Polypeptide, hydrogel prepared from polypeptide and application of polypeptide in preparation of biomacromolecular drug transdermal delivery system

Abstract

The invention discloses a polypeptide, hydrogel prepared from the polypeptide and application of the polypeptide in preparation of a biological macromolecular drug transdermal delivery system. The sequence of the polypeptide is as follows: the sequence of the polypeptide is as follows: (C18)-FGGRGGHGGGG or (C12)-FGGRGGHGGGG. Wherein C18 represents saturated fatty acid with 18 carbon atoms, C12 represents lauric acid, F represents phenylalanine, G represents glycine, R represents arginine, and H represents histidine. The polypeptide disclosed by the invention can be self-assembled in an aqueous solution through chemical modification to form supramolecular hydrogel, and experiments find that the polypeptide hydrogel disclosed by the invention can be used for reducing the expression of tightly connected protein ZO-1 on a cell membrane within a certain time, so that the permeability of the cell membrane is increased, the transdermal delivery of a biological macromolecular drug is realized, and the application prospect is wide. The problem that existing biological macromolecular drugs are difficult to transdermally deliver to exert treatment potential is solved, and the use safety is high.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine and new drug development, and in particular to polypeptides and hydrogels made therefrom, as well as their applications in preparing biomacromolecule drug transdermal delivery systems. Background Art

[0002] Biomacromolecules (such as proteins, peptides, nucleic acids and polysaccharides) are highly specific, biocompatible and have therapeutic potential, and are widely used in the treatment of major diseases such as tumors, cardiovascular and cerebrovascular diseases and autoimmune diseases. However, these molecules usually face the problem of difficulty in penetrating the skin barrier. Transdermal delivery, as a non-invasive and patient-friendly delivery method, has received great attention in recent years, but the barrier function of the skin and the inherent properties of biomacromolecules have brought many challenges to this field.

[0003] In order to achieve transdermal delivery of biomacromolecule drugs, especially protein drugs, researchers have developed many measures, including: novel chemical enhancers such as membrane-penetrating peptides, and various physical enhancement devices, including cavitation ultrasound, electroporation, thermal ablation, microdermabrasion, and microneedles. Although these strategies can be used for transdermal delivery of various macromolecules, they are still faced with many problems in terms of delivery efficiency and side effects after practical application. For example, membrane-penetrating peptides can deliver small molecule proteins such as insulin transdermally, but the delivery efficiency is not ideal. More importantly, it is still ineffective for transdermal delivery of macromolecular proteins. Microneedles refer to patches with many small needles. In recent years, they have shown great potential in transdermal delivery of insulin and influenza vaccines. However, the manufacturing process and quality control of microneedle patches are very complicated, especially the control of the effective load of biomacromolecules. In addition, microneedles may also cause certain skin damage, thereby increasing the risk of infection.

[0004] Therefore, developing a biomacromolecule transdermal delivery carrier with high safety, high delivery efficiency and simple preparation process is a technical problem that needs to be solved urgently in this field. Summary of the invention

[0005] The present invention aims at the problem that biomacromolecule drugs are difficult to deliver transdermally to exert their therapeutic potential in the prior art, and provides a polypeptide and a hydrogel made thereof, as well as the use in preparing a drug transdermal delivery system. The hydrogel prepared by the polypeptide can reduce the expression of tight junction protein ZO-1 on the cell membrane within a certain period of time, thereby increasing the permeability of the cell membrane and realizing the transdermal delivery of biomacromolecule drugs. The hydrogel has high safety in use and has good market prospects and application potential.

[0006] Based on the above, the present invention first provides a polypeptide, the structure of which is shown in Formula I or Formula II:

[0007]

[0008] Another aspect of the present invention provides a hydrogel for transdermal drug delivery, wherein the hydrogel comprises the aforementioned polypeptide.

[0009] Preferably, the hydrogel is formed by dissolving an aqueous solution containing the polypeptide by ultrasonication and allowing it to stand.

[0010] Preferably, the content of the polypeptide in the aqueous solution is 5 mg / mL-40 mg / mL.

[0011] Another aspect of the present invention also provides use of the aforementioned polypeptide or the aforementioned hydrogel in preparing a drug transdermal delivery system.

[0012] Another aspect of the present invention provides a drug transdermal delivery system, which comprises: the aforementioned hydrogel, wherein the drug to be delivered is loaded in the hydrogel.

[0013] Preferably, the drug is a biomacromolecule drug.

[0014] Preferably, the biomacromolecule drug includes at least one of protein drugs, polypeptide drugs, nucleic acid drugs or polysaccharide drugs.

[0015] Preferably, the hydrogel can reduce the expression of tight junction protein ZO-1 on the cell membrane, thereby increasing the permeability of the cell membrane and achieving transdermal delivery of biomacromolecule drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention include at least:

[0017] The present invention provides a polypeptide, the sequence of which is: (C18)-FGGRGGHGGGG or (C12)-FGGRGGHGGGG. The polypeptide of the present invention can be self-assembled in an aqueous solution by chemical modification to form a supramolecular hydrogel, and experiments have shown that the polypeptide hydrogel of the present invention can reduce the expression of tight junction protein ZO-1 on the cell membrane within a certain period of time, thereby increasing the permeability of the cell membrane and realizing the transdermal delivery of biomacromolecule drugs, solving the problem that existing biomacromolecule drugs are difficult to deliver transdermally to exert their therapeutic potential, and has high safety in use, and has good market prospects and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a photo of an inverted experiment of a polypeptide hydrogel according to an embodiment of the present invention.

[0019] Figure 2 This is a transmission electron microscopy image of a polypeptide hydrogel according to an embodiment of the present invention.

[0020] Figure 3The infrared spectra of the polypeptide hydrogel before and after gelation according to an embodiment of the present invention are shown; wherein D represents polypeptide freeze-dried powder; and D-GEL represents hydrogel freeze-dried powder.

[0021] Figure 4 This is a diagram showing the cytotoxicity experiment of the polypeptide hydrogel according to an embodiment of the present invention.

[0022] Figure 5 This is a diagram showing the effect of transdermal antibody delivery of polypeptide hydrogels detected by immunofluorescence according to an embodiment of the present invention.

[0023] Figure 6 This is the immunoblotting result of the polypeptide hydrogel in one embodiment of the present invention affecting the expression of tight junction protein ZO-1.

[0024] Figure 7 This is the immunofluorescence result of the polypeptide hydrogel affecting the expression of tight junction protein ZO-1 according to one embodiment of the present invention; wherein A is the immunofluorescence image, and B is the quantitative result of A.

[0025] Figure 8 This is a diagram showing the expression of tight junction proteins after the polypeptide hydrogel according to an embodiment of the present invention acts on mouse skin; wherein A is an immunofluorescence image, and B is the quantitative result of A.

[0026] Fig. 9 This is a graph showing the expression changes of tight junction protein ZO-1 after the polypeptide hydrogel according to one embodiment of the present invention acts on mouse skin for 24h, 48h, and 72h.

[0027] Fig.10 The figure is an evaluation of the therapeutic effect of delivering IL-17 antibody by the polypeptide hydrogel of the present invention in psoriasis mice; wherein A is HE staining, and B is the quantitative result of A. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0029] As mentioned above, biomacromolecules (including peptides, proteins, RNA, etc.) are expected to play an important role in the treatment of various diseases by virtue of their key advantages such as high efficacy, specificity and safety. However, due to their weak cell membrane permeability, the therapeutic potential of biomacromolecules has not been fully utilized. Although the prior art has developed many measures for achieving transdermal delivery of biomacromolecules, there are problems such as low delivery efficiency, and even ineffective delivery of large molecule protein drugs; or complex preparation process, difficult to control drug quality; or infection risk, low safety, etc. Therefore, the development of biomacromolecule transdermal delivery carriers with high safety, high delivery efficiency and simple preparation process is a technical problem that needs to be solved urgently in this field.

[0030] To solve this problem, the present invention has, after a lot of research and experiments, finally provided a supramolecular hydrogel formed by self-assembly of polypeptides in an aqueous solution through chemical modification. The hydrogel can reduce the expression of tight junction protein ZO-1 on the cell membrane, thereby opening the tight junction protein ZO-1 and increasing the permeability of the cell membrane, so that biomacromolecules can pass through the skin barrier and realize the transdermal delivery of biomacromolecule drugs (such as IL-23 antibodies, IL-17 antibodies, etc.), solving the problem that existing biomacromolecule drugs, especially protein drugs, are difficult to deliver transdermally to exert their therapeutic potential.

[0031] Therefore, the first object of the present invention is to provide a polypeptide having a sequence of (C18)-FGGRGGHGGGG, with a structure as shown in the following formula I; or (C12)-FGGRGGHGGGG, with a structure as shown in the following formula II. Wherein, C18 represents a saturated fatty acid with 18 carbon atoms, C12 represents lauric acid, F represents phenylalanine, G represents glycine, R represents arginine, and H represents histidine.

[0032]

[0033] The second object of the present invention is to provide a hydrogel for transdermal drug delivery, wherein the hydrogel comprises: the aforementioned polypeptide.

[0034] In some embodiments, the hydrogel is formed by ultrasonically dissolving an aqueous solution containing the aforementioned polypeptide and allowing it to stand.

[0035] In some embodiments, the concentration of the polypeptide in the aqueous solution is 5 mg / mL-15 mg / mL.

[0036] The third object of the present invention is to provide the use of the aforementioned polypeptide or the aforementioned hydrogel in preparing a drug transdermal delivery system.

[0037] The fourth object of the present invention is to provide a drug transdermal delivery system, which comprises: the aforementioned hydrogel, wherein the drug to be delivered is loaded in the hydrogel.

[0038] In some embodiments, the drug is a biomacromolecule drug.

[0039] Furthermore, the biomacromolecule drug includes at least one of protein drugs, polypeptide drugs, nucleic acid drugs or polysaccharide drugs.

[0040] In some embodiments, the hydrogel can reduce the expression of tight junction protein ZO-1 on the cell membrane, thereby increasing the permeability of the cell membrane and achieving transdermal delivery of biomacromolecule drugs.

[0041] Next, the process and results of the present invention are described in detail in combination with experimental data:

[0042] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. The experimental methods used in the following examples, unless otherwise specified, were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0043] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0044] Example 1

[0045] (I) Peptide synthesis

[0046] The polypeptide sequence used in this example is: (C18)-FGGRGGHGGGG, and the structure is shown below:

[0047]

[0048] The polypeptide with the structure shown above is synthesized by solid phase synthesis technology, which is completed by the synthesis service provider according to the structure provided, and is delivered in the form of lyophilized powder (purity ≥ 95%).

[0049] (II) Preparation and characterization of hydrogel

[0050] 1. Preparation of C18 peptide hydrogel

[0051] Weigh 5 mg of the above-obtained polypeptide freeze-dried powder, add 0.5 mL of deionized water and mix; then ultrasonically dissolve it at room temperature, with an ultrasonic power of 100 w and an ultrasonic time of 30 s; after the ultrasonic dissolution is completed, let it stand for 10 minutes, and observe whether a hydrogel is formed by inverting the vial.

[0052] The results are as follows Figure 1 As shown, the inverted vial proves that the polypeptide aqueous solution of the present invention can form a hydrogel state that no longer flows after being dissolved by ultrasound and left to stand.

[0053] 2. Characterization of hydrogel

[0054] (1) TEM morphology characterization

[0055] The hydrogel prepared by the above method was shaken and 10 μL was dropped on a 200-mesh carbon support film. After drying, it was stained with 1% uranyl acetate solution for about 30 seconds, washed with ultrapure water for 3 times, and dried for TEM testing.

[0056] The results are as follows Figure 2 As shown, it can be seen that the polypeptide hydrogel of the present invention is formed by dense nanofibers entangled with each other.

[0057] (2) Infrared spectroscopy

[0058] The hydrogel prepared by the above method was freeze-dried at -50°C to obtain a hydrogel freeze-dried powder, which was pressed into tablets with potassium bromide (ratio of freeze-dried powder to potassium bromide: 1:20), and infrared spectra were measured. At the same time, polypeptide freeze-dried powder was included as a control.

[0059] The results are as follows Figure 3 As shown, compared with the polypeptide freeze-dried powder, the infrared spectrum absorption wavelength of the hydrogel freeze-dried powder is red-shifted, indicating that hydrogen bonds between molecules are formed in the gel state.

[0060] (III) Evaluation of cytotoxicity of hydrogels

[0061] Human immortalized epidermal cells (HaCaT cells) were used to investigate the cytotoxicity of the hydrogel. The peptide hydrogel solution was prepared according to the above method and sterilized by ultraviolet irradiation for 12 h before use. HaCaT cells (human immortalized epidermal cells) were cultured at 2×10 4 The cells / well density was inoculated in a 96-well plate and placed in a CO2 incubator. 10% fetal bovine serum and double-antibody (DMEM) were used as complete growth medium for culture. After 24 hours of culture, the hydrogel was attached to the wells and the final concentrations were 72.5 μg / mL, 125 μg / mL, 250 μg / mL, 500 μg / mL, and 20 μg / mL, respectively. After 1, 3, and 5 days of culture, the cell culture medium was replaced with 100 μL of fresh DMEM medium. After 5 days of culture, 10% (v / v) CCK-8 reagent (Beyotime, China) was also added. After culturing at 37°C for 24, 48, and 72 hours, the cell viability was quantified using a 450 nm microplate reader (TECAN, Switzerland).

[0062] The results are as follows Figure 4 As shown, the hydrogel of the present invention has no obvious toxic effect on cells and has good biocompatibility.

[0063] (III) Evaluation of the transdermal delivery effect of hydrogels on biomacromolecules and exploration of their mechanism

[0064] (1) Evaluation of transdermal delivery of biomacromolecules

[0065] Weigh 20 mg of the above-obtained polypeptide freeze-dried powder, add 0.5 mL of deionized water and mix; then perform ultrasonic dissolution at room temperature, with an ultrasonic power of 100 w and an ultrasonic time of 30 s; after the ultrasonic dissolution is completed, add Cy5.5 fluorescently labeled IL-23 antibody, mix well, and let stand for 10 minutes to obtain a hydrogel loaded with IL-23 antibody (pre-labeled with Cy5.5 fluorescent protein) (hereinafter referred to as Cy5.5-IL23-hydrogel).

[0066] Several 6-8 week old C57 mice were taken, and the back hair was removed after anesthesia. An area of ​​1 cm × 2 cm was taken and 25 μL of the Cy5.5-IL23-hydrogel prepared above was applied. At the same time, blank hydrogel (40 mg / mL) and IL-23 antibody alone (pre-labeled with Cy5.5 fluorescence) were included as controls. After 5, 20, 60, and 120 minutes of application, the samples were embedded in OCT, transferred to -80 ° C overnight, and then ice-cut. The slices were sealed and photographed using laser confocal microscopy, and the transdermal delivery effect of IL-23 antibody was compared by fluorescence intensity.

[0067] The results are as follows Figure 5 As shown, compared with the IL-23 antibody group alone (IL-23Ab only group), the fluorescence of the subcutaneous tissue of mice in the Cy5.5-IL23-hydrogel group (Gel+IL-23Ab group) was significantly enhanced, indicating that the subcutaneous tissue absorbed more IL-23 antibodies. This result shows that the hydrogel provided by the present invention helps biomacromolecules to pass through the skin barrier and enter the subcutaneous tissue, thereby achieving transdermal delivery of biomacromolecule drugs.

[0068] (2) Mechanism of hydrogel transdermal delivery of biomacromolecules

[0069] Furthermore, the present invention experimentally explores the delivery mechanism of hydrogel transdermal delivery of biomacromolecules.

[0070] First, the effect of the hydrogel of the present invention on the expression of tight junction protein ZO-1 on the cell membrane was investigated by cell experiments. The experimental process is as follows: HaCaT cells were cultured at 2×10 4 Cells / well density were inoculated in a 6-well plate. After 24 hours of culture, the hydrogel was added to the wells and incubated for another 24 hours. When the cell fusion was ideal, the cell culture was terminated, the culture medium was aspirated, and the cells were washed three times with PBS to remove the excess culture medium. 100 μL of RAPI lysis buffer and protease inhibitors were added to extract the total protein, which was quantified using a BCA protein assay kit. Then 30 μg of protein sample was dissolved with 10% SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with ZO-1 primary antibody (1:500, Abcam) at 4°C overnight, with GAPDH as the internal reference. After the PVDF membrane was soaked in 5% skim milk for 1 hour, it was incubated with secondary antibody at room temperature for 2 hours, and finally washed with TBST three times. The results of the immunoblotting experiment were observed in an enhanced chemiluminescence detection system. At the same time, immunofluorescence was used to detect the expression of ZO-1 protein in different groups of cells.

[0071] The results are as follows Figure 6 As shown in FIG. , the results of the immunoblotting experiment showed that compared with the blank control group (UT group), the expression level of the tight junction protein ZO-1 in the cells co-cultured with the hydrogel of the present invention was significantly reduced. Figure 7As shown, the immunofluorescence results also showed that compared with the blank control group (UT group), the fluorescence intensity of ZO-1 in the experimental group (C18 group) co-cultured with the hydrogel was significantly weakened, indicating that the hydrogel of the present invention can reduce the expression of tight junction protein ZO-1 on the cell membrane, thereby opening the tight junction protein ZO-1 and increasing the permeability of the cell membrane.

[0072] The effect of the hydrogel of the present invention on the expression of tight junction protein ZO-1 on the cell membrane was also evaluated by animal experiments. Figure 8 As shown in the figure, compared with the blank control group (UT group), the ZO-1 fluorescence intensity of the back skin of the mice in the Hydrogel group (i.e., the mice coated with hydrogel) was significantly reduced. In addition, the cycle of the hydrogel provided by the present invention to open tight junction proteins was further investigated. The results are shown in Fig. 9 As shown, 24 hours after the hydrogel of the present invention was applied to the back of mice, the fluorescence intensity of ZO-1 was significantly reduced, but 48 hours later, the fluorescence intensity of ZO-1 was significantly increased, indicating that the tight junction proteins opened by the hydrogel of the present invention can be restored after 48 hours, and will not make the cell membrane permanently permeable, nor cause damage to the tissue, and the use safety is high.

[0073] Example 2

[0074] 48 nude mice (C57, 4-6 weeks) were ordered and divided into four groups, blank group (UT group), polypeptide hydrogel group (C18 group), IL-17 antibody group and IL-17 antibody hydrogel group, 12 mice in each group, and mice in each group were used to construct a psoriasis mouse model: imiquimod ointment was applied to the back skin for 7 consecutive days, and symptoms such as rashes and dandruff appeared at the site. Among them, after applying imiquimod ointment every day, the nude mice in the polypeptide hydrogel group will apply the C18 peptide hydrogel of the present invention to the same site, the nude mice in the IL-17 antibody group will apply the IL-17 antibody solution to the same site, and the nude mice in the IL-17 antibody hydrogel group will apply the C18 peptide hydrogel loaded with IL-17 antibody to the same site (500 μg of IL-17 antibody per mL of gel hydrogel), and the blank group will not be treated in any way.

[0075] The results are as follows Fig.10 As shown, compared with the IL-17 antibody group, the IL-17 antibody hydrogel group had a significant therapeutic effect on psoriasis in nude mice, indicating that the polypeptide hydrogel of the present invention can deliver IL-17 antibodies to penetrate the skin barrier and exert better therapeutic effects.

[0076] In summary, the present invention provides a polypeptide that can self-assemble to form a supramolecular hydrogel in an aqueous solution through chemical modification. More importantly, the hydrogel provided by the present invention can reduce the expression of tight junction protein ZO-1 on the cell membrane, increase the permeability of the cell membrane, and achieve transdermal delivery of biomacromolecule drugs, thereby solving the problem that existing biomacromolecule drugs, especially protein drugs, are difficult to deliver transdermally to exert their therapeutic potential. The hydrogel has high safety in use and has broad market prospects and application potential.

[0077] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A polypeptide, characterized in that The structure of the polypeptide is shown in Formula I or Formula II:

2. A hydrogel for transdermal drug delivery, characterized in that: The hydrogel comprises the polypeptide of claim 1.

3. The hydrogel according to claim 2, characterized in that The hydrogel is formed by ultrasonically dissolving an aqueous solution containing the polypeptide and then standing it.

4. The hydrogel according to claim 3, characterized in that In the aqueous solution, the content of the polypeptide is 5 mg / mL-40 mg / mL.

5. Use of the polypeptide according to claim 1 or the hydrogel according to any one of claims 2 to 4 in preparing a drug transdermal delivery system.

6. A drug transdermal delivery system, characterized in that: The drug transdermal delivery system comprises: the hydrogel according to any one of claims 2 to 4, wherein the drug to be delivered is loaded in the hydrogel.

7. The drug transdermal delivery system according to claim 6, characterized in that: The drug is a biomacromolecule drug.

8. The use according to claim 7, characterized in that The biomacromolecule drug includes at least one of protein drugs, polypeptide drugs, nucleic acid drugs or polysaccharide drugs.

9. The use according to claim 6, characterized in that The hydrogel can reduce the expression of tight junction protein ZO-1 on the cell membrane, thereby increasing the permeability of the cell membrane and achieving transdermal delivery of biomacromolecule drugs.

Images