CN107261124A - A kind of preparation method and the application of angiogenesis inhibitor peptide and its hyaluronic acid decorated thing - Google Patents
A kind of preparation method and the application of angiogenesis inhibitor peptide and its hyaluronic acid decorated thing Download PDFInfo
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- CN107261124A CN107261124A CN201710432512.9A CN201710432512A CN107261124A CN 107261124 A CN107261124 A CN 107261124A CN 201710432512 A CN201710432512 A CN 201710432512A CN 107261124 A CN107261124 A CN 107261124A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
Abstract
The invention discloses preparation method and the application of a kind of angiogenesis inhibitor peptide and its hyaluronic acid decorated thing, the amino acid sequence of the AF peptides of ES 2 is as shown in SEQ ID NO.1 in sequence table；The hyaluronic acid trim of the AF peptides of ES 2 is that the carboxyl on the amino and hyaluronan molecule of the above-mentioned AF peptides of ES 2 is combined into by amido link, and structural formula is：HA‑CO‑NH‑(ES‑2‑AF)n.The AF peptides of ES 2 of the present invention retain the ability for even enhancing and suppressing new vessels generation compared with Anti flt1 peptides, ES2 peptides.The hyaluronic acid trim of the AF peptides of ES 2 of the present invention is compared with the AF peptides of ES 2, remain the AF peptides Cancer therapies of ES 2 and antitumor activity, incorporate the characteristics such as macromolecular hyaluronic acid Cancer therapy, the characteristics such as stability is higher, hydrophily is stronger, targeting is stronger are made it have, therefore with more preferable using effect and application value.
Description
Technical field
The present invention relates to biomedicine technical field, more particularly to a kind of angiogenesis inhibitor peptide and its hyalomitome
The preparation method of sour trim and application.
Background technology
Endostatin (Endostatin, ES) is a kind of polypeptide of isolated 20KDa from nemendothelioma, true
Think collagen XV III C-terminal part.Endostatin can be directly targeted the endothelial cell of tumor vicinity without to normal
Cell produces overt toxicity.It can equally suppress endotheli ocytosis, migration, cause Apoptosis.Its anti-angiogenic proliferation activity
Realized by adjusting VEGF VEGF expression.ES-2 (IVRRADRAAVP) be in ES structures one have
The small peptide section of obvious Cancer therapy, antitumor activity, it is easier to enter cell interior, and be easier to obtain and transform,
But it is the same with ES, ES-2 is there is also the shortcomings of Half-life in vivo is short and stability is poor, and these shortcomings greatly limit ES-2
Further application.
Anti-flt1 peptides (GGNQWFI) can specifically with VEGFR1 (fms-like tyrosine kinase-1or
Flt1) combine, so as to play the combination that antagonism prevents VEGFR1 and VEGFA, VEGFB, placenta growth factor PIGF etc..With it
His VEGFR1 monoclonal antibody or RNA antagonists is different, and Anti-flt1 peptides are a kind of lower-cost non-immunogenics of synthesis
Small peptide.But compared with other polypeptides such as ES-2, Anti-flt1 peptides it is water-soluble poor, this has been largely affected by it
The performance of effect.
To sum up, the problems such as half-life short, stability are poor in the existing many bodies of polypeptide neovascularization inhibitor, limitation
Application of the polypeptide drug in neovascularization inhibitor is prepared.Therefore, this area is a kind of new more in the urgent need to developing
Peptide neomatal vasoinhibitor.
The content of the invention
For above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of ES-2-AF peptides, the ES-2-AF peptides
It is improved in activity, dissolubility, stability and in terms of half-life period, with the effect of obvious Cancer therapy.
Another object of the present invention is to provide a kind of hyaluronic acid trim of ES-2-AF peptides.
To achieve the above object, the present invention is adopted the following technical scheme that：
The first aspect of the present invention is there is provided a kind of ES-2-AF peptides, SEQ ID NO.1 in its amino acid sequence such as sequence table
It is shown, it is specific as follows：
ES-2-AF peptides：IVRRADRAAVPGGGGGGNQWFI；(SEQ ID NO.1).
Anti-flt1 peptides are merged by the present invention with ES-2 peptides, resulting ES-2-AF peptides activity, dissolubility,
Stability and good improvement is obtained in terms of half-life period.
The ES-2-AF peptides of the present invention are purified through Fmoc synthesis in solid state by efficient liquid phase, and mass spectrum, high-efficient liquid phase color
The means such as spectrum have carried out the confirmation of structure, and purity is up to more than 95%.
The second aspect of the present invention is there is provided a kind of hyaluronic acid trim of ES-2-AF peptides, on ES-2-AF peptides
Amino is combined with the carboxyl on hyaluronan molecule by amido link, and structural formula is as follows：
HA-CO-NH-(ES-2-AF)n；
In formula, n=30~60；ES-2-AF molecular weight is 2197.5Da, and HA molecular weight is 240000Da.
ES-2-AF quantity delivered (i.e. n value in structure above) can influence the conjugation with hyaluronic acid (HA), this
Investigation is optimized to ES-2-AF quantity delivered in invention, considered influence to HA space conformations and ES-2-AF with
The conjugation of hyaluronic acid, preferably n=60.
There is provided the preparation method of the hyaluronic acid trim of above-mentioned ES-2-AF peptides for the third aspect of the present invention.
The preparation method of the hyaluronic acid trim for the ES-2-AF peptides that the present invention is provided, step is as follows：
(1) HA-TBA conjugates are prepared, improves HA dissolubility, HA is dissolved in organic solvent；
(2) HA-TBA conjugates are dissolved in organic solvent, the special condensing agent of card are added, to activate HA carboxyl；Then will
After HA-TBA, ES-2-AF peptide and DIPEA (DIPEA) mixing after activated carboxyl, reaction 24h, NaOH is added
Solution；The pH for adjusting reaction system again is down to 3.0 with terminating reaction；Then the pH of reaction system is risen into 7.0, reaction product again
Through dialysis, dry, produce the hyaluronic acid trim of ES-2-AF peptides.
In step (1), the preparation method of the HA-TBA conjugates is：Added into Dowex ion exchange resin excessive
TBAH (TBA-OH), mix, obtain Dowex-TBA resins, be filtered to remove supernatant；By Sodium Hyaluronate NaHA
It is soluble in water, in the Dowex-TBA resins for pouring into preparation, mix, be filtered to remove Dowex resins, the HA-TBA clarified is molten
Liquid, freezes, produces HA-TBA conjugates.
In step (2), the organic solvent is DMSO.
In step (2), HA-TBA is 1 with ES-2-AF mol ratios:(4~100).
In step (2), the concentration of the NaOH solution is 1M.
In step (2), 3.0 are down to using 1M HCl regulations pH；PH is risen to 7.0 using 1M NaOH.
In step (2), the method for reaction product dialysis is：Reaction product is poured into the bag filter (10kDa) of pretreatment, point
Yong not NaCl solution, the dialysis of 25% second alcohol and water.
The present invention improves HA dissolubility by preparing HA-TBA conjugates, enables the HA originally insoluble in organic solvent
Enough it is dissolved in DMSO.Excessive BOP makes HA carboxyl fully activate, and the amino being allowed to peptide chain reacts to form amido link.Ka Te contracts
Mixture is the method commonly used in protein, peptide chain reaction, but the factor such as pH, reaction time of reaction temperature, reaction system is all
Have a significant impact to reaction efficiency.The present invention by optimize the quantity delivered of the special condensing agent of card, reaction temperature, the pH of reaction system,
The conditions such as reaction time, successfully prepare the hyaluronic acid ES-2-AF peptide trims that half-life period increases, activity is stronger.
The fourth aspect of the present invention exists there is provided the hyaluronic acid trim of above-mentioned ES-2-AF peptides and/or ES-2-AF peptides
Prepare the purposes in the medicine for suppressing new vessels generation.
Further, the present invention also provides above-mentioned ES-2-AF peptides and/or the hyaluronic acid trim of ES-2-AF peptides exists
Prepare the purposes in medicine and/or antineoplastic that disease is generated with new vessels.
It is described to include diabetic retinopathy, treating senile maculopathy and arthritis with new vessels generation disease
Deng.
Beneficial effects of the present invention：
(1) bar such as pH, the reaction time of quantity delivered, reaction temperature, reaction system of the present invention by adjusting ES-2-AF peptides
Part, can prepare the hyaluronic acid-ES-2-AF peptide conjugates of different conjugations.
(2) ES-2-AF peptides prepared by the present invention are compared with Anti-flt1, ES-2 peptide, and its molecular level resists with cellular level
The effect of new vessels generation is superior to both rear.
(3) the ES-2-AF peptide hyaluronic acid trims that prepare of the present invention are compared with ES-2-AF peptides, its stability is higher,
Bioactivity is stronger.
Brief description of the drawings
The Figure of description for constituting the part of the application is used for providing further understanding of the present application, and the application's shows
Meaning property embodiment and its illustrate be used for explain the application, do not constitute the improper restriction to the application.
Fig. 1：The nuclear magnetic resonance of ES-2-AF peptide hyaluronic acid trims1H collection of illustrative plates；
Fig. 2：The inhibitory action of Anti-flt1, ES-2, ES-2-AF endothelial cell proliferation；
Fig. 3：Anti-flt1, ES-2, ES-2-AF can suppress VEGF and its acceptor VEGFR1 (Flt-1) and combine；
Fig. 4：ES-2-AF, HA＆ES-2-AF, HA-ES-2-AF endothelial cell proliferation inhibitory action；
Fig. 5：ES-2-AF, HA＆ES-2-AF, HA-ES-2-AF can suppress VEGF and its acceptor VEGFR1 (Flt-1) and combine；
Fig. 6：The inhibitory action of ES-2-AF, HA＆ES-2-AF, HA-ES-2-AF endothelial cell transfer.
Embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the application.Unless another
Indicate, all technologies used herein and scientific terminology are with usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation, device, component and/or combinations thereof.
It is half-life short in the existing many bodies of polypeptide neovascularization inhibitor, steady as background technology is introduced
The problems such as qualitative difference and poorly water-soluble.Based on this, the present invention proposes a kind of angiogenesis inhibitor peptide and its hyaluronic acid
Trim.
There is provided a kind of ES-2-AF peptides in a kind of embodiment of the application, its isoelectric point is 11.54, its amino acid
Sequence is as follows：
ES-2-AF peptides：IVRRADRAAVPGGGGGGNQWFI.
Due to Anti-flt1 and ES-2 in terms of activity existing similarity, have many differences again, by Anti-flt1 and
The difficult point that ES-2 is merged is：How to ensure by the respective advantage fusions of Anti-flt1 and ES-2 in same peptide, together
When overcome the intrinsic shortcoming of each bar polypeptide before fusion.The application realizes Anti-flt1 and ES- by designing linker (GGGG)
2 fusion, the ES-2-AF peptides after fusion have more preferable bioactivity, more specific mechanism of action, more preferable stability and more
Long half-life period.
In the another embodiment of the application, a kind of hyaluronic acid trim of ES-2-AF peptides is additionally provided, by
Amino on ES-2-AF peptides is combined with the carboxyl on hyaluronan molecule by amido link, and structural formula is as follows：
HA-CO-NH-(ES-2-AF)n；
In formula, n=30~60；ES-2-AF molecular weight is 2197.5Da, and HA molecular weight is 240000Da.
The hyaluronic acid trim of the ES-2-AF peptides of the present invention remains ES-2-AF peptides and resisted compared with ES-2-AF peptides
New vessels is generated and antitumor activity, incorporates the characteristics such as macromolecular hyaluronic acid Cancer therapy, is made it have steady
The characteristic such as qualitative higher, hydrophily is stronger, targeting is stronger, therefore with more preferable using effect and application value.
In order that the technical scheme of the application can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes the technical scheme of the application in detail.
Test material used is the conventional test material in this area in the embodiment of the present invention, can pass through commercial channel
It is commercially available.
Embodiment 1：The preparation of ES-2-AF peptides
The molecular weight of the ES-2-AF peptides of the present invention is 2197.5Da, using Fmoc synthesis in solid state, is carried out by efficient liquid phase pure
Change, and the means such as mass spectrum, high performance liquid chromatography have carried out the confirmation of structure, purity is up to more than 95%.
Embodiment 2：The preparation of ES-2-AF peptide hyaluronic acid trims (HA-ES-2-AF)
Preparation process is as follows：
(1) Dowex ion exchange resin is cleaned with water, adds excessive tetrabutylammonium hydroxide amine TBA-OH
(24.5ml), is mixed.Dowex-TBA resins are obtained, supernatant is filtered to remove.Sodium Hyaluronate NaHA (1g) is soluble in water, fall
Enter in ready Dowex-TBA resins (10g).Mix after 3h, obtained with 0.45 μm of syringe needle filter filtering with removing Dowex resins
To the HA-TBA solution of clarification, freeze 3 days.
(2) obtained HA-TBA in step (1) is dissolved in DMSO respectively with ES-2-AF peptides.After HA-TBA fully dissolves, plus
Enter excessive card spy condensing agent BOP to activate HA carboxyls, mix 30min.Then by HA-TBA solution and peptide solution, equimolar amounts
DIPEA mixing be dissolved in DMSO, after reaction 24h, add isometric 1M NaOH aqueous solution.PH is down to 3.0 with end with 1M HCl
Only react, pH is then risen to 7.0 with 1M NaOH.Reaction product pours into the bag filter (10kDa) of pretreatment, with substantial amounts of
NaCl solution, 25% ethanol, water dialysis.It is lyophilized 3 days.
Using1H nuclear magnetic resonance identifies structure, as a result as shown in figure 1, as a result showing that hyaluronic acid has successfully modified ES-
2-AF peptides, average connection rate is 93.75%.
Test example 1：ES-2-AF peptides suppressing cell reproduction acts on the comparison with Anti-flt1, ES-2 peptide
Experimentation is as follows：
(1) trial drug：Peptide is dense in ES-2-AF peptides prepared by Anti-flt1 peptides, ES-2 peptides and embodiment 1, three groups of medicines
Degree is consistent.
(2) test method：
Logarithmic phase Human umbilical vein endothelial cells EAhy926 is collected, adjustment concentration of cell suspension is 7000/ hole, is divided in 96 holes
Plate, per the μ L of hole 200 (the DMEM culture mediums containing 10%FBS), is placed in 37 DEG C, 5%CO2Constant incubator in culture paste cell
Wall；The trial drug of various concentrations gradient is added, every kind of 5 multiple holes of medicine continue to cultivate 48h；The μ of MTT solution 20 is added per hole
L, 37 DEG C are continued to cultivate termination culture after 4h, and enzyme-linked immunosorbent assay instrument determines each hole absorbance (OD) value at wavelength 490nm, and
Calculate its inhibiting rate：[(experimental group-blank group)/(control-blank group) of inhibiting rate=1-.Experiment three times is repeated, is averaged.
The result of anti-endothelial cell proliferation experiment is shown in Fig. 2, as seen from Figure 2, the Anti-flt1 of various concentrations gradient
Peptide, ES-2 peptides and ES-2-AF peptides are after effect 48h, and the suppressing cell reproduction action effect of ES-2-AF peptides is totally higher than Anti-
Flt1 peptides and ES-2 peptides.
Test example 2：External Anti-flt1, ES-2, ES-2-AF peptide suppresses VEGF and its acceptor VEGFR1 (Flt-1) and combined
The test (ELISA method) of ability
(1) trial drug：Peptide is dense in ES-2-AF peptides prepared by Anti-flt1 peptides, ES-2 peptides and embodiment 1, three groups of medicines
Degree is consistent.
(2) test method：
VEGF165It is dissolved in PBS and is made into 0.5 μ g/mL, spreads on per the μ L of hole 50 in 96 orifice plates, 4 DEG C overnight.Phosphate buffer
PBS board-washings, 300 μ L*3 times, every time 5 minutes.The μ L/ holes of 3wt%BSA in PBS 250,37 DEG C are closed 2 hours.Phosphate-buffered
Liquid PBS board-washings, 300 μ L*3 times, every time 5 minutes.Add testing sample.Per 3 repetitions of concentration.Incubation at room temperature 1 hour.It is positive right
According to：500ng/mL Flt-Fc in BSA 1wt%in PBS.Experimental group：500ng/mL Flt-Fc+Anti-flt1 groups, ES-2
Group, ES-2-AF groups, peptide concentration is 5,25,50,100,200 μ g/mL in three groups.PBS board-washings containing 0.05wt%Tween20
300 μ L*3 times, every time 5 minutes.Add secondary antibody anti-human IgG-HRP-Fc in 0.3wt%BSA in PBS (1:
1000,50 μ L), it is incubated at room temperature 1 hour.300 μ of PBS board-washings containing 0.05wt%Tween 20 L*2 times, 5 minutes every time, then
300 μ of PBS board-washings L*1 times, 5 minutes.Suck dry moisture is rapped on absorbent cloth.TMB nitrite ions are per the μ L of hole 200, and lucifuge is incubated, 10 points
Enzyme exempts from instrument 450nm tests after clock.
Experimental result is shown in Fig. 3, when concentration is more than 25 μ g/mL, and ES-2-AF suppresses VEGF and its ability of acceptor combination is equal
Better than Anti-flt1, ES-2 peptide.
Test example 3：ES-2-AF peptide hyaluronic acid trims suppressing cell reproduction acts on the comparative experiments with ES-2-AF peptides
Process is as follows：
(1) trial drug：The ES-2-AF peptide hyaluronic acid trims (HA-ES-2-AF) of the preparation of embodiment 2, embodiment
The 1 ES-2-AF peptides prepared and ES-2-AF peptides are with hyaluronic acid by the mixture (HA＆ES-2-AF) linked than mixing, three groups
Peptide concentration is consistent in medicine.
(2) test method：
Logarithmic phase Human umbilical vein endothelial cells EAhy926 is collected, adjustment concentration of cell suspension is 7000/ hole, is divided in 96 holes
Plate, per the μ L of hole 200 (the DMEM culture mediums containing 10%FBS), is placed in 37 DEG C, 5%CO2Constant incubator in culture paste cell
Wall；The trial drug of various concentrations gradient is added, every kind of 5 multiple holes of medicine continue to cultivate 48h；The μ of MTT solution 20 is added per hole
L, 37 DEG C are continued to cultivate termination culture after 4h, and enzyme-linked immunosorbent assay instrument determines each hole absorbance (OD) value at wavelength 490nm, and
Calculate its inhibiting rate：[(experimental group-blank group)/(control-blank group) of inhibiting rate=1-.Experiment three times is repeated, is averaged.
The result of anti-endothelial cell proliferation experiment is shown in Fig. 4, and as seen from Figure 4, the ES-2-AF peptides of various concentrations gradient are saturating
Bright matter acidifying trim and ES-2-AF peptides are after effect 48h, and the suppressing cell reproduction of ES-2-AF peptide hyaluronic acid trims is made
ES-2-AF peptides and both mixtures with effect substantially than identical peptide concentration are eager to excel a lot.
Test example 4：External HA-ES-2-AF conjugates suppress the survey of VEGF and its acceptor VEGFR1 (Flt-1) binding ability
Try (ELISA method)
(1) trial drug：The ES-2-AF peptide hyaluronic acid trims (HA-ES-2-AF) of the preparation of embodiment 2, embodiment
The 1 ES-2-AF peptides prepared and ES-2-AF peptides are with hyaluronic acid by the mixture (HA＆ES-2-AF) linked than mixing, three groups
Peptide concentration is consistent in medicine.
(2) test method：
VEGF165It is dissolved in PBS and is made into 0.5 μ g/mL, spreads on per the μ L of hole 50 in 96 orifice plates, 4 DEG C overnight.Phosphate buffer
PBS board-washings, 300 μ L*3 times, every time 5 minutes.The μ L/ holes of 3wt%BSA in PBS 250,37 DEG C are closed 2 hours.Phosphate-buffered
Liquid PBS board-washings, 300 μ L*3 times, every time 5 minutes.Add testing sample.Per 3 repetitions of concentration.Incubation at room temperature 1 hour.It is positive right
According to：500ng/mL Flt-Fc in BSA 1wt%in PBS.Experimental group：500ng/mL Flt-Fc+ES-2-AF groups, HA＆ES-
2-AF groups, HA-ES-2-AF groups, peptide concentration is 5,25,50,100,200 μ g/mL in three groups.Containing 0.05wt%Tween's 20
300 μ of PBS board-washings L*3 times, every time 5 minutes.Add secondary antibody anti-human IgG-HRP-Fc in 0.3wt%BSA in PBS
(1:1000,50 μ L), it is incubated at room temperature 1 hour.300 μ of PBS board-washings containing 0.05wt%Tween 20 L*2 times, 5 minutes every time, so
300 μ of PBS board-washings L*1 times, 5 minutes afterwards.Suck dry moisture is rapped on absorbent cloth.TMB nitrite ions are per the μ L of hole 200, and lucifuge is incubated, and 10
Enzyme exempts from instrument 450nm tests after minute.
Experimental result is shown in Fig. 5, ES-2-AF peptides, HA＆ES-2-AF mixtures, HA-ES-2-AF conjugates inhibitory action it is equal
Increase in concentration dependent.And mixture group inhibitory action is better than ES-2-AF peptides, HA-ES- when peptide concentration is higher than 25 μ g/mL
The inhibitory action of 2-AF conjugate groups is most obvious in three.
Test example 5：Transwell cells method is determined for transcellular inhibitory action
(1) trial drug：ES-2- prepared by the ES-2-AF peptide hyaluronic acids trim of the preparation of embodiment 2, embodiment 1
It is consistent that AF peptides and ES-2-AF peptides press peptide concentration in the mixture linked than mixing, three groups of medicines with hyaluronic acid.
(2) test method：
Sterile yellow pipette tips are put into refrigerated overnight in 4 DEG C of refrigerators.Matrigel glue is moved to and melted about in 4 DEG C of refrigerators
40min.DMEM dilution 1 of the Matrigel glue without FBS antibiotic-frees:6, add 50 μ l per hole in Transwell cells upper strata, put
Enter incubator and be incubated 1h, take out Transwell cells, suction out not solidified liquid in upper chamber, culture medium DMEM flushings are obtained with double nothings
Cell is once.Cell is inverted, and the μ l of FN (FN) 10 uniformly smear in pellicle lower floor, is placed in super-clean bench and is air-dried.24 orifice plates
Add 600 μ l (complete medium containing DMEM) per hole, cell is put into wherein；The endothelial cell EAhy926 of exponential phase is collected,
Adjustment concentration of cell suspension is 5 × 104/ holes, takes the μ l of cell 100 to be inoculated in the small interior in upper strata of Transwell cells, and add
Drug-treated cell, is incubated 24h in incubator, takes out cell, sucks upper strata culture medium, wiped away with swab stick Matrigel glue and
The cell of film is not passed through, PBS embathes one time.Cell is in fixer (methanol：Glacial acetic acid=3:1) 25min is fixed in, PBS washes one
Time, 25min is dyed with 0.1% crystal violet dye liquor, PBS is washed twice, dried, is placed under inverted fluorescence microscope and takes pictures, counting is worn
Cross the cell that Matrigel glue is colored to cell lower floor, and the figure that takes statistics.Experiment is repeated 5 times, and is averaged.
Experimental result is shown in Fig. 6, and each dosing group is compared with control group to be played the role of substantially to suppress endothelial cell migration.With
ES-2-AF peptides, HA＆ES-2-AF mixtures are minimum compared to the cell quantity that HA-ES-2-AF conjugate groups are migrated, explanation
The effect that HA-ES-2-AF suppresses cell migration is most strong.
The preferred embodiment of the application is the foregoing is only, the application is not limited to, for the skill of this area
For art personnel, the application can have various modifications and variations.It is all within spirit herein and principle, made any repair
Change, equivalent substitution, improvement etc., should be included within the protection domain of the application.
Claims (10)
1. a kind of ES-2-AF peptides, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.1 in sequence table.
2. a kind of hyaluronic acid trim of ES-2-AF peptides, it is characterised in that as the ES-2-AF peptides described in claim 1
Amino is combined into the carboxyl on hyaluronan molecule by amido link, and structural formula is as follows：
HA-CO-NH-(ES-2-AF)n；
In formula, n=30~60.
3. the hyaluronic acid trim of ES-2-AF peptides according to claim 2, it is characterised in that n=in structural formula
60。
4. the preparation method of the hyaluronic acid trim of ES-2-AF peptides according to claim 2, it is characterised in that step
It is rapid as follows：
(1) HA-TBA conjugates are prepared, improves HA dissolubility, HA is dissolved in organic solvent；
(2) HA-TBA conjugates are dissolved in organic solvent, the special condensing agent of card are added, to activate HA carboxyl；Then will activation
After HA-TBA, ES-2-AF peptide and DIPEA mixing after carboxyl, reaction 24h, NaOH solution is added；The pH of reaction system is adjusted again
3.0 are down to terminating reaction；Then the pH of reaction system is risen to 7.0 again, reaction product produces ES-2-AF through dialysing, drying
The hyaluronic acid trim of peptide.
5. preparation method according to claim 4, it is characterised in that in step (1), the preparation of the HA-TBA conjugates
Method is：Excessive TBAH is added into Dowex ion exchange resin, mixes, obtains Dowex-TBA resins,
It is filtered to remove supernatant；Sodium Hyaluronate NaHA is soluble in water, in the Dowex-TBA resins for pouring into preparation, mix, be filtered to remove
Dowex resins, the HA-TBA solution clarified freezes, produces HA-TBA conjugates.
6. preparation method according to claim 4, it is characterised in that in step (2), HA-TBA and ES-2-AF mol ratios
For 1:(4~100).
7. preparation method according to claim 4, it is characterised in that in step (2), is down to using 1M HCl regulations pH
3.0；PH is risen to 7.0 using 1M NaOH.
8. preparation method according to claim 4, it is characterised in that in step (2), the method for reaction product dialysis is：
Reaction product is poured into the bag filter of pretreatment, dialysed respectively with NaCl solution, 25% second alcohol and water.
9. the hyaluronic acid trim of the ES-2-AF peptides described in ES-2-AF peptides and/or claim 2 described in claim 1
Purposes in the medicine for suppressing new vessels generation is prepared.
10. the hyaluronic acid modification of the ES-2-AF peptides described in ES-2-AF peptides and/or claim 2 described in claim 1
Thing is preparing the purposes in generating the medicine and/or antineoplastic of disease with new vessels.
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| CN110302389A (en) * | 2019-06-18 | 2019-10-08 | 西南医科大学附属医院 | A kind of hydrogel sustained release preparation of anti-angiogenesis and its application |
| CN110358799A (en) * | 2019-07-26 | 2019-10-22 | 山东大学 | A method of based on enzymatic hyaluronic acid pointed decoration polypeptide |
| CN110713517A (en) * | 2019-10-11 | 2020-01-21 | 润辉生物技术（威海）有限公司 | Hyaluronan oligopeptide and preparation and application methods thereof |
| CN112618714A (en) * | 2020-11-30 | 2021-04-09 | 四川大学 | Anti-FLT1 polypeptide mediated synthetic gold cluster and preparation method and application thereof |
| CN113648427A (en) * | 2021-08-20 | 2021-11-16 | 山东大学 | Hyaluronic acid-ES 2-AF peptide conjugate and preparation method and application thereof |
| CN117050146A (en) * | 2023-10-11 | 2023-11-14 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modified cosmetic peptide, preparation method and application thereof |
| CN117050145A (en) * | 2023-10-11 | 2023-11-14 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modifier of cosmetic peptide and application thereof |
| CN117050145B (en) * | 2023-10-11 | 2024-05-03 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modifier of cosmetic peptide and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120294945A1 (en) * | 2011-05-16 | 2012-11-22 | Postech Academy-Industry Foundation | Drug delivery system using hyaluronic acid-peptide conjugate micelle |
| CN105669857A (en) * | 2016-03-14 | 2016-06-15 | 山东大学 | Quaternization chitosan oligosaccharide trim of ES-2 peptides and preparation method and application thereof |
| WO2017062407A1 (en) * | 2015-10-07 | 2017-04-13 | Genentech, Inc. | Systems and methods for predicting vitreal half-life of therapeutic agent-polymer conjugates |
-
2017
- 2017-06-09 CN CN201710432512.9A patent/CN107261124B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120294945A1 (en) * | 2011-05-16 | 2012-11-22 | Postech Academy-Industry Foundation | Drug delivery system using hyaluronic acid-peptide conjugate micelle |
| WO2017062407A1 (en) * | 2015-10-07 | 2017-04-13 | Genentech, Inc. | Systems and methods for predicting vitreal half-life of therapeutic agent-polymer conjugates |
| CN105669857A (en) * | 2016-03-14 | 2016-06-15 | 山东大学 | Quaternization chitosan oligosaccharide trim of ES-2 peptides and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| OH EJ等: "Anti- Flt1 peptide- hyaluronate conjugate for the treatment of retinal neovascularization and diabetic retinopathy", 《BIOMATERIALS》 * |
| 孙政伟等: "透明质酸修饰药物的研究进展", 《中国新药与临床杂志》 * |
| 徐寒梅: "人内皮抑素部分序列—ES-2的生物活性研究", 《中国药科大学学报》 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110302389A (en) * | 2019-06-18 | 2019-10-08 | 西南医科大学附属医院 | A kind of hydrogel sustained release preparation of anti-angiogenesis and its application |
| CN110302389B (en) * | 2019-06-18 | 2020-05-05 | 西南医科大学附属医院 | Anti-angiogenesis hydrogel sustained-release preparation and application thereof |
| CN110358799A (en) * | 2019-07-26 | 2019-10-22 | 山东大学 | A method of based on enzymatic hyaluronic acid pointed decoration polypeptide |
| CN110358799B (en) * | 2019-07-26 | 2021-02-26 | 山东大学 | Method for site-directed modification of polypeptide based on enzyme-catalyzed hyaluronic acid |
| CN110713517A (en) * | 2019-10-11 | 2020-01-21 | 润辉生物技术（威海）有限公司 | Hyaluronan oligopeptide and preparation and application methods thereof |
| CN110713517B (en) * | 2019-10-11 | 2022-11-01 | 润辉生物技术（威海）有限公司 | Hyaluronan oligopeptide and preparation and application methods thereof |
| CN112618714A (en) * | 2020-11-30 | 2021-04-09 | 四川大学 | Anti-FLT1 polypeptide mediated synthetic gold cluster and preparation method and application thereof |
| CN112618714B (en) * | 2020-11-30 | 2022-02-22 | 四川大学 | Anti-FLT1 polypeptide mediated synthetic gold cluster and preparation method and application thereof |
| CN113648427A (en) * | 2021-08-20 | 2021-11-16 | 山东大学 | Hyaluronic acid-ES 2-AF peptide conjugate and preparation method and application thereof |
| CN117050146A (en) * | 2023-10-11 | 2023-11-14 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modified cosmetic peptide, preparation method and application thereof |
| CN117050145A (en) * | 2023-10-11 | 2023-11-14 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modifier of cosmetic peptide and application thereof |
| CN117050145B (en) * | 2023-10-11 | 2024-05-03 | 杭州湃肽生化科技有限公司 | Hyaluronic acid modifier of cosmetic peptide and application thereof |
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