimifugin, Ci) as the cimicifugin extract is loaded in the ferritin, such that the additional anti-inflammatory and tissue protection functions are provided so as to achieve the more accurate and effective treatment effect. After being injected through a retroauricular round window membrane (RWM), the Ci-Fn is conveyed to a basilar membrane along with flowing of exolymph and endolymph and finally targets inner ear hair cells (HCs). After entering the hair cells, the Ci-Fn is absorbed and degraded by lysosome, and the cimicifugin (Ci) is released, so that the protection effect on the hair cells is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a hair cell protectant based on a ferritin nano-platform and its application in treating hearing loss. Background Art

[0002] Hearing loss and deafness are the most common sensory organ disabilities globally, having a significant impact on the quality of life of patients and on the social economy. However, current treatment methods for hearing loss mainly focus on using hearing aids, cochlear implants, or steroid drugs. However, these methods have many limitations, such as limited efficacy, obvious potential side effects, and the inability to reverse hair cell damage. Therefore, there is an urgent need to develop new treatment strategies to achieve more precise and efficient hearing protection and repair.

[0003] In terms of drug treatment, existing studies have shown that small molecule inhibitors and plant extracts exhibit certain hair cell protection effects. Among them, round window membrane (RWM) drug delivery has been explored as a potential inner ear delivery method. For example, the combined use of antioxidants to reduce cisplatin ototoxicity. However, the main drawbacks of these methods are: 1. Poor targeting: Existing small molecule drugs are difficult to effectively concentrate on cochlear hair cells, resulting in limited efficacy; 2. High off-target toxicity: The drug may act on non-target cells, producing side effects; 3. Poor stability: The drug is easily degraded in the cochlear internal environment, affecting the efficacy; 4. Short residence time: Existing delivery systems cannot extend the residence time of the drug in the cochlea, reducing the treatment effect. In recent years, nanocarriers have been widely studied as drug delivery platforms. For example, poly(lactic-co-glycolic acid) (PLGA) nanoparticles. However, such nanosystems still face challenges such as limited biocompatibility, low delivery efficiency, and difficulty in controlling the release rate. Therefore, developing an efficient and precise cochlear delivery system to improve drug targeting, stability, and bioavailability is the focus of current research. Summary of the Invention

[0004] The technical problem to be solved by the present invention is: Aiming at the problems of poor targeting, high off-target toxicity, insufficient stability, and short residence time existing in the prior art, the present invention aims to develop a drug delivery system based on a ferritin nano-platform to achieve precise protection of cochlear hair cells.

[0005] The technical solution of the present invention is: A ferritin nanoparticle (Ci@Fn) loaded with cimifugin (Ci), this nano-system uses ferritin (Fn) as a carrier and loads cimifugin with anti-inflammatory and antioxidant effects. The cimifugin is coated inside the ferritin to form a core-shell structure complex with ferritin as the shell and cimifugin as the core.

[0006] Further, the ferritin is human heavy chain ferritin

[0007] Furthermore, the amino acid sequence of the ferritin is as shown in SEQ ID NO.1.

[0008] Furthermore, the molar ratio of the ferritin to cimifugin is 33.8.

[0009] For the preparation method of the above-mentioned ferritin nanoparticles, ferritin and cimifugin are stirred under acidic conditions, then restored to neutrality, centrifuged, the supernatant is collected, and the supernatant is dialyzed to obtain ferritin nanoparticles loaded with cimifugin.

[0010] Furthermore, it specifically includes the following steps:

[0011] (1) Dissolve ferritin in Tris-HCl buffer with a pH of 7.4, adjust the pH to 2 with HCl under continuous stirring, then drop in the cimifugin solution, continue stirring for 1 hour, add NaOH to restore the pH to 7, and then stir for another 30 minutes;

[0012] (2) After the reaction is completed, centrifuge the solution at 10000×g for 15 minutes to collect the supernatant;

[0013] (3) Dialyze the supernatant with a dialysis bag with a molecular weight of 2 kDa for 24 hours to obtain ferritin nanoparticles loaded with cimifugin.

[0014] Use of the above-mentioned ferritin nanoparticles in the preparation of a drug for treating or preventing inner ear hair cell damage.

[0015] Furthermore, the hair cell damage refers to hearing loss or mechanical damage caused by drug toxicity.

[0016] Furthermore, the hearing loss caused by drug toxicity refers to the hearing loss caused by cisplatin.

[0017] Furthermore, the mechanical damage refers to noise-induced hearing loss.

[0018] The Ci@Fn nanodelivery system developed in this study is administered by retroauricular round window membrane (RWM) injection. The drug reaches the cochlear basilar membrane with the flow of endolymph in the inner ear and is precisely delivered to hair cells with the help of the targeting property of ferritin. After being endocytosed by hair cells, Ci@Fn enters lysosomes and releases cimifugin, exerting anti-inflammatory and anti-apoptotic effects intracellularly.

[0019] In a neonatal mouse model of cisplatin-induced hearing loss, we found that Ci@Fn can effectively reduce hair cell apoptosis and significantly reduce hearing loss. In addition, in a noise-induced hearing loss model of adult mice, the results of auditory brainstem response (ABR) measurement showed that Ci@Fn can provide significant hearing protection and has high safety.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Efficient targeted delivery: The ferritin nanoplatform can enhance the enrichment of drugs in cochlear hair cells and improve the therapeutic effect.

[0022] 2. Long-term protective effect: Prolong the residence time of drugs in the cochlea, achieve sustained release, and improve the treatment efficiency.

[0023] 3. Enhanced therapeutic effect: By regulating the PI3K-Akt signaling pathway, reduce the hair cell damage induced by cisplatin and noise.

[0024] 4. High safety: Ferritin has good biocompatibility, reducing the risk of immune response and toxic and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the characterization and performance diagrams of ferritin purification and Ci@Fn synthesis;

[0026] Figure 2 It is the in vitro and in vivo delivery research of Ci@Fn;

[0027] Figure 3 It is the evaluation of the protective effect of Ci@Fn on cisplatin-induced ototoxicity in neonatal rats;

[0028] Figure 4 It is the evaluation of the protective effect of Ci@Fn on noise-induced hearing loss in adult rats;

[0029] Figure 5 It is the research on the protective mechanism of Ci@Fn. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the relevant descriptions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present application.

[0031] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0032] Cimifugin (Ci) is a substance extracted from the plant Cimicifuga, with the molecular formula C 16 H 18 O 6 , and the structural formula is:

[0033]

[0034] Ferritin (Fn) is an iron storage protein widely expressed in the human body and has received extensive attention as a drug carrier in recent years. The unique cage-like structure of Fn (pore size about 8 - 12 nm) provides natural advantages for drug loading.

[0035] This application provides a hair cell protectant based on a ferritin nanoplatform. The protectant includes ferritin (Fn) as a carrier and cimifugin (Ci) loaded in ferritin, forming a complex Ci@Fn. By loading cimifugin, the ferritin nanoparticles are endowed with additional anti-inflammatory and tissue protection functions. After the ferritin nanoparticles (Fn) are injected through the round window membrane behind the ear, they can be transported to the basilar membrane along with the flow of perilymph and endolymph, and with the targeting function of ferritin, precisely target the inner ear hair cells. Subsequently, Ci@Fn is endocytosed by the hair cells, and Ci is released in the lysosome, playing a protective role on the hair cells.

[0036] Example 1: Ferritin purification and synthesis and characterization of Ci@Fn

[0037] By genetic engineering means, we constructed a heavy chain ferritin (Fn) expression system and highly expressed it in Escherichia coli. The specific steps are as follows: The gene of human heavy chain ferritin (amino acid sequence shown in SEQ ID NO.1) was directionally cloned into the pET-30a(+) vector to construct a heavy chain ferritin expression plasmid. The recombinant plasmid was transformed into BL21(DE3) competent cells, and positive clones were obtained through resistance screening.

[0038] Select monoclonal strains and culture them in a shaking flask at 37°C and 220 rpm until OD 600 ≈0.6 - 0.8, add 0.5 mM IPTG to induce protein expression, and culture at 25°C for 12 h. Collect the bacteria, and after ultrasonic lysis, perform protein purification by methods such as affinity chromatography, ion exchange chromatography, and gel filtration chromatography to ensure obtaining high-purity Fn. Detect the purity of Fn by SDS-PAGE electrophoresis and determine its particle size distribution by dynamic light scattering (DLS). The results show that the purified Fn is mainly in a monodisperse state, with a particle size of about 12 nm, indicating that its structure is complete and suitable for subsequent drug loading research ( Figure 1 in A, B, C).

[0039] Under acidic conditions, cimifugin (Ci) was successfully loaded into the Fn cavity by pH induction method. The specific steps are as follows: Dissolve 20 mg of Fn in 2 mL of 20 mM Tris-HCl buffer (pH 7.4). Under stirring conditions, slowly add 0.1 M HCl to adjust the pH to 2 to open the cage structure of Fn. Add 10 mM Ci solution dropwise and continue stirring for 1 h to allow Ci molecules to fully enter the Fn cavity. Restore the pH to 7 by slowly adding 0.1 M NaOH and continue stirring for 30 min to close the Fn structure and fix Ci. Remove unencapsulated Ci by centrifugation at 10,000×g for 15 min, and collect the supernatant. Dialyze for 24 h using a dialysis bag with a molecular weight cut-off of 2 kDa to remove free Ci, and finally obtain the Ci@Fn nanocomposite.

[0040] After successful loading of Ci, we evaluated the effect of the loading process on the properties of Fn. Through transmission electron microscopy (TEM) and dynamic light scattering (DLS), we found that there were no obvious changes in the morphology and size of Fn after loading Ci ( Figure 1 in D, E), indicating that the drug-loading process did not affect the structural stability of Fn. In addition, through circular dichroism spectroscopy analysis, it was confirmed that the protein structure of Fn remained stable after hydrothermal synthesis without conformational changes ( Figure 1 in F). Next, we established a concentration profile by high-performance liquid chromatography (HPLC) to evaluate the drug-loading capacity of Fn (1 mg / mL). The analysis showed that the synthesized drug-loading concentration was 67.52 μM ( Figure 1 in G), and the molar ratio of Fn to Ci was 33.8.

[0041] Example 2: In vitro and in vivo delivery studies of Ci@Fn

[0042] In vitro studies can preliminarily verify the targeting and delivery efficiency of Ci@Fn nanoparticles to target cells (such as hair cells), providing a theoretical basis for in vivo studies. Through in vitro experiments, the intracellular behavior of nanoparticles (such as endocytic pathways, lysosomal escape, etc.) can be revealed. In vitro studies help to deeply understand the mechanism of action of Ci@Fn nanoparticles, such as its ability to scavenge reactive oxygen species (ROS) and its impact on cell metabolism. To verify whether Ci@Fn can be effectively taken up by hair cells, we used HEI-OC1 cells for confocal microscopy imaging experiments. Seed HEI-OC1 cells in 24-well plates and culture to a density of 70%. Label Ci@Fn with Cy5-SE and incubate HEI-OC1 cells for 4 h. Stain lysosomes with LysoTracker Green, and then observe the intracellular fluorescence signal with a confocal microscope ( Figure 2 in A). The results showed that Cy5-labeled Ci@Fn co-localized with lysosomes, indicating that Ci was released after being phagocytosed by the cells, suggesting that it could enter hair cells through endocytosis.

[0043] In vivo studies are a key step to verify the effects of Ci@Fn nanoparticles in actual physiological environments. Through in vivo experiments, the stability, targeting, distribution and metabolism of nanoparticles in complex biological environments, as well as their protective effects on target tissues (such as inner ear hair cells) can be evaluated. Therefore, through systematic in vitro and in vivo experiments, the therapeutic potential of Ci@Fn nanoparticles can be comprehensively evaluated, providing reliable data support for their future clinical transformation. To evaluate the distribution of Ci@Fn in the mouse cochlea, we delivered Cy5-labeled Ci@Fn to the mouse cochlea via retroauricular round window membrane (RWM) injection. Under anesthesia, the RWM was exposed through a retroauricular approach, and Ci@Fn was slowly injected with a microsyringe. 10 hours after the injection, the cochlea was removed, fixed and decalcified, and the basement membrane was separated and fluorescent imaging was performed ( Figure 2 The results showed that Ci@Fn was significantly enriched in the inner and outer hair cells of the cochlear basilar membrane ( Figure 2 C), indicating its superior targeting ability.

[0044] In summary, our results indicate that Ci@Fn can simultaneously target inner and outer hair cells in the mouse cochlea, providing strong support for the application of Ci@Fn in hearing protection and treatment.

[0045] Example 3: Evaluation of the protective effect of Ci@Fn on cisplatin-induced ototoxicity in neonatal mice

[0046] In order to evaluate the protective effect of Ci@Fn on different types of hearing loss, we first constructed an in vitro model of cisplatin injury. Two-day-old C57 mice were selected and the basement membrane was dissected under a body microscope. After incubation for 24 hours, the basement membranes of the mice were randomly divided into 6 groups: control group, cis injury group, Fn group, dexamethasone (DEX) group, Ci group, and Ci@Fn group. They were incubated for 2 hours and then incubated with 20μM Cis for another 48 hours. The basement membrane was evaluated by different types of staining ( Figure 3 Using confocal laser scanning microscopy, we evaluated the morphology and number of hair cells in each group ( Figure 3 (B). We observed significant hair cell damage and a dramatic decrease in the number of HCs in the Cis group. Significant cell damage was also observed in the Fn and DEX groups, and Fn or DEX alone lacked protective effects on hair cells. In contrast, the hair cells in the Ci@Fn group had good morphology and no significant damage, confirming that Ci@Fn has a significant protective ability on hair cells. In addition, statistical analysis showed that hair cells were more severely damaged in the basal circle of the basement membrane, while the damage was less severe in the apical circle. Compared with the other groups, the Ci@Fn group had less hair cell damage in the basal circle due to the ability of Fn to target hair cells.

[0047] Example 4: Evaluation of the protective effect of Ci@Fn on noise-induced hearing loss in adult mice

[0048] Adult C57BL / 6 mice were selected. After RWM surgery, the Ci@Fn nano-drug was injected behind the ear. Two days after the surgery, the mice were exposed to a noise signal of 115 dB, 2 - 20 kHz for 2 hours to establish a noise-induced hair cell damage model. The ABR test method was used to measure the hearing thresholds of the mice at 4, 8, 16, 24, and 32 kHz. Before treatment, the normal hearing of the mice was ensured by otoscopy ( Figure 4 in A). The hearing thresholds of the mice in the Ci@Fn group were significantly lower than those in the noise group at each frequency, showing an obvious hearing protection effect ( Figure 4 in B). Generally, noise-induced hearing loss is related to synaptic reduction and nerve fiber damage. To verify whether Ci@Fn can increase the number of synapses, we stained the cochlea with anti-c-terminal binding protein 2 (CtBP2). The results showed that the number of synapses in the Ci@Fn group was significantly higher than that in the noise group, indicating that Ci@Fn can effectively prevent synaptic loss ( Figure 4 in C). To further explore the role of Ci@Fn in neuroprotection, we performed immunofluorescence co-staining of neurofilament (NF) and myosin 7a. The neurofilaments in the Fn group, DEX group, and Ci group were also sparsely arranged. However, most of the neurogenic fibers survived in the Ci@Fn group, and the axons extended to the outer hair cells, and the morphology of the original nerve fibers was partially restored ( Figure 4 in D). Compared with the Fn, DEX, and Ci groups, the number of NF-positive neurogenic fibers in the outer hair cells of the Ci@Fn group was significantly increased, indicating that Ci@Fn significantly reduced noise-induced neuronal fiber damage. In addition, to study whether Ci@Fn has an anti-apoptotic effect, we analyzed its mechanism of action by Cleaved Caspase-3 staining. Quantitative analysis of the double-positive hair cells of Cleaved Caspase-3 / Myosin 7a showed that the number of double-positive labeled cells in the Ci@Fn group was significantly lower than that in the control group, suggesting that Ci@Fn effectively reduced noise exposure-induced apoptosis by inhibiting the activation of pro-apoptotic signals ( Figure 4 in E). These findings reveal the molecular mechanism of Ci@Fn in noise-induced hearing loss and provide important theoretical and experimental support for its potential application in the treatment of hearing loss.

[0049] Example 5: Study on the protection mechanism of Ci@Fn

[0050] To deeply explore the mechanism of action of Ci@Fn in cisplatin-induced ototoxicity, we selected the widely used and easily accessible HEI-OC1 cell model of auditory hair cells for RNA sequencing (RNA-seq) analysis. Transcriptome profiling was used to analyze differentially expressed genes (DEGs) in the control group (Con), cisplatin-treated group (Cis), and cisplatin and Ci@Fn co-treated group (Ci@Fn+Cis). Through RNA-seq analysis, DEGs meeting the criteria of p<0.05 and fold change ≥2 were screened out. Compared with the Cis group, the expression of 3498 genes was significantly changed in the Ci@Fn+Cis group, including 2205 up-regulated genes (such as PI3Kca and Bcl2) and 1293 down-regulated genes (such as AKT1). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the change in the PI3K-Akt signaling pathway was the most significant, suggesting that this pathway might be an important route for Ci@Fn to attenuate cisplatin-induced damage to HEI-OC1 cells ( Figure 5 in A). Heatmap analysis further demonstrated the differential expression of genes related to the PI3K-Akt signaling pathway between the Ci@Fn+Cis group and the Cis group ( Figure 5 in B). To verify the RNA-seq results, qRT-PCR was used to quantitatively analyze the mRNA expression of PI3Kca, AKT1, AKT3, Bcl2, Bcl2l1, and Foxo3. The results showed that the Ci@Fn group significantly up-regulated the expression of PI3Kca, AKT3, Bcl2, Bcl2l1, and Foxo3, while down-regulating the expression of AKT1 ( Figure 5 in C). Western blotting was used to analyze the expression of proliferation-related proteins in the PI3K-Akt signaling pathway. Compared with the control group, cisplatin treatment significantly decreased the protein levels of p-PI3K, PI3K, p-AKT, AKT, Bcl2, Bcl2l1, and Foxo3, while Ci@Fn treatment significantly restored and up-regulated the expression of these proteins, further demonstrating its mechanism of action ( Figure 5 in D). After using the PI3K-Akt inhibitor LY294002, it was found that LY294002 partially attenuated the anti-apoptotic effect of Ci@Fn on cisplatin-treated HEI-OC1 cells, indicating the important role of the PI3K-Akt signaling pathway in Ci@Fn reducing cisplatin ototoxicity ( Figure 5 in E). Ci@Fn activates proliferation-promoting genes and reduces cisplatin-induced cell death by significantly up-regulating the expression of genes and proteins related to the PI3K-Akt signaling pathway, showing potential anti-ototoxic effects.

Claims

1. A ferritin nanoparticle loaded with cimicifuga, characterized in that: The invention comprises ferritin as a carrier and cimicifuga as an active ingredient. The cimicifuga is encapsulated in the ferritin to form a core-shell structure complex with the ferritin as a shell and the cimicifuga as a core.

2. The ferritin nanoparticles according to claim 1, characterized in that The ferritin is human heavy chain ferritin.

3. The ferritin nanoparticles according to claim 1, characterized in that The amino acid sequence of the ferritin is shown in SEQ ID NO.

1.

4. The ferritin nanoparticles according to claim 1, characterized in that The molar ratio of ferritin to cimicifuga is 33.

8.

5. The method for preparing ferritin nanoparticles according to any one of claims 1 to 4, characterized in that: Ferritin and cimicifuga are stirred under acidic conditions, then restored to neutrality, centrifuged, the supernatant is collected, and the supernatant is dialyzed to obtain ferritin nanoparticles loaded with cimicifuga.

6. The preparation method according to claim 5, characterized in that: The steps include: (1) Ferritin was dissolved in a Tris-HCl buffer with a pH of 7.4, and the pH was adjusted to 2 with HCl under continuous stirring. Then, the cimicifuga solution was added dropwise. After continuous stirring for 1 hour, NaOH was added to restore the pH to 7, and the mixture was stirred for another 30 minutes. (2) After the reaction, the solution was centrifuged at 10,000 × g for 15 minutes and the supernatant was collected; (3) The supernatant was dialyzed for 24 hours using a dialysis bag with a molecular weight of 2 kDa to obtain ferritin nanoparticles loaded with cimicifuga.

7. Use of the ferritin nanoparticles according to any one of claims 1 to 4 in the preparation of a medicament for treating or preventing inner ear hair cell damage.

8. The use according to claim 7, characterized in that The hair cell damage refers to hearing loss or mechanical damage caused by drug toxicity.

9. The use according to claim 7, characterized in that: The hearing loss caused by drug toxicity refers to hearing loss caused by cisplatin.

10. The use according to claim 7, characterized in that The mechanical damage refers to noise-induced hearing loss.

Images