CN102008733A - Anti-tumor controlled release nanocomposite and preparation method thereof - Google Patents
Anti-tumor controlled release nanocomposite and preparation method thereof Download PDFInfo
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- NWXMGUDVXFXRIG-WESIUVDSSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O NWXMGUDVXFXRIG-WESIUVDSSA-N 0.000 claims description 31
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- NXLNNXIXOYSCMB-UHFFFAOYSA-N (4-nitrophenyl) carbonochloridate Chemical compound [O-][N+](=O)C1=CC=C(OC(Cl)=O)C=C1 NXLNNXIXOYSCMB-UHFFFAOYSA-N 0.000 claims description 4
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Abstract
The invention discloses an anti-tumor controlled release nanocomposite and a preparation method thereof. The nanocomposite utilizes nano diamonds after carboxylation treatment as carriers, and comprises adsorption enzyme self degradation chains and cell-penetrating peptides. The preparation method is implemented as follows: dispersing nano diamonds after carboxylation treatment of a strong acid in a dimethyl sulfoxide aqueous solution, adding an anti-tumor drug connected with a self degradation chain, and stirring under room temperature to obtain a composite; and dispersing the obtained composite in water, adding the cell-penetrating peptides, and stirring and drying at room temperature to obtain the anti-tumor controlled release nanocomposite. The nano carrier composite in the invention is simple and convenient in preparation, has no toxicity and pollution, and is easy for industrialization. In vitro research shows that the composite can realize controlled release of an anti-tumor drug, enhance the killing effect of the drug on tumors, and provide a new material and method for drug transport.
Description
Technical field
The present invention relates to a kind of anti cancer sustained releasing nano-complex.
The invention still further relates to the preparation method of above-mentioned anti cancer sustained releasing nano-complex.
Background technology
(doxorubicin is a non-specific antitumor antibiotics of a kind of cycle DOX) to amycin, has very strong active anticancer, and antitumor spectra is wider.But because its toxic and side effects is restricted its application clinically.
Phenylalanine (Phe)-lysine (Lys) is the specific substrate of cathepsin B (cathepsin B).Cathepsin B can overexpression in tumor.When this substrate linked to each other with amycin, drug release mainly occurred in tumor locus.Yet because the substrate chain is too short, when linking to each other with the bigger amycin of volume, enzyme weakens because of sterically hindered the effect meeting of substrate.Between substrate and amycin, introduce one and will reduce the sterically hindered of enzyme effect, realize that the medicine may command discharges from degrading chain (PABOH).Therefore, enzyme is from degrading chain, i.e. the combination of the zymolyte-chain-amycin of degrading certainly both can keep medicine to discharge in the fixed point of tumor, can realize that again the may command of medicine discharges.But this chemical compound generally is soluble in organic solvent, and poorly water-soluble is unfavorable for biologic applications.
Nanometer diamond can be improved the water solublity of insoluble drug greatly.In addition, its surface activity center is many, and high adsorption capacity is easy to carry out finishing, and physical property is stable, and good biocompatibility is nontoxic, is easy to carry out transport of drug.Nanometer diamond is combined by physical absorption from the chain (Phe-Lys-PABC-DOX) of degrading with enzyme, simple and easy to do, do not cause environmental pollution, be easy to industrialization.Realize the external biological application of Phe-Lys-PABC-DOX, realized the may command drug release of amycin, reduced the toxicity of amycin.Nanometer diamond is in absorption Phe-Lys-PABC-DOX, wear film peptide TAT in the absorption, strengthen the ability that it passes through tumor cell membrane, increased intracellular complex concentration, improve curative effect of medication greatly, and the combination of wearing film peptide TAT also can increase the lethal effect to the resistant tumors cell.
The nanometer diamond while is in conjunction with Phe-Lys-PABC-DOX and wear film peptide TAT, overcome antitumor drug amycin toxic and side effects important disadvantages, both made carrier complexes optionally be gathered in tumor locus, increase the ability that it passes through tumor cell membrane again, weakened the toxicity of amycin, strengthened the lethal effect of medicine, provide theoretical basis for realizing the drug disposition transhipment at tumor locus.
Summary of the invention
The objective of the invention is to the anti cancer sustained releasing nano-complex.
Another purpose of the present invention is to provide the method for the above-mentioned anti cancer sustained releasing nano-complex of preparation.
For achieving the above object, antitumor drug controlled release nanometer complex provided by the invention is a carrier with the nanometer diamond of carboxylated processing, and the physical absorption enzyme is from degrading chain and wear the film peptide; Obtain by following method:
The nanometer diamond of carboxylated processing is scattered in dimethyl sulfoxide (DMSO) aqueous solution, and adding is connected with the antitumor drug of enzyme from the chain of degrading, and stirs under the room temperature and obtains complex; The complex that obtains is scattered in the water, adds and wear the film peptide, obtain the anti cancer sustained releasing nano-complex after the stirring drying under the room temperature;
Antitumor drug is amycin, paclitaxel or methotrexate.
Described anti cancer sustained releasing nano-complex, wherein the nanometer diamond of carboxylated processing is the nanometer diamond through the carboxylated processing of strong acid.
Described anti cancer sustained releasing nano-complex, wherein enzyme from the chain of degrading successively by phenylalanine, lysine, aminobenzyl alcohol, p-nitrophenyl chloro-formate, antitumor drug covalent bond are formed.
Described anti cancer sustained releasing nano-complex is wherein worn the film peptide for strengthening the polypeptide of antitumor activity: tyrosine-glycine-arginine-lysine-lysine-arginine-arginine-glutamine-arginine-arginine-arginine, Arg-Gln-isoleucine-Lys-Ile-tryptophan-Phe-Gln-asparagine-arginine-arginine-methionine-lysine-tryptophan-lysine-lysine, Asp-Ala-alanine-threonine-alanine-arginine-glycine-arginine-glycine-arginine-serine-Ala-Ala-Vitro By Serine/arginine-proline-TE-arginine-Ala-Pro-alanine-arginine-serine-alanine-Vitro By Serine/arginine-Pro-Arg-Arg-Pro-valine-aspartic acid, arginine-arginine-arginine-arginine-arginine-arginine-arginine or glycine-tryptophan-threonine-leucine-aspartic acid-tryptophan-Ala-Gly-Tyr-Leu-leucine-Lys-Ile-Asp-Leu-lysine-Ala-Leu-Ala-Ala-Leu-Ala-lysine-Lys-Ile-leucine or valine-threonine-Val-Leu-Ala-Leu-Gly-Ala-Leu-Ala-Gly-Val-glycine-valine-glycine.
The method of the above-mentioned anti cancer sustained releasing nano-complex of preparation provided by the invention, key step is:
1) carboxylated processing nanometer diamond;
2) carboxylated nanometer diamond is joined in the dimethyl sulphoxide aqueous solution room temperature ultra-sonic dispersion;
3) adding is connected with the antitumor drug of enzyme from the chain of degrading in scattered nanometer diamond solution, stirs under the room temperature, makes enzyme from degrading chain combination on nanometer diamond, the centrifugal precipitate that obtains;
Antitumor drug is amycin, paclitaxel or methotrexate;
4) precipitate with step disperses to make aqueous solution, adds the aqueous solution of wearing the film peptide, stirs under the room temperature, and the centrifugal precipitate that obtains prepares target product after the drying.
Described method, wherein carboxylated processing nanometer diamond is to adopt the carboxylated processing of strong acid in the step 1, the carboxylated processing procedure of strong acid is: nanometer diamond is added in the blended strong acid stirs, carry out alkali cleaning and pickling more respectively; Blended strong acid is H
2SO
4-HNO
3, H
2SO
4-HF or HF-HNO
3Volume ratio is 9-1: 1.
Described method, wherein the concentration of dimethyl sulfoxide is 40-60% by volume in the step 2.
Described method, wherein in the step 3 enzyme of amycin from the chain of degrading successively by phenylalanine, lysine, aminobenzyl alcohol, p-nitrophenyl chloro-formate, amycin covalent bond are formed.
Described method is wherein worn the film peptide for strengthening the polypeptide of antitumor activity: tyrosine-glycine-arginine-lysine-lysine-arginine-arginine-glutamine-arginine-arginine-arginine, Arg-Gln-isoleucine-Lys-Ile-tryptophan-Phe-Gln-asparagine-arginine-arginine-methionine-lysine-tryptophan-lysine-lysine, Asp-Ala-alanine-threonine-alanine-arginine-glycine-arginine-glycine-arginine-serine-Ala-Ala-Vitro By Serine/arginine-proline-TE-arginine-Ala-Pro-alanine-arginine-serine-alanine-Vitro By Serine/arginine-Pro-Arg-Arg-Pro-valine-aspartic acid, arginine-arginine-arginine-arginine-arginine-arginine-arginine or glycine-tryptophan-threonine-leucine-aspartic acid-tryptophan-Ala-Gly-Tyr-Leu-leucine-Lys-Ile-Asp-Leu-lysine-Ala-Leu-Ala-Ala-Leu-Ala-lysine-Lys-Ile-leucine or valine-threonine-Val-Leu-Ala-Leu-Gly-Ala-Leu-Ala-Gly-Val-glycine-valine-glycine.
The present invention respectively with nanometer diamond and enzyme from degrading chain, antitumor drug and wear film peptide TAT physical absorption and combine, make drug release mainly occur in tumor locus, control the dispose procedure of medicine simultaneously.Antitumor drug controlled release nanometer complex of the present invention has increased the inhibitory action to tumor cell.
Description of drawings
Fig. 1 is the structural representation of amycin controlled release nanometer complex of the present invention.
Fig. 2 is the supernatant uv absorption trendgram of nanometer diamond desmoenzyme of the present invention before and after the chain reaction of degrading; Wherein a is the supernatant uv absorption trendgram of pure enzyme from the chain of degrading, b is that enzyme is from degrading chain combination behind the supernatant uv absorption trendgram on the nanometer diamond, c is an enzyme from degrading chain combination after on the nanometer diamond, the supernatant uv absorption after washing for the first time with 50%DMSO.
Fig. 3 is the transmission electron microscope figure of amycin controlled release nanometer complex of the present invention; A is known products nanometer diamond (ND) among the figure; B is TAT-ND-Phe-Lys-PABC-DOX.
Fig. 4 is the infrared figure (FITR) of carrier of the present invention, intermediate product and end product; Wherein:
A is known products ND;
B is the product ND-COOH of ND through the carboxylated processing of strong acid;
C is intermediate product ND-Phe-Lys-PABC-DOX of the present invention;
D is Phe-Lys-PABC-DOX among the present invention;
E is end-product TAT-ND-Phe-Lys-PABC-DOX of the present invention;
F is that known products is worn film peptide TAT.
Fig. 5 is the Zeta electric potential figure of amycin controlled release nanometer complex of the present invention;
A is known products nanometer diamond (ND) among the figure; B is the ND of carboxylated processing, and c is ND-Phe-Lys-PABC-DOX, and d is TAT-ND-Phe-Lys-PABC-DOX.
Fig. 6 is the cytotoxicity result of amycin controlled release nanometer complex of the present invention
A is known products nanometer diamond (ND) among the figure; B is the ND of carboxylated processing, and c is ND-Phe-Lys-PABC-DOX, and d is TAT-ND-Phe-Lys-PABC-DOX.
Fig. 7 is the result that influences to the tumor cell migration ability of amycin controlled release nanometer complex of the present invention; A is contrast, and b is the DOX group, and c is the ND-Phe-Lys-PABC-DOX group, and d is the TAT-ND-Phe-Lys-PABC-DOX group.
The specific embodiment
The invention provides a kind of may command drug release, the nano-complex that anti-tumor activity is higher is to improve the present application limit of antitumor drug.Below lifting amycin is that example describes.
Anti cancer sustained releasing nano-complex provided by the invention is a carrier with the nanometer diamond of diameter＜10nm, physical absorption Phe-Lys-PABC-DOX with wear film peptide TAT.
The above-mentioned antitumor drug controlled release nanometer of preparation provided by the invention complex, its key step is: through the carboxylated processing of strong acid, ultra-sonic dispersion is in 50%DMSO/H with nanometer diamond
2O, adding is connected with the amycin (also can be paclitaxel or methotrexate etc.) of enzyme from the chain of degrading, and the room temperature lower magnetic force stirs.The complex that obtains is scattered in the tri-distilled water, adds and wear film peptide TAT, the room temperature lower magnetic force stirs, and drying obtains amycin controlled release nanometer complex (TAT-ND-Phe-Lys-PABC-DOX).
The present invention prepares in the method for above-mentioned amycin controlled release nanometer complex, and nanometer diamond through the carboxylated processing procedure of strong acid is: nanometer diamond is joined H
2SO
4-HClO
4In (3: 1) strong acid mixed solution, 40 ℃ are stirred 24h, 90 ℃ of backflows in 0.1M NaOH aqueous solution, 90 ℃ of backflows in 0.1M HCl aqueous solution again.
In the method for the above-mentioned amycin controlled release nanometer of preparation provided by the invention complex, nanometer diamond and the Phe-Lys-PABC-DOX principal character in physical adsorption process is:
A) adsorption isotherm accords with Freundlich equation, the inconsistent distribution of adsorbent surface heat of adsorption.
B) adsorption process meets the Lagergren first _ order kinetics equation.
C) free energy in the adsorption process is for negative, and absorption can spontaneously be carried out; Enthalpy is for negative, and adsorption reaction is exothermic reaction; Entropy is for negative, and adsorption process is unordered.
D) adsorbance is subjected to the influence of temperature, pH value and ionic strength.
The present invention is described further to lift specific embodiment below again.
Embodiment 1
1) preparation of carboxylated nanometer diamond:
The 0.5g nanometer diamond is joined 20mL H
2SO
4-HClO
4In (3: 1) mixed solution, 40 ℃ are stirred 24h, centrifugal, 90 ℃ of backflow 2h in 0.1M NaOH aqueous solution, and centrifugal treating, 90 ℃ of backflow 2h in the 0.1MHCl aqueous solution again, centrifugal, tri-distilled water repeatedly washs, vacuum drying.
2) preparation of ND-Phe-Lys-PABC-DOX nano-complex:
The nanometer diamond that 4mg is carboxylated joins in the aqueous solution of 50% dimethyl sulfoxide, and the ultrasonic 4h of room temperature makes its dispersion.Add 0.4mgPhe-Lys-PABC-DOX in scattered nanometer diamond solution, the room temperature lower magnetic force stirs 24h, and Phe-Lys-PABC-DOX is combined on the nanometer diamond fully, and is centrifugal, abandons supernatant, and tri-distilled water is washed.
3) preparation of TAT-ND-Phe-Lys-PABC-DOX nano-complex:
Behind ND-Phe-Lys-PABC-DOX nano-complex ultra-sonic dispersion, make the aqueous solution of 1mg/mL, add the aqueous solution of TAT, the room temperature lower magnetic force stirs 24h, and is centrifugal, abandons supernatant, and tri-distilled water is washed, vacuum drying.
Fig. 1 is the amycin controlled release nanometer composite structure sketch map of the embodiment of the invention 1 preparation, among the figure:
-NH
2Be Phe-Lys-PAB C-DOX;
The combination stability of Phe-Lys-PABC-DOX and nanometer diamond is described among the present invention as seen in Figure 2.This figure is the supernatant uv absorption trendgram before and after the synthetic reaction of the present invention.A is the supernatant uv absorption trendgram of pure enzyme from the chain of degrading among the figure, b is that enzyme is from degrading chain combination behind the supernatant uv absorption trendgram on the nanometer diamond, c is an enzyme from degrading chain combination after on the nanometer diamond, the supernatant uv absorption after washing for the first time with 50%DMSO.Illustrate that Phe-Lys-PABC-DOX reduces in the supernatant of reaction back, promptly be combined on the nanometer diamond.Substantially do not have uv absorption in the supernatant after washing, illustrated that Phe-Lys-PABC-DOX was stable with combining of nanometer diamond.
Fig. 3 is the transmission electron microscope figure of amycin controlled release nanometer complex of the present invention; A is known products nanometer diamond (ND) among the figure; B is TAT-ND-Phe-Lys-PABC-DOX.Nanometer diamond clean mark before as can be seen from Figure 3 modifying, it is fuzzy to modify the back texture, illustrates that the Phe-Lys-PABC-DOX on modifying has covered the texture of nanometer diamond.
Fig. 4 is the infrared figure (FITR) of carrier of the present invention, intermediate product and end product.As can be seen from the figure b-the OH peak strengthens, the characteristic peak of Phe-Lys-PABC-DOX has appearred in c, the characteristic peak of TAT has appearred in e.
Fig. 5 is the Zeta electric potential figure of amycin controlled release nanometer complex of the present invention; As can be seen from Figure 5 the nanometer diamond carrier surface is modified the corresponding change of back Zeta electric potential, also proves the combination of carrier surface enzyme from degrade chain and TAT.
Embodiment 2
Nanometer diamond and the research of Phe-Lys-PABC-DOX adsorption isotherm:
In centrifuge tube, respectively add the 1mg nanometer diamond respectively, in centrifuge tube, add mass concentration successively and be 0.08,0.11,0.14,0.17,0.20, the Phe-Lys-PABC-DOX of 0.23mg/mL, 27 ℃ of constant temperature shaking tables, 80 change, vibration 28h, centrifugalize, get supernatant in cuvette, in the uv absorption of 492nm mensuration Phe-Lys-PABC-DOX, obtain the supernatant amount according to its standard curve, further ask and calculate adsorbance.According to formula
Langmuir isotherm equation:
Right with Ce
Do linear equation: y=1.3323x+0.3279, R
2=0.3666.According to formula F reundlich isotherm equation
With LogC
eTo Logq
eDo linear equation: y=0.8654x+0.2243R
2=0.9112.
Embodiment 3
Nanometer diamond and the research of Phe-Lys-PABC-DOX adsorption dynamics adsorption kinetics:
In centrifuge tube, add the 1mg nanometer diamond, to the Phe-Lys-PABC-DOX that wherein adds mass concentration 0.16mg/mL, 27 ℃, 80 change, and the vibration of constant temperature shaking table is in the particular point in time centrifugalize, get supernatant in cuvette, measure its uv absorption at 492nm, obtain the supernatant amount, further ask and calculate adsorbance according to standard curve.According to Lagergren first order kinetics adsoption equation ln (q
e-q)=ln q
e-k1t carries out the first _ order kinetics equation match to experimental data:
y＝-0.1967x-2.4207R
2＝0.9711。According to revising pseudo-first _ order kinetics equation
Experimental data is revised pseudo-first _ order kinetics equation match: y=-0.1712x-1.7765 R
2=0.9706.According to model
Experimental data is carried out pseudo-second-order kinetics equation model: y=0.0815x-2.2409 R
2=0.4968.
Embodiment 4
Nanometer diamond and the research of Phe-Lys-PABC-DOX Adsorption thermodynamics:
In centrifuge tube, add the 1mg nanometer diamond, to the Phe-Lys-PABC-DOX that wherein adds mass concentration 0.16mg/mL, respectively at 27 ℃, 37 ℃ of constant temperature shaking table vibrations, get supernatant behind the 28h in cuvette, measure its uv absorption at 492nm, obtain the supernatant amount, further ask and calculate adsorbance according to standard curve.According to
Experimental data is handled: Δ G=-4.25KJ/mol in the time of 27 ℃, Δ H=-24.045KJ/mol, Δ S=-0.066KJ/molK.Δ G=-3.59KJ/mol in the time of 37 ℃.
Cell experiment:
1) Study of cytotoxicity:
The take the logarithm C6 of trophophase, digestive inoculation be in 96 orifice plates, and 37 ℃, 5%CO
2Hatch 24h, add DOX, ND-Phe-lys-PABC-DOX, each 5 μ g/mL (being as the criterion) of TAT-ND-Phe-lys-PABC-DOX respectively, continue to hatch 24h with DOX concentration, every hole adds MTT 100uL, hatches 4h for 37 ℃, and every hole adds 150uLDMSO, microplate reader 490nm place is detected, with formula
Fig. 6 is the Study of cytotoxicity result of the carrier of present embodiment amycin controlled release nanometer complex, as can be seen from Figure 6 Phe-Lys-PABC-DOX itself is very little to the toxicity of tumor cell, the transhipment of nanometer diamond increases the cytotoxicity of ND-Phe-Lys-PABC-DOX, further increase in conjunction with cytotoxicity behind the last TAT, and greater than the DOX without any modification.
2) cell migration:
The take the logarithm C6 of trophophase, digestive inoculation be in 24 orifice plates, and 37 ℃, 5%CO
2Hatch 24h, every hole 200uL rifle head cut gently, PBS cleans damaging cells, adds DOX, ND-Phe-Lys-PABC-DOX, each 5 μ g/mL (being as the criterion with DOX concentration) of TAT-ND-Phe-Lys-PABC-DOX more respectively, continues to hatch 24h, and microscopically is observed.
Fig. 7 is the influence result of present embodiment amycin controlled release nanometer complex to the tumor cell migration ability; As can be seen from Figure 7 cut is apart from TAT-ND-Phe-Lys-PABC-DOX＞ND-Phe-Lys-PABC-DOX＞DOX＞contrast.The migration influence that the TAT-ND-Phe-Lys-PABC-DOX pair cell is described is bigger.
Claims (9)
1. anti cancer sustained releasing nano-complex is a carrier with the nanometer diamond of carboxylated processing, and the physical absorption enzyme is from degrading chain and wear the film peptide; Obtain by following method:
The nanometer diamond of carboxylated processing is scattered in the dimethyl sulphoxide aqueous solution, and adding is connected with the antitumor drug of enzyme from the chain of degrading, and stirs under the room temperature and obtains complex; Antitumor drug is amycin, paclitaxel or methotrexate;
The complex that obtains is scattered in the water, adds and wear the film peptide, obtain the anti cancer sustained releasing nano-complex after the stirring drying under the room temperature.
2. anti cancer sustained releasing nano-complex as claimed in claim 1, wherein, the nanometer diamond of described carboxylated processing is the nanometer diamond through the carboxylated processing of strong acid.
3. anti cancer sustained releasing nano-complex as claimed in claim 1, wherein, enzyme from the chain of degrading successively by phenylalanine, lysine, aminobenzyl alcohol, p-nitrophenyl chloro-formate, antitumor drug covalent bond are formed.
4. anti cancer sustained releasing nano-complex as claimed in claim 1, wherein, wearing the film peptide is tyrosine-glycine-arginine-lysine-lysine-arginine-arginine-glutamine-arginine-arginine-arginine, arginine-glutamine-isoleucine-lysine-isoleucine-tryptophan-phenylalanine-glutamine-agedoite-arginine-arginine-methionine-lysine-tryptophan-lysine-lysine, aspartic acid-Ala-Ala-threonine-alanine-arginine-glycine-arginine-glycine-arginine-serine-Ala-Ala-serine-Arg-Pro-threonine-glutamic acid-arginine-alanine-proline-alanine-arginine-serine-alanine-serine-Arg-Pro-arginine-Arg-Pro-valine-aspartic acid, arginine-arginine-arginine-arginine-arginine-arginine-arginine or glycine-tryptophan-threonine-leucine-aspartic acid-tryptophan-alanine-glycine-tyrosine-leucine-leucine-lysine-isoleucine-aspartic acid-leucine-lysine-alanine-leucine-Ala-Ala-leucine-alanine-lysine-lysine-isoleucine-leucine or valine-threonine-Val-Leu-alanine-leucine-glycine-alanine-leucine-alanine-glycine-valine-glycine-valine-glycine.
5. method for preparing the described anti cancer sustained releasing nano-complex of claim 1, key step is:
1) carboxylated processing nanometer diamond;
2) carboxylated nanometer diamond is joined in the dimethyl sulphoxide aqueous solution room temperature ultra-sonic dispersion;
3) adding is connected with the antitumor drug of enzyme from the chain of degrading in scattered nanometer diamond solution, stirs under the room temperature, makes enzyme from degrading chain combination on nanometer diamond, the centrifugal precipitate that obtains; Antitumor drug is amycin, paclitaxel or methotrexate;
4) precipitate with step disperses to make aqueous solution, adds the aqueous solution of wearing the film peptide, stirs under the room temperature, and the centrifugal precipitate that obtains prepares target product after the drying.
6. method as claimed in claim 5, wherein, carboxylated processing nanometer diamond is to adopt the carboxylated processing of strong acid in the step 1, the carboxylated processing procedure of strong acid is: nanometer diamond is added in the blended strong acid stirs, carry out alkali cleaning and pickling more respectively;
Blended strong acid is H
2SO
4-HNO
3, H
2SO
4-HF or HF-HNO
3Volume ratio is 9-1: 1.
7. method as claimed in claim 5, wherein, the concentration of dimethyl sulfoxide is 40-60% by volume in the step 2.
8. method as claimed in claim 5, wherein, in the step 3 enzyme of amycin from the chain of degrading successively by phenylalanine, lysine, aminobenzyl alcohol, p-nitrophenyl chloro-formate, amycin covalent bond are formed.
9. method as claimed in claim 5, wherein, wearing the film peptide is tyrosine-glycine-arginine-lysine-lysine-arginine-arginine-glutamine-arginine-arginine-arginine, arginine-glutamine-isoleucine-lysine-isoleucine-tryptophan-phenylalanine-glutamine-agedoite-arginine-arginine-methionine-lysine-tryptophan-lysine-lysine, aspartic acid-Ala-Ala-threonine-alanine-arginine-glycine-arginine-glycine-arginine-serine-Ala-Ala-serine-Arg-Pro-threonine-glutamic acid-arginine-alanine-proline-alanine-arginine-serine-alanine-serine-Arg-Pro-arginine-Arg-Pro-valine-aspartic acid, arginine-arginine-arginine-arginine-arginine-arginine-arginine or glycine-tryptophan-threonine-leucine-aspartic acid-tryptophan-alanine-glycine-tyrosine-leucine-leucine-lysine-isoleucine-aspartic acid-leucine-lysine-alanine-leucine-Ala-Ala-leucine-alanine-lysine-lysine-isoleucine-leucine or valine-threonine-Val-Leu-alanine-leucine-glycine-alanine-leucine-alanine-glycine-valine-glycine-valine-glycine.
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