A hair cell protectant based on a ferritin nanoplatform and its application in the treatment of hearing loss.

Abstract

This invention discloses a hair cell protectant based on a ferritin nanoplatform and its application in treating hearing loss, belonging to the field of biomedicine. The ferritin nanoparticles of this invention, loaded with cimicifugin, include ferritin (Fn) as a carrier and cimicifugin (Ci@Fn) loaded within the ferritin. By loading cimicifugin (Ci), an extract of cimicifugin, into ferritin, it is endowed with additional anti-inflammatory and tissue-protective functions, thereby achieving a more precise and effective therapeutic effect. After injection through the round window membrane (RWM) behind the ear, Ci@Fn is transported to the basement membrane along with the flow of perilymph and endolymph, ultimately targeting the inner ear hair cells (HCs). After entering the hair cells, Ci@Fn is taken up and degraded by lysosomes, releasing cimicifugin (Ci), thereby exerting a protective effect on the hair cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a hair cell protectant based on a ferritin nanoplatform and its application in the treatment of hearing loss. Background Technology

[0002] Hearing loss and deafness are the most common sensory organ disabilities worldwide, significantly impacting patients' quality of life and socioeconomic status. However, current treatments for hearing loss primarily focus on hearing aids, cochlear implants, or steroid medications. These methods, however, have numerous limitations, such as limited efficacy, significant potential side effects, and the inability to reverse hair cell damage. Therefore, there is an urgent need to develop novel treatment strategies to achieve more precise and effective hearing protection and restoration.

[0003] In terms of drug therapy, studies have shown that small molecule inhibitors and plant extracts exhibit certain hair cell protective effects. Round window membrane (RWM) administration has been explored as a potential inner ear delivery method, for example, in combination with 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: Drugs may act on non-target cells, producing side effects; 3. Poor stability: Drugs are easily degraded in the cochlear environment, affecting efficacy; 4. Short residence time: Existing delivery systems cannot prolong the residence time of drugs in the cochlea, reducing therapeutic effects. In recent years, nanocarriers have been widely studied as drug delivery platforms. For example, polylactic acid-glycolic acid (PLGA) nanoparticles. However, these 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 a current research focus. Summary of the Invention

[0004] The technical problem to be solved by this invention is: addressing the problems of poor targeting, high off-target toxicity, insufficient stability and short retention time in the existing technology, this invention aims to develop a drug delivery system based on a ferritin nanoplatform to achieve precise protection of cochlear hair cells.

[0005] The technical solution of the present invention is: a ferritin nanoparticle (Ci@Fn) loaded with cimicifugin (Ci). The nanosystem uses ferritin (Fn) as a carrier and loads cimicifugin, which has anti-inflammatory and antioxidant effects. The cimicifugin is encapsulated in the ferritin to form a shell-core structure complex with ferritin as the shell and cimicifugin as the core.

[0006] Furthermore, the ferritin is human heavy chain ferritin.

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

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

[0009] The above-described method for preparing ferritin nanoparticles involves stirring ferritin and cimicifugain under acidic conditions, then restoring the mixture to neutral, centrifuging, collecting the supernatant, dialyzing the supernatant, and obtaining ferritin nanoparticles loaded with cimicifugain.

[0010] Furthermore, the specific steps include the following:

[0011] (1) Dissolve ferritin in Tris-HCl buffer at pH 7.4, adjust the pH to 2 with HCl under continuous stirring, then add cimicifugain solution dropwise, stir continuously for 1 hour, add NaOH to restore the pH to 7, and stir for another 30 minutes.

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

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

[0014] The use of the ferritin nanoparticles described above in the preparation of drugs for treating or preventing damage to inner ear hair cells.

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

[0016] Furthermore, the hearing loss caused by the drug toxicity refers to 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 administers the drug via retro-auricular RWM injection. The drug travels with the inner ear lymph to the cochlear basilar membrane and is precisely delivered to hair cells using the targeting ability of ferritin. After being internalized by the hair cells, Ci@Fn enters the lysosomes and releases cimicifugain, exerting anti-inflammatory and anti-apoptotic effects within the cells.

[0019] In a neonatal mouse model of cisplatin-induced hearing loss, we found that Ci@Fn effectively reduced hair cell apoptosis and significantly reduced hearing loss. Furthermore, in an adult mouse model of noise-induced hearing loss, ABR (auditory brainstem response) measurements showed that Ci@Fn provides significant hearing protection with high safety.

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

[0021] 1. Highly efficient targeted delivery: Ferritin nanoplatform can enhance drug accumulation in cochlear hair cells and improve treatment efficacy.

[0022] 2. Long-lasting protective effect: It prolongs the residence time of the drug in the cochlea, achieving continuous release and improving treatment efficiency.

[0023] 3. Enhance treatment efficacy: By regulating the PI3K-Akt signaling pathway, it reduces cisplatin- and noise-induced hair cell damage.

[0024] 4. High safety: Ferritin has good biocompatibility, reducing the risk of immune response and toxic side effects. Attached Figure Description

[0025] Figure 1 Characterization and performance of ferritin purification and Ci@Fn synthesis;

[0026] Figure 2 For in vitro and in vivo delivery studies of Ci@Fn;

[0027] Figure 3 To evaluate the protective effect of Ci@Fn against cisplatin-induced ototoxicity in newborn mice;

[0028] Figure 4 To evaluate the protective effect of Ci@Fn against noise-induced hearing loss in adult rats;

[0029] Figure 5 Research on the protection mechanism of Ci@Fn. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below in conjunction with the relevant descriptions in the embodiments of this application. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0032] Cimicifugin (Ci) is a substance extracted from the plant Cimicifuga, with the molecular formula C16-123. 16 H 18 O6, the structural formula is:

[0033]

[0034] Ferritin (Fn) is an iron storage protein widely expressed in the human body and has attracted much attention in recent years as a drug carrier. Fn's unique cage-like structure (pore size of approximately 8-12 nm) provides a natural advantage for drug delivery.

[0035] This application provides a hair cell protectant based on a ferritin nanoplatform. The protectant comprises ferritin (Fn) as a carrier and cimifugin (Ci) loaded within the ferritin, forming a complex Ci@Fn. By loading cimifugin, the ferritin nanoparticles are endowed with additional anti-inflammatory and tissue-protective functions. After injection through the round window membrane behind the ear, the ferritin nanoparticles (Fn) are transported to the basement membrane with the flow of perilymph and endolymph, and precisely target the inner ear hair cells by virtue of the targeting function of ferritin. Subsequently, Ci@Fn is internalized by the hair cells, and Ci is released in the lysosomes, exerting a protective effect on the hair cells.

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

[0037] We constructed a heavy chain ferritin (Fn) expression system using genetic engineering techniques and achieved high-efficiency expression in *E. coli*. The specific steps are as follows: The human heavy chain ferritin gene (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 antibiotic selection.

[0038] Select a single clone strain and culture it in a shake flask at 37°C and 220 rpm until OD. 600 ≈0.6-0.8 μL, 0.5 mM IPTG was added to induce protein expression, and the mixture was cultured at 25 °C for 12 h. Bacterial cells were collected, lysed by sonication, and purified using affinity chromatography, ion exchange chromatography, and gel filtration chromatography to ensure high-purity Fn. Fn purity was detected by SDS-PAGE electrophoresis, and its particle size distribution was determined by dynamic light scattering (DLS). Results showed that the purified Fn was mainly monodisperse with a particle size of approximately 12 nm, indicating its intact structure and suitability for subsequent drug loading studies. Figure 1 (A, B, C)

[0039] Cimicifugain (Ci) was successfully loaded into the cavity of Fn under acidic conditions via a pH-induced method. The specific steps are as follows: 20 mg of Fn was dissolved in 2 mL of 20 mM Tris-HCl buffer (pH 7.4). Under stirring, 0.1 M HCl was slowly added dropwise to adjust the pH to 2, opening the cage-like structure of Fn. 10 mM Ci solution was added dropwise, and stirring was continued for 1 h to allow Ci molecules to fully enter the Fn cavity. The pH was restored to 7 by slowly adding 0.1 M NaOH, and stirring was continued for 30 min to seal the Fn structure and immobilize Ci. Unencapsulated Ci was removed by centrifugation at 10,000 × g for 15 min, and the supernatant was collected. Dialysis was performed for 24 h using a 2 kDa molecular weight cutoff dialysis bag to remove free Ci, finally obtaining the Ci@Fn nanocomposite.

[0040] After successful Ci loading, we evaluated the impact of the loading process on the Fn properties. Using transmission electron microscopy (TEM) and dynamic light scattering (DLS), we found that the morphology and size of the Fn did not change significantly after Ci loading. Figure 1 The presence of D and E in the data indicates that the drug loading process did not affect the structural stability of Fn. Furthermore, circular dichroism spectroscopy analysis confirmed that the protein structure of Fn remained stable after hydrothermal synthesis, without any conformational changes. Figure 1 Next, we established a concentration profile using high-performance liquid chromatography (HPLC) to evaluate the drug loading of Fn (1 mg / mL). The analysis showed that the synthesized drug loading concentration was 67.52 μM (Fn). Figure 1 In G), the molar ratio of Fn to Ci is 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. In vitro experiments can reveal the intracellular behavior of nanoparticles (such as endocytosis and lysosomal escape), contributing to a deeper understanding of the mechanism of action of Ci@Fn nanoparticles, such as their ability to scavenge reactive oxygen species (ROS) and their effects 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. HEI-OC1 cells were seeded in 24-well plates and cultured to a density of 70%. Ci@Fn was labeled with Cy5-SE, and HEI-OC1 cells were incubated for 4 hours. Lysosomes were labeled with LysoTracker green staining, and then intracellular fluorescence signals were observed using confocal microscopy. Figure 2 (A). The results showed that Cy5-labeled Ci@Fn co-localized with lysosomes, indicating that it releases Ci after being phagocytosed by cells, suggesting that it can enter hair cells through endocytosis.

[0043] In vivo studies are a crucial step in validating the efficacy of Ci@Fn nanoparticles in real physiological environments. In vivo experiments can assess 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). Therefore, systematic in vitro and in vivo experiments can comprehensively evaluate the therapeutic potential of Ci@Fn nanoparticles, providing reliable data support for their future clinical translation. To assess the distribution of Ci@Fn in the mouse cochlea, we delivered Cy5-labeled Ci@Fn to the mouse cochlea via postauricular round window membrane (RWM) injection. Under anesthesia, the RWM was exposed via a postauricular approach, and Ci@Fn was slowly injected using a microinjector. Ten hours after injection, the cochlea was removed, fixed, decalcified, the basilar membrane was separated, and fluorescence imaging was performed. Figure 2 (B). The results showed that Ci@Fn was significantly enriched in both the inner and outer hair cells of the cochlear basilar membrane. Figure 2 The presence of C indicates that it has superior targeting capabilities.

[0044] In summary, our findings indicate that Ci@Fn can simultaneously target both 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 against cisplatin-induced ototoxicity in newborn mice

[0046] To evaluate the protective effect of Ci@Fn against different types of hearing loss, we first constructed an in vitro model of cisplatin-induced hearing loss. Two-day-old C57 mice were selected, and the basement membrane was dissected under a stereomicroscope. After 24 hours of incubation, the mouse basement membranes were randomly divided into six groups: control group, cis-induced hearing loss group, Fn group, dexamethasone (DEX) group, Ci group, and Ci@Fn group. After 2 hours of incubation, the mice were further incubated with 20 μM Cis for 48 hours. The basement membrane was evaluated using different types of staining. Figure 3 (A). Using laser confocal microscopy, we assessed the morphology and number of hair cells in each group ( Figure 3 (B) We observed significant hair cell damage, with a sharp decrease in the number of HC cells in the Cis group. Significant cell damage was also observed in the Fn and DEX groups, with Fn or DEX alone offering no protective effect on hair cells. In contrast, the hair cells in the Ci@Fn group exhibited good morphology and no significant damage, confirming the significant protective ability of Ci@Fn on hair cells. Furthermore, statistical analysis showed that hair cell damage was more severe in the basal ring of the basement membrane, while damage was less severe in the apical ring. Compared to other groups, the Ci@Fn group showed less hair cell damage in the basal ring due to Fn's ability to target hair cells.

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

[0048] Adult C57BL / 6 mice were selected and Ci@Fn nanomedicine was injected behind the ear via RWM surgery. Two days post-surgery, the mice were exposed to 115 dB, 2-20 kHz noise signals for 2 hours to establish a noise-induced hair cell damage model. Hearing thresholds were measured at 4, 8, 16, 24, and 32 kHz using the ABR test. Before treatment, otoscopy was performed to ensure normal hearing in the mice. Figure 4 (A). The Ci@Fn group mice showed significantly lower hearing thresholds at all frequencies compared to the noise group, demonstrating a clear hearing protection effect. Figure 4 (B) Generally, noise-induced hearing loss is associated with 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 (C). To further investigate the role of Ci@Fn in neuroprotection, we performed immunofluorescence co-staining of neurofilaments (NF) and myosin 7a. Neurofilaments in the Fn, DEX, and Ci groups also showed a sparse arrangement. However, in the Ci@Fn group, most neurofibrils survived, axons extended to outer hair cells, and the original neurofibril morphology was partially restored. Figure 4 Compared with the Fn, DEX, and Ci groups, the Ci@Fn group showed a significant increase in the number of NF-positive neural progenitor fibers in outer hair cells, indicating that Ci@Fn significantly reduced noise-induced neurofibrillary damage. Furthermore, to investigate whether Ci@Fn has an anti-apoptotic effect, we analyzed its mechanism of action using cleaved Caspase-3 staining. Quantitative analysis of lysed Caspase-3 / Myosin 7a double-positive hair cells revealed 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 alleviates noise exposure-induced apoptosis by inhibiting the activation of pro-apoptotic signals. Figure 4 These findings reveal the molecular mechanism of Ci@Fn in noise-induced hearing loss, providing important theoretical and experimental support for its potential application in the treatment of hearing loss.

[0049] Example 5: Research on Ci@Fn Protection Mechanism

[0050] To further explore the mechanism of action of Ci@Fn in cisplatin-induced ototoxicity, we selected HEI-OC1 cells, a widely used and readily available auditory hair cell model, for RNA sequencing (RNA-seq) analysis. Transcriptome profiling was performed on differentially expressed genes (DEGs) from the control group (Con), the cisplatin-treated group (Cis), and the cisplatin + Ci@Fn combined treatment group (Ci@Fn+Cis). RNA-seq analysis identified differentially expressed genes (DEGs) meeting the criteria of p < 0.05 and fold change ≥ 2. Compared to the Cis group, the Ci@Fn+Cis group showed significant changes in the expression of 3498 genes, including 2205 upregulated genes (such as PI3Kca and Bcl2) and 1293 downregulated genes (such as AKT1). Furthermore, Kyoto Genome Encyclopedia (KEGG) pathway analysis revealed that the PI3K-Akt signaling pathway showed the most significant changes, suggesting that this pathway may be an important way for Ci@Fn to reduce cisplatin-induced damage to HEI-OC1 cells. Figure 5 (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 (B) To validate the RNA-seq results, the mRNA expression of PI3Kca, AKT1, AKT3, Bcl2, Bcl2l1, and Foxo3 was quantitatively analyzed by qRT-PCR. The results showed that the Ci@Fn group significantly upregulated the expression of PI3Kca, AKT3, Bcl2, Bcl2l1, and Foxo3, while downregulating the expression of AKT1. Figure 5 (C). The expression of proliferative proteins in the PI3K-Akt signaling pathway was analyzed by Western blotting. Compared with the control group, Cis treatment significantly reduced the protein levels of p-PI3K, PI3K, p-AKT, AKT, Bcl2, Bcl2l1, and Foxo3, while Ci@Fn treatment significantly restored and upregulated the expression of these proteins, further demonstrating its mechanism of action. Figure 5 (D). After using the PI3K-Akt inhibitor LY294002, it was found that LY294002 partially attenuated the anti-apoptotic effect of Ci@Fn on Cis-treated HEI-OC1 cells, indicating the important role of the PI3K-Akt signaling pathway in Ci@Fn's reduction of cisplatin ototoxicity. Figure 5 Ci@Fn has potential anti-ototoxic effects by significantly upregulating the expression of genes and proteins related to the PI3K-Akt signaling pathway, activating proliferative genes, and alleviating cisplatin-induced cell death.

Claims

1. A ferritin nanoparticle loaded with cimicifugain, characterized in that, It includes ferritin as a carrier and cimicifugain as an active ingredient, wherein cimicifugain is encapsulated within ferritin to form a shell-core structure complex with ferritin as the shell and cimicifugain as the core, and the amino acid sequence of the ferritin is shown in SEQ ID NO.

1.

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

8.

3. The method for preparing ferritin nanoparticles according to claim 1 or 2, characterized in that, Ferritin and cimicifugain were stirred under acidic conditions, then restored to neutral, centrifuged, and the supernatant was collected and dialyzed to obtain ferritin nanoparticles loaded with cimicifugain.

4. The preparation method according to claim 3, characterized in that, Includes the following steps: (1) Dissolve ferritin in Tris-HCl buffer at pH 7.4, adjust the pH to 2 with HCl under continuous stirring, then add cimicifugain solution dropwise, stir continuously for 1 hour, add NaOH to restore the pH to 7, and stir for another 30 minutes. (2) After the reaction is complete, centrifuge the solution at 10000×g for 15 minutes and collect the supernatant; (3) Dialyze the supernatant with a dialysis bag with a molecular weight of 2 kDa for 24 hours to obtain ferritin nanoparticles loaded with cimicifugain.

5. Use of the ferritin nanoparticles according to claim 1 or 2 in the preparation of a medicament for preventing damage to inner ear hair cells; wherein the hair cell damage refers to hearing loss or mechanical damage caused by drug toxicity, wherein the hearing loss caused by drug toxicity refers to hearing loss caused by cisplatin, and wherein the mechanical damage refers to noise-induced hearing loss.

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