CN113425852B - A conjugate that can pass through the blood labyrinth barrier and its preparation method - Google Patents

Abstract

The invention relates to a conjugate capable of passing through a blood labyrinth barrier and a preparation method thereof, wherein the conjugate is formed by coupling peptide and active molecules with inner ear diagnostic or therapeutic activity, and the peptide has the following structural general formula TFYGGRX1KRNNFX2X3X4X5X6X 7. The conjugate can be administered by intravenous drip, can carry active molecules to enter inner ear without wound, and breaks through the technical bottleneck that drugs in the prior art are difficult to enter inner ear. The invention also specifically provides a peptide-curcumin compound, which has an inner ear targeting function verified on an animal experiment level, can effectively cross the labyrinth barrier of the inner ear of a mouse, treats nerve deafness and plays a role in protecting the hearing after noise exposure.

Description

A kind of conjugate that can pass through blood labyrinth barrier and preparation method thereof

technical field

The invention relates to the technical fields of bioengineering and medicine, in particular to a conjugate that can pass through the blood labyrinth barrier and a preparation method thereof.

Background technique

The anatomical structure of the human ear is mainly divided into the outer ear, the middle ear and the inner ear. The inner ear is a relatively closed and extremely complex acoustic-electrical conversion and balance sensing organ. It is one of the most sophisticated sensory organs in the human body. It is filled with two kinds of lymph fluids, which contain different ion concentrations and circulate independently, in order to provide the necessary potential difference for the action potential generated by the hair cells during acoustoelectric conversion. To ensure the normal operation of this mechanism, the material exchange and ion balance between the inner ear lymphatic system and the peripheral blood circulatory system need to be strictly controlled by the blood-labyrinth barrier (BLB). However, this barrier system, which is necessary to ensure the special function of the inner ear, has also become an important obstacle to restrict the entry of drug molecules into the inner ear and the treatment of related diseases. Therefore, a series of hearing and balance medical-related inner ear diseases, including deafness, tinnitus, and vertigo, are worldwide. For difficult diseases, there is currently no effective drug delivery measures to the inner ear except surgery, and surgery has many inconveniences in preventive drug delivery and multiple drug use, and the risks are high. Therefore, in the field of inner ear disease treatment, there is an urgent need for a drug-carrying system that can non-invasively carry drug molecules across the BLB into the inner ear under physiological conditions. If this technical "bottleneck" is broken, huge social and economic benefits can be obtained.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a conjugate that can pass through the blood labyrinth barrier and a preparation method thereof. The conjugate realizes non-invasive inner ear administration by coupling a peptide with an active molecule with inner ear diagnostic or therapeutic activity, and develops a novel inner ear targeted drug-loading system suitable for intravenous drip administration.

To this end, the first aspect of the present invention provides a conjugate that can pass through the blood labyrinth barrier, the conjugate is formed by coupling a peptide and an active molecule; wherein the peptide is selected from the group consisting of the following structural generalities Peptides of the formula:

TFYGGRX1KRNNFX2X3X4X5X6X7,

in,

X1 is P, V, S or R;

X2 is L, A, P or T;

X3 is R, L, K or A;

X4 is G, S, L or V;

X5 is I, L, H or S;

X6 is R, W, R or A;

X7 is SRGD or not present

The active molecule has inner ear diagnostic or therapeutic activity.

Further, the peptide is selected from the group consisting of TFYGGRPKRNNFLRGIR, TFYGGRVKRNNFALSLW, TFYGGRSKRNNFPKLHR, TFYGGRRKRNNFTAVSA, TFYGGRPKRNNFLRGIRSRGD, TFYGGRVKRNNFALSLWSRGD, TFYGGRSKRNNFPKLHRSRGD, TFYGGRRKRNNFTAVSASRGD.

Further, the active molecule is coupled with the peptide through a connecting bond selected from the group consisting of: disulfide bond, hydrazone bond, amide bond, ester bond, ether bond, carbonyl bond, thioester bond, mercapto-maleic bond imide bond.

Further, the active molecule is selected from the group consisting of small molecule drugs, dyes, polypeptides, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, viruses, quantum dots, and magnetic resonance imaging contrast agents.

In a specific embodiment, the active molecule is curcumin.

Further, the curcumin is coupled to an NHS active ester via glutaric acid, and coupled to the peptide via a sulfhydryl-maleimide bond.

A second aspect of the present invention provides the use of a peptide in the preparation of a drug for penetrating the blood labyrinth barrier and/or a targeting drug for the inner ear, the peptide is selected from peptides with the following general structural formula:

TFYGGRX1KRNNFX2X3X4X5X6X7,

in,

X1 is P, V, S or R;

X2 is L, A, P or T;

X3 is R, L, K or A;

X4 is G, S, L or V;

X5 is I, L, H or S;

X6 is R, W, R or A;

X7 is SRGD or not present.

Further, the peptide is selected from the group consisting of TFYGGRPKRNNFLRGIR, TFYGGRVKRNNFALSLW, TFYGGRSKRNNFPKLHR, TFYGGRRKRNNFTAVSA, TFYGGRPKRNNFLRGIRSRGD, TFYGGRVKRNNFALSLWSRGD, TFYGGRSKRNNFPKLHRSRGD, TFYGGRRKRNNFTAVSASRGD.

A third aspect of the present invention provides a method for preparing the conjugate, comprising: (1) modifying a coupling group to the active molecule, wherein the coupling group is selected from the group consisting of a thiol reactive group, Amine reactive group, maleimide group, thiol group, aldehyde group, carbodiimide group, NHS-ester group, NHS-maleimide group; (2) the modified active molecule Conjugation with the peptide.

Further, the preparation method includes: performing a condensation reaction with glutaric anhydride and the free hydroxyl group of the active molecule to introduce free carboxyl groups, adding DCC and NHS to obtain the active molecule-glutaric acid-NHS active ester; converting the active molecule-glutaric acid An acid-NHS active ester is coupled to the peptide.

Further, the molar ratio of the peptide to the active molecule-glutaric acid-NHS active ester is 1:5-10, preferably 1:7.5.

In a specific embodiment, the active molecule is curcumin, and the preparation method comprises: performing a condensation reaction with glutaric anhydride and curcumin, adding DCC and NHS to obtain curcumin-glutaric acid-NHS active ester; The curcumin-glutaric acid-NHS active ester is coupled to the peptide.

Further, the molar ratio of the peptide to the curcumin-glutaric acid-NHS active ester is 1:5-10, preferably 1:7.5.

The fourth aspect of the present invention provides the use of the conjugate in the preparation of a drug for penetrating the blood labyrinth barrier and/or a targeting drug for the inner ear.

Further, the drugs include detection drugs and treatment drugs.

Further, the medicament is used for the treatment of neural deafness or noise-induced deafness, or for hearing protection.

In Chinese patent CN 109666973 A, the applicant discloses a peptide library that crosses the blood-brain barrier, a method for screening blood-brain-barrier-penetrating peptides using the peptide library, and the obtained blood-brain-barrier-penetrating peptide. The present invention further proposes a new function of diagnosing and treating diseases of the inner ear based on the enrichment phenomenon of the polypeptide in the inner ear. By coupling the peptide with an active molecule for diagnosis or treatment of the inner ear, a drug that can be delivered through the blood labyrinth barrier can be obtained, A conjugate for treating inner ear diseases, specifically taking curcumin as an example, provides a peptide-curcumin conjugate, which has an inner ear targeting function, can be used for the treatment of neural deafness, and has a significant hearing protection effect .

Compared with the prior art, the technical solution of the present invention has the following advantages:

(1) The conjugate provided by the present invention can pass through the blood labyrinth barrier for drug delivery, is a novel inner ear targeted drug loading system suitable for systemic administration, and can carry active molecules into the inner ear non-invasively. It breaks through the technical bottleneck that the drug is difficult to enter the inner ear in the prior art.

(2) The present invention provides a peptide-curcumin conjugate, which has been verified at the level of animal experiments that it can effectively cross the labyrinth barrier of the mouse inner ear and enter the lymph fluid, and has good inner ear targeting and enrichment functions, And can effectively treat neural deafness, and play a role in hearing protection after noise exposure.

Description of drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are for the purpose of illustrating preferred embodiments only and are not to be considered limiting of the invention. In the attached image:

Figure 1. Plaque counts of peptide library gradient dilution

A: Dilution 10e2; B: Dilution 10e4; C: Dilution 10e6

Figure 2 shows the optimal enrichment time after administration of phage-displayed peptides in mouse brain

Figure 3 shows the mass spectrometry detection map of synthetic M1

Figure 4 shows the detection map of M1-labeled cy5.5 fluorescence mass spectrometry

Figure 5 shows the in vivo imaging detection of M1-cy5.5 4h after intravenous injection of M1-cy5.5

A: M1-cy5.5; B: negative control.

Figure 6 is a graph of organ fluorescence distribution

Figure 7 shows the organ fluorescence distribution values

Fig. 8 is the mass spectrometry detection map of the condensation product of glutaric anhydride and curcumin

Figure 9 shows the mass spectrometry of the synthesis and purification of M1-RGD-Cur

Figure 10 shows the evaluation of the enrichment effect of M1-RGD-Cur in the inner ear of mice

Figure 11 shows the evaluation of the enrichment effect of M1-RGD-Cur in the inner ear of minipigs

Figure 12 shows the results of ABR sequencing

Figure 13 shows the results of statistical analysis of ABR data

Detailed ways

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure will be more thoroughly understood, and will fully convey the scope of the present disclosure to those skilled in the art.

In the description of the present invention, the one-letter codes for amino acids are used, and the amino acids mentioned herein are abbreviated as follows according to the nomenclature of the IUPAC-IUB:

Alanine (Ala, A) Arginine (Arg, R)

Asparagine (Asn, N) Aspartic acid (Asp, D)

Cysteine (Cys, C) Glutamate (Glu, E)

Glutamine (Gln, Q) Glycine (Gly, G)

Histidine (His, H) Isoleucine (Ile, I)

Leucine (Leu, L) Lysine (Lys, K)

Methionine (Met, M) Phenylalanine (Phe, F)

Proline (Pro, P) Serine (Ser, S)

Threonine (Thr, T) Tryptophan (Trp, W)

Tyrosine (Tyr, Y) Valine (Val, V)

According to the embodiment of the present invention, with reference to the conserved active center kunitz region of bovine islet inhibitor and β-amyloid peptide across the blood-brain barrier protein, the backbone was selected, and the phage display technology was used to construct a high-throughput phage display library in vitro. Basic acquisition of new independent intellectual property rights for efficient blood-brain barrier short peptides, using mouse in vivo screening technology, optimizing the screening process, and obtaining high-efficiency blood-brain barrier short peptides through three rounds of screening.

Example 1 Building a library

1. Based on the sequence analysis of the active center kunitz region, the following central backbone sequence was found, and the construction of the phage-displayed polypeptide library kun-M was designed and completed.

Build method:

Library 1: TFYGGRXKRNNF XXXXX (SEQ ID NO: 1)

X is a random amino acid.

Long primers:

Library1

5'-cccaggtgcag ctgcag ACCTTTTATGGTGGTCGTNNKAAACGTAATAATTTTNNKNNKNNKNNKNNK tctaga ggggacccaggtc-3' (SEQ ID NO: 2)

Described N is A, T, C or G; Described K is G or T

The restriction sites are underlined, and the capital letters are the nucleic acid sequences encoding the peptide library.

Design the first pair of primers NAG-F: GCCCAGGTGCAGCTG (Tm 57.24) (SEQ ID NO: 3)

Second pair of primers NAG-R: GACCTGGGTCCCCTCTAG (Tm 57.29) (SEQ ID NO: 4)

Primer cross-primer synthesis company synthesis.

2. Amplification of the target fragment

PCR amplification conditions:

PCR conditions:

The amplified target fragment is about 100bp, and the PCR product is recovered by Tiangen PCR purification and recovery kit. The concentration of the recovered product was determined.

3. Enzyme digestion, recovery and purification

The PMESY 4-plasmid was recovered by enzyme digestion, and the PCR target fragment was obtained.

Enzyme cleavage system:

Run a 1% agarose gel and cut the gel to recover the enzyme-cut plasmid fragments.

The PCR target fragment ang2nw was digested with the same enzyme digestion system, and the target fragment was recovered using the Sanitary PCR purification and recovery kit.

4. Target fragment connection

In the ligation system, the plasmid and the target fragment are added in a molar amount of 1:3:

At 16°C overnight, the ligation product was recovered according to the product recovery kit, and eluted with ultrapure water at 70°C.

5. Preparation of transcompetent:

Prepare competent

(1) Transfer the overnight cultured ER2738 bacteria to 500 ml of LB+TET medium at a ratio of 1:100, cultivate to log phase 0.6, and incubate on ice for 30 min.

(2) The E. coli solution was centrifuged at 4°C and 4000 rpm for 10 min, and the supernatant was discarded.

(3) Discard the supernatant, add a small amount of ddH ₂ O to the centrifuge tube, gently suspend the pellet, fill the centrifuge tube with 100 ml of water, and centrifuge at 4°C and 4000 rpm for 10 minutes.

(4) Repeat step (3) once.

(5) Carefully discard the supernatant (precipitate may be very loose), then add 100 ml of 10% glycerol (sterilized, pre-cooled) to the centrifuge tube, resuspend the cells, and centrifuge at 4000rpm for 10min at 4°C. Repeat step 5 once

(6) Resuspend the cells with 10% glycerol to a final volume of 2 ml, put the cells in 100 μl aliquots into microcentrifuge tubes, and store them at -80°C.

6. Electric conversion:

The electroporation cup was pre-cooled on ice for 30 min, and the connected product was taken for electroporation.

Electric to 2.5kv, 5ms, 20uF.

Add 0.9ml 2YT medium for electroporation, culture at 37°C for 1h, and spread on amp plate.

Take 10ul of gradient dilution, dilute and coat on small plates, and incubate at 37 degrees overnight. The results are shown in Figure 1.

The phage clones were picked, and the partial sequencing results were shown in the sequences of SEQ ID NOs: 9-21.

Example 2 In vivo screening of peptide libraries

Before screening, a pre-experiment was conducted to determine the optimal enrichment time for phage-displayed peptides in the mouse brain. The results are shown in Figure 2. The phage brain-to-blood ratio was the highest at 24h after inoculation, indicating the highest enrichment degree in the brain.

The filtering method is as follows:

1. Adult balb/c mice (18-22g), take the phage library TBS and dilute it to 100ul/1011PFU, and inject it into the tail vein. According to the pre-experiment, the enrichment of phage in the brain is the highest at 24h and the brain-to-blood ratio.

2. Take the 24h time point, anesthetize the mice with 5% chloral hydrate, keep the mice sterile, flow 100 ml of normal saline through the heart, dissect the brains, dissect the brain reticulum, smash the cerebral meshes with ultrasonic waves, centrifuge, and pass through a 0.45 μm filter membrane , Take the supernatant solution and mix it with the ER2738 bacteria cultured to the logarithmic phase, infect and culture at 37 degrees for 4h.

3. Centrifuge at 12000 rpm for 20 min, take the supernatant of the bacterial liquid, enrich the phage by the method of PEG/NaCl, and use the enriched library for the next round of screening.

4. Repeat the above for at least three rounds. After seeing the obvious enrichment of the titer of the output phage, it is advisable to infect the clone with the phage and send it to sequence analysis to display the sequence of the polypeptide nanobody. The clones were picked and sequenced, and the results are shown in Table 1:

Table 1: High-frequency sequences obtained by clone sequencing

label sequence Frequency No. M1 TFYGGRPKRNNFLRGIR 3 SEQ ID NO: 5 M2 TFYGGRVKRNNFALSLW 2 SEQ ID NO: 6 M3 TFYGGRSKRNNFPKLHR 2 SEQ ID NO: 7 M4 TFYGGRRKRNNFTAVSA 2 SEQ ID NO: 8

Example 3 In vivo verification in animals

1. Peptide synthesis and fluorescent labeling

M1 was selected for animal in vivo verification, and the peptide sequence TFYGGRPKRNNFLRGIR (MW: 2053) was sent to a peptide synthesis company for synthetic peptide synthesis and labeling fluorescence purification. A pure polypeptide with a purity of more than 95% was obtained.

The detection results of label-free peptide mass spectrometry are shown in Figure 3. The molecular weight of the peptide is 1027.86*2-2=2053.6, which is consistent with the molecular weight.

Figure 4 shows the results of mass spectrometry detection of the labeled CY5.5 fluorescent peptides. CY5.5 is labeled with a fluorescent molecular weight of 873.22*3-3-2053=564, which is the molecular weight after the reaction of the cy5.5 fluorescent reagent. Explain that the M1 short body is connected to a fluorescent molecule.

2. In vivo detection of fluorescently labeled peptides

Linked fluorescent molecular peptides were used for subsequent in vivo detection in mice. Accurately weigh the linked short peptide, dissolve it with normal saline, adjust the dilution of the linked fluorescent short peptide to a fluorescence equivalent of 5uM, and inject 100ul of nude mice into the tail vein. At different time points, use the IVIS small animal live imaging system to divide the time points Observational imaging (Figure 5).

M1-CY5.5 was clearly observed to be enriched in the brain at 4h after tail vein injection. The mice were taken, and 100ml of normal saline was flowed through the heart at a rate of 5ml/min to wash away the fluorescence interference in the blood.

Taking the mouse brain and other organs for observation, it can be seen that the brain has obvious fluorescence, which is higher than that of the muscle heart, etc. The brain/muscle fluorescence ratio is about 3:1 (Figure 6, 7). It shows that the short peptide can pass through the block of the blood-brain barrier, has the ability to cross the blood-brain barrier efficiently, and has a higher enrichment than the parts without the barrier, such as muscle.

Example 4 Preparation of curcumin-glutaric acid-NHS active ester

Using glutaric acid as a linker, glutaric anhydride and the phenolic hydroxyl group of Curcumin are condensed to form an ester, and the map is shown in Figure 8. Then DCC (dicyclohexylcarbodiimide) is used to activate the carboxyl group, and the NHS (N-hydroxysuccinimide) active ester is coupled with the carboxyl group to obtain curcumin-glutaric acid-NHS active ester, which can be directly combined with protein/ The lysine residues of the short peptide are linked. Confirmed by mass spectrometry, curcumin-glutaric acid-NHS active ester product was obtained. Theoretical molecular weight: 579, MS peak 1: 580 (M+H ⁺ ), MS peak 2: 602M+Na ⁺ .

Example 5 Preparation of M1-RGD-Cur

In this example, a polypeptide (M1-RGD) whose sequence is TFYGGRPKRNNFLRGIRSRGD is used to prepare a peptide-curcumin, which is specifically named as M1-RGD-Cur.

M1-RGD and curcumin-glutaric acid-NHS active ester were mixed in a substance amount of 1:7.5, dissolved in DMF, added with 7.5 times equivalent of triethylamine as a base, and reacted at 37 °C for 3 h.

Purification was carried out by high performance preparative liquid chromatography, the mobile phase was acetonitrile (0.1% formic acid)-water (0.1% formic acid) gradient elution. The chromatogram and identification mass spectrum are shown in Figure 9.

Example 6 Inner ear enrichment effect evaluation

Take 9 mice, randomly divide them into 3 groups according to 3 mice/group, and perform tail vein injection according to different groups: experimental group, M1-RGD-Cur prepared in Example 5, dosage: 3mg/ kg; control group, curcumin 3 mg/kg; blank group, equal volume of normal saline.

30min after the injection, the mice were sacrificed, the cochlear tissue was separated, and the cochlear lymph fluid was drawn under a dissecting microscope (the same group of mice were mixed in the same ER tube for collection), and the collected inner ear lymph fluid of different groups of mice was analyzed by HPLC , the results are shown in Figure 10.

As shown in Figure 10, Cur standard can be effectively detected in the inner ear lymph fluid of mice (upper left), while no curcumin-specific peak appeared in the inner ear lymph fluid of mice in the blank group (lower left), while the mice injected with curcumin alone The relevant curcumin-specific peaks (upper right) could not be detected in the inner ear lymph fluid, indicating that the inner ear drug concentration failed to reach the detection baseline 30 minutes after the injection of curcumin alone; while the inner ear lymph fluid of the M1-RGD-Cur group could detect more The curcumin characteristic peak with a shorter retention time of curcumin is determined by preliminary analysis, and this peak is the Linker-Cur characteristic peak. This result fully proves that the inner ear targeted drug loading system (M1-RGD-Cur) involved in the present invention can be successfully A small molecule compound (taking curcumin as an example) was transported into the inner ear lymph of mice, and the effective separation of the short peptide and the loaded active molecule was successfully achieved after entry.

Further, the above experimental conclusions were further verified in a large animal model (Diannan small-eared pig), and the experimental results are shown in Figure 11. This shows that the drug delivery system provided by the present invention has wide applicability.

Example 7 Evaluation of the effect of treating noise-induced deafness

Twelve 5-week-old c57 mice with normal initial hearing were randomly divided into 4 groups according to 3 mice/group. The drugs used in the low-dose group, middle-dose group and high-dose group were M1-RGD-Cur prepared in Example 2 , the control group used normal saline. The mice in each group were placed in a detonation soundproof shielding room, and after being exposed to 120dB narrow-band white noise for 2 hours for 2 consecutive days, the mice were administered continuously for 14 days after the earthquake. 1 mg/kg/day; middle-dose group, 3 mg/kg/day; high-dose group, 9 mg/kg/day; control group, equal volume of normal saline. The mode of administration is tail vein injection. The auditory brainstem evoked potential (ABR) sequencing was performed on 4d, 7d, and 14d after the earthquake, respectively, to judge whether the auditory function of the mice was abnormal according to the changes of the auditory thresholds at different frequencies.

The ABR sequencing results are shown in Figure 12, and the ABR data statistical analysis results are shown in Figure 13. According to Figure 12, M1-RGD-Cur in the middle-dose group can superimpose to form better brainstem evoked potential waves, and the differentiation of auditory potential waves is relatively clear, while the potential waves in the control group and low-dose group are disordered and abnormally differentiated. As shown in Figure 13, the statistical analysis of auditory threshold interpretation based on the above ABR data found that after 4 days of M1-RGD-Cur treatment, the auditory threshold of each treatment group (low-dose group, middle-dose group, and high-dose group) was significantly lower than that of the control group. group, 7d and 14d after the earthquake, the auditory threshold of the middle-dose group was significantly lower than that of the other groups, indicating that M1-RGD-Cur can quantify the treatment of neural deafness, and play a role in auditory protection after noise exposure.

The above description is only a preferred embodiment of the present invention, but the protection scope of the present invention is not limited to this. Substitutions should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

SEQUENCE LISTING

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<400> 17

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Thr Phe Tyr Gly Gly

20 25 30

Arg Arg Lys Arg Asn Asn Phe His Gln Arg Arg Leu Ser Arg Gly Asp

35 40 45

Pro Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg Thr

50 55 60

<210> 18

<211> 62

<212> PRT

<213> Artificial Sequence

<400> 18

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Thr Phe Tyr Gly Gly

20 25 30

Arg Ile Lys Arg Asn Asn Phe Lys Met Ser Cys Asn Ser Arg Gly Asp

35 40 45

Pro Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg Thr

50 55 60

<210> 19

<211> 62

<212> PRT

<213> Artificial Sequence

<400> 19

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Thr Phe Tyr Gly Gly

20 25 30

Arg Leu Lys Arg Asn Asn Phe Ser Arg Leu Tyr Asp Ser Arg Gly Asp

35 40 45

Pro Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg Thr

50 55 60

<210> 20

<211> 64

<212> PRT

<213> Artificial Sequence

<400> 20

Met Lys Phe Ile Leu Pro Ser Ala Ala Val Ser Leu Leu Leu Leu Lys Ile

1 5 10 15

Thr Thr Ser Thr Thr Thr Ile Lys Gly Leu Gln Thr Phe Tyr Gly Gly

20 25 30

Asp Lys Arg Asn Asn Phe Ala Ser Met Ser Trp Ser Arg Gly Asp Pro

35 40 45

Gly Arg Pro Gly Ala Ala Ala Asp Leu Leu Trp Trp Ser Trp Glu Thr

50 55 60

<210> 21

<211> 62

<212> PRT

<213> Artificial Sequence

<400> 21

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Gln Val Gln Leu Gln Thr Phe Tyr Gly Gly

20 25 30

Arg Ile Lys Arg Asn Asn Phe Leu Ala Val Gly Val Ser Arg Gly Asp

35 40 45

Pro Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg Thr

50 55 60

<210> 22

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 22

Thr Phe Tyr Gly Gly Arg Pro Lys Arg Asn Asn Phe Leu Arg Gly Ile

1 5 10 15

Arg Ser Arg Gly Asp

20

<210> 23

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 23

Thr Phe Tyr Gly Gly Arg Val Lys Arg Asn Asn Phe Ala Leu Ser Leu

1 5 10 15

Trp Ser Arg Gly Asp

20

<210> 24

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 24

Thr Phe Tyr Gly Gly Arg Ser Lys Arg Asn Asn Phe Pro Lys Leu His

1 5 10 15

Arg Ser Arg Gly Asp

20

<210> 25

<211> 21

<212> PRT

<213> Artificial Sequence

<400> 25

Thr Phe Tyr Gly Gly Arg Arg Lys Arg Asn Asn Phe Thr Ala Val Ser

1 5 10 15

Ala Ser Arg Gly Asp

20

Claims (9)

1. A conjugate formed by coupling a peptide and an active molecule, wherein the peptide is selected from the group consisting of: TFYGGRPKRNNFLRGIR, TFYGGRVKRNNFALSLW, TFYGGRSKRNNFPKLHR, TFYGGRRKRNNFTAVSA, TFYGGRPKRNNFLRGIRSRGD, TFYGGRVKRNNFALSLWSRGD, TFYGGRSKRNNFPKLHRSRGD, TFYGGRRKRNNFTAVSASRGD, respectively;

the active molecule is curcumin.

2. The conjugate of claim 1, wherein the active molecule is conjugated to the peptide via a linkage selected from the group consisting of: disulfide bond, hydrazone bond, amide bond, ester bond, ether bond, carbonyl bond, thioester bond, mercapto-maleimide bond.

3. Use of a peptide for the preparation of a medicament for crossing the blood labyrinth barrier and/or for targeting the inner ear, the peptide being selected from the group consisting of: TFYGGRPKRNNFLRGIR, TFYGGRVKRNNFALSLW, TFYGGRSKRNNFPKLHR, TFYGGRRKRNNFTAVSA, TFYGGRPKRNNFLRGIRSRGD, TFYGGRVKRNNFALSLWSRGD, TFYGGRSKRNNFPKLHRSRGD, TFYGGRRKRNNFTAVSASRGD are provided.

4. The method of preparing a conjugate according to any one of claims 1-2, comprising, (1) modifying the reactive molecule with a coupling group selected from the group consisting of: thiol-reactive groups, amine-reactive groups, maleimide groups, thiol groups, aldehyde groups, carbodiimide groups, NHS-ester groups, NHS-maleimide groups; (2) coupling the modified active molecule to the peptide.

5. The method of claim 4, wherein free carboxyl is introduced by condensation reaction of glutaric anhydride and free hydroxyl of active molecule, DCC and NHS are added to obtain active molecule-glutaric acid-NHS active ester; coupling the active molecule, glutarate-NHS active ester, to the peptide.

6. The method of claim 5, wherein the molar ratio of the peptide to the active molecule, glutarate-NHS active ester, is from 1:5 to 10.

7. Use of a conjugate according to any of claims 1-2 for the preparation of a medicament for crossing the blood labyrinth barrier and/or for targeting the inner ear.

8. The use of claim 7, wherein the medicament comprises a test medicament or a therapeutic medicament.

9. The use of claim 7, wherein the medicament is a medicament for the treatment of nerve deafness or noise deafness.

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