CN107226846A - Novel transparent matter acid binding peptide and Transdermal absorption and subcutaneous Targeting delivery preparation - Google Patents

Abstract

The present invention is a kind of sour binding peptide of novel transparent matter of design and Transdermal absorption and subcutaneous Targeting delivery preparation, it is designed to the HaBP specifically bound with hyaluronic acid, the HaBP is incubated with cell-penetrating peptides, HA with certain condition and mixed, and HaBP, cell-penetrating peptides, the subcutaneous part of endogenous protease restriction enzyme site three are linked together be configured to after fusion protein and HA be incubated, the transmembrane transport and Transdermal absorption of hyaluronic acid can be remarkably promoted, and realizes HA subcutaneous Targeting delivery.The low defect of the percutaneous absorbability that has as large biological molecule material the present invention overcomes hyaluronic acid, hyaluronic acid is set efficiently to cross over keratoderma, and promotion cell propagation is played to play its actual efficacy in terms of skin care, moisturizing, anti-wrinkle, whitening in subcutaneous Targeting delivery, provide a kind of new approaches and new method for the Development and Production of related cosmetics and bio-pharmaceutical.

Description

Novel transparent matter acid binding peptide and Transdermal absorption and subcutaneous Targeting delivery preparation

Technical field

It is a kind of sour binding peptide of novel transparent matter and its promotion hyalomitome the invention belongs to cosmetics and biomedicine field Method of the sour Transdermal absorption with subcutaneous Targeting delivery and its application in beauty and biological medicine.

Background technology

Hyaluronic acid (Hyaluronic acid, HA) is a kind of straight chain polymer polysaccharide, universally present in organism In connective tissue, it is to constitute one of several glycosaminoglycans of extracellular matrix, contains in the dermal layer of the skin and knuckle synovia of people Amount is most, with the important physiological action such as water conservation, lubrication.Its natural sex, the biology good without anaphylaxis and with tissue Compatibility, is widely used in cosmetics, health food, beauty treatment and field of medicaments.However, as a kind of biological big Molecule, low skin permeation rate is always the significant problem for perplexing its practical application.Cell-penetrating peptide (cell-penetrating Peptides, CPP) be it is a kind of can carry a variety of exogenous materials such as protein enter cell micromolecule polypeptide, realize thing Cell plastid internalization, efficient transdermal characteristic.Method to obtain a kind of efficient Transdermal absorptions of promotion HA and subcutaneous Targeting delivery, the present invention A kind of novel transparent matter acid binding peptide HaBP and its further derivative fusion protein PMH are devised, and establishes it and is promoted thoroughly The sour Transdermal absorption of bright matter and the method for subcutaneous Targeting delivery.

The content of the invention

It is an object of the invention to overcome the defect of the low transdermal characteristics of large biological molecule material HA, a kind of novel transparent matter is designed Sour binding peptide HaBP and its further derivative fusion protein PMH, and establish its promotion hyaluronic acid Transdermal absorption and subcutaneous The method of Targeting delivery.The HaBP and PMH that the present invention is obtained can promote the efficient transmembrane transports of HA, through skin keratin confluent monolayer cells, And be biological beautiful so as to strengthen its performance effect for improving the effects such as skin elasticity, water lock go wrinkle in subcutaneous targeting positioning release The production for holding product and bio-pharmaceutical provides a kind of new approaches and new method.

The present invention realizes that the technical scheme of purpose is as follows：

A kind of hyaluronic acid binding peptide HaBP, shown in its protein sequence as sequence 1.

A kind of fusion protein PMH, shown in its protein sequence as sequence 2.

A kind of hyaluronic acid binding peptide Transdermal absorption and subcutaneous targeting preparation, HaBP is mixed with cell-penetrating peptide, HA three Close to be incubated and obtain, incubation conditions are：HaBP:Cell-penetrating peptide:HA ratio is 1:1:8, first HaBP is mixed with cell-penetrating peptide Close uniform rear addition HA, 37 DEG C of incubation 1h.

A kind of hyaluronic acid binding peptide Transdermal absorption and subcutaneous targeting preparation, it is characterised in that：By hyaluronic acid binding peptide HaBP, cell-penetrating peptide, the subcutaneous part of endogenous protease restriction enzyme site three link together and are configured to fusion protein, Ran Houyu HA is obtained after being incubated.

Moreover, the cell-penetrating peptide is to include Pep-1, TAT, MPG or many poly arginine.

Moreover, fusion protein PMH basic composition situation is the N-terminal introducing cell-penetrating peptide in HaBP, with subcutaneous endogenous Property expression protease cleavage site as the linking arm of the two, three is stitched together.

Moreover, the protease of the subcutaneous endogenous expression be matrix metalloproteinase MMP-2, it is MMP-9, clostridiopetidase A, bright Glue enzyme, stromelysin, keratinase or cathepsin.

Hyaluronic acid binding peptide as be formed with skin care, moisturizing, anti-wrinkle, skin-lightening cosmetic application.

Fusion protein PMH as be formed with skin care, moisturizing, anti-wrinkle, skin-lightening cosmetic application.

Advantages and advantages of the invention are：

1st, the present invention successfully devises a kind of novel transparent matter acid binding peptide HaBP and its further derivative fusion egg White PMH, and its promotion hyaluronic acid Transdermal absorption and the method for subcutaneous Targeting delivery are established, HA can be overcome well as life The defect under transdermal absorption factor present in thing macromolecular, the development ＆ production for biological cosmetics and bio-pharmaceutical is provided A kind of new method and New Century Planned Textbook.

2nd, the present invention establishes this high Transdermal absorption, subcutaneous Targeting delivery type PMH gene engineering expression method, is it Industrialization is laid a good foundation.

3rd, the low defect of the transdermal characteristic that has as large biological molecule material the present invention overcomes hyaluronic acid, makes Hyaluronic acid can efficiently cross over keratoderma, and play promotion cell propagation to play it in shield in subcutaneous Targeting delivery Actual efficacy in terms of skin, moisturizing, anti-wrinkle, whitening, the Development and Production for related cosmetics and bio-pharmaceutical provides one Plant new approaches and new method.

Brief description of the drawings

Fig. 1 is recombinant plasmid of the present invention (by taking colibacillus expression plasmid pGST-6P-3-PMH as an example) collection of illustrative plates；

Fig. 2 is recombinant plasmid of the present invention (by taking colibacillus expression plasmid pGST-6P-3-PMH as an example) double digestion proof diagram, Swimming lane 1 be pGST-6P-3 empty plasmids by EcoR I and Xho I double digestion results, swimming lane 2 is recombinant plasmid pGST-6P-3- PMH is that swimming lane 3 is PMH gene PCR products by EcoR I and Xho I double digestion results.

Fig. 3 is the carrier Optimal Expression of fusion protein, and swimming lane 1,2,3,4,5,6 is that pSUMO distinguishes for carrier PMH respectively Expression in the Host Strains such as BL21 (DE3), BL21 (DE3) pLysS, Rossetta (DE3), swimming lane 7,8,9,10,11,12 points It is not that pGST-6P-3 is carrier PMH respectively in the Host Strains such as BL21 (DE3), BL21 (DE3) pLysS, Rossetta (DE3) In expression.

Fig. 4 is that HaBP promotes the turn-over capacity of HA across horn cell to analyze.

Fig. 5 is Pushing Function Analysis results of the HaBP to HA Transdermal absorption abilities in mouse experiment (red is HA).

Fig. 6 is SERUM IgE detection 1 in mouse experiment：Control group；2：Only CPP groups；3：Only HaBP groups；4：Only HA groups；5： PMH groups.

Embodiment

Below by specific embodiment, the invention will be further described, and following examples are descriptive, is not limit Qualitatively, it is impossible to which protection scope of the present invention is limited with this.

The present invention is a kind of sour binding peptide (HaBP) of novel transparent matter of design and its promotes hyaluronic acid Transdermal absorption and skin The method of lower Targeting delivery.According to the design feature of HABP, it is designed to and hyaluronic acid (HA) specificity With reference to HaBP, the HaBP and cell-penetrating peptide, HA are incubated with certain condition and mixed, and by HaBP, cell-penetrating peptide, skins The lower part of endogenous protease restriction enzyme site three, which links together, to be configured to after fusion protein be incubated with HA, can be remarkably promoted The transmembrane transport and Transdermal absorption of bright matter acid, and realize HA subcutaneous Targeting delivery.

The present invention designs a kind of according to the design feature of HABP under the guidance of bioinformatic analysis Novel transparent matter acid binding peptide HaBP, is mixed by the way that HaBP is incubated with cell-penetrating peptide, HA three with certain condition, or will HaBP, cell-penetrating peptide, the subcutaneous part of endogenous protease restriction enzyme site three link together and are configured to fusion protein PMH, so The method being incubated afterwards with HA, realizes HA efficient transmembrane transport, through skin keratin confluent monolayer cells, and is released in subcutaneously targeting positioning Put, so as to strengthen its performance effect for improving the effects such as skin elasticity, water lock go wrinkle.Wherein HaBP, cell-penetrating peptide and fusion Albumen PMH's prepares, and can use chemical synthesis, it would however also be possible to employ gene engineering expression production method.

First, high Transdermal absorption, subcutaneous Targeting delivery type PMH construction method, fusion protein PMH basic composition situation is Cell-penetrating peptide is introduced in HaBP N-terminal, will using the protease cleavage site of subcutaneous endogenous expression as the linking arm of the two Three is stitched together.

Comprise the following steps that：

Primer enters performing PCR amplification in following table

Introduce EcoR I and XhoI restriction enzyme site (underscore part) respectively in primer P1, P2.PCR steps：With artificial The cell-penetrating peptide and HaBP genes of synthesis are template (sequence refers to annex hereinafter), are carried out using primer 1 and primer 2 for primer PCR amplifications obtain target gene PMH.Specific system and amplification condition are as follows：

Expand PMH PCR reaction systems

2nd, PMH genetic engineering induced expression (by taking escherichia expression system as an example)

The 1st, PMH genes are inserted into expression vector (by taking pGST-6P-3 as an example) multiple cloning sites, structure obtains PMH weights Group plasmid.

By PMH genes and pGST-6P-3 plasmids with after EcoR I and Xho I double digestions, through the 16 DEG C of water-baths of T4DNA ligases Connect overnight, convert bacillus coli DH 5 alpha, on the LB culture medium flat plates containing 50 μ g/mL ampicillins, chosen after culture 12h Monoclonal is taken, plasmid is extracted and is verified with EcoR I and XhoI double digestion, agarose gel electrophoresis detects the piece for having 266bp PMH mesh Section recombinant plasmid through sequencing identification it is correct after, be named as pGST-6P-3-PMH (see Fig. 1).

Build linked system needed for plasmid

2nd, by pGST-6P-3-PMH recombinant plasmid transformed Escherichia coli, PMH recombination bacillus colis are obtained

Using robin is changed, by pGST-6P-3-PMH Transformed E .coli DH5 α competent escherichia coli cells, PMH weights are obtained Group Escherichia coli, change robin and comprise the following steps that：

E.coli DH5 α competent cells are dissolved on ice, 10 μ l connection products are added in competent cell, ice bath 30min, notices that competence must be placed on ice, is not touched with hand.42 DEG C of water-bath thermal shock 90s, should not vibrate.Mixture is turned rapidly Ice bath 2min is put, allows cell to close completely, 900 μ l LB fluid nutrient mediums are added after 37 DEG C to each EP pipes, 220r/min shakes Swing culture 40min.8000r/min centrifugations 1min removes 900 μ l supernatants, and remaining 100 μ l blow outstanding mixing converted product, are coated on and contain On the LB solid plates of antibiotic, flat board is just being put to inversion 37 DEG C of overnight incubations of plate after 20min.At the same time, competence is thin Born of the same parents' strain will carry out check experiment, it is ensured that it is unable to normal growth in the flat board containing antibiotic.Picking positive monoclonal is passed through Cross plasmid enzyme restriction checking and sequencing determines correctness (Fig. 1).

3rd, induced expression

By recombinant plasmid pGST-6P-3-PMH be transferred to E. coli expression strains BL21 (DE3), BL21 (DE3) pLysS, In Rossetta (DE3) etc., connect bacterium amount with 1% and be inoculated in 100ml LB culture mediums (containing 2% glycerine) middle culture to OD600 ≈ When 0.8, add 0.1mM IPTG and induce its protein expression.Inductive condition is：30 DEG C, 200r/min incubated overnights.Expression is laggard Row SDS-PAGE detects (Fig. 2).

3rd, recombinant protein is isolated and purified

1st, the washing of albumen can be melted

(1) after the expression of induction destination protein, 10000r/min centrifuges 15min and collects thalline in 4 DEG C of centrifuges, by every Thalline, and ultrasonic degradation thalline is resuspended with 50ml PBS (pH 7.4) in 1.5g wet thallus.

(2) 4 DEG C of 12000r/min centrifuge 15min, collect supernatant, and with 0.45 μm of membrane filtration.

2nd, the purifying of albumen can be melted

Because destination protein carries GST labels, therefore can be with covalently close by sulfide linkage with fixed glutathione (GSH) Purified with the method for chromatography.Here the main Glutathione Sepharose 4B chromatographic columns using GE companies are purified, Its purification process is as follows：

(1) column equilibration：Being balanced with the Binding Buffer of 5 times of volumes can loading after pillar, balance.

(2) loading：Sample loads upper prop, and flow velocity is 10 times of column volume/hours, and collection flows through liquid.

(3) the washing buffer of 10 times of column volumes are added after sample flows to end；

(4) every milliliter of cylinder adds 0.5ml elution buffer to elute, and in triplicate, and collects eluent；

(5) eluent being collected into is subjected to buffer displacements and concentration with retention molecular dimension 10kD super filter tube；

(6) albumen that each eluting peak is collected is detected with SDS-PAGE, therefrom obtains the PMH albumen (Fig. 2) of GST fusions.

(7) using build introduced again between GST and PMH during plasmid restriction enzyme site (Xa factor, enterokinase, fibrin ferment, TEV enzymes etc.) digestion processing is carried out to the fusion protein obtained, GST affinity chromatographys are then reused by the GST cut Label and because of the incomplete and remaining fusion protein of digestion, comes with the PMH Protein Separations that remove GST labels, is purified PMH albumen afterwards.

4th, Immunofluorescence test hyaluronic acid crosses over the ability that horn cell is transported (by taking HaBP as an example)

(1) progenitor cells culture：Hacat people immortalizes complete culture solution MEM of the epidermal cell containing 10% peptide cow's serum In 37 DEG C, 5%CO₂Under the conditions of cultivate.Take 500 μ l to be inoculated in 24 orifice plates after culture to certain density digestion, cultivate 24h.

(2) dosing：By materials such as Pep-1, HaBP and HA by different proportion or by the PMH albumen of amalgamation and expression with without blood Clear culture medium gradient dilution, discards old nutrient solution, and the sample of 500 μ l gradient dilutions is added per hole, continues to cultivate 24h.

(2) wash：Cell culture fluid is discarded, with cell PBSs 3 times, each 5min.

(3) it is fixed：4% paraformaldehyde cell is fixed room temperature 45min, then with cell PBSs 3 times, every time 5min。

(4) it is permeabilized：0.3%Triton permeable membrane agent covering cell, permeable membrane processing 30min, then with cell PBSs 3 It is secondary, each 5min.

(5) close：Rabbit anteserum confining liquid (1:20 dilutions) 37 DEG C of closing 1h.

(6) primary antibody is incubated the antibody (1 with HA:200 dilutions) 4 DEG C of night incubations, next day places 1h at room temperature, then uses PBS 3 times, each 5min.

(7) secondary antibody is incubated and dye core：Use AlexaMark rabbit-anti sheep lgG antibody (1:200 dilutions)+DAPI (1: 1000 dilutions) mixed solution, add 37 DEG C of lucifuges in 24 orifice plates and be incubated 1h, then with cell PBSs 3 times, each 5min.

(8) taken a picture with Laser Scanning Confocal Microscope, the distribution situation (Fig. 3) of observation hyaluronic acid in the cell.

Test result indicates that：Pep-1 can carry HaBP and penetrate corneocyte, and HaBP is a part of ammonia of HA acceptors Base acid sequence, therefore HaBP can connect Pep-1 and HA in the form of similar to bridge tie in theory, so that with Pep-1 concertednesses promote HA transmembrane transports.Compared with control and the only experimental group containing HA, Pep-1 can promote HA cross-film to turn Capacity power, and HaBP presence can then further enhance this effect.

5th, raw material of the present invention and method are analyzed the Transdermal absorption ability and sensitization of mouse skin

(1) 20 Kunming mouses of healthy week old 4 week, male and female half and half make its ad lib and drinking-water, fit first week it Answer environment.

(2) lose hair or feathers：The lower back portion and buttocks hair of mouse uniformly smear people with depilatory cream (common vetch is graceful), act on three minutes, use Medical cotton dips in clear water and cleaned up, and recovery starts experiment for three days, notices that mouse is warming.

(3) experiment packet：5 groups, respectively 1 group of negative control group, 2 groups of Pep-1 groups, HaBP groups are randomly divided into according to body weight 5 groups of 3 groups, 4 groups of hyaluronic acid group and sample sets (Pep-1+HaBP+HA), carry out mark by every group four.

(4) coating：0.1ml amytal is injected for mouse, it is anaesthetized.Region is separated on skin after depilation, The 0.9% μ l of NaCl solution 200 are smeared on the left side, and each 200 μ l of correspondence medicine are smeared on the right respectively.

(5) Transdermal absorption ability and sensitization analysis：Daily monitoring skin appearance situation of change, continuous seven days.At the 7th day When, blood is taken by eye socket, upper serum will be taken after blood plasma Quick spin (3000 revs/min, 20 minutes), -80 DEG C freeze and treat immediately Inspection.Experiment area skin sample is cut with scalpel simultaneously, FFPE is prepared after histotomy, is immunized using HA antibody Histochemical analysis, using lgE ELISA kits detect serum in IgE levels (Fig. 4, Fig. 5).

Test result indicates that：Compared with control group, individually smear Pep-1, HaBP and HA and three mixes small after smearing The IgE of mouse is not changed significantly, and sensitization positive controls IgE contents add about 1.2 times.These results point out me ：Pep-1/HaBP is when collaboration promotes HA Transdermal absorptions, and the mixture of three will not produce certain sensitization risk to skin.

SEQUENCE LISTING

<110>University Of Science and Technology Of Tianjin

<120>Novel transparent matter acid binding peptide and Transdermal absorption and subcutaneous Targeting delivery preparation

<130> 2017-03-27

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 31

<212> PRT

<213>HaBP amino acid sequence

<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>PMH amino acid sequence

<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（By taking Pep-1 as an example）Gene order

<400> 3

aaagaaacct ggtgggaaac ctggtggacc gaatggtctc agccgaaaaa aaaacgtaaa 60

gtg 63

<210> 4

<211> 93

<212> DNA

<213>HaBP gene orders

<400> 4

aaacagaaaa ttaaacatgt ggtgaaactg aaagatgaaa atagccagct gaaaagcgaa 60

gtgagcaaac tgcgttgcca gctggcgaaa aaa 93

Claims (9)

1. a kind of hyaluronic acid binding peptide HaBP, it is characterised in that：Shown in its protein sequence as sequence 1.

2. a kind of fusion protein PMH, it is characterised in that：Shown in its protein sequence as sequence 2.

3. a kind of hyaluronic acid binding peptide Transdermal absorption and subcutaneous targeting preparation, it is characterised in that：By HaBP and cell-penetrating peptide, HA three's mixing, which is incubated, to be obtained, and incubation conditions are：HaBP:Cell-penetrating peptide:HA ratio is 1:1:8, first by HaBP and cell HA, 37 DEG C of incubation 1h are added after cell-penetrating peptide is well mixed.

4. a kind of hyaluronic acid binding peptide Transdermal absorption and subcutaneous targeting preparation, it is characterised in that：By hyaluronic acid binding peptide HaBP, cell-penetrating peptide, the subcutaneous part of endogenous protease restriction enzyme site three link together and are configured to fusion protein, Ran Houyu HA is obtained after being incubated.

5. hyaluronic acid binding peptide Transdermal absorption and subcutaneous targeting preparation according to claim 3 or 4, it is characterised in that： The cell-penetrating peptide is to include Pep-1, TAT, MPG or many poly arginine.

6. hyaluronic acid binding peptide Transdermal absorption and subcutaneous targeting preparation according to claim 3 or 4, it is characterised in that： Fusion protein PMH basic composition situation is the N-terminal introducing cell-penetrating peptide in HaBP, with the protease of subcutaneous endogenous expression Three is stitched together by restriction enzyme site as the linking arm of the two.

7. hyaluronic acid binding peptide Transdermal absorption and subcutaneous targeting preparation according to claim 3 or 4, it is characterised in that： The protease of the subcutaneous endogenous expression be matrix metalloproteinase MMP-2, MMP-9, clostridiopetidase A, gelatinase, stromelysin, Keratinase or cathepsin.

8. hyaluronic acid binding peptide described in claim 1 as be formed with skin care, moisturizing, anti-wrinkle, skin-lightening cosmetic application.

9. fusion protein PMH described in claim 1 as be formed with skin care, moisturizing, anti-wrinkle, skin-lightening cosmetic application.