CN107226846B

CN107226846B - Novel hyaluronic acid-binding peptide and transdermal absorption and subcutaneous targeted release preparation

Info

Publication number: CN107226846B
Application number: CN201710187705.2A
Authority: CN
Inventors: 罗学刚; 张同存; 闫莉华; 王玥; 张宇洁; 乔丽萍
Original assignee: Tianjin University of Science and Technology
Current assignee: Shenzhen Heniya Industrial Co., Ltd.
Priority date: 2017-03-27
Filing date: 2017-03-27
Publication date: 2020-05-19
Anticipated expiration: 2037-03-27
Also published as: CN107226846A

Abstract

The present invention is to design a novel hyaluronic acid-binding peptide and a transdermal absorption and subcutaneous targeted release preparation, design HaBP that can specifically bind to hyaluronic acid, and incubate and mix the HaBP with cell-penetrating peptide and HA under certain conditions , and the three parts of HaBP, cell penetrating peptide, and subcutaneous endogenous protease cleavage site are linked together to construct a fusion protein and then incubated with HA, which can significantly promote the transmembrane transport and transdermal absorption of hyaluronic acid, and Subcutaneous targeted release of HA is achieved. The method of the invention overcomes the defect of low transdermal absorption of hyaluronic acid as a biological macromolecular substance, so that hyaluronic acid can efficiently cross the stratum corneum of the skin, and is targeted to release subcutaneously to promote cell proliferation, so as to exert its role in skin care. , moisturizing, anti-wrinkle, whitening and other aspects of the actual effect, provides a new idea and new method for the development and production of related beauty products and biological drugs.

Description

Novel hyaluronic acid binding peptide and transdermal absorption and subcutaneous targeted release preparation

Technical Field

The invention belongs to the field of cosmetics and biomedicine, and relates to a novel hyaluronic acid binding peptide, a method for promoting transdermal absorption and subcutaneous targeted release of hyaluronic acid and application of the hyaluronic acid binding peptide in cosmetology and biomedicine.

Background

Hyaluronic Acid (HA) is a straight-chain high-molecular polysaccharide, widely present in connective tissues of organisms, one of several glycosaminoglycans constituting extracellular matrix, and HAs the highest content in human dermal layers and joint synovial fluid, and HAs important physiological effects of water retention, lubrication and the like. The natural and non-allergic biological collagen peptide has good natural property, no allergy and good biocompatibility with human tissues, and can be widely applied to the fields of cosmetics, health-care food, cosmetic surgery and medicines. However, as a biological macromolecule, low transdermal rate has been a significant problem that plagues its practical application. Cell-penetrating peptides (CPPs) are small molecular polypeptides capable of carrying various exogenous substances such as proteins and the like into cells, and can realize substance cell internalization and high transdermal property. In order to obtain a method for promoting high-efficiency transdermal absorption and subcutaneous targeted release of HA, the invention designs a novel hyaluronic acid binding peptide HaBP and a fusion protein PMH further derived from the same, and establishes a method for promoting the transdermal absorption and the subcutaneous targeted release of the hyaluronic acid.

Disclosure of Invention

The invention aims to overcome the defect of low transdermal property of a biomacromolecule substance HA, designs a novel hyaluronic acid binding peptide HaBP and a fusion protein PMH further derived from the same, and establishes a method for promoting transdermal absorption and subcutaneous targeted release of hyaluronic acid. The HaBP and PMH obtained by the invention can promote HA to efficiently transport across membranes, penetrate through skin stratum corneum cells and be targeted and released subcutaneously, thereby enhancing the exertion effects of the effects of improving skin elasticity, locking water, removing wrinkles and the like, and providing a new idea and a new method for the production of biological beauty products and biological medicines.

The technical scheme for realizing the purpose of the invention is as follows:

a protein sequence of the hyaluronic acid binding peptide HaBP is shown in a sequence 1.

The protein sequence of the fusion protein PMH is shown in sequence 2.

A hyaluronic acid binding peptide transdermal absorption and subcutaneous targeting preparation is obtained by mixing and incubating HaBP, cell-penetrating peptide and HA, wherein the incubation conditions are as follows: HaBP, cell-penetrating peptide and HA in a ratio of 1:1:8, the HaBP and the cell-penetrating peptide are uniformly mixed, the HA is added, and incubation is carried out for 1h at 37 ℃.

A hyaluronic acid binding peptide transdermal absorption and subcutaneous targeting preparation is characterized in that: the hyaluronic acid binding peptide HaBP, the cell-penetrating peptide and the subcutaneous endogenous protease enzyme cutting site are connected together to construct fusion protein, and then the fusion protein is obtained after incubation with HA.

Furthermore, the cell-penetrating peptide includes Pep-1, TAT, MPG or polyarginine.

Moreover, the basic composition of the fusion protein PMH is that cell-penetrating peptide is introduced into the N end of HaBP, and the protease enzyme cutting site expressed endogenously under the skin is used as the connecting arm of the cell-penetrating peptide and the cell-penetrating peptide, so that the three are spliced together.

Furthermore, the subcutaneously endogenously expressed protease is a matrix metalloproteinase MMP-2, MMP-9, collagenase, gelatinase, stromelysin, keratinase or cathepsin.

The hyaluronic acid binding peptide can be used for preparing cosmetics for caring skin, keeping moisture, removing wrinkle, and whitening skin.

The fusion protein PMH is applied to preparing cosmetics for protecting skin, moisturizing, removing wrinkles and whitening.

The invention has the advantages that:

1. the invention successfully designs a novel hyaluronic acid binding peptide HaBP and a fusion protein PMH further derived from the same, establishes a method for promoting the transdermal absorption and subcutaneous targeted release of hyaluronic acid, can well overcome the defect of low transdermal absorption rate of HA serving as a biological macromolecule, and provides a new method and a new visual angle for the research and development and production of biological beauty products and biological medicines.

2. The invention establishes the gene engineering expression method of the PMH with high transdermal absorption and subcutaneous targeted release, and lays a foundation for the industrialization of the PMH.

3. The method overcomes the defect of low transdermal property of hyaluronic acid as a biological macromolecular substance, enables the hyaluronic acid to efficiently cross over the stratum corneum of the skin, and performs the actual effects of promoting cell proliferation in the aspects of skin care, moisture preservation, wrinkle removal, whitening and the like by subcutaneous targeted release, and provides a new thought and a new method for the development and production of related beauty products and biological medicines.

Drawings

FIG. 1 is a map of a recombinant plasmid of the present invention (exemplified by Escherichia coli expression plasmid pGST-6P-3-PMH);

FIG. 2 shows the double restriction enzyme digestion verification chart of the recombinant plasmid of the present invention (taking Escherichia coli expression plasmid pGST-6P-3-PMH as an example), lane 1 shows the result of double restriction of pGST-6P-3 empty plasmid by EcoR I and Xho I, lane 2 shows the result of double restriction of pGST-6P-3-PMH recombinant plasmid by EcoR I and Xho I, and lane 3 shows the PCR product of PMH gene.

FIG. 3 shows the optimized expression of the fusion protein vector, wherein lanes 1, 2, 3, 4, 5 and 6 show the expression of the vector PMH from pSUMO in host bacteria such as BL21(DE3), BL21(DE3) pLysS and Rosseta (DE3), and

lanes

7, 8, 9, 10, 11 and 12 show the expression of the vector PMH from pGST-6P-3 in host bacteria such as BL21(DE3), BL21(DE3) pLysS and Rosseta (DE 3).

FIG. 4 is an analysis of the ability of HaBP to promote the transport of HA across keratinocytes.

FIG. 5 shows the result of analysis of the effect of HaBP on the transdermal absorption capacity of HA in the mouse experiment (HA is red).

FIG. 6 is serum IgE detection in mouse experiments 1: a control group; 2: CPP group only; 3: HaBP-only groups; 4: HA group only; 5: and (4) PMH group.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.

The invention relates to a novel hyaluronic acid binding peptide (HaBP) and a method for promoting hyaluronic acid transdermal absorption and subcutaneous targeted release thereof. According to the structural characteristics of hyaluronic acid binding protein, HaBP capable of being specifically bound with Hyaluronic Acid (HA) is designed, the HaBP, cell-penetrating peptide and HA are incubated and mixed under certain conditions, three parts of the HaBP, the cell-penetrating peptide and subcutaneous endogenous protease enzyme cutting sites are connected together to construct fusion protein, and then the fusion protein is incubated with the HA, so that transmembrane transport and transdermal absorption of the hyaluronic acid can be remarkably promoted, and subcutaneous targeted release of the HA is realized.

According to the structural characteristics of hyaluronic acid binding protein, a novel hyaluronic acid binding peptide HaBP is designed under the guidance of bioinformatics analysis, and the high-efficiency transmembrane transport of HA, the permeation of skin stratum corneum cells and the targeted release under the skin are realized by incubating and mixing the HaBP, the cell-penetrating peptide and the HA under certain conditions or connecting the HaBP, the cell-penetrating peptide and the enzyme cutting site of subcutaneous endogenous protease together to construct a fusion protein PMH, and then incubating the fusion protein PMH with the HA, so that the effects of improving the skin elasticity, locking water and removing wrinkles of the HA and the like are enhanced. The preparation of HaBP, cell-penetrating peptide and fusion protein PMH may be chemical synthesis process or gene engineering expression process.

A construction method of PMH with high percutaneous absorption and subcutaneous target release is characterized in that the fusion protein PMH is basically formed by introducing cell-penetrating peptide into the N end of HaBP, and splicing the cell-penetrating peptide and the polypeptide together by taking a protease enzyme cutting site expressed endogenously under the skin as a connecting arm of the cell-penetrating peptide and the polypeptide.

The method comprises the following specific steps:

PCR amplification with primers in the following Table

EcoR I and XhoI cleavage sites (underlined) were introduced into primers P1 and P2, respectively. A PCR step: the target gene PMH is obtained by PCR amplification using artificially synthesized cell-penetrating peptide and HaBP gene as templates (see the appendix below for the sequence details) and using primer 1 and primer 2 as primers. The specific system and amplification conditions are as follows:

PCR reaction system for amplifying PMH

Secondly, PMH gene engineering induced expression (taking an escherichia coli expression system as an example)

1. The PMH gene is inserted into the multiple cloning site of an expression vector (taking pGST-6P-3 as an example) to construct a PMH recombinant plasmid.

after the PMH gene and pGST-6P-3 plasmid are subjected to double enzyme digestion by EcoR I and Xho I, the enzyme digestion is carried out by T4DNA ligase at 16 ℃ in water bath overnight to transform escherichia coli DH5 alpha, a single clone is picked up after being cultured for 12h on an LB medium plate containing 50 mug/mL ampicillin, the extracted plasmid is verified by double enzyme digestion of the EcoR I and the Xho I, and the recombinant plasmid with 266bp of PMH target fragment detected by agarose gel electrophoresis is named as pGST-6P-3-PMH after being verified to be correct by sequencing (see figure 1).

Construction of the ligation System required for plasmids

2. Transforming the pGST-6P-3-PMH recombinant plasmid into escherichia coli to obtain PMH recombinant escherichia coli

pGST-6P-3-PMH is transformed into E.coli DH5 α escherichia coli competent cells by adopting a transformation method to obtain PMH recombinant escherichia coli, and the transformation method comprises the following specific steps:

coli DH5 α competent cells were lysed on ice, 10 μ l of the ligation product was added to the competent cells, ice-cooled for 30min, taking care that the competence was necessary to be kept on ice, hot-shocked in a 42 ℃ water bath for 90s without touching, without shaking, the mixture was quickly transferred to ice-cooled for 2min, the cells were allowed to close completely, 900 μ l of LB liquid medium was added to each EP tube, followed by shaking culture at 37 ℃ and 220r/min for 40 min.8000r/min, centrifugation for 1min to remove 900 μ l of supernatant, 100 μ l of the remaining transformation product was suspended and mixed well, spread on an antibiotic-containing LB solid plate, the plate was set upright for 20min and then inverted to plate 37 ℃ overnight, while the competent cell strain was subjected to a control test to ensure that it did not grow normally in the antibiotic-containing plate, positive monoclonals were picked for confirmation by plasmid digestion and sequencing (FIG. 1).

3. Inducible expression

The recombinant plasmid pGST-6P-3-PMH is transferred into Escherichia coli expression strains BL21(DE3), BL21(DE3) pLysS, Rossetta (DE3) and the like, inoculated in 100ml of LB medium (containing 2% glycerol) at an inoculation amount of 1% and cultured until OD600 is approximately equal to 0.8, and 0.1mM IPTG is added to induce protein expression. The induction conditions were: the culture was carried out overnight at 30 ℃ and 200 r/min. SDS-PAGE detection was performed after expression (FIG. 2).

Thirdly, separation and purification of recombinant protein

1. Washing of meltable proteins

(1) After inducing the expression of the target protein, the cells were collected by centrifugation at 10000r/min for 15min in a 4 ℃ centrifuge, and the cells were resuspended in 50ml of PBS (pH 7.4) per 1.5g of wet cells, and then lysed by sonication.

(2) Centrifugation was carried out at 12000r/min at 4 ℃ for 15min, and the supernatant was collected and filtered through a 0.45 μm filter.

2. Purification of meltable proteins

Since the target protein has a GST tag, it can be purified by covalent affinity chromatography with immobilized Glutathione (GSH) via a sulfur bond. The purification was carried out mainly using a Glutathione Sepharose 4B column from GE, according to the following method:

(1) column balancing: the column was equilibrated with 5 volumes of Binding Buffer and loaded after equilibration.

(2) Loading: the sample was loaded onto the column at a flow rate of 10 column volumes per hour and the flow-through was collected.

(3) After the sample is drained, adding washing buffer with 10 times of column volume;

(4) eluting with 0.5ml of elution buffer per ml of column, repeating for three times, and collecting the eluate;

(5) performing buffer displacement and concentration on the collected eluent by using an ultrafiltration tube with the cut-off molecular size of 10 kD;

(6) the proteins collected from each elution peak were examined by SDS-PAGE to obtain GST-fused PMH protein (FIG. 2).

(7) The obtained fusion protein is subjected to enzyme digestion treatment by utilizing enzyme digestion sites (Xa factor, enterokinase, thrombin, TEV enzyme and the like) introduced between GST and PMH during plasmid construction, and then the cut GST tag and the fusion protein left due to incomplete enzyme digestion are separated from the PMH protein from which the GST tag is removed by utilizing GST affinity chromatography to obtain the purified PMH protein.

Fourth, immunofluorescence measures the ability of hyaluronic acid to transport across keratinocytes (taking HaBP as an example)

(1) Early-stage cell culture: hacat immortalized epidermal cells were cultured in MEM (complete culture medium) containing 10% peptide bovine serum at 37 ℃ in 5% CO₂Culturing under the condition. Culturing to digest at a certain density, inoculating 500 μ l into 24-well plate, and culturing for 24 h.

(2) Adding medicine: and (3) diluting the Pep-1, HaBP, HA and other substances according to different proportions or the fusion expressed PMH protein by using a serum-free culture medium in a gradient manner, discarding an old culture solution, adding 500 mu l of a sample diluted in a gradient manner into each hole, and continuously culturing for 24 h.

(2) Washing: the cell culture medium was discarded and washed 3 times with cell PBS for 5min each.

(3) Fixing: cells were fixed with 4% paraformaldehyde at room temperature for 45min and washed with cell PBS 3 times for 5min each.

(4) Penetration: the cells were covered with 0.3% Triton permeabilizing agent, permeabilized for 30min, and washed 3 times with cell PBS, 5min each.

(5) And (3) sealing: rabbit serum blocking solution (1:20 dilution) was blocked for 1h at 37 ℃.

(6) The primary antibody was incubated with HA antibody (1:200 diluted) overnight at 4 ℃ the next day for 1h at room temperature, followed by 3 washes with PBS for 5min each.

(7) Secondary antibody incubation and staining: by Alexa

The mixed solution of the labeled rabbit anti-sheep lgG antibody (diluted 1: 200) + DAPI (diluted 1: 1000) is added into a 24-well plate and incubated for 1h at 37 ℃ in the dark, and then washed 3 times with cell PBS for 5min each time.

(8) The distribution of hyaluronic acid in cells was observed by confocal microscopy (FIG. 3).

The experimental results show that: pep-1 can carry HaBP to penetrate stratum corneum cells, and HaBP is a part of an amino acid sequence of an HA receptor, so that the HaBP can connect Pep-1 and HA in a form similar to a bridge ligament in theory, thereby promoting HA transmembrane transport in synergy with Pep-1. Pep-1 promoted transmembrane transport of HA, which was further enhanced by the presence of HaBP, compared to control and HA-only experimental groups.

Fifthly, analyzing the transdermal absorption capacity and sensitization of the raw materials and the method of the invention on the skin of the mouse

(1) Healthy 4 weeks old 20 Kunming mice, male and female half each, were allowed to eat and drink water freely, and acclimatized in the first week.

(2) Depilation: the lower back and the buttock hair of mouse evenly paint the human cream that moults (wei ting), and the effect is three minutes, dips in the clear water sanitization with medical cotton, resumes three days and begins the experiment, notices the mouse and keeps warm.

(3) Grouping experiments: the test samples were randomly divided into 5 groups according to body weight, namely a negative control group 1 group, a Pep-1 group 2 group, a HaBP group 3 group, a hyaluronic acid group 4 group and a sample group (Pep-1+ HaBP + HA)5 group, and four groups were marked.

(4) Coating the medicine: mice were anesthetized with 0.1ml of amobarbital. The area of the skin after depilation was divided, 200. mu.l of 0.9% NaCl solution was applied to the left and 200. mu.l of the corresponding drug was applied to the right, respectively.

(5) Analysis of transdermal absorption capacity and sensitization: changes in skin appearance were monitored daily for seven consecutive days. On day 7, blood was drawn through the orbit, plasma was rapidly centrifuged (3000 rpm, 20 minutes) and the supernatant serum was immediately frozen at-80 ℃ for examination. Meanwhile, skin samples of the test parts are cut by a scalpel, after tissue sections are prepared by paraffin embedding, immunohistochemical analysis is carried out by using an HA antibody, and the IgE level in serum is detected by using an lgE ELISA kit (figure 4 and figure 5).

The experimental results show that: compared with a control group, the IgE of the mice coated with the Pep-1, the HaBP and the HA alone and the IgE of the mice coated with the three mixed materials have no obvious change, and the content of the IgE of the sensitized positive control group is increased by about 1.2 times. These results suggest that we: when Pep-1/HaBP synergistically promotes HA transdermal absorption, the mixture of the three does not generate a certain sensitization risk to skin.

SEQUENCE LISTING

<110> Tianjin science and technology university

<120> novel hyaluronic acid binding peptide and transdermal absorption and subcutaneous targeted release preparation

<130>2017-03-27

<160>4

<170>PatentIn version 3.3

<210>1

<211>31

<212>PRT

<213> amino acid sequence of HaBP

<400>1

Lys Gln Lys Ile Lys His Val Val Lys Leu Lys Asp Glu Asn Ser Gln

1 5 10 15

Leu Lys Ser Glu Val Ser Lys Leu Arg Cys Gln Leu Ala Lys Lys

20 25 30

<210>2

<211>73

<212>PRT

<213> amino acid sequence of PMH

<400>2

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

20 25 30

Gly Gly Gly Thr Pro Leu Gly Leu Ala Gly Lys Gln Lys Ile Lys His

35 40 45

Val Val Lys Leu Lys Asp Glu Asn Ser Gln Leu Lys Ser Glu Val Ser

50 55 60

Lys Leu Arg Cys Gln Leu Ala Lys Lys

65 70

<210>3

<211>63

<212>DNA

<213> cell-penetrating peptide (Pep-1 as an example) Gene sequence

<400>3

aaagaaacct ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa 60

gtg 63

<210>4

<211>93

<212>DNA

<213> HaBP Gene sequence

<400>4

aaacagaaaa ttaaacatgt ggtgaaactg aaagatgaaa atagccagct gaaaagcgaa 60

gtgagcaaac tgcgttgcca gctggcgaaa aaa 93

Claims

1. A hyaluronic acid binding peptide transdermal absorption and subcutaneous targeting preparation is characterized in that: HaBP, cell-penetrating peptide and HA are mixed and incubated to obtain the cell-penetrating peptide, and the incubation conditions are as follows: the ratio of HaBP to cell-penetrating peptide to HA is 1:1:8, HaBP and cell-penetrating peptide are uniformly mixed, HA is added, incubation is carried out for 1h at 37 ℃, the cell-penetrating peptide is Pep-1, and the protein sequence of HaBP is shown in sequence 1.

2. The use of the hyaluronic acid binding peptide transdermal absorption and subcutaneous targeting preparation of claim 1 in the preparation of skin care, moisturizing, wrinkle removing and whitening cosmetics.