CN102199195B - Half-path charge matching amphiphilic self assembling short peptide, and application thereof as nanometer hemostatic material and hydrophobic drug carrier - Google Patents
Half-path charge matching amphiphilic self assembling short peptide, and application thereof 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 Nano Medicine Materials field, be specifically related to an amphipathic self-assembled short peptide of class half way charge matching and in the application aspect hydrophobic drug carrier and hemostatic material.
Background technology
Peptide molecule self-assembling technique is a wonderful work in the middle of current biological nano technology, and it has become the joint of the 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 small peptide self-assembly has potential use widely in productive life, and has been successfully applied to nanometer biological field particularly cell engineering, organizational project, medicine controlled releasing, regenerating medicine, membranin and energy project etc.Though there are clinically a lot of curative effects good but be subject to using the medicine of restriction because of the problem of solubleness, amphipathic self-assembled short peptide material can, by interacting and improve the water-soluble of medicine with hydrophobic drug, be expected to become a kind of novel hydrophobic drug solid support material.In operation common parenchymal viscera hemorrhage be a great problem facing clinically, if sometimes bleeding is difficult to control according to conventional hemostasis mode with conventional hemostatic material, and the haemostatic medicament having needs higher operative technique, bleeding stopping period is longer, there is untoward reaction or complication, even threaten to life.The blood interaction Rapid self assembly that both at home and abroad existing small peptide material is located with blutpunkte becomes nanofiber hydrogel and plays the report of anastalsis, but the self-assembly of the disclosed energy of currently available technology forms the self-assembled short peptide of nanometer water gel timbering material structurally has the features of omnidistance charge matching more, even there is the amphipathic small peptide of half way charge matching in nearly two annual report roads, but be also to only limit to one or two limited peptide sequence, the present invention has designed the amphipathic small peptide of a class half way charge matching, their peptide chain structure and different (the Ruan LP of the amphipathic small peptide of half way charge matching that 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 the amphipathic self-assembled short peptide of a class half way charge matching, form the netted apparatus derivatorius of nanofiber after such small peptide self-assembly, the self-assembled short peptide kind building to increase amino acid, promotes the application of self-assembled short peptide.
Object of the present invention is achieved through the following technical solutions:
The amphipathic self-assembled short peptide of one class half way charge matching, 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
2for amination C end, wherein X
1, X
2for polarity neutral amino acid, X
3for 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 be spontaneously combined to form that a kind of structure is clear and definite, construction of stable, the molecule aggregates with certain physicochemical property or supramolecular structure, the introducing of charge residue is to form antiparallel and be wound around in order to produce charge matching.
Small peptide of the present invention can adopt solid-phase peptide synthesis synthesize and produce: the amino acid of the Fmoc of take protection is raw material; Rink Amide-MBHA Resin (Rake acid amides-mbha resin) is carrier; 20% piperidines/DMF solution is deprotection agent; HBTU is peptide bond condensing agent; 50% aceticanhydride/pyridine (V: V) solution is aminoterminal acetylize solvent; coupling is synthetic successively for the order of holding N to hold from C by known array, N-terminal acetylize.End of synthesis takes off reaction flask, uses washed with methanol resin 8 times, removes DMF.Nitrogen dries up and with TFA strong acid lysate, will synthesize polypeptide cracking from resin and get off afterwards, sloughs all blocking groups simultaneously.Reaction finishes the lysate that rear collection is dissolved with synthetic peptide, and then filtration under diminished pressure, collects filtrate, uses the polypeptide of cold diethyl ether resolution of precipitate in filtrate, obtains the crude product of synthetic peptide.Synthetic peptide crude product, through high-pressure liquid phase reversed phase chromatography column separating purification, is collected main peak, after lyophilize, obtains the synthetic small peptide of target.
The amphipathic self-assembled short peptide of half way charge matching of the present invention is applied in hydrophobic drug solid support material and nanometer hemostatic material.In small peptide self assembling process, interact with lyophobic dust, can improve the water-soluble of lyophobic dust, thereby can become a kind of novel hydrophobic drug carrier.Experiment also shows the peptide aqueous solution of the lyophilized powder of amphipathic small peptide or different mass volume ratio to act on after damaged animal soft tissue tangent plane, form immediately hydrogel, sealing blood outlet, play the effect of quick-acting haemostatic powder and wound care, and small peptide consists of natural amino acid, be easy in vivo degraded, product non-immunogenicity, histocompatibility is good, therefore by adding pharmaceutically acceptable stablizer or vehicle, small peptide material can be prepared into a kind of nanometer hemostatic medicine that is suitable for using clinically.
The present invention has following usefulness:
1, the amphipathic small peptide of half way charge matching of one class formation novelty is provided, 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 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 technique 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.
Accompanying drawing explanation
Fig. 1 is the 1. high performance liquid phase of sample (HPLC) color atlas of the amphipathic small peptide of half way charge matching of the present invention, and result shows its purity >=95%.
Fig. 2 is the 1. mass spectrum of sample (MS) figure of the amphipathic small peptide of half way charge matching of the present invention, and 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 the 1. three-dimensional molecular structure model schematic diagram of sample of the amphipathic small peptide of half way charge matching of the present invention, employing be that MDL ISIS Draw 2.5 softwares are drawn based on minimum energy principle.
Fig. 4 is 1. circular dichroism (CD) spectrogram of sample at 25 ℃ of the amphipathic small peptide of half way charge matching of the present invention.
Fig. 5 is 1. sample circular dichroism spectrogram at 25 ℃ after ultrasonic of the amphipathic small peptide of half way charge matching of the present invention.
Fig. 6 is the 1. shape appearance figure of sample ultrapure water solution under atomic force microscope of the amphipathic small peptide of half way charge matching of the present invention.
Fig. 7 be the amphipathic small peptide of half way charge matching of the present invention 1. sample and hydrophobic drug model pyrene ultrasonic after shape appearance figure under atomic force microscope.
Fig. 8 is the 1. transmission electron microscope picture of sample ultrapure water solution of the amphipathic small peptide of half way charge matching of the present invention.With ultrapure water, its preparation is become to 0.3mg/ml, adopt phospho-wolframic acid negative staining to prepare sample, under the pattern showing in figure and atomic force microscope, viewed morphological structure matches.
Fig. 9 is that the pyrene aqueous solution of 0.4mg/ml stirs the shape appearance figure of watching under atomic force microscope after three days on magnetic stirring apparatus.
Figure 10 be the amphipathic small peptide of half way charge matching of the present invention 1. sample for the hemostasis lab diagram of SD rats'liver tangent plane.1% (w/v) peptide water liquid is injected to wound surface, form immediately a transparent spawn, seal blood outlet, reach the object of rapid hemostasis.
Embodiment
For a better understanding of the present invention, below in conjunction with embodiment, the invention will be further described, 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
1for Thr, X
2for 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) purchased 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 purchased from Tianjin Fu Yu Fine Chemical Co., Ltd.
2, preparation method
The solid-phase synthesis that adopts Fmoc (fluorenylmethyloxycarbonyl) protection, its processing step is as follows:
(1) the Rink Amide-MBHA Resin that takes 20g 0.5mmol/g is in peptide synthesizer ware, get 200ml DMF swelling resin 30min, then suction filtration, take again 300ml DMF washing resin, divide and carry out for three times, each washing time is 2min, in the backward peptide synthesizer of suction filtration dry cleaning liquid, add 100ml20% piperidines/DMF (V: V) concussion reaction 30min, after reaction finishes, suction filtration goes out reaction solution again, with 400ml DMF, divide washing resin four times again, washing the resin that takes a morsel after finishing does triketohydrindene hydrate and detects test, resin is positive, to adding following raw material in peptide synthesizer ware:
After the above raw material adds, concussion reaction 30min, after reaction finishes, divides washing resin four times with 300ml DMF, and each washing time 2 minutes is got a little resin and done ninhydrin reaction detection, and resin is negative.
(2) in peptide synthesizer ware, add 5ml 20% piperidines/DMF (V: V) concussion reaction 30min, reaction finishes rear suction filtration and goes out reaction solution, with 40ml DMF, divide washing resin four times again, wash complete a little resin of getting and do ninhydrin reaction detection, result resin is positive, to adding following raw material in reaction vessels:
After above-mentioned raw materials adds, concussion reaction 40min, after reaction finishes, divides washing resin four times with 40ml DMF, and each washing time is 2min, gets a little resin and does triketohydrindene hydrate detection, and resin is negative.
(3) (a) raw material in shift step (2), (b) (c) (d) (e) raw material and add-on 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), convert a kind of (a) raw material, until above-mentioned 7 kinds of raw materials are all used once;
Repeat once the operation of (1) (2) (3) step, the raw material of each step and raw materials used amount are all constant again; After last Pro end of synthesis, deviate from 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 again methanol wash resin 8 times, remove DMF.After nitrogen dries up; with TFA (trifluoroacetic acid)/DCM strong acid lysate; cracking 3 hours, will synthesize polypeptide cracking from resin and get off, so slough blocking group simultaneously; collection is dissolved with the lysate of synthetic peptide; then filtration under diminished pressure, collects filtrate, the polypeptide with cold diethyl ether resolution of precipitate in filtrate; suction filtration obtains white solid again, obtains the crude product of synthetic peptide.Synthetic peptide crude product, through HPLC (high performance liquid chromatography) purifying, is collected main peak, after lyophilize, obtains target small peptide.
By above-mentioned same program, can also synthesize following sequence small peptide:
X
1for Thr, X
2for Ser, X
3during for His, sequence is as follows:
②CH
3CO-Pro-Thr-Phe-Ser-Phe-His-Phe-Glu-Pro-NH
2
X
1for Asn, X
2for Asn, X
3during for His, sequence is as follows:
③CH
3CO-Pro-Asn-Phe-Asn-Phe-His-Phe-Glu-Pro-NH
2
X
1for Ser, X
2for Ser, X
3during for Arg, sequence is as follows:
④CH
3CO-Pro-Ser-Phe-Ser-Phe-Arg-Phe-Glu-Pro-NH
2
Fig. 1 is synthesized small peptide purifying HPLC collection of illustrative plates 1., and Fig. 2 is synthetic small peptide MS collection of illustrative plates 1., and Fig. 3 is small peptide three-dimensional molecular model 1., and carbon atom is white, and Sauerstoffatom is red, and nitrogen-atoms is blue, by known each the amino acid whose spatial distribution of this figure.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, at 25 ℃, carry out scanning collection signal, use the light path quartz cuvette of 1mm, first sweep blank quartz cuvette to check the baseline of CD signal, and the CD signal of scanning ultrapure water quartz cuvette is usingd as 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 the note of the mean value in the bandwidth range of 1nm with 1s, and data results represents with average residue molar ellipticity.Fig. 5 be small peptide solution of the same race under same concentration after continuous ultrasound 2h, place the CD collection of illustrative plates after spending the night, therefrom can find out what considerable change is its secondary structure do not have.
Embodiment 2
Small peptide is CH 1.
3cO-Pro-Thr-Phe-Asn-Phe-Arg-Phe-Glu-Pro-NH
2nanostructure in the aqueous solution after self-assembly and the application in preparing dewatering medicament carrier thereof
1) atomic force microscope (AFM) detects
Application atomic force microscope observe amphipathic small peptide solution under different concns 1. itself and with the ultrasonic mixed nanostructure of appropriate pyrene.Method: get and be stored in 1. solution of small peptide that 4 ℃ of concentration in refrigerator are 1mg/ml, with 18.2K Ω/cm
2ultrapure water is mixed with its dilution the peptide aqueous solution of 0.5mg/ml, 0.3mg/ml, 0.1mg/ml, and without any processing, the pattern of they threes under atomic power is as shown in a, b, c in Fig. 6; Get and be stored in 1. solution of small peptide that 4 ℃ of concentration in refrigerator are 1mg/ml, with 18.2K Ω/cm
2ultrapure water is mixed with small peptide dilution after the peptide aqueous solution of 0.5mg/ml, 0.3mg/ml, 0.1mg/ml, and they are spent the night with the rearmounted dark place of pyrene mixing continuous ultrasound 3h with amount respectively.The small peptide solution of different concns is with the pyrene with amount after ultrasonic mixing, and the structure and morphology under atomic force microscope is different, as shown in a in Fig. 7, b, c.Wherein 7 (a) figure is the peptide aqueous solution and 10 of 0.5mg/ml
-6aspect graph after M pyrene is ultrasonic under atomic force microscope, what in figure, show is the mesh nano fibrous texture being 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 in figure, show is the netted apparatus derivatorius figure of nanofiber.7 (c) figure is the peptide aqueous solution and 10 of ultrasonic rear 0.1mg/ml
-6the aspect graph of M pyrene under atomic force microscope, what in figure, show is still the netted apparatus derivatorius figure of nanofiber, compares fiber apparatus derivatorius intensive like that with 7 (b) figure.We can find out a of Fig. 6, b, c tri-figure in the small peptide aqueous solution of same concentration and add after the pyrene of equivalent after comparing with a, b, c tri-figure of Fig. 7 respectively, the width average of their netted apparatus derivatoriuses of nanofiber under atomic power fiber is than not adding large before pyrene, and small peptide concentration is higher, its width average is larger (as following table) also.This is because small peptide material and hydrophobic drug model pyrene overlay on the surface of model drug pyrene after by hydrophobic interaction self-assembly, thereby makes its nanometer particle size become large.
| 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 that the pyrene aqueous solution of 0.4mg/ml stirs the shape appearance figure of watching under atomic force microscope after three days on magnetic stirring apparatus.It is a random non-nano fibrous web-like apparatus derivatorius, and further 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.
While preparing afm scan sample, first on the mica of newly opening, with liquid-transfering gun, pipette the solution liquid to be measured of 8-10ul, after standing 1min, with the ultrapure water of 200ul, rinse the solution not adsorbing on this sheet mica surface, then with the culture dish after sterilizing, cover mark and put dried overnight under room temperature good number.Atomic force microscope is at room temperature to scan, use be the NanoScope of U.S. Veeco company, with the pattern of rapping, carry out, the size of the Sample Scan in Fig. 6 and Fig. 7 adopts respectively 5 μ mx5 μ m to observe, Fig. 8 is the range of observation of 3 μ mx3 μ m.
2) transmission electron microscope (TEM) detects
The Jeol Jem-1400 transmission electron microscope of application NEC company is observed the 1. nanostructure of the aqueous solution of small peptide under the acceleration voltage of 120kv.By 18.2K Ω/cm for the peptide solution of 1mg/ml
2milli-Q ultrapure water is diluted to concentration 0.3mg/ml to be measured.The peptide aqueous sample that first pipettes 10ul with liquid-transfering gun while preparing transmission electron microscope scanning samples is in clean surface, the copper mesh that then will be covered with polyvinyl formal film (Formvar) immerses twice each 10s of peptide solution, then sucks the unnecessary liquid of copper mesh periphery with filter paper.1% phospho-wolframic acid of pH6.5 is dripped in clean surface, simultaneously by the wiping surface negative staining 15s of copper mesh, then with filter paper, suck unnecessary liquid immediately, room temperature is naturally dried the rear electron microscope that utilizes, under the acceleration voltage of 120kv, the sample with phospho-wolframic acid dyeing is carried out to light field observation.Under transmission electron microscope, the small peptide solution morphology figure of 0.3mg/ml as shown in Figure 8, magnification is 3.3 ten thousand times, what show is that what after the amphipathic small peptide self-assembly of this half way charge matching, to form is the netted apparatus derivatorius of nanofiber, and this result is with concentration small peptide solution, the aspect graph under atomic force microscope can match well.
Meanwhile, 4. 3. 2. other half way charge matching short peptide sequences in the present invention all can be self-assembled into the netted apparatus derivatorius of nanofiber in the aqueous solution, with the 1. the same solid support material that all can be used as dewatering medicament of small peptide.
According to amount of bleeding and bleeding stopping period, judge the haemostatic effect of four kinds of small peptides.In the present embodiment, laboratory animal is the SPF level SD rat between 290g～310g, male and female stochastic distribution, and all laboratory animal are all purchased from Nanfang Medical Univ's experimentation on animals center.All laboratory animal are fasting 24h (freely drinking water) before operation, and all animals are injected the vetanarcol liquid of various dose before test by body weight.In experiment, cotton swab used is medical cotton stick, cotton yarn autoclaving is processed, cotton swab, cotton are placed on respectively in sterilizing clean vial and before experiment and weigh, put back to bottle after using cotton swab cotton in, seal, in anti-Hemostatic Oral Liquid, water loss is in air, the gross weight that weighs the rear bottle of experiment, can draw amount of bleeding, records the weight of amount of bleeding=bottle finally weighing and the weight of suck blood cotton swab and cotton yarn-dry cotton swab and cotton yarn and bottle.All data in this experiment all adopt (
) represent, data processing adopts Wilks ' s λ check (processing of SAS 8.0for Windows statistical package).
In experimental group, small peptide hemostatic material used can be the peptide aqueous solution of small peptide lyophilized powder or different mass volume ratio, and the peptide aqueous solution is with 18.2K Ω/cm
2milli-Q ultrapure water is mixed with and forms, and this peptide aqueous solution ultrasonic several minutes in ultrasonic washing instrument can be removed to viscosity, and bubble can be removed by high speed centrifugation, 4 ℃ of preservations.
1, by the small peptide of 1% (w/v)
1. (CH
3cO-Pro-Thr-Phe-Asn-Phe-Arg-Phe-Glu-Pro-NH
2) for SD rat damaged liver section, stop blooding
According to hemostasis mode, be divided into following three groups:
1) cotton yarn group (n=10 for experiment, blank group): after rat anesthesia, careful dissection, with the suitably large minor cut or wound of sterilization blade cutting-up on its lobe of the liver, wound is bled after 5s naturally, with cotton swab, blood is wiped, with shearing big or small sterilizing cotton yarn, push down wound again, after applying light 30s, with cotton swab, draw the blood oozing out around during this time, remove cotton yarn and observe the hemorrhage of wound surface, as wound in 10s continues hemorrhagely, continue with cotton yarn pressing haemostatic until stopped blooding.If total time surpasses 10min, look this group hemostasis the failure of an experiment.
2) 1% (w/V) peptide water group (n=10, experimental group): peptide is 1. Ac-Pro-Thr-Phe-Asn-Phe-Arg-Phe-Glu-Pro-NH of the amphipathic small peptide of half way charge matching chosen from the present invention
2sample, after rat anesthesia, carefully dissects, and with the suitably large minor cut or wound of blade cutting-up on its lobe of the liver, wound is bled after 5s naturally, with cotton swab, blood is wiped, now by off-the-shelf small peptide 1. solution inject wound, observe bleeding.Find in damaged liver surface, to form a spawn when peptide liquid and wound rigidly connect to touch, adhere to damaged surface, as shown in figure 10, reach fast haemostatic effect, during with cotton swab, draw the blood oozing out around until the experimentation that stops blooding completes.
3) hemostasis gelatin group (n=10, experiment contrast group) experimentation is the same.Record bleeding stopping period (hemostasis completely), by weighting method, weigh the weight of gauze and cotton swab, calculate blood volume.
The different hemostatic materials of table 1 are to the hemorrhage anastalsis of SD rat damaged liver section
| Hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| |
10 | 125±19 | 1.49±0.22 |
| 1% (w/v) |
10 | 13±3 | 0.31±0.15 |
| |
10 | 20±2 | 0.82±0.21 |
Result shows (in 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, by 2. (CH of small peptide
3cO-Pro-Thr-Phe-Ser-Phe-His-Phe-Glu-Pro-NH
2) lyophilized powder for SD rat damaged liver section hemostasis
Hemostasis experimental implementation as above 1, has changed the peptide aqueous solution into the lyophilized powder of peptide, and control group is constant, with stopwatch, records the bleeding time (bleeding stopping period completely), in hemostasis, with cotton swab, draws the blood that oozes out around until hemostasis experimentation completes.Finally by weighting method, weigh the weight of gauze and cotton swab, calculate blood volume.
The different hemostatic materials of table 2 are to the hemorrhage anastalsis of SD rat damaged liver section
| Hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| |
10 | 128±17 | 1.51±0.21 |
| Small peptide lyophilized |
10 | 10±2 | 0.28±0.11 |
| |
10 | 21±2 | 0.83±0.20 |
Result shows (in Table 2), the small peptide anastalsis that 2. lyophilized powder is compared generation with control group (common cotton yarn group and hemostasis gelatin group) 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, by 3. (CH of the small peptide of 0.7% (w/V)
3cO-Pro-Asn-Phe-Asn-Phe-His-Phe-Glu-Pro-NH
2) for the hemorrhage hemostasis of SD rat damaged liver section
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, with stopwatch, record the bleeding time (bleeding stopping period completely), in hemostasis, with cotton swab, draw the blood oozing out around until the experimentation that stops blooding completes.Finally by weighting method, weigh the weight of gauze and cotton swab, calculate blood volume.
The different hemostatic materials of table 3 are to the hemorrhage anastalsis of SD rat damaged liver section
| Hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| |
10 | 125±19 | 1.49±0.22 |
| 0.7% (w/V) |
10 | 16±2 | 0.42±0.15 |
| |
10 | 19±2 | 0.82±0.21 |
Result shows (in 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, by 4. (CH of the small peptide of 2% (w/V)
3cO-Pro-Ser-Phe-Ser-Phe-Arg-Phe-Glu-Pro-NH
2) for the hemorrhage hemostasis of SD rat damaged liver section
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, with stopwatch, record the bleeding time (bleeding stopping period completely), in hemostasis, with cotton swab, draw the blood oozing out around until the experimentation that stops blooding completes.Finally by weighting method, weigh the weight of gauze and cotton swab, calculate blood volume.
The different hemostatic materials of table 4 are to the hemorrhage anastalsis of SD rat damaged liver section
| Hemostasis mode | Rat quantity (N) | Bleeding stopping period (s) | Amount of bleeding (g) |
| |
10 | 124±18 | 1.48±0.19 |
| 2% (w/V) |
10 | 10±2 | 0.27±0.12 |
| |
10 | 21±2 | 0.81±0.22 |
Result shows (in 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.
From four groups of above hemostasis experiments, 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 higher its haemostatic effect of the concentration of peptide water is better, and the lyophilized powder of small peptide material is better than the haemostatic effect of the peptide aqueous solution to a certain extent.
Above-described embodiment is preferably embodiment 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 principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (3)
1. the amphipathic self-assembled short peptide of a class half way charge matching, is characterized in that, the general formula of its aminoacid sequence is as follows:
Ac-Pro-X
1-Phe-X
2-Phe-Arg-Phe-Glu-Pro-NH
2
X wherein
1, X
2for Thr, Ser, Cys or Asn.
2. the application of small peptide in preparing hydrophobic drug solid support material described in claim 1.
3. the application of small peptide in preparing nanometer hemostatic material described in claim 1.
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| CN103012558B (en) * | 2012-06-28 | 2014-12-03 | 华南理工大学 | Cation symmetrical amphipathic self-assembled polypeptide and application thereof |
| CN103788177B (en) * | 2014-01-27 | 2016-02-17 | 四川大学 | A kind of amphipathic nonapeptide and the application as pharmaceutical carrier thereof |
| CN105085622B (en) * | 2015-09-16 | 2016-05-04 | 中国石油大学(华东) | The ultrashort peptide nanometer hemostatic material of a kind of amphipathic self assembly |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
Non-Patent Citations (3)
| Title |
|---|
| Parallel b-sheet assemblies at interfaces;Sneer, Ronit等;《ChemPhysChem》;20041231;第5卷(第5期);第747-750页 * |
| RUAN Liping等人.Application research of a novel designed peptide as a potential carrier.《Science in China Series B:Chemistry》.2009,第52卷(第5期),摘要. * |
| Sneer, Ronit等.Parallel b-sheet assemblies at interfaces.《ChemPhysChem》.2004,第5卷(第5期),第747-750页. |
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