Transdermal peptide with nuclear localization ability and use thereof

Abstract

A transdermal peptide with a nuclear localization ability and having an amino acid sequence as shown in SEQ ID NO: 1 is disclosed. A fusion protein including a macromolecular protein with one end being linked to the transdermal peptide is also disclosed. The transdermal peptide can be used in the preparation of a medicament or a transdermal preparation for treating skin diseases. A medicament for treating a skin disease includes the transdermal peptide and a pharmaceutically acceptable excipient. The transdermal peptide enters the cells autonomously to locate in the nuclei, and can penetrate through the stratum corneum of the skin into cells in the dermis. The peptide is conveniently synthesized artificially and suitable for transdermal administration, and has a therapeutic potential via transdermal administration by carrying a drug for treating skin diseases.

Claims

1. A fusion protein with a nuclear localization ability, comprising a macromolecular protein, one end of which is linked to an isolated transdermal peptide with a nuclear localization ability, having an amino acid sequence as shown in SEQ ID NO: 1 and being linked to a fluorescein, wherein the transdermal peptide enters the eukaryotic cells autonomously.

2. The fusion protein with a nuclear localization ability according to claim 1, wherein the other end of the macromolecular protein is linked to a fluorescent protein.

3. The fusion protein with a nuclear localization ability according to claim 1, wherein the macromolecular protein has a molecular weight of 10-50 KD.

4. The fusion protein with a nuclear localization ability according to claim 1, wherein the macromolecular protein has an amino acid sequence as shown in SEQ ID NO: 2, and the macromolecular protein is human superoxide dismutase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the detection results of abilities to enter HaCaT cells and localize in the nuclei of the transdermal peptide of the present invention and the control peptide after labeling with FITC;

(2) FIG. 2 shows the detection results of abilities to enter WS1 cells and localize in the nuclei of the transdermal peptide of the present invention and the control peptide after labeling with FITC;

(3) FIG. 3 shows the detection results of abilities to enter TE-1 cells and localize in the nuclei of the transdermal peptide of the present invention and the control peptide after labeling with FITC;

(4) FIG. 4 shows the detection results of the dose and time dependence of the percentages of the transdermal peptide of the present invention entering HaCaT cells after labeling with FITC;

(5) FIG. 5 shows the test result of the fusion protein of the present invention entering HaCaT cells and being localized into the nuclei; and

(6) FIG. 6 is a schematic view showing the results of the transdermal penetration effect of the transdermal peptide of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(7) The invention will be further illustrated in more detail with reference to the accompanying drawings and embodiments. It is noted that, the following embodiments only are intended for purposes of illustration, but are not intended to limit the scope of the present invention.

Example 1

(8) The raw materials and reagents for the transdermal peptide with a nuclear localization ability provided in the present invention are commercially available.

(9) A peptide having an N terminus labeled with FITC was synthesized by the fmoc method, which has an amino acid sequence as shown in SEQ ID NO: 1. The synthesized peptide was dissolved in a phosphate buffer (PBS, pH=7.4) to prepare a solution having a concentration of 500 μmol/L. HaCaT cells were cultured in vitro, and when the cells were grown to a cell density of 50%, the polypeptide solution was added to the medium at a final concentration of 10 μmol/L. After 4 hrs, DAPI was added for nuclear staining for 30 min. The culture medium was discarded, the cells were rinsed three times with PBS, and photographed under a confocal fluorescence microscope after the free fluorescent polypeptide was removed. For comparison, a control polypeptide as shown in SEQ ID NO: 3 was synthesized using the above method, where the N-terminus of the control polypeptide was also labeled with FITC. The fluorescence intensity was tested by culturing the cells with the control polypeptide according to the above method. The results of comparison with the untreated control cells are shown in FIG. 1. FIG. 1 shows that a green fluorescent signal can be observed in the nuclei of the cells (the treatment group with the target polypeptide) to which the peptide of the present invention is added, and there is no green fluorescence in the cells added with the irrelevant control polypeptide (the treatment group with the irrelevant polypeptide), indicating that the peptide of the present invention has a function of entering HaCaT cells.

Example 2

(10) The transdermal peptide of the present invention and the control polypeptide were synthesized according to the method as described in Example 1, and the N-termini of both polypeptides were labeled with FITC. The human fibroblasts WS1 were cultured following the method as described in Example 1, and when the cells were grown to a cell density of 50%, the polypeptide solution or the control polypeptide solution was added to the medium at a final concentration of 10 μmol/L. After 4 hrs, DAPI was added for nuclear staining for 30 min. The culture medium was discarded, the cells were rinsed three times with PBS, and photographed under a confocal fluorescence microscope after the free fluorescent polypeptide was removed. The results of comparison with the untreated control cells are shown in FIG. 2. FIG. 2 shows that a green fluorescent signal can be observed in the nuclei of the cells (the treatment group with the target polypeptide) to which the peptide of the present invention is added, and there is no green fluorescence in the cells added with the irrelevant control polypeptide (the treatment group with the irrelevant polypeptide), indicating that the peptide of the present invention has a function of entering WS1 cells.

Example 3

(11) The transdermal peptide of the present invention and the control polypeptide were synthesized according to the method as described in Example 1, and the N-termini of both polypeptides were labeled with FITC. The TE-1 cells were cultured following the method as described in Example 1, and when the cells were grown to a cell density of 50%, the polypeptide solution or the control polypeptide solution was added to the medium at a final concentration of 10 μmol/L. After 4 hrs, DAPI was added for nuclear staining for 30 min. The culture medium was discarded, the cells were rinsed three times with PBS, and photographed under a confocal fluorescence microscope after the free fluorescent polypeptide was removed. The results of comparison with the untreated control cells are shown in FIG. 3. FIG. 3 shows that a green fluorescent signal can be observed in the nuclei of the cells (the treatment group with the target polypeptide) to which the peptide of the present invention is added, and there is no green fluorescence in the cells added with the irrelevant control polypeptide (the treatment group with the irrelevant polypeptide), indicating that the peptide of the present invention has a function of entering TE-1 cells.

Example 4

(12) The transdermal peptide of the present invention was synthesized according to the method as described in Example 1, and the N-terminal of the polypeptide was labeled with FITC. The HaCaT cells were cultured in multiple petri dishes following the method as described in Example 1, and when the cells were grown to a cell density of 50%, the polypeptide solution was added to the medium at a final concentration of 0 μmol/L, 3 μmol/L, 6 μmon, and 10 μmol/L respectively. After 4 hrs, DAPI was added for nuclear staining for 30 min. The culture medium was discarded, the cells were rinsed three times with PBS, and photographed under a confocal fluorescence microscope after the free fluorescent polypeptide was removed.

(13) Alternatively, after the cells were grown to a cell density of 50%, the polypeptide solution was added to the medium at a final concentration of 10 μmol/L, and after 0, 60, 120, and 240 min, DAPI was added for nuclear staining for 30 min. The culture medium was discarded, the cells were rinsed three times with PBS, and photographed under a confocal fluorescence microscope after the free fluorescent polypeptide was removed. The results are shown in FIG. 4. The percentage of cells having fluorescence signal in the nuclei of the cells to which the peptide of the present invention is added is dose-dependent (shown in FIG. 4A) and time dependent (shown in FIG. 4B). As the dose of the polypeptide increases and the time elapse, more polypeptide enters the cells.

Example 5

(14) This example provides a fusion protein and the abilities of the fusion protein to enter the cells and localize in the nuclei are detected. The specific method is as follows.

(15) 1. Construction of Gene of Coupled Protein Fused with Peptide with Nuclear Localization and Transdermal Penetration Abilities

(16) A peptide (having an amino acid sequence as shown in SEQ ID NO: 1) with nuclear localization and transdermal penetration abilities, and a fusion protein of a protein as shown in SEQ ID NO: 2 with enhanced green fluorescent protein (EGFP) were constructed. The gene encoding the above protein is in the middle, the EGFP encoding gene is located at the N terminus, and the gene encoding the peptide with nuclear localization and transdermal penetration abilities is located at the C terminus. The nucleic acid was designed and synthesized according to the sequence of the polypeptide and the protein, and then inserted into the pET-28a vector (Novagen, USA) to construct pET-28a-EGFP-pep, and the inserted sequence was sequenced and identified to be correct.

(17) 2. Expression of Fusion Gene in E. coli

(18) E. coli BL21 (DE3) was transformed with the constructed vector pET-28a-EGFP-pep. When the bacterial solution was grown to an OD600 of 0.6, IPTG (final concentration: 1 mmol/L) was added to perform induction at 30° C. for 10 hrs.

(19) 3. Purification of Fusion Protein

(20) The bacteria solution induced to expression was treated for 1 hr by adding a lysozyme, ultrasonically homogenized, and centrifuged at 12,000 rpm for 30 minutes at 4° C. The expression product was dissolved in the supernatant. After centrifugation, the supernatant was transferred to a 10 mL Eppendorf tube, and Ni-NTA (Novagen, USA) was added, and shaken at 50 rpm for 2 h at 4° C. The above mixture was transferred to a chromatographic column, and 4 ml of Wash Buffer (containing PBS and 0.1 M imidazole) was added to wash the chromatographic column when the liquid was running out. When the liquid was almost completely flowed through, 300 μL of an eluate (containing PBS and 0.4 M imidazole) was added, and the efflux protein peak was collected under a nucleic acid/protein detector. The purified liquid was subjected to SDS-PAGE analysis and the molecular weight was about 50 KD, which was consistent with the calculated value. Through the above method, a fusion protein with a purity of over 90% can be obtained.

(21) 4. Detection of Abilities to Enter Cells and Localize in Nuclei of Fusion Protein

(22) HaCaT cells were cultured in vitro, and when the cells were grown to a cell density of 50%, the purified fusion protein was added to the medium at a final concentration of 10 μmol/L. After 4 hrs, DAPI was added for nuclear staining for 30 min. The cells were rinsed three times with PBS, and photographed under a confocal fluorescence microscope after the free fusion protein was removed. The results show that after adding the peptide of the present invention fused to the fusion protein of the polypeptide and EGFP, a green fluorescent signal is observed in the nuclei of HaCaT cells (FIG. 5), indicating that the peptide has the function of carrying a macromolecular protein molecule attached thereto to enter the cells and localize in the nuclei.

Example 6

(23) In this example, the transdermal penetration effect of the peptide was detected through the following specific method.

(24) The experiments were performed using 8-week old SD rats (body weight 250 g). The back of SD rats was shaved, and the FITC-labeled peptide and control peptide prepared in Example 1 were each dissolved in a phosphate buffer (PBS, pH=7.4) to prepare a solution having a concentration of 500 μmol/L. The SD rats were divided into three groups, including: 1) a control group, in which no polypeptide solution was applied on the back; 2) a control group, in which the peptide as shown in SEQ ID NO: 3 was applied on the dorsal epidermis of rats; and 3) a treatment group, in which the peptide as shown in SEQ ID NO: 1 was applied on the back skin of rats. The rats in Groups 2) and 3) were each applied with 50 μL of the polypeptide solution. After three hours, the polypeptide in the applied area was washed off; the SD rats were sacrificed, and the skin applied with the polypeptide was taken, frozen, sectioned, and then observed under a fluorescence microscope. The test results show that there is no green fluorescence in the dermal tissue of the skin of the control group (untreated rat skin) and the control peptide group (control group applied with irrelevant polypeptide) (the arrow in FIG. 6 indicates the stratum corneum with autofluorescence). In the skin section of rats coated with the peptide of the present invention (the polypeptide of interest), a fluorescent signal (the fluorescent signal shown in the ellipse in FIG. 6) is clearly observed in the skin appendages of the dermis, indicating that the peptide can transdermally penetrate into the dermal tissue.

Example 7

(25) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was used in the preparation of a medicament for treating skin diseases, and was prepared as a water extract after adding a conventional excipient.

(26) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was fused with human superoxide dismutase 1 (SOD1), expressed, and then applied once a day to the mice from which the epidermis on the back was removed. The control group was applied with SOD1 not linked to the polypeptide of the present invention. The results show that after three days, the wounds of the rats in the experimental group are reduced by 38% compared with the control group, indicating that the peptide of the present invention mediates the entry of SOD1 into cells to effectively treat skin wounds.

Example 8

(27) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was used in the preparation of a medicament for treating skin diseases, and was prepared as a powder after adding a conventional excipient.

Example 9

(28) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was used in the preparation of a medicament for treating skin diseases, and was prepared as an oily agent after adding a conventional excipient.

Example 10

(29) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was used in the preparation of a medicament for treating skin diseases, and was prepared as a cream after adding a conventional excipient.

Example 11

(30) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was used in the preparation of a medicament for treating skin diseases, and was prepared as an ointment after adding a conventional excipient.

Example 12

(31) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was used in the preparation of a medicament for treating skin diseases, and was prepared as a plaster after adding a conventional excipient.

Example 13

(32) The transdermal peptide (with an amino acid sequence as shown in SEQ ID NO: 1) of the present invention was used in the preparation of a medicament for treating skin diseases, and was prepared as an aerosol after adding a conventional excipient.

(33) The above description is only preferred embodiments of the present invention and not intended to limit the present invention, it should be noted that those of ordinary skill in the art can further make various modifications and variations without departing from the technical principles of the present invention, and these modifications and variations also should be considered to be within the scope of protection of the present invention.