CN102199195A - Half-path charge matching amphiphilic self assembling short peptide, and its application as nanometer hemostatic material and hydrophobic drug carrier - Google Patents
Half-path charge matching amphiphilic self assembling short peptide, and its application as nanometer hemostatic material and hydrophobic drug carrier Download PDFInfo
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Abstract
The invention discloses a half-path charge matching amphiphilic self assembling short peptide, which is characterized in that: the general formula of amino acid sequences of the short peptide is Ac-Pro-X1-Phe-X2-Phe-X3-Phe-Glu-Pro-NH2, wherein X1 and X2 are polar uncharged amino acids, X3 is an amino acid with positive charges, and X1, X2 and X3 all belong to L-amino acids. The half-path charge matching amphiphilic self assembling short peptide provided in the invention can be used as a carrier material of hydrophobic drugs so as to enhance water-solubility of hydrophobic drugs, and can also be used as a nanometer hemostatic material for hemorrhagic wounds by forming a nanometer fiber barrier through self assembling. The half-path charge matching amphiphilic self assembling short peptide nano-material has the advantages of a simple and practicable production process and low cost; application of the nano-material not only enriches the species of existing nanometer medical materials, but also is of important realistic significance for life and health of mankind.
Description
Technical field
The invention belongs to nanosecond medical science material field, be specifically related to that a class half way electric charge mates amphipathic self-assembled short peptide and in the application aspect hydrophobic drug carrier and hemostatic material.
Background technology
The peptide molecule self-assembling technique is a wonderful work in the middle of the current biological nano technology, and it has become the joint of subjects such as molecular biology, chemistry, medical science, materialogy and engineering, is the important means of creating novel substance and producing new function.Nano-sized materials after the small peptide self-assembly has potential use widely in productive life, and has been successfully applied to nano biological field particularly cell engineering, organizational project, medicine controlled releasing, regenerating medicine, membranin and energy project etc.Though there are a lot of curative effects good but be subjected to using the medicine of restriction because of the problem of solubleness clinically, amphipathic self-assembled short peptide material can improve the water-soluble of medicine by interacting with hydrophobic drug, is expected to become a kind of novel hydrophobic drug solid support material.In the operation common parenchymal viscera hemorrhage be a great problem that faces clinically, if sometimes hemorrhage situation is difficult to control according to hemostasis mode commonly used with hemostatic material commonly used, and the haemostatic medicament that has needs higher operative technique, bleeding stopping period is longer, there are untoward reaction or complication, even threaten to life.The blood interaction at existing both at home and abroad small peptide material and place, blutpunkte is self-assembled into the nanofiber hydrogel fast and plays the report of anastalsis, but the disclosed energy of prior art self-assembly at present forms the structurally characteristics with omnidistance electric charge coupling of the self-assembled short peptide of nanometer water gel timbering material more, even the amphipathic small peptide of half way electric charge coupling has appearred in nearly two annual report roads, but also be to only limit to one or two limited peptide sequence, the present invention has designed a class half way electric charge mates amphipathic small peptide, their peptide chain structure mates different (the Ruan LP of amphipathic small peptide with the half way electric charge of having reported, Zhang HY, Luo HL, et al.Proc Natl Acad Sci U SA.2009,106 (13): 5105-5110; Zhao Xiaojun, Ruan Liping, Zhang Hangyu, Luo Hanlin. a kind of half electric charge matching nonapeptide and application thereof. the patent No.: CN 100543033C).
Summary of the invention
The object of the present invention is to provide a class half way electric charge to mate amphipathic self-assembled short peptide, form the netted apparatus derivatorius of nanofiber after such small peptide self-assembly,, promote the application of self-assembled short peptide to increase the self-assembled short peptide kind that amino acid makes up.
Purpose of the present invention is achieved through the following technical solutions:
One class half way electric charge mates amphipathic self-assembled short peptide, and the general formula of its aminoacid sequence is as follows:
Ac-Pro-X
1-Phe-X
2-Phe-X
3-Phe-Glu-Pro-NH
2
Ac is acetylize N end, NH
2Be amination C end, wherein X
1, X
2Be polarity neutral amino acid, X
3Be positively charged amino acid, X
1, X
2, X
3Complete is L type amino acid, X
1, X
2For identical or different amino acid all can.
Preferably, described polarity neutral amino acid is Thr, Ser, Cys or Asn.
Preferably, described positively charged amino acid is Arg or His.
Its self-assembly principle be small peptide intermolecular by non-covalent interaction spontaneously be combined to form that a kind of structure is clear and definite, construction of stable, molecule aggregates or supramolecular structure with certain physicochemical property, the introducing of charge residue is to form antiparallel and twine in order to produce the electric charge coupling.
Small peptide of the present invention can adopt solid-phase peptide synthesis to synthesize and produce: the amino acid with the Fmoc protection is raw material; Rink Amide-MBHA Resin (Rake acid amides-mbha resin) is a carrier; 20% piperidines/DMF solution is deprotection agent; HBTU is the peptide bond condensing agent; (V: V) solution is aminoterminal acetylize solvent to 50% aceticanhydride/pyridine; coupling is synthetic successively to hold the order of N end by known array from C, the N-terminal acetylize.End of synthesis takes off reaction flask, uses washed with methanol resin 8 times, removes DMF.Nitrogen dries up the back and will synthesize polypeptide cracking from the resin with TFA strong acid lysate and get off, and sloughs all blocking groups simultaneously.Reaction finishes the back and collects the lysate that is dissolved with synthetic peptide, and filtration under diminished pressure is collected filtrate then, with the polypeptide of cold diethyl ether resolution of precipitate in filtrate, promptly gets the crude product that synthesizes peptide.Synthetic peptide crude product is collected main peak through high-pressure liquid phase reversed phase chromatography column separating purification, after the lyophilize, obtains the synthetic small peptide of target.
Half way electric charge of the present invention mates amphipathic self-assembled short peptide and is applied in hydrophobic drug solid support material and the nanometer hemostatic material.Interact with lyophobic dust in the small peptide self assembling process, can improve the water-soluble of lyophobic dust, thereby can become a kind of novel hydrophobic drug carrier.After experiment shows that also the peptide aqueous solution with the lyophilized powder of amphipathic small peptide or different mass volume ratio acts on damaged animal soft tissue tangent plane, form hydrogel immediately, the sealing blood outlet, play the effect of quick-acting haemostatic powder and wound care, and small peptide consists of natural amino acid, be easy to degraded in vivo, the product non-immunogenicity, histocompatibility is good, therefore the small peptide material preparation can be become a kind of nanometer haemostatic medicament that is suitable for using clinically by adding pharmaceutically acceptable stablizer or vehicle.
The present invention has following usefulness:
1, provide the half way electric charge of a class formation novelty to mate amphipathic small peptide, enriched the kind of self-assembled short peptide nano material, described amphipathic small peptide both can be used as the solid support material of hydrophobic drug and had improved that it is water-soluble, can form the nano-scale fiber barrier by self-assembly again, as the nanometer hemostatic material of the hemorrhage surface of a wound, can play the effect of quick-acting haemostatic powder and wound care.
2, amphipathic self-assembled short peptide material produce technology simple possible of the present invention, cost are relatively low, and its application can not only be enriched existing nanometer medical material kind, and significant to human life's health.
Description of drawings
Fig. 1 is that half way electric charge of the present invention mates the 1. high performance liquid phase of sample (HPLC) color atlas of amphipathic small peptide, and the result shows its purity 〉=95%.
Fig. 2 is that half way electric charge of the present invention mates the 1. mass spectrum of sample (MS) figure of amphipathic small peptide, and the result shows that its molecular weight is 1195.7, and the theoretical value (1195.3) of this and it is very close.
Fig. 3 is that half way electric charge of the present invention mates the 1. three-dimensional molecular structure model synoptic diagram of sample of amphipathic small peptide, employing to be MDL ISIS Draw 2.5 softwares draw based on the minimum principle of energy.
Fig. 4 is that half way electric charge of the present invention mates 1. circular dichroism (CD) spectrogram of sample under 25 ℃ of amphipathic small peptide.
Fig. 5 be half way electric charge of the present invention mate amphipathic small peptide 1. sample through the circular dichroism spectrogram under ultrasonic back 25 ℃.
Fig. 6 is that half way electric charge of the present invention mates the 1. shape appearance figure of sample ultrapure water solution under atomic force microscope of amphipathic small peptide.
Fig. 7 is that half way electric charge of the present invention mates 1. sample and the ultrasonic back of the hydrophobic drug model pyrene shape appearance figure under atomic force microscope of amphipathic small peptide.
Fig. 8 is that half way electric charge of the present invention mates the 1. transmission electron microscope picture of sample ultrapure water solution of amphipathic small peptide.With ultrapure water its preparation is become 0.3mg/ml, adopt the phospho-wolframic acid negative staining to prepare sample, viewed morphological structure matches under pattern that shows among the figure and the atomic force microscope.
Fig. 9 is the shape appearance figure that the pyrene aqueous solution of 0.4mg/ml is watched under atomic force microscope after stirring three days on the magnetic stirring apparatus.
Figure 10 be half way electric charge of the present invention mate amphipathic small peptide 1. sample be used for the hemostasis lab diagram of SD rats'liver tangent plane.1% (w/v) peptide water liquid is injected wound surface, form a transparent spawn immediately, the sealing blood outlet reaches rapid hematostatic purpose.
Embodiment
For a better understanding of the present invention, the invention will be further described below in conjunction with embodiment, and embodiment further explains of the present invention, but the scope of protection of present invention is not limited to the scope that embodiment represents.
Work as X
1Be Thr, X
2Be Asn, X
3During for Arg, sequence is as follows:
①CH
3CO-Pro-Thr-Phe-Asn-Phe-Arg-Phe-Glu-Pro-NH
2
1. synthetic of self-assembled short peptide:
1, material
Fmoc-Pro-OH (N-fluorenylmethyloxycarbonyl-proline(Pro)); Fmoc-Thr (tBu)-OH (fluorenylmethoxycarb-nyl-nyl O-tert-butyl-Threonine); Fmoc-Phe-OH (N-fluorenylmethyloxycarbonyl-phenylalanine); Fmoc-Asn (Trt)-OH (fluorenylmethyloxycarbonyl-N-trityl-l-asparagine); Fmoc-Glu (OtBu)-OH (fluorenylmethoxycarb-nyl-nyl O-tert-butyl-L-glutamic acid); Fmoc-Arg (pbf)-OH (N-fluorenes methoxy carbonyl acyl group-2; 2; 4; 6,7-pentamethyl-Dihydrobenzofuranes-5-sulphonyl-arginine); Rink Amide-MBHA Resin (resin); HBTU (O-benzotriazole-1-base-N; N; N; N-tetramethyl-urine phosphofluoric acid fat) and HOBT (1-hydroxy benzo triazole) available from Shanghai gill biochemistry company limited.Piperidines, acetic anhydride, DMF (N, dinethylformamide), TFA (trifluoroacetic acid), NMM (N-methylmorpholine), ether, methyl alcohol and DCM (methylene dichloride) are available from Tianjin Fu Yu Fine Chemical Co., Ltd.
2, preparation method
Adopt the solid-phase synthesis of Fmoc (fluorenylmethyloxycarbonyl) protection, its processing step is as follows:
(1) takes by weighing the Rink Amide-MBHA Resin of 20g 0.5mmol/g in the peptide synthesizer ware, get 200ml DMF swelling resin 30min, suction filtration then, take 300ml DMF washing resin again, divide and carry out for three times, each washing time is 2min, in peptide synthesizer, add 100ml20% piperidines/DMF (V: V) concussion reaction 30min behind the suction filtration dry cleaning liquid, after reaction finishes, suction filtration goes out reaction solution again, divides washing resin four times with 400ml DMF again, washes the resin that takes a morsel after finishing and does triketohydrindene hydrate and detect test, resin is positive, and adds following raw material in the peptide synthesizer ware:
After the above raw material adds, shake reaction 30min, reaction divides washing resin four times with 300ml DMF after finishing, and each washing time 2 minutes is got a little resin and done the ninhydrin reaction detection, and resin is negative.
(2) in the peptide synthesizer ware, add 5ml 20% piperidines/DMF (V: V) concussion reaction 30min, reaction finishes the back suction filtration and goes out reaction solution, divide washing resin four times with 40ml DMF again, wash complete a little resin of getting and do the ninhydrin reaction detection, resin is positive as a result, adds following raw material in reaction vessels:
After above-mentioned raw materials adds, shake reaction 40min, reaction divides washing resin four times with 40ml DMF after finishing, and each washing time is 2min, gets a little resin and does the triketohydrindene hydrate detection, and resin is negative.
(3) (a) raw material in the shift step (2), (b) (c) (d) (e) raw material and add-on are constant, the operation of repeating step (2); (a) raw material replaces with Fmoc-Phe-OH (15.48g), Fmoc-Arg (pbf)-OH (25.93g), Fmoc-Phe-OH (15.48g), Fmoc-Asn (Trt)-OH (23.85g), Fmoc-Phe-OH (15.48g), Fmoc-Thr (tBu)-OH (15.89g), Fmoc-Pro-OH (13.49g) successively.Be the operation of a step of every repetition (2), conversion a kind of (a) raw material is till all using above-mentioned 7 kinds of raw materials once;
Repeat once the operation of (1) (2) (3) step again, the raw material of each step and raw materials used amount are all constant; Behind last Pro end of synthesis, deviate from the Fmoc-protecting group, add 20% piperidines/DMF (V: V) reaction 30min; eluted resin adds 160ml 50% acetic anhydride/DMF (V: V) react 30min, with 40ml DMF eluted resin; use the methanol wash resin again 8 times, remove DMF.After nitrogen dries up; with TFA (trifluoroacetic acid)/DCM strong acid lysate; cracking 3 hours will be synthesized polypeptide cracking from the resin and get off, so sloughed blocking group simultaneously; collection is dissolved with the lysate of synthetic peptide; filtration under diminished pressure is collected filtrate then, with the polypeptide of cold diethyl ether resolution of precipitate in filtrate; suction filtration obtains white solid again, promptly gets the crude product that synthesizes peptide.Synthetic peptide crude product is collected main peak through HPLC (high performance liquid chromatography) purifying, through after the lyophilize, promptly obtains the target small peptide.
Can also synthesize following sequence small peptide by above-mentioned same program:
X
1Be Thr, X
2Be Ser, X
3During for His, sequence is as follows:
②CH
3CO-Pro-Thr-Phe-Ser-Phe-His-Phe-Glu-Pro-NH
2
X
1Be Asn, X
2Be Asn, X
3During for His, sequence is as follows:
③CH
3CO-Pro-Asn-Phe-Asn-Phe-His-Phe-Glu-Pro-NH
2
X
1Be Ser, X
2Be Ser, X
3During for Arg, sequence is as follows:
④CH
3CO-Pro-Ser-Phe-Ser-Phe-Arg-Phe-Glu-Pro-NH
2
Fig. 1 be synthetic small peptide purifying HPLC collection of illustrative plates 1., Fig. 2 is synthetic small peptide MS collection of illustrative plates 1., Fig. 3 is a small peptide three-dimensional molecular model 1., carbon atom be a white, Sauerstoffatom is a redness, the nitrogen-atoms blueness is by this figure each amino acid whose spatial distribution as can be known.Fig. 4 is the circular dichroism spectrogram of this synthetic small peptide 0.21mg/ml aqueous solution 1..CD spectrum adopts Applied photophysics CD spectrograph, under 25 ℃, carry out the scanning collection signal, use the light path quartz cuvette of 1mm, sweep blank quartz cuvette earlier to check the baseline of CD signal, and the CD signal of scanning ultrapure water quartz cuvette is with as the sample background signal, and all data all deduct time background signal by it and revise.The wave spectrum scope of data gathering is 190-260nm, and step-length is 1nm, and data value is that data results is represented with average residue molar ellipticity with the mean value note of 1s in the bandwidth range of 1nm.Fig. 5 be with the small peptide solution of the same race under the concentration through behind the continuous ultrasound 2h, place the CD collection of illustrative plates after spending the night, therefrom what considerable change is its secondary structure do not have as can be seen.
Small peptide is CH 1.
3CO-Pro-Thr-Phe-Asn-Phe-Arg-Phe-Glu-Pro-NH
2Nanostructure in the aqueous solution after the self-assembly and the application in preparation dewatering medicament carrier thereof
1) atomic force microscope (AFM) detects
Use atomic force microscope AFM observe amphipathic small peptide solution different concns under 1. itself reach with the ultrasonic mixed nanostructure of an amount of pyrene.Method: get and be stored in 4 ℃ of concentration in the refrigerator 1. solution of the small peptide that is 1mg/ml, with 18.2K Ω/cm
2Ultrapure water is mixed with the peptide aqueous solution of 0.5mg/ml, 0.3mg/ml, 0.1mg/ml with its dilution, and without any processing, the pattern of they threes under atomic power is shown in a, b, c among Fig. 6; Get and be stored in 4 ℃ of concentration in the refrigerator 1. solution of the small peptide that is 1mg/ml, with 18.2K Ω/cm
2After ultrapure water is mixed with the peptide aqueous solution of 0.5mg/ml, 0.3mg/ml, 0.1mg/ml with the small peptide dilution, they are spent the night respectively with the rearmounted dark place of the pyrene mixing continuous ultrasound 3h of amount.The small peptide solution of different concns and pyrene after ultrasonic mixing the with amount, the structure and morphology under atomic force microscope is different, shown in a among Fig. 7, b, c.Wherein 7 (a) figure is the peptide aqueous solution and 10 of 0.5mg/ml
-6The aspect graph of the ultrasonic back of M pyrene under atomic force microscope, what show among the figure is the mesh nano fibrous texture that is joined together to form by spheroidal aggravation.7 (b) figure is the peptide aqueous solution and 10 of the 0.3mg/ml after ultrasonic
-6The aspect graph of M pyrene under atomic force microscope, that show among the figure is the netted apparatus derivatorius figure of nanofiber.7 (c) figure is the peptide aqueous solution and 10 of ultrasonic back 0.1mg/ml
-6The aspect graph of M pyrene under atomic force microscope, what show among the figure is still the netted apparatus derivatorius figure of nanofiber, and compare the fiber apparatus derivatorius with 7 (b) figure intensive like that.We with a, the b of Fig. 6, c three figure respectively with add the pyrene of equivalent in the small peptide aqueous solution of concentration as can be seen after a, b, c three figure of Fig. 7 compare after, the width average of their netted apparatus derivatoriuses of nanofiber under the atomic power fiber big than before not adding pyrene, and small peptide concentration is high more, its width average also big more (as following table).This is because small peptide material and hydrophobic drug model pyrene overlay on the surface of model drug pyrene after by the hydrophobic interaction self-assembly, thereby its nanometer particle size is become greatly.
| Different sample solutions | Width average (nm) |
| 0.5mg/ml peptide+10-6M pyrene | 103.77±2.63 |
| 0.3mg/ml peptide+10-6M pyrene | 70.62±2.14 |
| 0.1mg/ml peptide+10-6M pyrene | 68.92±1.89 |
| 0.5mg/ml peptide | 81.92±2.85 |
| 0.3mg/ml peptide | 24.35±1.79 |
| 0.1mg/ml peptide | 25.68±1.36 |
Fig. 9 is the shape appearance figure that the pyrene aqueous solution of 0.4mg/ml is watched under atomic force microscope after stirring three days on the magnetic stirring apparatus.It is a random non-nano fibrous web-like apparatus derivatorius, and further the small peptide material can interact with dewatering medicament model pyrene in self assembling process certainly, and medicine carrying is more obvious, i.e. the water-soluble remarkable enhancing of hydrophobic drug model pyrene.
On the mica of newly opening, pipette earlier the solution liquid to be measured of 8-10ul during preparation afm scan sample with liquid-transfering gun, after leaving standstill 1min, ultrapure water with 200ul washes the solution that does not adsorb on this sheet mica surface, covers mark with the culture dish after the sterilization then and puts dried overnight under the room temperature good number.Atomic force microscope is at room temperature to scan, use be the NanoScope of U.S. Veeco company, carry out with the pattern of rapping, the size of sample among Fig. 6 and Fig. 7 scanning adopts 5 μ mx5 μ m to observe respectively, Fig. 8 is the range of observation of 3 μ mx3 μ m.
2) transmission electron microscope (TEM) detects
Use the Jeol Jem-1400 transmission electron microscope of NEC company and under the acceleration voltage of 120kv, observe the 1. nanostructure of the aqueous solution of small peptide.Peptide solution 18.2K Ω/cm with 1mg/ml
2The Milli-Q ultrapure water is diluted to concentration 0.3mg/ml to be measured.Earlier pipette the peptide aqueous sample of 10ul in the surface of cleaning during preparation transmission electron microscope scanning samples with liquid-transfering gun, the copper mesh that will be covered with polyvinyl formal film (Formvar) then immerses twice each 10s of peptide solution, inhales with filter paper and goes copper mesh periphery excess liquid.1% phospho-wolframic acid of pH6.5 is dripped in clean surface, simultaneously with the wiping surface negative staining 15s of copper mesh, inhale with filter paper immediately then and remove unnecessary liquid, utilize electron microscope under the acceleration voltage of 120kv, to observe after room temperature is dried naturally carry out light field with the painted sample of phospho-wolframic acid.The small peptide solution morphology figure of 0.3mg/ml as shown in Figure 8 under the transmission electron microscope, magnification is 3.3 ten thousand times, show be this half way electric charge to mate what form after the amphipathic small peptide self-assembly be the netted apparatus derivatorius of nanofiber, this result with can match well with the aspect graph of concentration small peptide solution under atomic force microscope.
Simultaneously, 4. 3. 2. other half way electric charge coupling short peptide sequences among the present invention all can be self-assembled into the netted apparatus derivatorius of nanofiber, the 1. the same solid support material that all can be used as dewatering medicament with small peptide in the aqueous solution.
Embodiment 3 small peptides 1., 2., 3., 4. in the application of preparation in the haemostatic medicament
Judge the haemostatic effect of four kinds of small peptides according to amount of bleeding and bleeding stopping period.In the present embodiment, laboratory animal is the SPF level SD rat between 290g～310g, the male and female stochastic distribution, and all laboratory animal are all available from Nanfang Medical Univ experimentation on animals center.All laboratory animal are fasting 24h (freely drinking water) before operation, and all animals are injected the vetanarcol soup of various dose by body weight before test.Used cotton swab is a medical cotton stick in the experiment, the cotton yarn autoclaving is handled, cotton swab, cotton are placed on respectively in the sterilization clean vial and before experiment and weigh, after using the cotton swab cotton, put back in the bottle and seal, prevent that water loss is in air in the blood, the gross weight of weighing experiment back bottle can draw amount of bleeding, the bottle of record amount of bleeding=last weighing and the suck blood weight-dry cotton swab of cotton swab and cotton yarn and the weight of cotton yarn and bottle.All data in this experiment all adopt (
) represent that data processing adopts Wilks ' s λ check (processing of SAS 8.0for Windows statistical package).
Used small peptide hemostatic material can be the peptide aqueous solution of small peptide lyophilized powder or different mass volume ratio in the experimental group, and the peptide aqueous solution is with 18.2K Ω/cm
2The Milli-Q ultrapure water is mixed with and forms, and this peptide aqueous solution ultrasonic several minutes in ultrasonic washing instrument can be removed viscosity, and bubble can be removed by high speed centrifugation, 4 ℃ of preservations.
1, with the small peptide of 1% (w/v)
1. (CH
3CO-Pro-Thr-Phe-Asn-Phe-Arg-Phe-Glu-Pro-NH
2) be used for SD rat damaged liver section hemostasis
Be divided into following three groups according to the hemostasis mode:
1) tests with cotton yarn group (n=10, blank group): behind the rat anesthesia, the careful dissection is with the suitably big minor cut or wound of sterilization blade cutting-up on its lobe of the liver, wound is bled behind the 5s naturally, with cotton swab blood is wiped, pushed down wound with the sterilization cotton yarn that shears size again, behind the applying light 30s, the blood that oozes out around drawing with cotton swab during this time, remove cotton yarn and observe the situation of bleeding of wound surface, hemorrhage as the continuation of wound in 10s, then continue to push hemostasis till hemostasis is finished with cotton yarn.If total time surpasses 10min, then look this group hemostasis the failure of an experiment.
2) 1% (w/V) peptide water group (n=10, experimental group): peptide mates 1. Ac-Pro-Thr-Phe-Asn-Phe-Arg-Phe-Glu-Pro-NH of amphipathic small peptide for the half way electric charge of choosing from the present invention
2Sample, behind the rat anesthesia, the careful dissection, with the suitably big minor cut or wound of blade cutting-up on its lobe of the liver, wound is bled behind the 5s naturally, with cotton swab blood is wiped, this moment with off-the-shelf small peptide 1. solution inject wound, observe hemorrhage situation.Find to form a spawn in the damaged liver surface, adhere to the damaged surface, as shown in figure 10, reach haemostatic effect fast when peptide liquid and wound rigidly connect to touch promptly, during the blood that oozes out around drawing with cotton swab finish until the experimentation that stops blooding.
3) hemostasis gelatin group (n=10, experiment contrast group) experimentation is the same.Record bleeding stopping period (hemostasis fully), the weight with weighting method weighing gauze and cotton swab calculates blood volume.
The different hemostatic materials of table 1 are to the hemorrhage anastalsis of SD rat damaged liver section
| The hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| Cotton yarn | ?10 | 125±19 | 1.49±0.22 |
| 1% (w/v) peptide water | ?10 | 13±3 | 0.31±0.15 |
| The hemostasis gelatin | ?10 | 20±2 | 0.82±0.21 |
The result shows (seeing Table 1), 1% (w/v) peptide water group is rapid-action with the anastalsis that control group (cotton yarn group and hemostasis gelatin group) is compared generation, haemostatic effect is definite, can significantly reduce the hemorrhage blood loss of rat damaged liver section, obviously shorten bleeding stopping period, therefore, 1. small peptide of the present invention can develop into a kind of hemorrhage novel hemostatic material of parenchymal viscera that is suitable for clinical use.
2, with 2. (CH of small peptide
3CO-Pro-Thr-Phe-Ser-Phe-His-Phe-Glu-Pro-NH
2) lyophilized powder be used for SD rat damaged liver section hemostasis
Hemostasis experimental implementation as above 1 has changed the peptide aqueous solution lyophilized powder of peptide into, and control group is constant, writes down the bleeding time (bleeding stopping period fully) with stopwatch, and the blood that oozes out around drawing with cotton swab in hemostasis is finished until the hemostasis experimentation.Use the weight of weighting method weighing gauze and cotton swab at last, calculate blood volume.
The different hemostatic materials of table 2 are to the hemorrhage anastalsis of SD rat damaged liver section
| The hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| Cotton yarn | ?10 | ?128±17 | 1.51±0.21 |
| The small peptide lyophilized powder | ?10 | ?10±2 | 0.28±0.11 |
| The hemostasis gelatin | ?10 | ?21±2 | 0.83±0.20 |
The result shows (seeing Table 2), small peptide 2. lyophilized powder and control group (common cotton yarn group and hemostasis gelatin group) compares that the anastalsis of generation is rapid-action, haemostatic effect is definite, can significantly reduce the hemorrhage blood loss of rat damaged liver section, obviously shorten bleeding stopping period, therefore, 2. small peptide of the present invention can develop into a kind of hemorrhage novel hemostatic material of parenchymal viscera that is suitable for clinical use.
3, with 3. (CH of the small peptide of 0.7% (w/V)
3CO-Pro-Asn-Phe-Asn-Phe-His-Phe-Glu-Pro-NH
2) be used for the hemorrhage hemostasis of SD rat damaged liver section
The hemostasis experimental implementation is as shown in 1, the 1. peptide aqueous solution of 1% (w/v) has been changed into the 3. peptide aqueous solution of 0.7% (w/v), control group is constant, writes down the bleeding time (bleeding stopping period fully) with stopwatch, draws the blood that oozes out on every side with cotton swab and finish until the hemostasis experimentation in hemostasis.Use the weight of weighting method weighing gauze and cotton swab at last, calculate blood volume.
The different hemostatic materials of table 3 are to the hemorrhage anastalsis of SD rat damaged liver section
| The hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| Cotton yarn | ?10 | 125±19 | 1.49±0.22 |
| 0.7% (w/V) peptide water | ?10 | 16±2 | 0.42±0.15 |
| The hemostasis gelatin | ?10 | 19±2 | 0.82±0.21 |
The result shows (seeing Table 3), 0.7% (w/V) peptide water group is rapid-action with the anastalsis that control group (cotton yarn group and hemostasis gelatin group) is compared generation, haemostatic effect is definite, can significantly reduce the hemorrhage blood loss of rat damaged liver section, obviously shorten bleeding stopping period, therefore, 3. small peptide of the present invention can develop into a kind of hemorrhage novel hemostatic material of parenchymal viscera that is suitable for clinical use.
4, with 4. (CH of the small peptide of 2% (w/V)
3CO-Pro-Ser-Phe-Ser-Phe-Arg-Phe-Glu-Pro-NH
2) be used for the hemorrhage hemostasis of SD rat damaged liver section
The hemostasis experimental implementation is as shown in 1, the 1. peptide aqueous solution of 1% (w/v) has been changed into the 4. peptide aqueous solution of 2% (w/v), control group is constant, writes down the bleeding time (bleeding stopping period fully) with stopwatch, draws the blood that oozes out on every side with cotton swab and finish until the hemostasis experimentation in hemostasis.Use the weight of weighting method weighing gauze and cotton swab at last, calculate blood volume.
The different hemostatic materials of table 4 are to the hemorrhage anastalsis of SD rat damaged liver section
| The hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| Cotton yarn | ?10 | 124±18 | 1.48±0.19 |
| 2% (w/V) small peptide | ?10 | 10±2 | 0.27±0.12 |
| The hemostasis gelatin | ?10 | 21±2 | 0.81±0.22 |
The result shows (seeing Table 4), 4. 2% (w/V) small peptide compares with control group (cotton yarn group and hemostasis gelatin group) that the anastalsis of generation is rapid-action, haemostatic effect is definite, can significantly reduce the hemorrhage blood loss of rat damaged liver section, obviously shorten bleeding stopping period, therefore, 4. small peptide of the present invention can develop into a kind of hemorrhage novel hemostatic material of parenchymal viscera that is suitable for clinical use.
By four groups of above hemostasis experiments as can be known, four kinds of small peptide materials all can be used as and are effective to the hemorrhage novel hemostatic material of SD rat damaged liver section, wherein high more its haemostatic effect of the concentration of peptide water is good more, and the lyophilized powder of small peptide material is better than the haemostatic effect of the peptide aqueous solution to a certain extent.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. a class half way electric charge mates amphipathic self-assembled short peptide, it is characterized in that the general formula of its aminoacid sequence is as follows:
Ac-Pro-X
1-Phe-X
2-Phe-X
3-Phe-Glu-Pro-NH
2
X wherein
1, X
2Be polarity neutral amino acid, X
3Be positively charged amino acid, X
1, X
2, X
3Complete is L type amino acid.
2. according to the described small peptide of claim 1, it is characterized in that described polarity neutral amino acid is Thr, Ser, Cys or Asn.
3. according to claim 1 or 2 described small peptides, it is characterized in that described positively charged amino acid is Arg or His.
4. small peptide described in any one of the claim 1～3 is as the application in the hydrophobic drug solid support material.
5. small peptide described in any one of the claim 1～3 is as the application in the nanometer hemostatic material.
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Cited By (4)
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| CN103012558A (en) * | 2012-06-28 | 2013-04-03 | 华南理工大学 | Cation symmetrical amphipathic self-assembled polypeptide and application thereof |
| CN103788177A (en) * | 2014-01-27 | 2014-05-14 | 四川大学 | Amphipathic nonapeptide and application thereof as drug carrier |
| CN105085622A (en) * | 2015-09-16 | 2015-11-25 | 中国石油大学(华东) | Amphipathic self-assembly ultra-short peptide nano hemostatic material |
| CN111848736A (en) * | 2020-08-05 | 2020-10-30 | 赛克赛斯生物科技股份有限公司 | Self-assembly polypeptide, preparation method, self-assembly polypeptide preparation and application |
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| CN101037471A (en) * | 2007-04-12 | 2007-09-19 | 四川大学 | Half electric charge matching nonapeptide and application thereof |
| CN101939329A (en) * | 2007-08-30 | 2011-01-05 | 滑铁卢大学 | Amino acid pairing-based self assembling peptides and methods |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103012558A (en) * | 2012-06-28 | 2013-04-03 | 华南理工大学 | Cation symmetrical amphipathic self-assembled polypeptide and application thereof |
| CN103012558B (en) * | 2012-06-28 | 2014-12-03 | 华南理工大学 | Cation symmetrical amphipathic self-assembled polypeptide and application thereof |
| CN103788177A (en) * | 2014-01-27 | 2014-05-14 | 四川大学 | Amphipathic nonapeptide and application thereof as drug carrier |
| CN103788177B (en) * | 2014-01-27 | 2016-02-17 | 四川大学 | A kind of amphipathic nonapeptide and the application as pharmaceutical carrier thereof |
| CN105085622A (en) * | 2015-09-16 | 2015-11-25 | 中国石油大学(华东) | Amphipathic self-assembly ultra-short peptide nano hemostatic material |
| CN111848736A (en) * | 2020-08-05 | 2020-10-30 | 赛克赛斯生物科技股份有限公司 | Self-assembly polypeptide, preparation method, self-assembly polypeptide preparation and application |
| CN111848736B (en) * | 2020-08-05 | 2021-12-31 | 赛克赛斯生物科技股份有限公司 | Self-assembly polypeptide, preparation method, self-assembly polypeptide preparation and application |
| WO2022028036A1 (en) * | 2020-08-05 | 2022-02-10 | 赛克赛斯生物科技股份有限公司 | Self-assembling polypeptide, preparation method therefor, self-assembling polypeptide preparation and use thereof |
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