Biotinylated insulin-like growth factor-I polypeptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicine, in particular to a biotinylated insulin-like growth factor-I polypeptide, a preparation method and application, the molecular formula of the biotinylated insulin-like growth factor-I polypeptide is C61H97N21O21S, and the biotinylated insulin-like growth factor-I polypeptide is obtained by blocking the N end of the amino acid sequence SEQ ID NO.1 of the insulin-like growth factor-I polypeptide with biotin. The biotinylated insulin-like growth factor-I polypeptide is prepared by adopting a classical Fmoc oligopeptide solid-phase synthesis method. The biotinylated insulin-like growth factor-I polypeptide prepared by the method is high in purity, simple to operate, good in reproducibility and capable of realizing large-scale production. The insulin-like growth factor-I polypeptide is endowed with targeting property by using biotin end capping, the stability and transdermal absorption rate of the insulin-like growth factor-I polypeptide are improved, and a good hair regeneration effect can be realized by directly applying the insulin-like growth factor-I polypeptide to the skin.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a biotinylated insulin-like growth factor-I polypeptide, a preparation method and an application thereof. Background Art

[0002] Hair loss creates a significant psychological burden on patients and reduces their self-esteem. Moreover, the number of patients with hair loss is increasing year by year. Currently, the main methods for treating hair loss include drugs and hair transplantation. However, the currently approved drugs for treating hair loss, minoxidil and finasteride, can only temporarily relieve symptoms. When patients stop taking the drugs, their hair will start to fall out again, while hair transplantation is limited by high costs, insufficient donors, and low transplant survival rates. Therefore, there is an urgent need to develop a safe, side-effect-free, low-cost, and donor-free strategy for treating hair loss.

[0003] So far, the direct use of growth factors to regulate hair regeneration has good application prospects. Among them, insulin-like growth factor (IGF) is an effective mitogen that participates in normal growth and development. IGF-I is synthesized in the liver under the stimulation of growth hormone released by the pituitary gland, and can act on peripheral tissues after redistribution. IGF-I is also a paracrine and autocrine mediator of growth. In addition, IGF-I plays a key regulatory role in tissue regeneration and acts at all stages of regeneration. However, the current IGF-I polypeptide has a low transdermal absorption rate and a short in vivo circulation half-life, and there is a problem that it cannot be directly administered through skin application. Summary of the invention

[0004] To solve the above problems, the present invention provides a biotinylated insulin-like growth factor-I polypeptide, a preparation method and an application thereof, which can be directly used for drug administration by skin application.

[0005] A biotinylated insulin-like growth factor-I polypeptide, wherein the molecular formula of the biotinylated insulin-like growth factor-I polypeptide is C 61 H 97 N 21 O 21 S is obtained by blocking the N-terminus of the amino acid sequence SEQ ID NO.1 of the insulin-like growth factor-I polypeptide with biotin.

[0006] The amino acid sequence of the insulin-like growth factor-I polypeptide is shown in SEQ ID NO.1, which is GYGSSSRRAPQT.

[0007] Preferably, the biotin is bound to the N-terminus of the polypeptide via an aminocarboxyl condensation reaction.

[0008] Preferably, the method for preparing the biotinylated insulin-like growth factor-I polypeptide comprises the following specific steps:

[0009] Based on the solid phase carrier dichlororesin and HATU as the polypeptide condensation agent, the synthesis starts from the C-terminus of the polypeptide under alkaline conditions, F-moc amino acids are added in sequence according to the SEQ ID NO.1 sequence for condensation reaction, the protecting groups are removed, biotin is added for aminocarboxyl condensation reaction, and the resin is cut to obtain the biotinylated insulin-like growth factor-I polypeptide.

[0010] Preferably, the pH of the alkaline condition is 8-10.

[0011] Preferably, the reagent for removing the protecting group is a piperidine solution with a volume concentration of 15% to 25%, and the reaction time is 20 minutes to 60 minutes.

[0012] Preferably, the cutting reagent is a mixed solution of trifluoroacetic acid, water and triisopropylsilane; the volume ratio of trifluoroacetic acid, water and triisopropylsilane is 94-96:2-3:2-3; and the cutting time is 40 minutes to 60 minutes.

[0013] Application of the biotinylated insulin-like growth factor-I polypeptide in promoting hair regeneration.

[0014] Preferably, the biotinylated insulin-like growth factor-I polypeptide is mixed with a solvent to prepare a polypeptide solution for direct use; the solvent is pure water.

[0015] Preferably, the concentration of the polypeptide solution is 0.1 μM to 100 μM.

[0016] Preferably, the polypeptide solution is used to prepare drugs for skin application.

[0017] The present invention is achieved through the following technical solutions:

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a biotinylated insulin-like growth factor-I polypeptide, wherein the molecular formula of the biotinylated insulin-like growth factor-I polypeptide is C 61 H 97 N 21 O 21S is obtained by capping the N-terminus of the amino acid sequence SEQ ID NO.1 of the insulin-like growth factor-I polypeptide with biotin. IGF-I plays a key regulatory role in tissue regeneration and works at all stages of regeneration. The biotin receptor is a glycoprotein that has a high affinity with biotin and its derivatives and mediates their endocytosis. Therefore, the present invention selects biotin as a capping group for the first time, caps the N-terminus of the insulin-like growth factor-I polypeptide, binds to the N-terminus of the polypeptide through an aminocarboxyl condensation reaction, biotinylates the IGF-I polypeptide, and gives the IGF-I polypeptide targeting, which can achieve a good targeting cell membrane effect, improve the stability and transdermal absorption rate of the IGF-I polypeptide, solve the problem that the prior art cannot be directly administered by skin application, and achieve a good hair regeneration effect, and the performance is stable, without side effects, and can also achieve good skin surface wound healing. In addition, the polypeptide described in the present invention has a simple structure, a simple preparation method, low cost, good reproducibility, can be prepared on a large scale, and mass production can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 is the structural formula of biotinylated IGF-I polypeptide;

[0022] Figure 2 This is a high-resolution mass spectrum of the biotinylated IGF-I peptide;

[0023] Figure 3 This is an image of hair regrowth in mice after biotinylated IGF-I peptide was applied to the skin. DETAILED DESCRIPTION

[0024] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0026] The inventive concept of the present invention is as follows:

[0027] Insulin-like growth factor-I, or IGF-I, is synthesized in the liver under the stimulation of growth hormone released by the pituitary gland and can act on peripheral tissues after redistribution. IGF-I is also a paracrine and autocrine mediator of growth. In addition, IGF-I plays a key regulatory role in tissue regeneration and acts at all stages of regeneration. However, the current IGF-I polypeptide has a low transdermal absorption rate and a short in vivo circulation half-life, which means that it cannot be directly administered through skin application.

[0028] Based on this, the present invention provides a biotinylated insulin-like growth factor-I polypeptide, wherein the molecular formula of the biotinylated insulin-like growth factor-I polypeptide is C 61 H 97 N 21 O 21 S is obtained by capping the N-terminus of the amino acid sequence SEQ ID NO.1 of the insulin-like growth factor-I polypeptide with biotin. IGF-I plays a key regulatory role in tissue regeneration and works at all stages of regeneration. The biotin receptor is a glycoprotein that has a high affinity with biotin and its derivatives and mediates their endocytosis. Therefore, the present invention selects biotin as a capping group for the first time, caps the N-terminus of the insulin-like growth factor-I polypeptide, binds to the N-terminus of the polypeptide through an aminocarboxyl condensation reaction, biotinylates the IGF-I polypeptide, and gives the IGF-I polypeptide targeting, which can achieve a good targeting cell membrane effect, improve the stability and transdermal absorption rate of the IGF-I polypeptide, solve the problem that the prior art cannot be directly administered by skin application, and achieve a good hair regeneration effect, and the performance is stable, without side effects, and can also achieve good skin surface wound healing. In addition, the polypeptide described in the present invention has a simple structure, a simple preparation method, low cost, good reproducibility, can be prepared on a large scale, and mass production can be achieved.

[0029] The beneficial effects of the present invention are described below by means of specific embodiments:

[0030] The reagents used in the embodiments of the present invention are as follows:

[0031] Various F-moc amino acids: purchased from Shanghai Jier Biochemical Co., Ltd., with a purity of 98%.

[0032] Dichlororesin: purchased from Shanghai Jier Biochemical Co., Ltd., with a substitution rate of 1.158 mmol / g.

[0033] N, N-diisopropylethylamine, abbreviated as DIEPA: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity of 99%.

[0034] 2-(7-Azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, abbreviated as HATU: purchased from Tianjin Xiensi Biochemical Technology Co., Ltd., with a purity of 98%.

[0035] Trifluoroacetic acid, abbreviated as TFA, was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a purity of 99%.

[0036] Triisopropylsilane, abbreviated as TIS in English, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. with a purity of 99%.

[0037] Anhydrous dichloromethane, abbreviated as DCM, was purchased from Tianjin Chemical Reagent Company.

[0038] N,N-Dimethylformamide, abbreviated as DMF, was purchased from Tianjin Chemical Reagent Company.

[0039] Piperidine: purchased from Tianjin Chemical Reagent Company.

[0040] Biotin: purchased from Tianjin Xiensi Biochemical Technology Co., Ltd., analytical grade.

[0041] C57BL / 6 mice, 6 weeks old, male: purchased from Henan Sikebes Biotechnology Co., Ltd.

[0042] Example 1. Preparation method of biotinylated insulin-like growth factor-I polypeptide

[0043] The biotinylated insulin-like growth factor-I polypeptide was prepared by the classic F-moc short peptide solid phase synthesis method. The obtained polypeptide is referred to as IGF-Biotin. The specific steps are as follows:

[0044] (1) Weigh 1 mmol of dichlororesin and add it into the reactor, then add an appropriate amount of DCM, swell at room temperature for 10 minutes, and then remove the DCM with an ear bulb.

[0045] (2) Weigh 1 mmol of F-moc threonine and add an appropriate amount of DCM to dissolve it, then add 2 mmol of DIEPA, mix well and add it to the reactor. Under alkaline conditions of pH 8, condensation reaction is carried out at room temperature for 1 hour.

[0046] (3) Wash with DCM three times, then add a mixed solution with a volume ratio of DCM: DIEPA: methanol of 17:1:2 to block for 30 min; after blocking, first wash with DCM three times, and then wash with DMF three times.

[0047] (4) Deprotection of the protecting group: Use a piperidine solution with a volume concentration of 20% for 30 min. After the reaction is completed, wash with DMF for 3 times.

[0048] (5) Weigh 1.5 mmol F-moc-glutamine and 0.57 g 1.5 mmol HATU, add an appropriate amount of DMF to dissolve, add 3 mmol DIEPA, mix well and add to the reactor, and carry out condensation reaction at room temperature under alkaline conditions of pH 8 for 1 hour; after the reaction, wash with DMF three times.

[0049] (6) Deprotection group. Use 20% piperidine solution to react for 30 min. After the reaction is completed, wash with DMF three times. Repeat steps 5 and 6 until the amino acid condensation reaction required by the peptide chain sequence is completed.

[0050] (7) Weigh 1.5 mmol biotin and 1.5 mmol HBTU, add an appropriate amount of DMF to dissolve, then add 3 mmol DIEPA, mix well and add to the reactor, and react at room temperature overnight; after the reaction, first wash with DMF 3 times, and then wash with DCM 3 times.

[0051] (8) Resin cleavage: Add 15 mL of a mixed solution of 95% TFA + 2.5% H2O + 2.5% TIS by volume and cleave for 40 minutes.

[0052] (9) Collect the cutting agent into a single-necked bottle, then wash the reactor with DCM three times, and collect the washing liquid into the above single-necked bottle; then use a rotary evaporator to remove the solvent.

[0053] (10) Anhydrous ether was added to the product, and after crystallization, it was filtered with a separatory funnel to collect the upper solid sample to obtain biotinylated insulin-like growth factor-I polypeptide, referred to as IGF-Biotin.

[0054] (11) The sample was separated and purified by reverse phase high performance liquid chromatography (HPLC). The solution obtained after separation was first subjected to rotary evaporation to remove the methanol solvent, and then the sample aqueous solution was placed in a -8°C refrigerator for freezing, and then transferred to a freeze dryer for freeze drying to obtain a powder sample, which was collected and placed in a -20°C refrigerator for storage for later use.

[0055] The structural formula of biotinylated insulin-like growth factor-I polypeptide is as follows Figure 1 As shown, its molecular formula is C 61 H 97 N 21 O21 S.

[0056] High-resolution mass spectrometry analysis of biotinylated insulin-like growth factor-I peptide was performed, and the results were as follows Figure 2 As shown, the molecular weight of the double peak of IGF-Biotin is 746.85, and there is only one single peak, indicating that the prepared biotinylated insulin-like growth factor-I polypeptide is successful and the sample purity is very high.

[0057] Example 2: Preparation of biotinylated insulin-like growth factor-I polypeptide

[0058] (1) Weigh 1 mmol of dichlororesin and add it into the reactor, then add an appropriate amount of DCM, swell at room temperature for 10 minutes, and then remove the DCM with an ear bulb.

[0059] (2) Weigh 1 mmol of F-moc threonine and add an appropriate amount of DCM to dissolve it, then add 2 mmol of DIEPA, mix well and add it to the reactor. Under alkaline conditions of pH 9, condensation reaction is carried out at room temperature for 1 hour.

[0060] (3) Wash with DCM three times, then add a mixed solution with a volume ratio of DCM: DIEPA: methanol of 17:1:2 to block for 30 min; after blocking, first wash with DCM three times, and then wash with DMF three times.

[0061] (4) Use 20% piperidine solution to react for 30 min. After the reaction is completed, wash with DMF three times.

[0062] (5) Weigh 1.5 mmol F-moc-glutamine and 0.57 g 1.5 mmol HATU, add an appropriate amount of DMF to dissolve, add 3 mmol DIEPA, mix well and add to the reactor, and carry out condensation reaction at room temperature under alkaline conditions of pH 8 for 1 hour; after the reaction, wash with DMF three times.

[0063] (6) React with 15% piperidine solution for 20 min. After the reaction is completed, wash with DMF 3 times. Repeat steps 5 and 6 until the amino acid condensation reaction required by the peptide chain sequence is completed.

[0064] (7) Weigh 1.5 mmol biotin and 1.5 mmol HBTU, add an appropriate amount of DMF to dissolve, then add 3 mmol DIEPA, mix well and add to the reactor, and react at room temperature overnight; after the reaction, first wash with DMF 3 times, and then wash with DCM 3 times.

[0065] (8) Resin cleavage: Add 15 mL of a mixed solution of 94% TFA + 3% H2O + 3% TIS by volume and cleave for 50 minutes.

[0066] (9) Collect the cutting agent into a single-necked bottle, then wash the reactor with DCM three times, and collect the washing liquid into the above single-necked bottle; then use a rotary evaporator to remove the solvent.

[0067] (10) Anhydrous ether was added to the product, and after crystallization, it was filtered with a separatory funnel to collect the upper solid sample to obtain biotinylated insulin-like growth factor-I polypeptide, referred to as IGF-Biotin.

[0068] (11) The sample was separated and purified by reverse phase high performance liquid chromatography (HPLC). The solution obtained after separation was first subjected to rotary evaporation to remove the methanol solvent, and then the sample aqueous solution was placed in a -8°C refrigerator for freezing, and then transferred to a freeze dryer for freeze drying to obtain a powder sample, which was collected and placed in a -20°C refrigerator for storage for later use.

[0069] The structural formula of biotinylated insulin-like growth factor-I polypeptide is as follows Figure 1 As shown, its molecular formula is C 61 H 97 N 21 O 21 S.

[0070] High-resolution mass spectrometry analysis of the biotinylated insulin-like growth factor-I polypeptide showed that the prepared biotinylated insulin-like growth factor-I polypeptide was successful and the sample purity was very high.

[0071] Example 3. Preparation of biotinylated insulin-like growth factor-I polypeptide

[0072] (1) Weigh 1 mmol of dichlororesin and add it into the reactor, then add an appropriate amount of DCM, swell at room temperature for 10 minutes, and then remove the DCM with an ear bulb.

[0073] (2) Weigh 1 mmol of F-moc-threonine and add an appropriate amount of DCM to dissolve it, then add 2 mmol of DIEPA, mix well and add it to the reactor. Under alkaline conditions of pH 10, condensation reaction is carried out at room temperature for 1 hour.

[0074] (3) Wash with DCM three times, then add a mixed solution with a volume ratio of DCM: DIEPA: methanol of 17:1:2 to block for 30 min; after blocking, first wash with DCM three times, and then wash with DMF three times.

[0075] (4) Use 25% piperidine solution to react for 60 min. After the reaction is completed, wash with DMF three times.

[0076] (5) Weigh 1.5 mmol F-moc-glutamine and 0.57 g 1.5 mmol HATU, add an appropriate amount of DMF to dissolve, add 3 mmol DIEPA, mix well and add to the reactor, and carry out condensation reaction at room temperature under alkaline conditions of pH 8 for 1 hour; after the reaction, wash with DMF three times.

[0077] (6) React with 20% piperidine solution for 30 min. After the reaction is completed, wash with DMF 3 times. Repeat steps 5 and 6 until the amino acid condensation reaction required by the peptide sequence is completed.

[0078] (7) Weigh 1.5 mmol biotin and 1.5 mmol HBTU, add an appropriate amount of DMF to dissolve, then add 3 mmol DIEPA, mix well and add to the reactor, and react at room temperature overnight; after the reaction, first wash with DMF 3 times, and then wash with DCM 3 times.

[0079] (8) Resin cleavage: Add 15 mL of a mixed solution of 96% TFA + 2% H2O + 2% TIS by volume and cleave for 60 min.

[0080] (9) Collect the cutting agent into a single-necked bottle, then wash the reactor with DCM three times, and collect the washing liquid into the above single-necked bottle; then use a rotary evaporator to remove the solvent.

[0081] (10) Anhydrous ether was added to the product, and after crystallization, it was filtered with a separatory funnel to collect the upper solid sample to obtain biotinylated insulin-like growth factor-I polypeptide, referred to as IGF-Biotin.

[0082] (11) The sample was separated and purified by reverse phase high performance liquid chromatography (HPLC). The solution obtained after separation was first subjected to rotary evaporation to remove the methanol solvent, and then the sample aqueous solution was placed in a -8°C refrigerator for freezing, and then transferred to a freeze dryer for freeze drying to obtain a powder sample, which was collected and placed in a -20°C refrigerator for storage for later use.

[0083] The structural formula of biotinylated insulin-like growth factor-I polypeptide is as follows Figure 1 As shown, its molecular formula is C 61 H 97 N 21 O 21 S.

[0084] High-resolution mass spectrometry analysis of the biotinylated insulin-like growth factor-I polypeptide showed that the prepared biotinylated insulin-like growth factor-I polypeptide was successful and the sample purity was very high.

[0085] Example 4: Application of biotinylated insulin-like growth factor-I polypeptide in hair regeneration

[0086] The biotinylated insulin-like growth factor-I polypeptide prepared in Example 1 was dissolved in pure water at a concentration ranging from 0.1 μM to 100 μM.

[0087] The depilated mice were randomly divided into two groups, with 3 mice in each group. One group was used as the control group, i.e., the other group was used as the experimental group, i.e., the IGF-Biotin group. Subsequently, pure water and biotinylated insulin-like growth factor-I peptide solution were evenly applied to the back of the depilated mice by skin application once a day, and photos were taken on the 1st, 10th, 13th, and 18th days to record the hair growth on the back of the mice. The results are shown in Figure 2. Figure 3 As shown, compared with the control group, after 18 consecutive days of administration by skin application, the skin area of ​​the mice in the IGF-Biotin group gradually changed from pink to dark black, and finally the hair grew out completely, which indicates that biotinylated IGF-I polypeptide can promote hair regeneration by direct skin application.

[0088] Example 5: Application of biotinylated insulin-like growth factor-I polypeptide in hair regeneration

[0089] The biotinylated insulin-like growth factor-I polypeptide prepared in Example 1 was dissolved in pure water at a concentration of 0.1 μM.

[0090] The depilatory mice were randomly divided into two groups, with 3 mice in each group, one group as the control group, i.e., the other group as the experimental group, i.e., the IGF-Biotin group. Subsequently, pure water and biotinylated insulin-like growth factor-I polypeptide solutions were evenly applied to the backs of the depilatory mice by skin application once a day, and photos were taken on the 1st, 10th, 13th and 18th days to record the hair growth on the backs of the mice. The results showed that compared with the control group, after 18 consecutive days of skin application, the skin area of ​​the mice in the IGF-Biotin group gradually changed from pink to dark black, and eventually the hair completely regrew, indicating that the biotinylated IGF-I polypeptide can promote hair regeneration by direct skin application.

[0091] Example 6: Application of biotinylated insulin-like growth factor-I polypeptide in hair regeneration

[0092] The biotinylated insulin-like growth factor-I polypeptide prepared in Example 1 was dissolved in pure water at a concentration of 10 μM.

[0093] The depilatory mice were randomly divided into two groups, with 3 mice in each group, one group as the control group, i.e., the other group as the experimental group, i.e., the IGF-Biotin group. Subsequently, pure water and biotinylated insulin-like growth factor-I polypeptide solutions were evenly applied to the backs of the depilatory mice by skin application once a day, and photos were taken on the 1st, 10th, 13th and 18th days to record the hair growth on the backs of the mice. The results showed that compared with the control group, after 18 consecutive days of skin application, the skin area of ​​the mice in the IGF-Biotin group gradually changed from pink to dark black, and eventually the hair completely regrew, indicating that the biotinylated IGF-I polypeptide can promote hair regeneration by direct skin application.

[0094] Example 7: Use of biotinylated insulin-like growth factor-I polypeptide in hair regeneration

[0095] The biotinylated insulin-like growth factor-I polypeptide prepared in Example 1 was dissolved in pure water at a concentration of 25 μM.

[0096] The depilatory mice were randomly divided into two groups, with 3 mice in each group, one group as the control group, i.e., the other group as the experimental group, i.e., the IGF-Biotin group. Subsequently, pure water and biotinylated insulin-like growth factor-I polypeptide solutions were evenly applied to the backs of the depilatory mice by skin application once a day, and photos were taken on the 1st, 10th, 13th and 18th days to record the hair growth on the backs of the mice. The results showed that compared with the control group, after 18 consecutive days of skin application, the skin area of ​​the mice in the IGF-Biotin group gradually changed from pink to dark black, and eventually the hair completely regrew, indicating that the biotinylated IGF-I polypeptide can promote hair regeneration by direct skin application.

[0097] Example 8: Use of biotinylated insulin-like growth factor-I polypeptide in hair regeneration

[0098] The biotinylated insulin-like growth factor-I polypeptide prepared in Example 1 was dissolved in pure water at a concentration of 50 μM.

[0099] The depilatory mice were randomly divided into two groups, with 3 mice in each group, one group as the control group, i.e., the other group as the experimental group, i.e., the IGF-Biotin group. Subsequently, pure water and biotinylated insulin-like growth factor-I polypeptide solutions were evenly applied to the backs of the depilatory mice by skin application once a day, and photos were taken on the 1st, 10th, 13th and 18th days to record the hair growth on the backs of the mice. The results showed that compared with the control group, after 18 consecutive days of skin application, the skin area of ​​the mice in the IGF-Biotin group gradually changed from pink to dark black, and eventually the hair completely regrew, indicating that the biotinylated IGF-I polypeptide can promote hair regeneration by direct skin application.

[0100] Example 9: Use of biotinylated insulin-like growth factor-I polypeptide in hair regeneration

[0101] The biotinylated insulin-like growth factor-I polypeptide prepared in Example 1 was dissolved in pure water at a concentration of 75 μM.

[0102] The depilatory mice were randomly divided into two groups, with 3 mice in each group, one group as the control group, i.e., the other group as the experimental group, i.e., the IGF-Biotin group. Subsequently, pure water and biotinylated insulin-like growth factor-I polypeptide solutions were evenly applied to the backs of the depilatory mice by skin application once a day, and photos were taken on the 1st, 10th, 13th and 18th days to record the hair growth on the backs of the mice. The results showed that compared with the control group, after 18 consecutive days of skin application, the skin area of ​​the mice in the IGF-Biotin group gradually changed from pink to dark black, and eventually the hair completely regrew, indicating that the biotinylated IGF-I polypeptide can promote hair regeneration by direct skin application.

[0103] Example 10: Use of biotinylated insulin-like growth factor-I polypeptide in hair regeneration

[0104] The biotinylated insulin-like growth factor-I polypeptide prepared in Example 1 was dissolved in pure water at a concentration of 100 μM.

[0105] The depilatory mice were randomly divided into two groups, with 3 mice in each group, one group as the control group, i.e., the other group as the experimental group, i.e., the IGF-Biotin group. Subsequently, pure water and biotinylated insulin-like growth factor-I polypeptide solutions were evenly applied to the backs of the depilatory mice by skin application once a day, and photos were taken on the 1st, 10th, 13th and 18th days to record the hair growth on the backs of the mice. The results showed that compared with the control group, after 18 consecutive days of skin application, the skin area of ​​the mice in the IGF-Biotin group gradually changed from pink to dark black, and eventually the hair completely regrew, indicating that the biotinylated IGF-I polypeptide can promote hair regeneration by direct skin application.

[0106] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A biotinylated insulin-like growth factor-I polypeptide, characterized in that The molecular formula of the biotinylated insulin-like growth factor-I polypeptide is C 61 H 97 N 21 O 21 S is obtained by blocking the N-terminus of the amino acid sequence SEQ ID NO.1 of the insulin-like growth factor-I polypeptide with biotin.

2. The biotinylated insulin-like growth factor-I polypeptide according to claim 1, characterized in that The biotin is combined with the N-terminus of the polypeptide through an aminocarboxyl condensation reaction.

3. The method for preparing the biotinylated insulin-like growth factor-I polypeptide according to claim 1, characterized in that: The specific steps are as follows: Based on the solid phase carrier dichlororesin and HATU as the polypeptide condensation agent, the synthesis starts from the C-terminus of the polypeptide under alkaline conditions, F-moc amino acids are added in sequence according to the SEQ ID NO.1 sequence for condensation reaction, the protecting groups are removed, biotin is added for aminocarboxyl condensation reaction, and the resin is cut to obtain the biotinylated insulin-like growth factor-I polypeptide.

4. The method for preparing the biotinylated insulin-like growth factor-I polypeptide according to claim 3, characterized in that: The pH of the alkaline condition is 8-10.

5. The method for preparing the biotinylated insulin-like growth factor-I polypeptide according to claim 3, characterized in that: The reagent used for the deprotection group is a piperidine solution with a volume concentration of 15% to 25%, and the reaction time is 20 minutes to 60 minutes.

6. The method for preparing the biotinylated insulin-like growth factor-I polypeptide according to claim 3, characterized in that: The cutting reagent is a mixed solution of trifluoroacetic acid, water and triisopropylsilane; the volume ratio of the trifluoroacetic acid, water and triisopropylsilane is 94-96:2-3:2-3; and the cutting time is 40 minutes to 60 minutes.

7. Use of the biotinylated insulin-like growth factor-I polypeptide as claimed in claim 1 in promoting hair regeneration.

8. The use according to claim 7, characterized in that The biotinylated insulin-like growth factor-I polypeptide is mixed with a solvent to prepare a polypeptide solution which is directly used; the solvent is pure water.

9. The use according to claim 8, characterized in that The concentration of the polypeptide solution is 0.1 μM to 100 μM.

10. The use according to claim 9, characterized in that: The polypeptide solution is used for preparing medicines for skin application.

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