Drug carrier acting on inner ear organ in targeting manner and application of drug carrier
By targeting the combination of the polypeptide, drug-loaded polypeptide and CD63 high-expression exosome, the safe, efficient and targeted delivery of drugs to the inner ear organs is achieved, and the problem of non-targeting drug delivery in the prior art is solved, and the effect of treating inner ear diseases is improved.
Patent Information
- Application Number
- CN202311102298.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-30
- Publication Date
- 2025-02-28
AI Technical Summary
The prior art is difficult to achieve safe, efficient and targeted drug delivery on the cochlear auditory epithelium, especially in avoiding local surgical injuries and drug side effects.
Using a combination of targeted linking polypeptides, drug-loading polypeptides and CD63 high-expression exosomes, the drug is delivered accurately and efficiently to the inner ear organs through intravenous channels, and targeted delivery is achieved using the natural nanocharacteristics of exosomes and the recognition ability of CD63 surface-specific proteins.
It realizes safe, efficient and targeted delivery of drugs to the inner ear organs, avoids local surgical injuries and drug side effects, and improves the effectiveness of treating inner ear diseases.
Smart Images
Figure 00000000_0000_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of novel drug carriers, and in particular to a drug carrier targeting inner ear organs and applications thereof. Background Art
[0002] Sensorineural hearing loss is considered the most common sensory disorder. With the aging of the population, the incidence of cancer and metabolic diseases is increasing year by year. According to the World Report On Hearing released by the World Health Organization in 2021, 466 million people in the world suffer from disabling hearing loss. About 42% of people over 60 years old have varying degrees of hearing loss, and the incidence rate increases exponentially with age. The cochlea is the end organ for recognizing and transducing sound. A variety of pathogenic factors damage the cochlear sensory epithelium and cause apoptosis of sensory cells such as hair cells, ultimately causing irreversible hearing damage.
[0003] Over the years, research on the treatment of inner ear diseases has been in full swing and has achieved fruitful results. A variety of drugs and other treatment methods have been developed, including drugs such as cortisol and antioxidants, as well as cochlear implants and electrodes. Inner ear drug delivery methods include systemic administration via vein, muscle or oral administration, perfusion penetration into the tympanic cavity, and local administration after cochlear surgery. The local administration of the latter two requires surgery or trauma to the middle ear, while systemic administration needs to consider the limitations of the blood-labyrinth barrier and other systemic side effects (Stefan K Plontke et al. Intratympanic corticosteroids for sudden sensorineural hearing loss. Cochrane Database Syst Rev. 2022 Jul 22; 7(7): CD008080.doi: 10.1002 / 14651858.CD008080.pub2; Sophie Nyberg et al. Delivery of therapeutics to the inner ear: The challenge of the blood-labyrinth barrier. Sci Transl Med. 2019 Mar 6; 11(482): eaao0935.doi: 10.1126 / scitranslmed.aao0935.).
[0004] Therefore, finding a drug carrier that can safely and efficiently target the cochlear auditory epithelium has become an urgent need in the research on inner ear damage repair. Summary of the invention
[0005] The purpose of the present invention is to provide a safe, efficient and inner ear targeted drug carrier to address the deficiencies of the prior art.
[0006] The carrier of the present invention can be used to accurately and efficiently deliver small molecule drugs or gene editing tools to the inner ear organs through the intravenous channel, avoiding the damage to the inner ear caused by local surgery and the side effects of drugs in non-target organs.
[0007] The drug carrier targeting the inner ear organs of the present invention comprises a targeting linker polypeptide, a drug-carrying polypeptide and CD63 highly expressed exosomes; wherein,
[0008] The targeting linker polypeptide and the drug-carrying polypeptide are respectively connected to the CD63 highly expressed exosomes.
[0009] According to the drug carrier of the present invention, preferably, the targeting connection polypeptide is a combination of polypeptide IETP2 and polypeptide CP5, and the drug-carrying polypeptide is polypeptide CP5.
[0010] Further preferably, the amino acid sequence of the combination of polypeptide IETP2 and polypeptide CP5 is shown in SEQ ID NO.1 (TFYGGRPKRNNFLRGIRSRGDCRHSQMTVTSRL), and the amino acid sequence of polypeptide CP5 is shown in SEQ ID NO.2 (CRHSQMTVTSRL).
[0011] According to the drug carrier of the present invention, the preparation of the CD63 highly expressed exosomes comprises the following steps:
[0012] The CD63 overexpression plasmid was lentivirally transfected into mesenchymal stem cells, and the positive cell lines were screened by Puro antibiotics to obtain the mesenchymal stem cell line overexpressing CD63; and then the exosomes in the cell supernatant were obtained by ultra-high speed centrifugation (not less than 30000r / min), thus obtaining the CD63 highly expressed exosomes.
[0013] The targeting linker polypeptide and the drug-carrying polypeptide of the present invention are both synthesized by a solid-liquid method.
[0014] The present invention also provides a method for preparing the drug carrier, comprising the following steps:
[0015] The targeting linker polypeptide and the drug-carrying polypeptide were incubated with CD63 highly expressed exosomes respectively to prepare a drug carrier that targets the inner ear organs.
[0016] The present invention also provides a drug, the drug includes any drug carrier described in the present invention, and the N-terminus of the last amino acid of the drug-carrying polypeptide can be connected to a small molecule drug or a gene editing tool by chemical coupling. The small molecule drugs that can be connected include but are not limited to glutathione, cortisol (such as dexamethasone and its derivatives, hydrocortisone and its derivatives, etc.), etc., and the gene editing tools that can be connected include but are not limited to microRNA, CRISPR / Cas system (such as CRISPR / Cas9, CRISPR / Cas10, CRISPR / Cas12 series, CRISPR / Cas13 series, etc.), etc.
[0017] According to the drug carrier of the present invention, the CD63 high-expressing exosomes can carry at least one connecting polypeptide and one drug-carrying polypeptide.
[0018] The present invention also provides the use of any of the above-mentioned drug carriers in the preparation of drugs for preventing or treating inner ear diseases.
[0019] Furthermore, the present invention also provides the application of any of the above-mentioned drug carriers in carrying drugs for non-therapeutic purposes. The targeting linked polypeptide, drug-carrying polypeptide and CD63 high-expressing exosomes can be independently connected with fluorescent dyes for in vivo or organ tracing.
[0020] IETP2 short peptide has the ability to cross the blood-brain barrier and blood-labyrinth barrier, but its drug loading capacity and drug loading amount are not as good as exosomes. Exosomes are extracellular vesicles secreted by various cells, mainly composed of lipid bilayers without organelles. As natural nanoparticles, exosomes play an important role in the body's busy and rapid intercellular information exchange. It has the advantages of low immunogenicity, good biodistribution and degradability, and long circulation half-life. It can also be engineered and modified to meet different needs, so as to adapt to the application of different diseases, different drugs and target organ tissues. At the same time, exosomes can cross the blood-brain barrier, and the surface of this exosome is rich in HSP-70 that can specifically bind to the inner ear capillary TLR4.
[0021] Therefore, the present invention combines IETP2 with exosomes as a drug delivery carrier for the inner ear based on the research of IETP2 to solve the problem of targeted drug delivery. However, previous studies have found that IETP2 cannot bind to exosomes ( Figure 6 ). Therefore, we modified IETP2 and added the recognition peptide CP05 of the exosome surface specific protein CD63 to form a new polypeptide. This new polypeptide can effectively bind to exosomes and deliver exosomes to the inner ear target organs ( Figure 5 ).
[0022] First, part of the targeted vector can enter the inner ear organs, part of it can enter the liver, and no enrichment is found in other parts. In addition, the inner ear target organs are small, and the drugs carried are mostly cortisol, antioxidants and other drugs or gene editing tools. The amount is small, and the impact on non-target organs is small.
[0023] The drug carrier of the present invention can carry small molecule drugs, gene editing tools and other small molecule substances through intravenous injection, through the blood-brain barrier and blood-labyrinth barrier to reach the inner ear target organs, thus avoiding the problem of local surgery damaging the inner ear and the side effects of drugs in non-target organs. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The figure is a schematic diagram of the preparation process of the drug carrier targeting the inner ear organs of the present invention;
[0025] Figure 2 This is a schematic diagram of the animal verification process of the drug carrier targeting the inner ear organs of the present invention;
[0026] Figure 3 This is the in vivo imaging of the blank container group and the exosome container group in the embodiment of the present invention;
[0027] Figure 4 The in vivo imaging of nude mice injected with exosomes at different time periods in the embodiment of the present invention;
[0028] Figure 5 The images are of the cochlear samples of nude mice injected with exosomes for 2.5 hours and the blank group in the present invention. The blank group is circled and no imaging is seen.
[0029] Figure 6 This is the imaging of exosomes without and with targeting peptides. DETAILED DESCRIPTION
[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and can be specifically carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide (4th Edition)" (author: MR Green, J. Sambrook), or according to the kits and product instructions; the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.
[0031] Example 1 Preparation of drug carriers targeting inner ear organs
[0032] Reference Figure 1 shown.
[0033] 1. Preparation of linked peptides, drug-loaded peptides and CD63 highly expressed exosomes
[0034] Referring to SEQ ID NO.1 (TFYGGRPKRNNFLRGIRSRGDCRHSQMTVTSRL) and SEQ ID NO.2 (CRHSQMTVTSRL), the linker polypeptide and the drug-carrying polypeptide were synthesized by the solid-liquid method. The specific synthesis process was completed by Beijing Bangfei Biotechnology Co., Ltd.
[0035] The CD63 overexpression plasmid lentivirus (plasmid: pMDLg / pRRE) was transfected into mesenchymal stem cells, and the positive cell lines were screened by Puro antibiotics to obtain the mesenchymal stem cell line overexpressing CD63; then the exosomes in the cell supernatant were obtained by ultra-high-speed centrifugation at 50,000 r / min to prepare CD63 highly expressed exosomes.
[0036] 2. Incubate to obtain drug carrier
[0037] a. Incubate the exosomes, linker peptides, and drug-carrying peptides at a ratio of 1:1:1 (by weight) in a rotator at 4°C overnight (12 hours). b. Add 10 times the volume of PBS at 4°C and blow gently twice. Centrifuge at 100,000 g for 70 minutes. Discard the supernatant. c. Repeat step 2 four times, and finally resuspend with 2 times the volume of exosomes in PBS (4°C) to obtain the drug carrier, and store at 4°C.
[0038] Embodiment 2
[0039] In this example, the drug carrier targeted to the inner ear organs prepared in Example 1 was labeled with DIR dye, and the exosomes were introduced into the nude mice by tail vein injection. The distribution of DIR dye-labeled exosomes in the nude mice was observed by an in vivo imaging system.
[0040] Reference Figure 2 As shown, specifically:
[0041] 1. Animal Selection for Tail Vein Injection
[0042] 1. In order to facilitate in vivo imaging, hairless nude mice (BALB / c Nude Mice) were selected.
[0043] 2. The age should be around 8 weeks, regardless of gender, and the weight should be around 30g.
[0044] 2. Experimental Grouping
[0045] 1. Baseline group: exosome container group and blank container group;
[0046] 2. Experimental group: set up exosome group and blank group (normal saline);
[0047] 3. Experimental Process
[0048] 1. First, 100ug (200ul) of exosomes were injected into the nude mice in group A through the tail vein as the exosome group;
[0049] 2. Inject the same volume of saline into the nude mice in group B through the tail vein as the blank group;
[0050] 3. Place the container containing exosomes and the blank container in the in vivo imaging system and set the baseline;
[0051] 4. Group A (exosome group) and Group B (blank group) were placed in in vivo imaging at 1h, 2h, 24h, and 48h.
[0052] 5. The nude mice treated in the same way as group A (exosome group) were taken as group C and the nude mice treated in the same way as group B (blank group) were taken as group D. 2.5 hours after the injection, the cochlear tissues of the two groups of nude mice were taken and placed in the imaging system.
[0053] IV. Experimental Results
[0054] 1. In vivo imaging of the blank container group and the exosome container group Figure 3 shown.
[0055] The container with exosomes on the right is visible in the imaging system, while the blank tube on the left is not.
[0056] 2. In vivo imaging of nude mice injected with exosomes at 1h, 2h, 24h, and 48h Figure 4 shown.
[0057] At 1 hour and 48 hours, exosomes were enriched in the cochlea and liver, and at 24 hours and 48 hours, exosomes were metabolized in the cochlea, while exosomes were not completely metabolized in the liver.
[0058] 3. Imaging of cochlear samples of nude mice injected with exosomes 2.5 hours after injection and the blank group Figure 5 shown.
[0059] The left side is the cochlea of the animal injected with exosomes, and the right side is the cochlea of the blank group. It can be seen that a large number of exosomes are enriched in the left cochlea and imaged in the imaging system, while the right side is the blank group and does not show imaging.
[0060] 4. Imaging of exosomes without targeting peptides Figure 6 shown.
[0061] The bright part on the left is the unlinked targeted peptide exosomes (IETP2-Ev) in the liver of nude mice, indicating that there is no enrichment in the inner ear. The blank group is not bright, and the whole body organs are not colored. For comparison, Figure 5It can be seen that a large number of exosomes (TETP2-Cp05-Ev) are enriched in the cochlea of the nude mouse on the left and are imaged in the imaging system, while the blank group on the right does not show any image.
[0062] The above experiments show that exosomes can be enriched in the inner ear after targeted modification.
[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention is described in detail with reference to the embodiments, it should be understood by those skilled in the art that any modification or equivalent replacement of the technical solutions of the present invention does not depart from the spirit and scope of the technical solutions of the present invention and should be included in the scope of the claims of the present invention.
Claims
1. A drug carrier targeting the inner ear organs, characterized in that: The drug carrier includes a targeting linker polypeptide, a drug-carrying polypeptide and CD63 highly expressed exosomes; wherein, The targeting linker polypeptide and the drug-carrying polypeptide are respectively connected to the CD63 highly expressed exosomes.
2. The drug carrier according to claim 1, characterized in that The targeting connection polypeptide is a combination of polypeptide IETP2 and polypeptide CP5 connected together, and the drug-carrying polypeptide is polypeptide CP5.
3. The drug carrier according to claim 2, characterized in that The amino acid sequence of the combination of polypeptide IETP2 and polypeptide CP5 is shown in SEQ ID NO.1, and the amino acid sequence of polypeptide CP5 is shown in SEQ ID NO.
2.
4. The drug carrier according to any one of claims 1 to 3, characterized in that The preparation of the CD63 high-expressing exosomes comprises the following steps: The CD63 overexpression plasmid was lentivirally transfected into mesenchymal stem cells, and the positive cell lines were screened by Puro antibiotics to obtain the mesenchymal stem cell line overexpressing CD63; and the exosomes in the cell supernatant were obtained by ultra-high-speed centrifugation.
5. The drug carrier according to any one of claims 1 to 3, characterized in that: The targeting connection polypeptide and the drug-carrying polypeptide are both synthesized by solid-liquid method.
6. A method for preparing the drug carrier according to any one of claims 1 to 5, comprising the following steps: The targeting linker polypeptide and the drug-carrying polypeptide were incubated with CD63 highly expressed exosomes respectively to prepare a drug carrier that targets the inner ear organs.
7. A drug, characterized in that The drug comprises the drug carrier according to any one of claims 1 to 5, and the N-terminus of the last amino acid of the drug-carrying polypeptide is connected to a small molecule drug or a gene editing tool.
8. Use of the drug carrier according to any one of claims 1 to 5 in the preparation of a drug for preventing or treating inner ear diseases.
9. Use of the drug carrier according to any one of claims 1 to 5 in carrying small molecule drugs and / or gene editing tools for non-therapeutic purposes.