CN102174078A

CN102174078A - Application of tumor cell membrane selectively penetrating peptide

Info

Publication number: CN102174078A
Application number: CN2011100035670A
Authority: CN
Inventors: 周建平; 吕慧侠; 孙博; 张振海; 张银龙; 姜天玥
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2011-01-10
Filing date: 2011-01-10
Publication date: 2011-09-07

Abstract

The invention belongs to membrane-penetrating peptides, in particular to tumor cell-selective membrane-penetrating peptides and applications thereof. Tumor cell-selective membrane-penetrating peptide, which contains a basic amino acid cluster composed of 5-12 consecutive arginine residues in its sequence, and is characterized in that the C-terminal or N-terminal of the basic amino acid cluster is connected with 3 histidines acid residues. The tumor cell selective membrane penetrating peptide of the present invention has cell membrane penetrating ability, can distinguish tumor cells from normal tissue cells, and can efficiently pass through tumor cell membranes while basically not penetrating normal tissue cell membranes.

Description

The tumour cell selectivity is worn the application of film peptide

Technical field:

The invention belongs to and wear the film peptide, particularly the tumour cell selectivity is worn film peptide and application thereof.

Background technology:

In the last few years, some had the polypeptide of cytolemma penetrativity, became the focus of biomedicine field research gradually as TAT, MPG, PEP-1, penetratin, oligomerization arginine etc.These positively charged peptide sequences can pass cytolemma and not destroy the structure of cytolemma.That these polypeptide natural or synthetic have is water-soluble, low cracking performance, and can enter various cells by non-phagolysis, so be referred to as to wear film peptide (cell-permeable peptides, CPP), because of wearing the transmembrane transport that the film peptide is found in full chain protein the earliest, so be referred to as again protein transduction domain (protein transduction domain, PTD).Still imperfectly understand at present though wear the film mechanism of wearing of film peptide, they are very high and do not consume energy to the transport efficacy of biomacromolecule.These cell-penetrating peptides are the polypeptide fragment of the varying length that has positive charge, wherein are rich in alkaline amino acid residues such as arginine.Utilize this characteristic, various novel cell-penetrating peptides are designed and are synthesized, and the octaarginine of synthetic etc. wears the film peptide and wears membrane efficiency far above the naturally occurring film peptide of wearing.Wear the film peptide and generally contain 11～30 amino-acid residues, can be divided three classes: the cationic film peptide of wearing is such as TAT peptide and octaarginine; Amphipathic cationic peptide such as penetratin and PEP-1; Hydrophobic peptide such as MTS etc.Wear the film peptide and can carry various biomacromolecule permeate through cell membranes by modes such as covalent linkage or non covalent bond couplings, the biomacromolecule of experiment confirm has gene, polypeptide, protein, nanoparticle and liposome etc.(Lain?Prochiantz?Protein?and?peptide?transduction，twenty?years?later?a?happy?birthday[J].Adv?Drug?Deliv?Rev，2008?Mar?1；60(4-5)：448-51.)。

Though the common film peptide of wearing has above many advantages, but when it assists the carrier of antitumor drug or its antitumor drug to enter cell, they can not be distinguished ordinary cells and tumour cell, can not bring into play membrane penetration effect selectively, therefore can cause antitumor drug drug effect and toxic side effect to be strengthened simultaneously, bring bigger misery to the patient.Therefore, make up and a kind of tumor tissues is had high selectivity, and the novel tumor cell selective that healthy tissues has a tight security is worn the film peptide be badly in need of by clinical.

Summary of the invention:

Goal of the invention

Purpose of the present invention aims to provide the tumour cell selectivity and wears film peptide and uses thereof.

Technical scheme

The tumour cell selectivity is worn the film peptide, and it contains basic aminoacids that 5-12 successive arginine residues form bunch, it is characterized in that the C end or the N end of described basic aminoacids bunch is connected with 3 histidine residues.Its peptide sequence comprises: RRRRRHHH (R ₅H ₃, R is at C end or N end), RRRRRRHHH (R ₆H ₃, R is at C end or N end), RRRRRRRHHH (R ₇H ₃, R is at C end or N end), RRRRRRRRHHH (R ₈H ₃, R is at C end or N end), RRRRRRRRRHHH (R ₉H ₃, R is at C end or N end), RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end or N end), RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end or N end), RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end or N end).

The tumour cell selectivity is worn the application of film peptide, it is characterized in that it being that preparation contains the described pharmaceutical composition of wearing the film peptide.

Described tumour cell selectivity is worn the application of film peptide, it is characterized in that described pharmaceutical composition Chinese traditional medicine is a taxol, Docetaxel, dactinomycin, Dx, epirubicin, daunorubicin, vincristine(VCR), vinorelbine, cis-platinum, oxaliplatin, methotrexate, Fluracil, mercaptopurine, cytosine arabinoside, doxifluridine, Etoposide, teniposide, camptothecine, hydroxycamptothecine, topotecan, irinotecan, mitoxantrone, endoxan, ifosfamide, ametycin, busulfan, lomustine, carmustine, semustine, nimustine, ranomustine, gemcitabine, capecitabine, Decitabine, Ancitabine, cis-platinum, bleomycin, Zhengguangmycin A5, morellic acid, the various pharmaceutical salts of neogambogic acid and these medicines.

Described tumour cell selectivity is worn the application of film peptide, it is characterized in that containing the various preparations that described tumour cell selectivity is worn the pharmaceutical composition of film peptide.

Useful achievement

Tumour cell selectivity of the present invention is worn the film peptide, has the cytolemma penetrativity, and can be distinguished tumour cell and normal tissue cell, can pass tumor cell membrane efficiently and normal cell membranes in tissue is not penetrated substantially; Innovation of the present invention is smart wants part promptly to be this, the peptide sequence of forming by arginine residues and histidine residues under different pH environment with the electric charge difference.Normal tissue cell is in the environment of pH7.4 usually, and the general slant acidity of its pH of tumor tissue cell is 5～6.5, and what have aminoacid sequence of the present invention wears the film peptide under normal pH environment, only have small positive charge, can't combine with the cell surface height and wear film; And the environment owing to its slant acidity makes Histidine and collaborative the showing of arginine have a large amount of positive charges when arriving the tumor tissue cell surface, thereby can be efficiently combine, embody the stronger effect that penetrates tumor cell membrane with electronegative cell surface.Tumour cell selectivity of the present invention wear the film peptide for the avidity of tumor cell membrane and to the carrying capacity of medicine and nano-medicament carrier by in the body and experiment in vitro proved.

Description of drawings:

Fig. 1 be 10 μ mol/L wear film peptide RRRRRRRRHHH (R ₈H ₃) fluorescence intensity that enters human acute leukemia cells strain HL60;

Fig. 2 be 10 μ mol/L wear film peptide RRRRRRRRHHH (R ₈H ₃) fluorescence intensity that enters mankind mastopathy cell's strain MCF-7;

Fig. 3 is that the free hydrochloric acid Dx of different concns, the doxorubicin hydrochloride phospholipid complex and the tumour cell selectivity of unmodified are worn the inhibiting rate experimental result of the doxorubicin hydrochloride phospholipid complex of film peptide modification to human oophoroma cell line SKOV3.

Embodiment:

R in the following example is an arginine, and H is a Histidine

Embodiment 1

With RRRRRHHH (R ₅H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 1.44g (2.22mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.46g (2.22mmol) dicyclohexylcarbodiimide and 0.30g (2.22mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.(protection) amino acid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRHHH.

The thick peptide of the RRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 1336, conforms to calculated value.RRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 2

With RRRRRRHHH (R ₆H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1.13gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 1.63g (2.51mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.52g (2.51mmol) dicyclohexylcarbodiimide and 0.34g (2.51mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.Oneself is connected in (protection) amino acid of C end on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRRHHH.

The thick peptide of the RRRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 1510, conforms to calculated value.RRRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 3

With RRRRRRRHHH (R ₇H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1.26gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 1.82g (2.80mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.58g (2.80mmol) dicyclohexylcarbodiimide and 0.38g (2.80mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.(protection) amino acid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRRRHHH.

The thick peptide of the RRRRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 1685, conforms to calculated value.RRRRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 4

With RRRRRRRRHHH (R ₈H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1.39gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 2.00g (3.09mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.64g (3.09mmol) dicyclohexylcarbodiimide and 0.42g (3.09mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.(protection) amino acid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRRRRHHH.

The thick peptide of the RRRRRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 1859, conforms to calculated value.RRRRRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 5

With RRRRRRRRRHHH (R ₉H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1.52gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 2.19g (3.38mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.70g (3.38mmol) dicyclohexylcarbodiimide and 0.46g (3.38mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.(protection) amino acid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRRRRRHHH.

The thick peptide of the RRRRRRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 2033, conforms to calculated value.RRRRRRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 6

With RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1.65gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 2.38g (3.67mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.76g (3.67mmol) dicyclohexylcarbodiimide and 0.50g (3.67mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.(protection) amino acid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRRRRRRHHH.

The thick peptide of the RRRRRRRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRRRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 2207, conforms to calculated value.RRRRRRRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 7

With RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1.78gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 2.57g (3.96mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.82g (3.96mmol) dicyclohexylcarbodiimide and 0.53g (3.96mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.(protection) amino acid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRRRRRRRHHH.

The thick peptide of the RRRRRRRRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRRRRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 2382, conforms to calculated value.RRRRRRRRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 8

With RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end and N end) and be example, the method that adopts solid-phase synthetic peptide is described.

Add 1.91gRinkamide-MBHA resin (0.74mmol/g) in 50mL side band arm, arm have the round-bottomed flask of sand plate filter core, added the 20mLDMF swelling 10 minutes, suction filtration removes solvent.Add 20mL 20% piperidines/DMF solution then, stirred suction filtration 30 minutes.With DMF washing resin 6 times, suction filtration removes solvent.(Pbf is 2 to add 2.75g (4.25mmol) Fmoc-Arg (Pbf)-OH in reaction flask; 2; 4; 6; 7-pentamethyl-Dihydrobenzofuranes-5-alkylsulfonyl), 0.88g (4.25mmol) dicyclohexylcarbodiimide and 0.57g (4.25mmol) HOBt (I-hydroxybenzotriazole) and 20mLDMF, stir under the room temperature and carry out condensation reaction.React the resin that takes a morsel after 2 hours and carry out the triketohydrindene hydrate color reaction, the result shows that condensation reaction is complete.Suction filtration is used DMF washing resin 6 times, and suction filtration removes solvent.(protection) amino acid of C end is connected on the resin.Reactions steps repeats top-operation to second of being connected with then that C held in back to last (protection) amino acid, promptly from 20% piperidines/DMF solution deprotection, after the triketohydrindene hydrate coloring test with the DMF washing only.If it is incomplete that the triketohydrindene hydrate coloring test shows condensation, then can prolong the condensation time until fully.All with the Fmoc protection, amino acid is followed successively by all amino acid whose a-amino: Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH, Fmoc-His (Trt)-OH.Last resin is washed for the third time vacuum-drying with methyl alcohol.

The above-mentioned resin that has connect peptide is joined in the 50mL round-bottomed flask, add 15mL reagent K (TFA/ thioanisole/EDT/ phenol/water=87.5/5.5/2.5/2.5/2.5 (v/v)), stirred 3 hours under the room temperature.Filter, collect filtrate.With give a baby a bath on the third day after its birth time resin of trifluoroacetic acid.Merging filtrate revolves steaming with filtrate and removes most trifluoroacetic acid, and adding volume is 8-10 ether sedimentation polypeptide doubly, puts into refrigerator overnight.Centrifugal, remove ether, vacuum-drying obtains the thick peptide of RRRRRRRRRRRRHHH.

The thick peptide of the RRRRRRRRRRRRHHH that 100mg is above-mentioned is dissolved in the 2mL pure water, with preparation reversed-phase HPLC purifying, collects the reservation main peak, collects liquid and contracts through revolving inspissation, and lyophilize gets the pure peptide of RRRRRRRRRRRRHHH then.The mass spectrum of pure peptide shows that its molecular weight is 2556, conforms to calculated value.RRRRRRRRRRRRHHH (R holds at N) can take identical preparation method and process to obtain.

Embodiment 9

With a series of 16 peptide species RRRRRHHH (R of this laboratory synthetic ₅H ₃, R is at C end or N end), RRRRRRHHH (R ₆H ₃, R is at C end or N end), RRRRRRRHHH (R ₇H ₃, R is at C end or N end), RRRRRRRRHHH (R ₈H ₃, R is at C end or N end), RRRRRRRRRHHH (R ₉H ₃, R is at C end or N end), RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end or N end), RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end or N end), RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end or N end) and for illustrating tumour cell selectivity of the present invention, example wears the wear film activity of film peptide to 2 kinds of common human tumor cells.16 kinds of tumour cell selectivity are worn film peptide N end flag F ITC fluorescein.RRRRRRRR (R ₈) available from AnaSpec company: N end flag F ITC fluorescein.

Get human acute leukemia cells strain HL60, centrifugal back adds the DMEM perfect medium, blows and beats into single cell suspension; Get 6 orifice plates, add the 0.9mL cell suspension in every hole, add 0.1mL then, the tumour cell selectivity of the FITC mark of 100 μ mol/L is worn film peptide or R8 culture medium solution, hatch (37 ℃, 5%CO ₂) after 1 hour, wash 3 times with the PBS solution centrifugal of 2mL, be resuspended in the PBS solution of 1mL, be used for flow cytometer quantitative analysis fluorescence intensity.

The mankind mastopathy cell's strain MCF-7 that takes the logarithm vegetative period, trysinization, centrifugal, add the DMEM perfect medium, blow and beat into single cell suspension; Take out the 2mL cell suspension and use the dilution of 10mL DMEM perfect medium.Get the every hole of 6 orifice plates and add 2mL cell suspension, CO ₂Incubator (37 ℃) is hatched to the cytogamy degree and is reached about 80%.Discard the upper strata nutrient solution, the tumour cell selectivity that adds the FITC mark of 1mL 10 μ mol/L is worn film peptide or R8 culture medium solution, hatch (37 ℃, 5%CO ₂) 1 hour.Discard the upper strata and wear the film peptide solution, wash 2 times, add 1mL trysinization 1min with cold PBS solution 1mL, every hole adds 1mL DMEM perfect medium again and stops digestion, blows and beats every porocyte and becomes single cell suspension, is transferred to then in the 10mL centrifuge tube, centrifugal 1000rpm, 5min.Discard supernatant liquid, add 2mL PBS solution re-suspended cell, be used for flow cytometer quantitative analysis fluorescence intensity.

The fluorescence intensity data analytical results as depicted in figs. 1 and 2.The result shows that 16 kinds of tumour cell selectivity of this laboratory synthetic are worn the film peptide all can bring into play membrane penetration effect well for human acute leukemia cells strain HL60 and mankind mastopathy cell's strain MCF-7, and effect obviously is better than classics and wears film peptide RRRRRRRR (R ₈).The tumour cell selectivity is worn film peptide R ₅H ₃, R ₆H ₃, R ₇H ₃, R ₈H ₃, R ₉H ₃, R ₁₀H ₃, R ₁₁H ₃, R ₁₂H ₃Two kinds of different sequences separately (R is at C end or N end) have the identical film activity of wearing.

Embodiment 10

With a series of 16 peptide species RRRRRHHH (R of this laboratory synthetic ₅H ₃, R is at C end or N end), RRRRRRHHH (R ₆H ₃, R is at C end or N end), RRRRRRRHHH (R ₇H ₃, R is at C end or N end), RRRRRRRRHHH (R ₈H ₃, R is at C end or N end), RRRRRRRRRHHH (R ₉H ₃, R is at C end or N end), RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end or N end), RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end or N end), RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end or N end) wear the film peptide normal cell of homologue and the selectivity of tumour cell are worn film activity for example illustrates tumour cell selectivity of the present invention.16 kinds of tumour cell selectivity are worn film peptide N end flag F ITC fluorescein.RRRRRRRR (R8) holds equal flag F ITC fluorescein available from AnaSpec company: N.

HepG-2 is incubated in the DMEM substratum with the human liver cancer cell strain, contains 10% foetal calf serum, the penbritin of each 100mg/L and Streptomycin sulphate.At 37 ℃, 5%CO ₂, normally cultivate under the saturated humidity fully, went down to posterity once in three days.

The human liver cancer cell strain HepG-2 that takes the logarithm vegetative period, trysinization, centrifugal, add the DMEM perfect medium, blow and beat into single cell suspension; Take out the 2mL cell suspension and use the dilution of 10mL DMEM perfect medium.Get 6 well culture plates, add cover glass, inoculation mankind mastopathy cell strain MCF-7, density is 2 * 10 ⁵Individual/hole, overnight incubation, treat cell attachment after, change fresh medium, behind the 30min, remove nutrient solution.The adding of every hole contains certain density fluorescently-labeled tumour cell selectivity wears film peptide or R8-FITC nutrient solution.After incubator is hatched certain hour, remove substratum, cell is washed 3 times with PBS again, and the Paraformaldehyde 96 stationary liquid with 3.7% (PBS preparation) is fixed cell 5min at room temperature, washes 3 times with PBS again.After glycerine with 50% (PBS preparation) mounting, under fluorescent microscope, observe.Adopt Image-Pro Plus (Version 4.5 for Windows ^TM) come the analysis of cells fluorescence intensity.

1:RRRRRHHH (R as a result ₅H ₃, R is at C end or N end), RRRRRRHHH (R ₆H ₃, R is at C end or N end), RRRRRRRHHH (R ₇H ₃, R is at C end or N end), RRRRRRRRHHH (R ₈H ₃, R is at C end or N end), RRRRRRRRRHHH (R ₉H ₃, R is at C end or N end), RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end or N end), RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end or N end), RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end or N end) all can effectively enter cell; Wherein especially with RRRRRRRRHHH (R ₈H ₃, R holds at C), RRRRRRRRHHH (R ₈H ₃, R holds at N) and to enter cell maximum; Next is RRRRRRRRRHHH (R ₉H ₃, R is at C end or N end), RRRRRRRHHH (R ₇H ₃, R is at C end or N end), RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end or N end), RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end or N end), RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end or N end), RRRRRRHHH (R ₆H ₃, R is at C end or N end), RRRRRHHH (R ₅H ₃, R is at C end or N end); And RRRRRRRR (R ₈) enter the minimum of cell.

In addition normal human subject liver cell line HL-7702 is incubated in the RPMI1640 substratum, contains 20% foetal calf serum, the penbritin of each 100mg/L and Streptomycin sulphate.At 37 ℃, 5%CO ₂, normally cultivate under the saturated humidity fully, went down to posterity once in per three days.

The normal human subject liver cell line HL-7702 that takes the logarithm vegetative period, trysinization, centrifugal, add the RPMI1640 substratum, blow and beat into single cell suspension; Take out the 2mL cell suspension and use the dilution of 10mL RPMI1640 substratum.Get 6 well culture plates, add cover glass, inoculation normal human subject liver cell line HL-7702, density is 2 * 10 ⁵Individual/hole, overnight incubation, treat cell attachment after, change fresh medium, behind the 30min, remove nutrient solution.The adding of every hole contains certain density fluorescently-labeled tumour cell selectivity wears film peptide or R8-FITC nutrient solution.After incubator is hatched certain hour, remove substratum, cell is washed 3 times with PBS again, and the Paraformaldehyde 96 stationary liquid with 3.7% (PBS preparation) is fixed cell 5min at room temperature, washes 3 times with PBS again.After glycerine with 50% (PBS preparation) mounting, under fluorescent microscope, observe.Adopt Image-ProPlus (Version 4.5 for Windows ^TM) come the analysis of cells fluorescence intensity.

2:RRRRRHHH (R as a result ₅H ₃, R is at C end or N end), RRRRRRHHH (R ₆H ₃, R is at C end or N end), RRRRRRRHHH (R ₇H ₃, R is at C end or N end), RRRRRRRRHHH (R ₈H ₃, R is at C end or N end), RRRRRRRRRHHH (R ₉H ₃, R is at C end or N end), RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end or N end), RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end or N end), RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end or N end) all can not enter cell effectively; Yet, RRRRRRRR (R ₈) can enter cell.

Based on the above results as can be known, tumour cell selectivity of the present invention is worn the film peptide and can be passed tumor cell membrane effectively and can not pass Normocellular cytolemma effectively, shows higher membrane efficiency and the higher selectivity of wearing.Other tumor cell lines have all drawn similar result with the test-results of normal cell system, do not enumerate one by one at this.

Embodiment 11

Wear film peptide RRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₅H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Take by weighing injection soybean phospholipid and taxol in eggplant type bottle according to recipe quantity, add a certain amount of dehydrated alcohol it is fully dissolved, and it is some to add granulated glass sphere.Vacuum rotary steam 1 hour is waved except that organic solvent formation medicine membrane of lipoprotein.Continue after the end to vacuumize to spend the night, to remove trace organic solvents.The hand hydration of 10mL pure water normal temperature, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.3000rpm is centrifugal to remove the ultrasonic scrap metal that may drop of probe then.Get supernatant liquor and cross 0.45 μ m respectively, each once can make the taxol phospholipid complex 0.22 μ m polycarbonate leaching film.UV spectrum, red external spectrum and dsc confirm that taxol and phosphatide have carried out combination effectively.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRHHH (R ₅H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRHHH (R ₅H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRHHH (R ₅H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 12

Wear film peptide RRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₅H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRHHH (R ₅H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRHHH (R ₅H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRHHH (R ₅H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 13

Wear film peptide RRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₆H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRHHH (R ₆H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRHHH (R ₆H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRHHH (R ₆H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 14

Wear film peptide RRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₆H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRHHH (R ₆H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRHHH (R ₆H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRHHH (R ₆H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 15

Wear film peptide RRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₇H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRHHH (R ₇H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRHHH (R ₇H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRHHH (R ₇H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 16

Wear film peptide RRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₇H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRHHH (R ₇H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRHHH (R ₇H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRHHH (R ₇H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 17

Wear film peptide RRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₈H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRHHH (R ₈H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRHHH (R ₈H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRHHH (R ₈H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 18

Wear film peptide RRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₈H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRHHH (R ₈H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRHHH (R ₈H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRHHH (R ₈H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 19

Wear film peptide RRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₉H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRHHH (R ₉H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRHHH (R ₉H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRHHH (R ₉H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 20

Wear film peptide RRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₉H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRHHH (R ₉H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRHHH (R ₉H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRHHH (R ₉H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 21

Wear film peptide RRRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₁₀H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRRHHH (R ₁₀H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRRHHH (R ₁₀H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRRHHH (R ₁₀H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 22

Wear film peptide RRRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₁₀H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRRHHH (R ₁₀H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRRHHH (R ₁₀H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRRHHH (R ₁₀H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 23

Wear film peptide RRRRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₁₁H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRRRHHH (R ₁₁H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRRRHHH (R ₁₁H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRRRHHH (R ₁₁H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 24

Wear film peptide RRRRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₁₁H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRRRHHH (R ₁₁H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRRRHHH (R ₁₁H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRRRHHH (R ₁₁H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 25

Wear film peptide RRRRRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₁₂H ₃, R holds at C) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRRRRHHH (R ₁₂H ₃, R holds at C), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRRRRHHH (R ₁₂H ₃, R holds at C) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRRRRHHH (R ₁₂H ₃, R holds at C) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 26

Wear film peptide RRRRRRRRRRRRHHH (R with the tumour cell selectivity that reaches of the present invention ₁₂H ₃, R holds at N) and the pharmaceutical composition formed with taxol illustrates its application method.

Precision takes by weighing 10mg tumour cell selectivity and wears film peptide RRRRRRRRRRRRHHH (R ₁₂H ₃, R holds at N), and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Drip to finish the back and continue to stir 30min, then this dispersion system is hatched at 4 ℃ and can make the tumour cell selectivity in 4 hours and wear film peptide RRRRRRRRRRRRHHH (R ₁₂H ₃, R holds at N) and the taxol phospholipid complex modified.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown that the tumour cell selectivity is worn film peptide RRRRRRRRRRRRHHH (R ₁₂H ₃, R holds at N) and the modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Embodiment 27

With of the present invention and the tumour cell selectivity pharmaceutical composition of wearing film peptide and taxol composition illustrate that this series wears promotion and the enhancement of film peptide to the external tumor killing effect of antitumor drug.

Precision takes by weighing 10mg tumour cell selectivity and wears the film peptide, and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the taxol phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Dropping finishes the back and continues to stir 30min, this dispersion system is hatched at 4 ℃ can make the taxol phospholipid complex that the tumour cell selectivity is worn the modification of film peptide in 4 hours then.The combination rate of taxol phospholipid complex before and after modifying and the investigation result of stability are shown, the tumour cell selectivity is worn the film peptide modification of taxol phospholipid complex is produced remarkably influenced to the physico-chemical property of common taxol phospholipid complex, the combination rate of taxol and phosphatide is greater than 97%, and normal temperature is placed 24 hours percolation ratios less than 3%.

Get human breast cancer cell strain MCF-7 and be incubated in DMEM (high sugar) substratum, contain 10% foetal calf serum, the penbritin of each 100mg/L and Streptomycin sulphate.At 37 ℃, 5%CO ₂, normally cultivate under the saturated humidity fully, go down to posterity once every other day.Choose the human breast cancer cell strain MCF-7 that is in logarithmic phase, trysinization, and be made into cell suspension with substratum dilution.In 96 orifice plates, every hole contains 1 * 10 approximately with cell inoculation ⁴Individual cell, at 37 ℃, 5%CO ₂Cultivated 24 hours in the environment, discard nutrient solution, dilute the common taxol phospholipid complex and the tumour cell selectivity of unmodified with the substratum that does not contain serum respectively and wear taxol phospholipid complex concentration to 1,4,8,10,15,20,30,50, the 100nmol/L that the film peptide is modified, each concentration group is provided with 3 multiple holes, every hole 200 μ L.At 37 ℃, 5%CO ₂Cultivated 72 hours in the environment.Fix with 4 ℃ Tricholroacetic Acid then, in 4 ℃ of placements 1 hour, distilled water was washed 5 times and is removed Tricholroacetic Acid, nutrient solution, meta-bolites and serum with 96 orifice plates, added 4g/L sulfo group rhodamine B (SRB) dyeing 15min after the dry air, washed 5 times with 1% acetic acid again.After the drying, add 10mmol/LTris liquid 150 μ L dissolving, use microplate reader to measure absorption value A, calculation of half inhibitory concentration (IC in wavelength 540nm place ₅₀).Adopt the rhodamine B method to investigate the cytotoxicity of two kinds of taxol phospholipid complex.For human breast cancer cell strain MCF-7, the IC of the taxol phospholipid complex of unmodified ₅₀Wear the IC of the taxol phospholipid complex that the film peptide modifies with the tumour cell selectivity ₅₀As shown in Table 1, the result shows, compares with the taxol phospholipid complex of unmodified, and the cytotoxicity that the tumour cell selectivity is worn the taxol phospholipid complex of film peptide modification obviously strengthens (P＜0.05).Matrix material and the equal no cytotoxicity of other auxiliary materials do not form interference to measuring.The tumour cell selectivity is worn film peptide R ₅H ₃, R ₆H ₃, R ₇H ₃, R ₈H ₃, R ₉H ₃, R ₁₀H ₃, R ₁₁H ₃, R ₁₂H ₃Not there are differences between the cytotoxicity of taxol phospholipid complex that two kinds of different sequences separately (R is at C end or N end) are modified human breast cancer cell strain MCF-7.

Each preparation of table one is to the half-inhibition concentration (IC of human breast cancer cell strain MCF-7 ₅₀)

Preparation IC ₅₀(nmol/L)

The common taxol phospholipid complex 49.6 ± 4.7 of unmodified

R ₅H ₃The taxol phospholipid complex of modifying 27.7 ± 2.8

R ₆H ₃The taxol phospholipid complex of modifying 24.3 ± 2.4

R ₇H ₃The taxol phospholipid complex of modifying 22.4 ± 3.1

R ₈H ₃The taxol phospholipid complex of modifying 20.8 ± 2.1

R ₉H ₃The taxol phospholipid complex of modifying 23.6 ± 2.5

R ₁₀H ₃The taxol phospholipid complex of modifying 26.1 ± 3.2

R ₁₁H ₃The taxol phospholipid complex of modifying 30.2 ± 4.6

R ₁₂H ₃The taxol phospholipid complex of modifying 32.3 ± 4.1

Embodiment 28

With of the present invention and the tumour cell selectivity pharmaceutical composition of wearing film peptide and doxorubicin hydrochloride composition illustrate that this series wears the film peptide to promotion and the enhancement of antitumor drug at external tumor killing effect.

Take by weighing injection Ovum Gallus domesticus Flavus lecithin and doxorubicin hydrochloride in round-bottomed flask according to recipe quantity, add a certain amount of tetrahydrofuran (THF) Ovum Gallus domesticus Flavus lecithin is fully dissolved, and it is some to add granulated glass sphere.Under the lucifuge condition, place the constant temperature water bath magnetic agitation in 40 ℃ of reactions 3 hours.Vacuum rotary steam is 1 hour then, waves except that organic solvent formation medicine membrane of lipoprotein.Continue after the end to vacuumize to spend the night, to remove trace organic solvents.Add the hand aquation medicine of phosphate buffer soln (pH6.5) normal temperature membrane of lipoprotein, till cleaning to eggplant type bottle wall, sample is uniform emulsion.Probe is ultrasonic to reduce particle diameter in the ice-water bath.Centrifugal 3000rpm is to remove the ultrasonic scrap metal that may drop of probe.Get supernatant liquor and cross 0.45 μ m respectively, 0.22 μ m polycarbonate leaching film respectively carries out whole grain for 5 times can make the doxorubicin hydrochloride phospholipid complex.UV spectrum, red external spectrum and dsc confirm that doxorubicin hydrochloride and Ovum Gallus domesticus Flavus lecithin have carried out combination effectively.

Precision takes by weighing 10mg tumour cell selectivity and wears the film peptide, and it fully is dissolved in the distilled water of 2mL.Under normal temperature, this solution slowly is added drop-wise in the above doxorubicin hydrochloride phospholipid complex dispersion system that makes, the dropping process keeps magnetic agitation constantly.Dropping finishes the back and continues to stir 30min, this dispersion system is hatched at 4 ℃ can make the doxorubicin hydrochloride phospholipid complex that the tumour cell selectivity is worn the modification of film peptide in 8 hours then.The combination rate of doxorubicin hydrochloride phospholipid complex before and after modifying and the investigation result of stability are shown, the tumour cell selectivity is worn the film peptide modification of doxorubicin hydrochloride phospholipid complex is produced remarkably influenced to the physico-chemical property of common doxorubicin hydrochloride phospholipid complex, combination rate is greater than 90%, and normal temperature is placed 24 hours percolation ratios less than 4%.

Get human oophoroma cell line SKOV3, containing the DMEN nutrient solution of 10% foetal calf serum, 5%CO ₂, normally cultivate under 37 ℃ of complete humidity.The cell of taking the logarithm vegetative period with the dilution of DMEM nutrient solution, is 2 * 10 by every hole density after the trysinization ⁵Individual/hole, overnight incubation, after treating cell attachment, in 24 orifice plates, add different amount free hydrochloric acid Dxs, unmodified doxorubicin hydrochloride phospholipid complex and tumour cell selectivity and wear each 1mL of doxorubicin hydrochloride phospholipid complex that the film peptide is modified, and with substratum as the blank group.Doxorubicin hydrochloride concentration is respectively 10,2,0.4,0.08 and 0.016 μ g/mL.Each concentration is done 3 multiple holes.After hatching 48 hours, each multiple hole adds MTT solution 100 μ L (5mg/mL), 5%CO ₂, continue under 37 ℃ of conditions to cultivate 4 hours, discard supernatant liquid, in each multiple hole, add dimethyl sulfoxide (DMSO) 600 μ L again, on microplate reader, measure the absorbancy in each hole, 570nm place behind the vibration 10min, average, with the substratum is blank, calculates cell inhibitory rate.Cell inhibitory rate (%)=[1-A _570nm(sample)/A _570nm(control)] * 100%, A _570nm(sample) be the absorbancy of cell behind the adding medicine, A _570nm(control) be the absorbancy of blank cell.In 10,2,0.4,0.08 and 0.016 μ g/mL concentration group, the tumour cell selectivity is worn the doxorubicin hydrochloride phospholipid complex of doxorubicin hydrochloride phospholipid complex that the film peptide modifies, unmodified and free hydrochloric acid Dx to the inhibiting rate of human oophoroma cell line SKOV3 as shown in Figure 3.By the result as can be known, the tumour cell selectivity wear doxorubicin hydrochloride phospholipid complex that the film peptide modifies to the inhibiting rate of human oophoroma cell line SKOV3 apparently higher than common doxorubicin hydrochloride phospholipid complex and free hydrochloric acid Dx (P＜0.05).The tumour cell selectivity is worn film peptide R ₅H ₃, R ₆H ₃, R ₇H ₃, R ₈H ₃, R ₉H ₃, R ₁₀H ₃, R ₁₁H ₃, R ₁₂H ₃Not there are differences between the inhibiting rate of doxorubicin hydrochloride phospholipid complex that two kinds of different sequences separately (R is at C end or N end) are modified human oophoroma cell line SKOV3.

Claims

1. the tumour cell selectivity is worn the film peptide, and it contains basic aminoacids that 5-12 successive arginine residues form bunch, it is characterized in that the C end or the N end of described basic aminoacids bunch also has 3 histidine residues.Its peptide sequence comprises: RRRRRHHH (R ₅H ₃, R is at C end or N end), RRRRRRHHH (R ₆H ₃, R is at C end or N end), RRRRRRRHHH (R ₇H ₃, R is at C end or N end), RRRRRRRRHHH (R ₈H ₃, R is at C end or N end), RRRRRRRRRHHH (R ₉H ₃, R is at C end or N end), RRRRRRRRRRHHH (R ₁₀H ₃, R is at C end or N end), RRRRRRRRRRRHHH (R ₁₁H ₃, R is at C end or N end), RRRRRRRRRRRRHHH (R ₁₂H ₃, R is at C end or N end).

2. tumour cell selectivity according to claim 1 is worn the application of film peptide, it is characterized in that it being that preparation contains the described pharmaceutical composition of wearing the film peptide.

3. tumour cell selectivity according to claim 2 is worn the application of film peptide, it is characterized in that described pharmaceutical composition Chinese traditional medicine is a taxol, Docetaxel, dactinomycin, Dx, epirubicin, daunorubicin, vincristine(VCR), vinorelbine, cis-platinum, oxaliplatin, methotrexate, Fluracil, mercaptopurine, cytosine arabinoside, doxifluridine, Etoposide, teniposide, camptothecine, hydroxycamptothecine, topotecan, irinotecan, mitoxantrone, endoxan, ifosfamide, ametycin, busulfan, lomustine, carmustine, semustine, nimustine, ranomustine, gemcitabine, capecitabine, Decitabine, Ancitabine, cis-platinum, bleomycin, Zhengguangmycin A5, morellic acid, the various pharmaceutical salts of neogambogic acid and these medicines.

4. tumour cell selectivity according to claim 2 is worn the application of film peptide, it is characterized in that containing the various preparations that described tumour cell selectivity is worn the pharmaceutical composition of film peptide.