Polypeptide with ovarian granular cell specific targeting property and application thereof

Abstract

The invention discloses a polypeptide with ovarian granular cell specificity targeting and application thereof, relates to Zhejiang Province natural science gold-based research project ZCLMS25H0401, and relates to the technical field of polypeptides. The key point of the technical scheme is as follows: the anti-mullerian hormone is designed and obtained by using a key site for binding a hormone anti-mullerian hormone (AMH) naturally existing in a human body and a specific receptor AMHRII thereof. The polypeptide is composed of 20 amino acid sequences, and the specific sequence of the polypeptide is FITC-C6-YAGKLLISLSEERISAHHVP. The polypeptide provided by the invention has good biocompatibility, almost has no biotoxicity, can realize specific targeting and internalization of ovarian granular cells, has proper length of a peptide fragment, can be widely applied to surface modification of a drug carrier for targeting ovary, and improves the targeting property and utilization rate of a drug.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptides, and more particularly to a polypeptide with ovarian granulosa cell specific targeting and application thereof. Background Art

[0002] Abnormal follicle development, quantity or quality caused by ovarian endocrine diseases is one of the most direct causes of low pregnancy rate. Premature ovarian insufficiency, which is a common ovarian endocrine disease in women before the age of 40, can cause low fertility. So far, there is still a lack of definite and effective treatment for ovarian reserve and premature ovarian insufficiency. Commonly used hormone replacement therapy can only improve clinical symptoms, but cannot reverse ovarian function or improve fertility outcomes. In addition, the natural barrier in the ovary, the blood-follicle barrier (BFB), will also hinder the passage of drug molecules, making it impossible for various drugs to achieve efficient bioavailability in the ovary. Therefore, more effective treatment methods are urgently needed to protect women's fertility. Among them, the design of nano drug delivery systems (NDDS) can achieve drug aggregation in target tissues through passive targeting and active targeting, and has good biological barrier penetration ability. Various peptides have become ideal ligands in targeted delivery systems due to their small molecular weight, good tissue penetration, and high affinity for cell surface receptors.

[0003] Anti-Mullerian hormone (AMH) is a glycoprotein secreted by ovarian granulosa cells. It belongs to the transforming growth factor-β (TGF-β) superfamily and has important value in assessing women's ovarian reserve function. AMH mainly exerts its biological effects by binding to its specific receptor AMHRⅡ, and the distribution of AMHRII has significant tissue specificity and is mainly distributed on the surface of ovarian granulosa cells.

[0004] Therefore, the present invention utilizes the binding site of AMH and its highly specific receptor AMHRII to screen out a polypeptide that can target and bind to AMHRII on the surface of ovarian granulosa cells, and verifies its targeting to examine its feasibility as a targeting ligand for delivering drugs to ovarian granulosa cells. Summary of the invention

[0005] The purpose of the present invention is to provide a polypeptide with ovarian granulosa cell-specific targeting and its application to solve the above problems.

[0006] Before describing the technical solution of the present invention, the terms used in this article are defined as follows:

[0007] The term "FITC" means: fluorescein isothiocyanate.

[0008] The above technical object of the present invention is achieved by the following technical solutions:

[0009] The first aspect of the present invention provides a polypeptide with ovarian granulosa cell-specific targeting. The amino acid sequence of the polypeptide is SEQ ID NO: 1. The polypeptide can specifically bind to and internalize with the ovarian granulosa cell line KGN, and the polypeptide can specifically bind to the AMH receptor AMHRII on the surface of KGN cells.

[0010] The second aspect of the present invention further provides a FITC-modified polypeptide. The amino acid sequence of the polypeptide is FITC-C6-YAGKLLISLSEERISAHHVP.

[0011] The third aspect of the present invention further provides an ovarian-targeted drug delivery carrier, and the carrier contains the polypeptide described in the first aspect or the second aspect.

[0012] The fourth aspect of the present invention provides the application of the polypeptide described in the first aspect in the preparation of drugs targeting the ovary.

[0013] In summary, the present invention has the following beneficial effects: the amino acid sequence of the polypeptide is FITC-C6-YAGKLLISLSEERISAHHVP; the polypeptide can effectively and specifically recognize and bind to the human granulosa cell line KGN; the polypeptide can specifically bind to AMHRII on the surface of KGN cells; the molecular weight of the polypeptide is small, and it has good biocompatibility and in vitro targeting ability. Description of the Drawings

[0014] Figure 1 is the HPLC identification of the synthesized polypeptide in Example 1 of the present invention;

[0015] Figure 2 is the identification of the targeting ability of the polypeptide in the KGN cell line after FITC fluorescence labeling in Example 2 of the present invention;

[0016] Figure 3 is the identification of the binding ability of the polypeptide to the AMHRI I receptor in the KGN cell line after Flag tag labeling in Example 3 of the present invention; Figure 4 is the specific targeting fluorescence imaging of the fluorescently labeled polypeptide in mice in Example 4 of the present invention. Detailed Embodiments

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] The reagents used in the following examples were purchased from the following sources and instrument models:

[0019] (1) Source of reagent purchase:

[0020]

[0021]

[0022] (2) Instrument model:

[0023] name Origin 1ml syringe Becton Dickinson Ophthalmic scissors, ophthalmic tweezers Shanghai Golden Bell Company Small animal in vivo optical imaging system (IVISLuminaIII) PerkinElmer, USA Cryostat (HM525NXU) Thermo Fisher Scientific Inc. Anti-slip slide (BP0510) Hubei Bioas Biotechnology Co., Ltd. Composition pen (PEN-0002) Fuzhou Maixin Biotechnology Development Co., Ltd. Laser confocal scanning microscope (STELLARIS5) Leica, Germany Cell culture incubator ThermoScientific, Inc. Biological Safety Cabinet (HEALforceHF200LC) Shanghai Likang Biomedical Technology Holdings Co., Ltd. High performance liquid chromatography (Agilent 6130 Quadrupole LC / MS) Agilent Technologies Peptide synthesizer (LibertyBlue) CEM Corporation Rotary evaporator (BuchiR-100) Buchi

[0024] Embodiment 1:

[0025] Preparation of peptides with specific targeting to ovarian granulosa cells

[0026] Using the solid phase synthesis method, first, weigh n equivalents of resin and put them into the reactor, add DCM (dichloromethane) to swell for half an hour, then remove the DCM, add 2n equivalents of the first amino acid (Proline) in the sequence, add 2n equivalents of DIEA, appropriate amounts of DMF, DCM, DIEA (diisopropylethylamine), DMF (dimethylformamide), DCM, and nitrogen bubbling reaction for 60 minutes. Then add about 5n equivalents of methanol, react for half an hour, remove the reaction liquid, and wash with DMF and MEOH. Add the second amino acid in the sequence (also 2n equivalents), 2n equivalents of HBTU (1-hydroxy, benzo, trichlorazole tetramethyl hexafluorophosphate) and DIEA to the reactor, N2 bubble reaction for half an hour, wash off the liquid, detect with ninhydrin, and then end-cap with pyridine and acetic anhydride. Finally, wash, add an appropriate amount of decapping liquid to remove the Fmoc (9-fluorenylmethoxycarbonyl) protecting group, wash, and detect with ninhydrin. Continue to add different amino acids in the sequence in sequence. Finally, the resin was blown dry with nitrogen, removed from the reaction column, poured into a flask, and then a certain amount of cutting fluid was added for shaking. The resin was filtered off, a large amount of ether was added to precipitate the crude product, and then centrifuged, purified by high performance liquid chromatography, and finally freeze-dried into a white powder.

[0027] The HPLC identification of synthetic peptides showed that Figure 1 The HPLC report shows that the main peak in the chromatogram is single and symmetrical, and the purity is high.

[0028] Embodiment 2:

[0029] Identification of the targeting ability of peptides after FITC fluorescence labeling in KGN cell line

[0030] The immunofluorescence method was used to verify whether the fluorescent tracer polypeptide could bind to and internalize ovarian granulosa cells. KGN cell lines were purchased and plated in 12-well plates after recovery. When the density reached about 60%, the culture medium was discarded and incubated at 37°C in a medium containing 10 to 80 μM FITC-C6-LRAERSVLIPETYQANNCQG polypeptide for 120 minutes in a dark environment. The cells were rinsed with PBS and fixed with 4% paraformaldehyde for 10 minutes. DAPI staining was performed and the slides were sealed. The fluorescence signal was observed under a laser confocal microscope at 488 nm excitation wavelength and 405 nm excitation wavelength. The results are shown in the figure. Figure 2 As shown, as the concentration of the peptide gradually increased, the green fluorescence signal corresponding to FITC gradually accumulated in the cells, indicating that the fluorescent missing peptide could bind to and internalize the ovarian granulosa cells.

[0031] Example 3

[0032] In vivo assay to identify the ability of fluorescent tracer peptides to target the ovaries in mice

[0033] Tail vein injection and in vitro organ imaging were used to verify whether the fluorescent tracer peptide could specifically target the ovarian organ in vivo. Healthy ICR female mice weighing about 25-30g were selected and randomly divided into an experimental group (injected with fluorescent tracer peptide) and a control group (injected with PBS solution). The experiment was carried out after 3-5 days of adaptive feeding. The FITC-C6-LRAERSVLIPETYQANNCQG polypeptide dry powder was dissolved in PBS buffer to prepare an injection solution of 1.25mg / ml. The polypeptide solution or PBS was injected into the mouse body by tail vein injection. The injection dose for each mouse was 10mg / kg. 30min after the injection, the mice were killed by cervical dislocation, and the mouse ovaries and heart, liver, spleen, lungs and other organs were collected after systemic PBS perfusion. The organs were immediately placed in the IVIS imaging system. The FITC fluorescence excitation wavelength (488nm) and emission wavelength (510-550nm) were set to observe the distribution of fluorescence signals. The results are as follows Figure 3 As shown, compared with the control group, the ovaries of mice injected with fluorescent tracer peptides showed obvious fluorescent signals, and the signal intensity was significantly stronger than that of other organs such as the liver and lungs, indicating that the polypeptide can achieve highly specific ovarian enrichment in mice.

[0034] Example 4

[0035] Histological analysis to identify the distribution of fluorescent tracer peptides in mouse ovarian tissue

[0036] Mice injected with FITC-C6-LRAERSVLIPETYQANNCQG peptide through the tail vein were killed by cervical dislocation at predetermined time points (15min, 30min, 1h, 2h). Under sterile operation, the mouse ovaries were exposed through abdominal incision and carefully dissected. The ex vivo tissue was quickly placed in OCT chemical fixative for complete infiltration, placed in a -20°C freezing rack, and frozen in an ultra-low temperature environment until the tissue was completely solidified. A 5μm thick tissue slice was cut using a freezing microtome, and the slices were carefully transferred to pre-cleaned and prepared slides. After the sectioning was completed, it was placed in a 4% paraformaldehyde (PFA) solution at room temperature for 10-15 minutes, and then the slices were washed with PBS buffer to remove excess fixative. The slices were immersed in DAPI staining solution and stained at room temperature for 5-10 minutes. The slices were gently washed with PBS buffer to remove unbound DAPI. Finally, the slices were covered and sealed with anti-fluorescence quenching sealing solution. The slices were observed using a confocal microscope. The results are as follows Figure 4 As shown, over time, the green fluorescent signal (i.e., fluorescent tracer polypeptide) in the mouse ovary gradually disperses evenly from the edge of the ovary to the inside of the ovary), further indicating that the fluorescent missing polypeptide can specifically target the ovary in mice.

[0037] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A polypeptide with ovarian granulosa cell-specific targeting, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1. The polypeptide can specifically bind to and internalize the ovarian granulosa cell line KGN. The polypeptide can specifically bind to the AMH receptor AMHRI I on the surface of KGN cells.

2. A FITC-modified polypeptide, characterized in that: The amino acid sequence of the polypeptide is FITC-C6-YAGKLLISLSEERISAHHVP.

3. An ovarian targeted drug delivery vector, characterized by: The vector contains the polypeptide according to any one of claims 1 to 2.

4. Use of the polypeptide according to any one of claims 1 to 2 in the preparation of a drug targeting ovary.

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