CN113425852B - Conjugate capable of penetrating blood labyrinth barrier and preparation method thereof - Google Patents
Conjugate capable of penetrating blood labyrinth barrier and preparation method thereof Download PDFInfo
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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
Technical Field
The invention relates to the technical field of bioengineering and medicines, in particular to a conjugate capable of passing through a blood labyrinth barrier and a preparation method thereof.
Background
The human ear anatomy structure is mainly divided into the external ear, the middle ear and the internal ear, wherein the internal ear is a relatively closed acoustoelectric conversion and balance induction organ with extremely complex structure, which is one of the most precise sense organs of the human body, and the internal complex membrane labyrinth system is filled with internal and external lymph fluids which contain different ion concentrations and are circulated independently so as to provide necessary potential difference for action potential generated when the hair cells perform 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 circulation system need to be strictly controlled by the blood-labyrinth barrier (BLB). However, the barrier system necessary for ensuring the special function of the inner ear also becomes an important obstacle for restricting the drug molecules from entering the inner ear and treating related diseases, so that a series of auditory and balance medical related inner ear diseases including deafness, tinnitus, vertigo and the like are all difficult and complicated diseases worldwide, no practical and effective inner ear administration measures are available except for operation, and the operation has great inconvenience and great risk in the aspects of preventive administration and multi-time administration. Therefore, a drug delivery system capable of non-invasively carrying drug molecules in a targeted manner across BLB into the inner ear under physiological conditions is urgently needed in the field of treatment of inner ear diseases. If the technology 'bottleneck' is broken through, huge social and economic benefits can be obtained.
Disclosure of Invention
The invention aims to provide a conjugate capable of passing through a blood labyrinth barrier and a preparation method thereof. The conjugate realizes noninvasive inner ear drug delivery by coupling a peptide and an active molecule with inner ear diagnosis or treatment activity, and develops a novel inner ear targeted drug delivery system suitable for intravenous drip drug delivery.
To this end, in a first aspect of the invention, there is provided a conjugate that can cross the blood labyrinth barrier, the conjugate being formed by coupling a peptide and an active molecule; wherein the peptide is selected from the group consisting of peptides having the following structural formula:
TFYGGRX1KRNNFX2X3X4X5X6X7，
wherein the content of the first and second substances,
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 absent
The active molecules have diagnostic or therapeutic activity in the inner ear.
Further, the peptide is selected from the group consisting of: TFYGGRPKRNNFLRGIR, TFYGGRVKRNNFALSLW, TFYGGRSKRNNFPKLHR, TFYGGRRKRNNFTAVSA, TFYGGRPKRNNFLRGIRSRGD, TFYGGRVKRNNFALSLWSRGD, TFYGGRSKRNNFPKLHRSRGD, TFYGGRRKRNNFTAVSASRGD is added.
Further, the active molecule is coupled 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.
Further, the active molecule is selected from: small molecule drugs, dyes, polypeptides, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, viruses, quantum dots, magnetic resonance imaging contrast agents.
In a specific embodiment, the active molecule is curcumin.
Further, the curcumin was coupled to the NHS active ester via glutaric acid and to the peptide via thiol-maleimide bond.
In a second aspect the present invention provides the use of a peptide selected from the group consisting of peptides having the general structural formula:
TFYGGRX1KRNNFX2X3X4X5X6X7，
wherein the content of the first and second substances,
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 absent.
Further, the peptide is selected from the group consisting of: TFYGGRPKRNNFLRGIR, TFYGGRVKRNNFALSLW, TFYGGRSKRNNFPKLHR, TFYGGRRKRNNFTAVSA, TFYGGRPKRNNFLRGIRSRGD, TFYGGRVKRNNFALSLWSRGD, TFYGGRSKRNNFPKLHRSRGD, TFYGGRRKRNNFTAVSASRGD are provided.
A third aspect of the invention provides a method for preparing the conjugate, comprising, (1) modifying the active 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.
Further, the preparation method comprises the steps of carrying out condensation reaction on glutaric anhydride and free hydroxyl of active molecules to introduce free carboxyl, and adding DCC and NHS to obtain active molecules, namely glutaric acid-NHS active ester; coupling the active molecule, glutarate-NHS active ester, 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 the steps of carrying out condensation reaction on glutaric anhydride and curcumin, and adding DCC and NHS to obtain curcumin-glutaric acid-NHS active ester; coupling the curcumin-glutarate-NHS active ester to the peptide.
Further, the molar ratio of the peptide to the curcumin-glutarate-NHS active ester is 1:5-10, preferably 1: 7.5.
The fourth aspect of the invention provides the application of the conjugate in preparing a blood-penetrating labyrinth barrier medicament and/or an inner ear targeting medicament.
Further, the drugs include detection drugs and treatment drugs.
Further, the medicament is used for treating nerve deafness or noise deafness, or for hearing protection.
The applicant discloses a peptide library crossing blood brain barrier, a method for screening peptides crossing blood brain barrier by using the peptide library and the obtained peptides crossing blood brain barrier in Chinese patent CN 109666973A. The invention further provides a new function of diagnosing and treating inner ear diseases based on the phenomenon of polypeptide enrichment in the inner ear, and a conjugate capable of delivering a medicament and treating the inner ear diseases through a blood labyrinth barrier is obtained by coupling the peptide and an inner ear diagnosis or treatment active molecule, particularly taking curcumin as an example, the invention provides a peptide-curcumin conjugate which has an inner ear targeting function, can be used for treating nerve deafness and has a remarkable hearing protection effect.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the conjugate provided by the invention can pass through a blood labyrinth barrier for drug delivery, is a novel inner ear targeted drug delivery system suitable for systemic drug delivery, and can carry active molecules to enter the inner ear without wound. Breaks through the technical bottleneck that the medicine is difficult to enter the inner ear in the prior art.
(2) The invention provides a peptide-curcumin conjugate which can effectively cross the mouse inner ear labyrinth barrier and enter lymph fluid, has good inner ear targeting and enriching functions, can effectively treat nerve deafness and plays a role in protecting hearing after noise exposure, and is verified on the animal experiment level.
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 only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:
FIG. 1 shows peptide library gradient dilution plaque counts
A: dilution 10e 2; b: dilution 10e 4; c: dilution 10e6
FIG. 2 shows the optimal enrichment time after administration of phage display peptide for mouse brain
FIG. 3 is a mass spectrometric view of Synthesis M1
FIG. 4 is a fluorescent mass spectrometric detection of M1-labeled cy5.5
FIG. 5 is an image of a 4h mouse in vivo after intravenous injection of M1-cy5.5
A: m1-cy 5.5; b: and (5) negative control.
FIG. 6 is a graph of organ fluorescence distribution
FIG. 7 is the values of organ fluorescence distributions
FIG. 8 is a mass spectrometric detection of glutaric anhydride and curcumin condensation products
FIG. 9 is a diagram of mass spectrometric detection of synthesis and purification of M1-RGD-Cur
FIG. 10 is a graph showing the evaluation of the effect of M1-RGD-Cur in enriching the inner ear of mouse
FIG. 11 is a graph showing the evaluation of the effect of M1-RGD-Cur in enrichment of inner ear of miniature pig
FIG. 12 is a graph showing the result of ABR sequencing
FIG. 13 is the result of statistical analysis of ABR data
Detailed Description
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 to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
In the description of the present invention, the one-letter codes of amino acids are used, and the amino acids mentioned herein are abbreviated as follows according to the IUPAC-IUB nomenclature rules:
alanine (Ala, A) arginine (Arg, R)
Asparagine (Asn, N) aspartic acid (Asp, D)
Cysteine (Cys, C) glutamic acid (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 implementation mode of the invention, a framework is selected by referring to a bovine pancreatic island inhibitor and a region of beta-amyloid peptide spanning a blood brain barrier protein conserved active center kunitz, a high-throughput phage display library is constructed by in vitro artificial synthesis by using a phage display technology, novel high-efficiency blood brain barrier-penetrating short peptide with independent intellectual property rights is obtained from the basis, a screening process is optimized by using a mouse in vivo screening technology, and the high-efficiency blood brain barrier-penetrating short peptide is obtained by three rounds of screening.
Example 1 library construction
1. Based on the sequence analysis of the kunitz region of the active center, the following central framework sequence is found, and the construction of a phage display polypeptide library kun-M is designed and completed.
The construction method comprises the following steps:
Library 1:TFYGGRXKRNNF XXXXX(SEQ ID NO:1)
x is a random amino acid.
Long primer:
Library1
5’-cccaggtgcagctgcagACCTTTTATGGTGGTCGTNNKAAACGTAATAATTTTNNKNNKNNKNNKNNKtctagaggggacccaggtc-3’(SEQ ID NO:2)
n is A, T, C or G; k is G or T
The restriction sites are underlined and the capital letters are the peptide library-encoding nucleic acid sequences.
Designing a first pair of primers NAG-F: GCCCAGGTGCAGCTG (Tm 57.24) (SEQ ID NO:3)
A second pair of primers NAG-R: GACCTGGGTCCCCTCTAG (Tm 57.29) (SEQ ID NO:4)
Primer Synthesis was performed.
2. Amplification of fragments of interest
PCR amplification conditions:
PCR conditions were as follows:
the amplified target fragment is about 100bp, and the PCR product is recovered by the Tiangen PCR purification recovery kit. The concentration of the recovered product was determined.
3. Enzyme digestion, recovery and purification
And (4) carrying out enzyme digestion to recover the PMESY 4-plasmid and a PCR target fragment.
Enzyme digestion system:
run 1% agarose gel, cut gel recovery enzyme cut plasmid fragment.
And (3) carrying out enzyme digestion on the PCR target fragment ang2nw, and recovering the target fragment by using the industrial PCR purification recovery kit by adopting the same enzyme digestion system.
4. Joining of fragments of interest
And (3) connecting the system, the plasmid and the target fragment according to the proportion of 1: 3 molar amount of addition:
after 16 ℃ overnight, the ligation product was recovered according to the product recovery kit and eluted with ultra pure water at 70 ℃.
5. B, trans-competence preparation:
preparation competence
(1) The overnight cultured ER2738 bacteria were grown in a 1: 100% of the culture volume overnight the cells were transferred to 500ml LB + TET medium, incubated to log phase 0.6 and incubated on ice for 30 min.
(2) The E.coli solution was centrifuged at 4000rpm for 10min at 4 ℃ and the supernatant was discarded.
(3) Abandoning the supernatant, adding a small amount of ddH into the centrifuge tube2O, after gentle suspension precipitation, 100ml of water was poured into the centrifuge tube and centrifuged at 4000rpm for 10min at 4 ℃.
(4) Repeating the step (3) for 1 time.
(5) The supernatant was carefully discarded (the pellet may be loose), 100ml of 10% glycerol was added to the tube (sterilized, pre-cooled), the cells were resuspended, and centrifuged at 4000rpm for 10min at 4 ℃. Repeat step 5 once
(6) Cells were resuspended in 10% glycerol to a final volume of 2ml, and 100. mu.l aliquots of cells were placed in microcentrifuge tubes and stored at-80 ℃.
6. And (3) electric conversion:
precooling the electric rotating cup on ice for 30min, taking the connecting product for electric rotation,
the electric conversion is 2.5kv, 5ms and 20uF.
0.9ml of 2YT medium was added for electroporation, incubated at 37 ℃ for 1h, and plated on amp plates.
10ul of the gradient was diluted, diluted and plated, and incubated at 37 ℃ overnight, as shown in FIG. 1.
Selecting phage for cloning, and partially sequencing the phage to obtain a sequence shown as SEQ ID NO. 9-21.
Example 2 in vivo screening of peptide libraries
The time for optimal enrichment of the phage-displayed peptides in the mouse brain was determined by preliminary experiments prior to screening, and the results are shown in FIG. 2. The phage cerebral blood ratio was highest 24h after inoculation, indicating the highest enrichment in brain.
The screening method comprises the following steps:
1. adult balb/c mice (18-22 g) were diluted to 100ul/1011PFU in phage library TBS and injected into tail vein, and the enrichment ratio of phage in brain was highest at 24h according to preliminary experiments.
2. And (3) anesthetizing 5% chloral hydrate at a time point of 24h, keeping the mouse sterile, flowing 100ml of normal saline through the heart, dissecting and taking the brain, ultrasonically breaking a brain reticulum, centrifuging, filtering through a 0.45-micron filter membrane, taking a supernatant solution, uniformly mixing with ER2738 bacterial solution cultured to logarithmic phase, and performing infection culture at 37 ℃ for 4 h.
3. Centrifuging at 12000rpm for 20min, collecting supernatant of the bacterial liquid, enriching phage by PEG/NaCl method, and using the enriched library for the next round of screening.
4. At least three rounds of the above are repeated. When the titer of the output phage is obviously enriched, phage infection clone can be taken, and the sequence of the displayed polypeptide nano antibody is analyzed by sequencing. The selected clones were sequenced, and the results are shown in table 1:
table 1: high frequency sequence obtained by clone sequencing
| label | sequence | Frequency | No. | |
| | TFYGGRPKRNNFLRGIR | 3 | SEQ ID NO:5 | |
| | TFYGGRVKRNNFALSLW | 2 | SEQ ID NO:6 | |
| | TFYGGRSKRNNFPKLHR | 2 | SEQ ID NO:7 | |
| | TFYGGRRKRNNFTAVSA | 2 | SEQ ID NO:8 |
Example 3 in vivo animal verification
1. Peptide synthesis and fluorescent labeling
M1 was selected for in vivo animal validation, and the peptide sequence TFYGGRPKRNNFLRGIR (MW:2053) was sent to peptide synthesis for peptide synthesis and fluorescent labeling purification. Obtain polypeptide with purity over 95%.
The results of mass spectrometric detection of the unlabeled polypeptides are shown in FIG. 3. The peptide has a molecular weight of 1027.86 x 2-2053.6, and the molecular weight is consistent.
The results of mass spectrometric detection of CY5.5 fluorescent polypeptides on the label are shown in FIG. 4. CY5.5 was labeled with fluorescent molecular weight 873.22 x 3-3-2053 ═ 564 and was the molecular weight of the CY5.5 fluorescent reagent after reaction. Indicating that the M1 short body is linked to a fluorescent molecule.
2. In vivo detection of fluorescently labeled peptides
The fluorescent molecule-connected short peptide is used for subsequent in vivo detection of mice. The ligation short peptide was accurately weighed, dissolved in physiological saline, diluted and ligated to a fluorescence short peptide of 5uM fluorescence equivalent, injected into nude mice via tail vein of 100ul, and observed at different time points using IVIS in vivo imaging system (FIG. 5).
After 4h of tail vein injection, the enrichment of M1-CY5.5 in brain can be obviously observed. Taking a mouse, flowing 100ml of physiological saline through a heart at a speed of 5ml/min, washing out fluorescence interference in blood,
the mouse brain and other organs are observed, the brain has obvious fluorescence which is higher than that of the muscle and the heart, and the fluorescence ratio of the brain/muscle is about 3: 1 (fig. 6, 7). The short peptide can penetrate the obstruction of blood-brain barrier, has high-efficiency capability of penetrating the blood-brain barrier, and has higher enrichment amount than the part without the barrier, such as muscle.
EXAMPLE 4 preparation of curcumin-glutarate-NHS active ester
Glutaric acid is used as a linker, glutaric anhydride and phenolic hydroxyl groups of Curcumin (Curcumin) are condensed into ester to be connected, and the map of the ester is shown in figure 8. Then DCC (dicyclohexylcarbodiimide) is used for activating carboxyl, and NHS (N-hydroxysuccinimide) active ester is coupled with the carboxyl to obtain curcumin-glutaric acid-NHS active ester which can be directly connected with lysine residue of protein/short peptide. The curcumin-glutaric acid-NHS active ester product is obtained after mass spectrum confirmation. Theoretical molecular weight: 579, mass spectrum peak 1: 580(M + H)+) Mass peak 2: 602M + Na+。
Example 5 preparation of M1-RGD-Cur
In the embodiment, the peptide-curcumin is prepared from the polypeptide (M1-RGD) with the sequence of TFYGGRPKRNNFLRGIRSRGD, and is specifically named as M1-RGD-Cur.
Mixing M1-RGD and curcumin-glutaric acid-NHS active ester in a mass ratio of 1:7.5, dissolving in DMF, adding 7.5 times of equivalent of triethylamine as base, and reacting at 37 ℃ for 3 h.
Purification was performed by hplc with acetonitrile (0.1% formic acid) water (0.1% formic acid) as mobile phase. The chromatogram and the identification mass spectrum are shown in FIG. 9.
Example 6 evaluation of inner ear enrichment Effect
9 mice were taken, randomly divided into 3 groups according to 3 mice/group, and tail vein injection was performed according to different groups: experimental group, M1-RGD-Cur prepared in example 5, dose: 3mg/kg calculated as curcumin; control group, curcumin 3 mg/kg; blank, equal volume of saline.
The mice were sacrificed 30min after the injection, cochlear tissues were separated, cochlear lymph fluid was aspirated under a dissecting microscope (the same group of mice was mixed in the same ER tube and collected), and collected inner ear lymph fluid of different groups of mice was subjected to HPLC detection analysis, and the results are shown in fig. 10.
As shown in fig. 10, Cur standard in the lymph fluid of the inner ear of the mouse can be effectively detected (upper left), while no curcumin specific peak appears in the lymph fluid of the inner ear of the mice in the blank group (lower left), while no related curcumin specific peak can be detected in the lymph fluid of the inner ear of the mice injected with curcumin alone (upper right), which indicates that the drug concentration of the inner ear can not reach the detection baseline after curcumin is injected alone for 30 min; the characteristic peak of curcumin, which is shorter than the retention time of pure curcumin, can be detected in M1-RGD-Cur group inner ear lymph fluid, and the peak is judged to be a Linker-Cur characteristic peak through preliminary analysis, and the result fully proves that the inner ear targeted drug delivery system (M1-RGD-Cur) provided by the invention can successfully carry small molecular compounds (taking curcumin as an example) to enter the inner ear lymph fluid of a mouse, and successfully realize effective separation of short peptides and loaded active molecules after entering.
Further, the above experimental findings were further verified in a large animal model (Yunnan Minus Sus domestica), and the results are shown in FIG. 11. This shows that the drug delivery system provided by the invention has wide applicability.
EXAMPLE 7 evaluation of the Effect of treating noise-induced deafness
12 mice (c 57, 5 weeks old) with initial hearing ability were selected and randomly divided into 4 groups of 3 mice/group, M1-RGD-Cur prepared in example 2 was used as the drug for the low dose group, the medium dose group and the high dose group, and normal saline was used for the control group. Placing each group of mice in a detonation sound insulation shielding chamber, continuously carrying out exposure treatment on 120dB narrow-band white noise for 2h for 2 days, and continuously administering for 14 days after the detonation, wherein the administration doses are respectively calculated by curcumin: low dose group, 1 mg/kg/day; medium dose group, 3 mg/kg/day; high dose group, 9 mg/kg/day; control group, equal volume of saline. The administration mode is tail vein injection. And respectively carrying out auditory brainstem evoked potential (ABR) sequencing at 4d, 7d and 14d after the earthquake, and judging whether the auditory function is abnormal or not according to the change condition of different frequencies of auditory thresholds of the mice.
The results of ABR sequencing are shown in FIG. 12, and the results of statistical analysis of ABR data are shown in FIG. 13. As can be seen from fig. 12, the M1-RGD-Cur in the medium dose group was superimposed to form a better brainstem evoked potential wave, the differentiation of the auditory potential wave was relatively clear, and the control group and the low dose group had disordered and abnormal differentiation of the potential wave. As shown in fig. 13, statistical analysis after hearing threshold interpretation according to the ABR data shows that after treatment for 4d by M1-RGD-Cur, the hearing threshold of each treatment group (low dose group, medium dose group, and high dose group) is significantly lower than that of the control group, and after 7d and 14d, the hearing threshold of the medium dose group is significantly lower than that of the other groups, indicating that M1-RGD-Cur can quantitatively treat nerve deafness and play a role in hearing protection after noise exposure.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
SEQUENCE LISTING
<110> Beijing university
<120> a conjugate capable of crossing blood labyrinthine barrier and a method for preparing the same
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> SITE
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<223> Xaa can be any naturally occurring amino acid
<220>
<221> SITE
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<400> 1
Thr Phe Tyr Gly Gly Arg Xaa Lys Arg Asn Asn Phe Xaa Xaa Xaa Xaa
1 5 10 15
Xaa
<210> 2
<211> 88
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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<221> misc_feature
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<223> n is a, c, g, or t
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<221> misc_feature
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gcccaggtgc agctgcagac cttttatggt ggtcgtnnka aacgtaataa ttttnnknnk 60
nnknnknnkt ctagagggga cccaggtc 88
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gcccaggtgc agctg 15
<210> 4
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Thr Phe Tyr Gly Gly Arg Pro Lys Arg Asn Asn Phe Leu Arg Gly Ile
1 5 10 15
Arg
<210> 6
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Thr Phe Tyr Gly Gly Arg Val Lys Arg Asn Asn Phe Ala Leu Ser Leu
1 5 10 15
Trp
<210> 7
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Thr Phe Tyr Gly Gly Arg Ser Lys Arg Asn Asn Phe Pro Lys Leu His
1 5 10 15
Arg
<210> 8
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Thr Phe Tyr Gly Gly Arg Arg Lys Arg Asn Asn Phe Thr Ala Val Ser
1 5 10 15
Ala
<210> 9
<211> 61
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
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 Phe Lys Arg Asn Asn Phe Phe Arg Val Thr Tyr Ser Arg Gly Asp
35 40 45
Pro Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg
50 55 60
<210> 10
<211> 61
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 10
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 Val Val Ser His Ser Arg Gly Asp Pro
35 40 45
Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg Thr
50 55 60
<210> 11
<211> 61
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 11
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 Lys Lys Arg Asn Asn Phe Val Arg Val Lys Ser Arg Gly Asp Pro
35 40 45
Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg Thr
50 55 60
<210> 12
<211> 62
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 12
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 Gly Phe Glu Asn 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> 13
<211> 62
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 13
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 His Lys Arg Asn Asn Phe Asp Asn Asn Gly Tyr 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> 14
<211> 62
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 14
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 Tyr Lys Arg Asn Asn Phe Leu Ser Phe Cys Tyr 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> 15
<211> 62
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
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 Gly Lys Arg Asn Asn Phe Thr Glu Arg Cys 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> 16
<211> 61
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
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 Gly Lys Arg Asn Asn Phe Gln Lys Asn Asp Ser Arg Gly Asp Pro
35 40 45
Gly His Arg Leu Leu Thr Pro Pro Ser Pro Ser Arg Thr
50 55 60
<210> 17
<211> 62
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<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 (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 (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 (Artificial Sequence)
<400> 20
Met Lys Phe Ile Leu Pro Ser Ala Ala Val Ser 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 (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 (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 (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 (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 (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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| CN202110566647.0A CN113425852B (en) | 2021-05-24 | 2021-05-24 | Conjugate capable of penetrating blood labyrinth barrier and preparation method thereof |
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| RU2499592C2 (en) * | 2008-04-21 | 2013-11-27 | Отономи, Инк. | Pharmaceutical composition for treating ear diseases |
| CN104337769A (en) * | 2013-08-06 | 2015-02-11 | 于湛 | Improved preparation technology of alhumin nanosphere and inner ear transmembrane administration sustained release microsphere drug carrier |
| CN109666973B (en) * | 2018-11-21 | 2022-11-04 | 北京大学 | Peptide library crossing blood brain barrier and screening method thereof |
| CN109678966B (en) * | 2018-11-21 | 2021-03-19 | 江苏集萃分子工程研究院有限公司 | Brain tumor targeting peptide and application thereof |
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