CN101732699A - Cyclopeptide nanotube medicinal composition and application thereof - Google Patents

Cyclopeptide nanotube medicinal composition and application thereof Download PDF

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CN101732699A
CN101732699A CN200910145249A CN200910145249A CN101732699A CN 101732699 A CN101732699 A CN 101732699A CN 200910145249 A CN200910145249 A CN 200910145249A CN 200910145249 A CN200910145249 A CN 200910145249A CN 101732699 A CN101732699 A CN 101732699A
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nanotube
cyclopeptide
cyclic peptide
medicinal composition
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吴伟
陈键
邱怡婷
张蓓
卢懿
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Fudan University
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Abstract

The invention relates to a cyclopeptide nanotube medicinal composition and application thereof. The medicinal composition consists of a cyclopeptide nanotube and medicaments. The cyclopeptide nanotube has a vander Waals inside diameter of 0.75 to 1.3nm and a length of 10 to 100nm. A basic unit of the cyclopeptide nanotube is cyclopeptide which is formed by cyclizing 8, 10 and 12 amino acids. The diameter of the medicament molecule is smaller than the vander Waals inside diameter of the cyclopeptide nanotube. The invention also provides application of the cyclopeptide nanotube medicinal composition. The cyclopeptide nanotube medicinal composition uses the cyclopeptide nanotube as a transmembrane artificial nano channel, so that micromolecular medicaments enter cells in a mode of passive diffusion. The invention discovers the new medical use of the cyclopeptide nanotube medicinal composition. The medicaments rapidly enter the cells at a high concentration for improving the cytotoxicity thereof. Due to the synergistic effect of the medicaments with the cyclopeptide nanotube, the cyclopeptide nanotube medicinal composition has application potential in the oncotherapy, bacteria resistance, virus resistance, and other pathogen infection aspects.

Description

A kind of Cyclopeptide nanotube medicinal composition and application thereof
[technical field]
The present invention relates to a kind of pharmaceutical composition, specifically, is about a kind of Cyclopeptide nanotube medicinal composition and application thereof.
[background technology]
Even number D, L-aminoacid alternately connect can form closed cyclic peptide.In order to make intramolecularly amino acid side chain and side chain and side and main interchain interaction minimum, cyclic peptide is taked plane configuration.Substituent group on the alpha-carbon is parallel to anchor ring and points to outside the ring and the anchor ring all vertical with amino of the carbonyl on the main chain backbone along the radius of ring.The cyclic peptide molecule of hollow forms intermolecular hydrogen bond network by the C=O in the main chain backbone acylamino-and N-H in the mode of β-lamella and is piled into stable in properties, open-ended, size tubular structure-self assembly Cyclopeptide nanotube (the J Am Chem Soc in molecular range, 1998,120 (35): 8949.).This β-lamella promptly can be that taking advantage of a situation parallel also can be that anti-gesture is parallel, but computer analysis and experiment all show, β-lamella hydrogen bond preferential anti-gesture parallel construction (MatSci Eng C, 1997,4 (4): 207.) selected on thermodynamics based on the Cyclopeptide nanotube of this principle design.Form nanotube by this way and have 2 particularly outstanding advantages: the aperture that 1. can regulate nanotube by the number of control loop peptide ammino acid; 2. can change the outer wall character of nanotube by the aminoacid kind of control cyclic peptide.
The aperture of the amino acid number scalable Cyclopeptide nanotube by changing cyclic peptide, but the size of cyclic peptide is too hour, produces huge tension force in the ring and is unfavorable for forming between subunit hydrogen bond network; When too big, because the laxity of ring skeleton also can't be self-assembled into stable tubular structure (J Am Chem Soc, 1996,118 (1): 43.).6,8,10,12 aminoacid cyclic peptide all can be self-assembled into the Cyclopeptide nanotube that the aperture increases successively, wherein the aperture of the Cyclopeptide nanotube formed of 8,10,12 aminoacid about 0.75,1.0,1.3nm (J PeptRes, 2001,57 (4): 301; Curr Opin Biotech, 1999,10 (1): 94; Angew Chem IntEd, 2001,40 (6): 988.).
The same with native protein, polypeptide, D, the amino acid residue order of L-cyclic peptide should be beneficial to it and be in stable space structure, and amino acid whose character all produces significant effects to the various self assemblies under the different condition in the cyclic peptide.The superiority of Cyclopeptide nanotube is and can regulates the surface nature of nanotube by selecting suitable aminoacid, this makes us can design the cyclic peptide that different aminoacids is formed according to different purposes, and it can be self-assembled into the different nanotube of character in different chemical environments.
The natural ion channel that protein or polypeptide constitute is being controlled the exchange and the signal transduction of ionic transhipment, the inside and outside material of cell, is therefore bringing into play extremely important biological function.Although we have had deep understanding to the structure and the function of ion channel, artificial ion channel remains one of current important research object.Theoretical, the bimolecular lipid membrane of low electric conductance constant is beneficial to cyclic peptide self assembly therein.Ghadirl in 1994 the 1st time has verified this probability by liposome model and a series of spectroscopy means.With enough concentration c yclo[-(Trp-D-Leu) 3-Gln-D-Leu-] be added in the suspension of liposome, the hydrophobic side chains of cyclic peptide is inserted in the lipid bilayer them rapidly, and relies on intermolecular hydrogen bond to be piled into the nanotube that internal diameter is about 0.75nm in adipose membrane.By with micro diaphragm pincers record ion single channel current, prove that Cyclopeptide nanotube is to K +, Na +The activity of transhipment is respectively 2.2 * 10 7, 1.8 * 10 7Ionss -1, be 3 times of natural analog gramicidin A under the same experimental conditions.And cyclo[-(Gln-D-Leu) 4-] and cyclo[-( MeN-D-Ala-Phe) 4-] under identical experiment condition, but can't form nanotube (J PeptRes, 2001,57 (4): 301.).This explanation cyclic peptide molecule will be self-assembled into nanotube in bimolecular lipid membrane, the cyclic peptide side chain must possess appropriate hydrophobicity, so that cyclic peptide can enter plasma membrane, and amino acid side chain is sterically hindered little, makes that cyclic peptide is intermolecular can form extensive and stable hydrogen bond.
The D of synthetic, L-α-cyclic peptide can embed bacterial cell membrane, and piles up the tubular conduit that forms hollow in adipose membrane.Internal and external test shows that all these Cyclopeptide nanotubes can effectively kill enterococcus faecalis and other pathogenic bacterium (Nature, 2001,412 (26): 452.) of methicillin resistant staphylococcus aureus, vancomycin resistance.The self assembly Cyclopeptide nanotube is the cyclic peptide flexible design, is easy to synthesize and protease is stablized as the advantage of new antibiotic.Can improve their selectivity and permeabilitys by selecting different aminoacid to form, reduce haemolysis the artificial red blood cells to bacterial cell membrane.Different with other antibiotic mechanism of action, Cyclopeptide nanotube does not produce chemical action, and only utilize on bacterial cell membrane the duct that forms nano-scale and bring into play antibacterial activity, the mechanism of action of this uniqueness has shortened sterilizing time, and can reduce chemical sproof generation.
Self assembly on cell membrane forms nanotube in view of cyclic peptide, allow diameter to diffuse in the cell by passive less than the small-molecule drug of its internal diameter, therefore can utilize this character of Cyclopeptide nanotube, make up the small-molecule drug induction system, make small-molecule drug enter in the cell fast with high concentration, improve its cytotoxicity,, have application potential aspect the pathogenic infections such as oncotherapy and anti-cell, virus with the Cyclopeptide nanotube synergism.
[summary of the invention]
The objective of the invention is, a kind of Cyclopeptide nanotube medicinal composition is provided.
One purpose more of the present invention is that a kind of purposes of Cyclopeptide nanotube medicinal composition is provided.
For achieving the above object, the technical scheme that the present invention takes is: a kind of Cyclopeptide nanotube medicinal composition, form by Cyclopeptide nanotube and medicine, described Cyclopeptide nanotube Van der Waals internal diameter is 0.75-1.3nm, pipe range is 10-100nm, Cyclopeptide nanotube basic composition unit is a cyclic peptide, and cyclic peptide is formed by 8,10,12 aminoacid cyclizations, and the diameter of described drug molecule is less than Cyclopeptide nanotube Van der Waals internal diameter.
Described Cyclopeptide nanotube Van der Waals internal diameter is 0.75-1.3nm, pipe range is 10-100nm, Cyclopeptide nanotube basic composition unit is a cyclic peptide, and cyclic peptide is formed by 8,10,12 aminoacid cyclizations, and the diameter of described drug molecule is less than Cyclopeptide nanotube Van der Waals internal diameter.
Cyclic peptide in the described Cyclopeptide nanotube replaces covalent bond by the D type of equal number and L type aminoacid and forms, and described drug molecule is a hydrophilic medicament.
The aminoacid sequence of cyclic peptide such as SEQ ID NO:1 in the described Cyclopeptide nanotube, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, shown in SEQ ID NO:23 or the SEQ ID NO:24, described medicine is selected from 5-fluorouracil, flucytosine, ftorafur, salicylic acid, para-aminosalicylic acid, cisplatin, carboplatin, chlormethine, cyclophosphamide, ifosfamide, formylmerphalan, glyforfin, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, Seninon, ethambutol, ribavirin, zidovudine or metronidazole.
The aminoacid sequence of cyclic peptide is shown in SEQ ID NO:23 or SEQ ID NO:24 in the described Cyclopeptide nanotube, and described medicine is selected from 5-fluorouracil, ftorafur, cisplatin or carboplatin.
For realizing above-mentioned second purpose, the technical scheme that the present invention takes is: the application of a kind of Cyclopeptide nanotube medicinal composition in preparation treatment tumor disease, bacterial infective diseases or virus infection medicine, described Cyclopeptide nanotube medicinal composition is made up of Cyclopeptide nanotube and medicine.
Described Cyclopeptide nanotube Van der Waals internal diameter is 0.75-1.3nm, pipe range is 10-100nm, Cyclopeptide nanotube basic composition unit is a cyclic peptide, and cyclic peptide is formed by 8,10,12 aminoacid cyclizations, and the diameter of described drug molecule is less than Cyclopeptide nanotube Van der Waals internal diameter.
Cyclic peptide in the described Cyclopeptide nanotube replaces covalent bond by the D type of equal number and L type aminoacid and forms, and described drug molecule is a hydrophilic medicament.
The aminoacid sequence of cyclic peptide such as SEQ ID NO:1 in the described Cyclopeptide nanotube, SEQ ID NO:2, SEQID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, shown in SEQ ID NO:23 or the SEQ ID NO:24, described medicine is selected from 5-fluorouracil, flucytosine, ftorafur, salicylic acid, para-aminosalicylic acid, cisplatin, carboplatin, chlormethine, cyclophosphamide, ifosfamide, formylmerphalan, glyforfin, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, Seninon, ethambutol, ribavirin, zidovudine or metronidazole.
The aminoacid sequence of cyclic peptide is shown in SEQ ID NO:23 or SEQ ID NO:24 in the described Cyclopeptide nanotube, and described medicine is selected from 5-fluorouracil, ftorafur, cisplatin or carboplatin.
Technique scheme realizes by following technical method:
Cyclopeptide nanotube is self assembly on cell membrane, can set up to run through the inside and outside passage of cell, mediation ion such as H +, Na +, K +Deng transmembrane transport, therefore break the inside and outside ionic equilibrium of cell, cause cell death, thereby have antibiotic, antivirus action.Still find that Cyclopeptide nanotube can carry life-critical molecule glucose and glutamic acid transmembrane transport.
The present inventor is by discovering, the self assembly Cyclopeptide nanotube can mediate small-molecule drug by cell membrane transporter, its speed greater than small-molecule drug without nanotube to intracellular diffusion rate.Find that Cyclopeptide nanotube and drug combination have collaborative tumor-inhibiting action in the activity research that provides in embodiments of the present invention to tumor cell.
The nanotube delivery system that the cyclic peptide self assembly forms among the present invention is made of jointly Cyclopeptide nanotube and medicine.
Based on the hydrophobic character of Cyclopeptide nanotube periphery, Cyclopeptide nanotube self assembly in the phospholipid bilayer of cell membrane, the thickness of its pipe range visual cell film or pathogen shell is between 10-100nm, preferably between 20-50nm.
The basic composition unit of Cyclopeptide nanotube is a cyclic peptide, the cyclic peptide sight structure of making even, intermolecular by hydrogen bond self assembly layer by layer formation nanotube, in order to help forming intermolecular hydrogen bonding and form the stabilized nano pipe, the total number of atnino acid of cyclic peptide is 8-12, and D type and L type aminoacid half and half alternately covalent bond form.Be subjected to the restriction of cyclic peptide total number of atnino acid, the internal diameter of Cyclopeptide nanotube is 0.75-1.3nm.
The aminoacid sequence that forms Cyclopeptide nanotube can be selected from S WF KT KS K-, SW FK TK SK-, S WF KH KS K-, S WBY KN KS K-, KK HK WL WK-, SK SW LW LW-, TH SW LW LW-, RG DW LW LW-, KQ RW LW LW-, K QR WL WL W-, RQ RW LW LW-, KQ KW LW LW-, KS KW LW LW-, SH KW LW LW-, SKHWLWLW-, EK HW LW LW-, KK KW LW LW-, RR KW LW LW-, KA KW LW LW-, RR RW LW LW-, HK HW LW LW-, KH KW LW LW-, W LW LW LQ L-, LW LW LW LW LQ-.According to the aminoacid single-letter code name of generally acknowledging, A, D, E, F, G, H, L, K, Q, R, S, T, W, Y, B represent alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, leucine, lysine, glutamine, arginine, serine, threonine, tryptophan, tyrosine, asparagine/aspartic acid respectively; The letter underscore is represented D configuration aminoacid, and all the other are L type aminoacid; On behalf of this aminoacid sequence, "-" behind the aminoacid sequence join end to end, and constitutes cyclic peptide.The aminoacid sequence of cyclic peptide especially is selected from W LW LW LQ L-, LW LW LW LW LQ-.
The synthetic of cyclic peptide of the present invention can be adopted general solid phase or liquid-phase synthesis process, carries out in general reactor or solid phase synthetic instrument.Synthetic cyclic peptide is by the preparation liquid phase separation, and purity is greater than 95%.Provided in the embodiment of the invention LW LW LW LW LThe liquid phase synthetic route of Q-.
Medicine is passive diffusion by Cyclopeptide nanotube transmembrane transport pattern, is subjected to the restriction of cyclic peptide internal diameter size, and the molecular diameter of medicine should be less than the internal diameter of Cyclopeptide nanotube.The cyclic decapeptide that provides in the embodiment of the invention LW LW LW LW LThe internal diameter of the nanotube that Q-forms is 1.0nm, can allow molecular diameter to pass through less than the micromolecule of 1.0nm.The nanotube internal diameter that the ring dodecapeptide forms is 1.3nm, can allow molecular diameter to pass through less than the medicine of 1.3nm.Consider that the big more resistance that runs into when penetrating of molecular diameter is big more, molecular diameter should be significantly less than the nanotube internal diameter.Cyclopeptide nanotube delivery system of the present invention, wherein the diameter of drug molecule is preferably less than 1.0nm less than 1.3nm.
Cyclopeptide nanotube delivery system of the present invention is applicable to that small-molecule drug mediates transmembrane transport through nanotube under the multiple situation, and the selection of medicine is not limited to its therapeutic purposes.From pressing down the angle analysis that kills cell, pathogen, the present invention is applicable to antitumor drug, antibacterial, antiviral agent.Its range of application can be extended according to know-why of the present invention.
The medicine that Cyclopeptide nanotube delivery system of the present invention is suitable for is selected from 5-fluorouracil, flucytosine, ftorafur, salicylic acid, para-aminosalicylic acid, cisplatin, carboplatin, chlormethine, cyclophosphamide, ifosfamide, formylmerphalan, glyforfin, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, Seninon, ethambutol, ribavirin, zidovudine, metronidazole.Especially be selected from 5-fluorouracil, ftorafur, cisplatin, carboplatin.
Provide Cyclopeptide nanotube mediation small-molecule drug to stride the effect of liposome artificial phospholipid bilayer film transhipment in the embodiments of the invention.Liposome artificial phospholipid bilayer film is acknowledged as the model of best analog cell film, is applicable to estimate delivery system of the present invention.Estimated successively in the embodiment of the invention under the different cyclic peptide consumption situations, the transmembrane transport effect of hydrion, 5-fluorouracil, ftorafur, cisplatin, the result shows the increase along with the cyclic peptide consumption, the transport velocity of hydrion or small-molecule drug is accelerated.The molecular diameter of cytosine arabinoside is 1.11nm, can not see through the cyclic decapeptide nanotube in the embodiment of the invention.The result confirms that cytosine arabinoside is difficult to see through liposome bimolecular tunic, increases the cyclic peptide consumption and also can't improve its transmembrane transport rate.
Select human hepatocellular carcinoma BEL-7402 cell's strain in the embodiment of the invention, after investigating Cyclopeptide nanotube and acting on tumor cell membrane, to the change situation of the sensitivity of 5-FU.During experimental implementation tumor cell line is inoculated on 96 orifice plates, 3000/hole, adds the culture fluid effect 48h that contains finite concentration CP and 5-FU, the cytotoxicity of mtt assay detection of drugs behind the 24h.The result shows that after cyclic peptide (64 μ g/ml) acted on human hepatocellular carcinoma BEL-7402 cell's strain, 5-FU was reduced to 25.02 μ g/ml to the IC50 of this cell from 47.93 μ g/ml.This explanation cyclic peptide can be on tumor cell membrane self assembly form Cyclopeptide nanotube, and the outside small-molecule drug 5-FU of mediation enters fast by this passage and produces cytotoxicity in the tumor cell.
The invention has the advantages that:
1, pharmaceutical composition of the present invention as striding film artificial nano passage, makes small-molecule drug enter in the cell by passive diffusion way with Cyclopeptide nanotube.
2, the present invention has excavated the new medical application of Cyclopeptide nanotube medicinal composition.
3, medicine enters in the cell fast with high concentration, improves its cytotoxicity, with the Cyclopeptide nanotube synergism, has application potential aspect the pathogenic infections such as oncotherapy and antibacterium, virus.
[description of drawings]
Accompanying drawing 1 cyclic decapeptide cyclo[ LW LW LW LW LQ] structural formula.
Accompanying drawing 2 cyclic decapeptide cyclo[ LW LW LW LW LQ] synthetic route chart.
Light microscopic (600 times) figure of accompanying drawing 3A pencil Cyclopeptide nanotube.
Transmission electron microscope (2000 times) figure of accompanying drawing 3B pencil Cyclopeptide nanotube.
Accompanying drawing 4 Cyclopeptide nanotubes mediation hydrion (H +) stride artificial lipid film transhipment figure.
Accompanying drawing 5 Cyclopeptide nanotubes mediation 5-FU strides artificial lipid film transhipment figure.
Accompanying drawing 6 Cyclopeptide nanotubes mediation cisplatin is striden artificial lipid film transhipment figure.
Accompanying drawing 7 Cyclopeptide nanotubes mediation ftorafur is striden artificial lipid film transhipment figure.
Accompanying drawing 8 peptide nanotubes mediation cytarabine is striden artificial lipid film transhipment figure.
[specific embodiment]
Elaborate below in conjunction with the specific embodiment of accompanying drawing to a kind of Cyclopeptide nanotube medicinal composition provided by the invention.
Embodiment 1
Synthesizing of cyclic peptide
Adopt the synthetic cyclo[of the bonded method of solid phase-liquid phase LW LW LW LW LQ], model Dehua internal diameter of this cyclic peptide is 1.0hm, its structural formula is as shown in Figure 1.
Synthetic method: adopt the synthetic polypeptide of the first solid phase method of Boc-Trp (For)-PAM resin (PAM resin), liquid phase method carries out cyclization again.Synthetic route chart as shown in Figure 2.
(substitution value: 0.6mmol/g 0.066mmol) places 10ml polypeptide reaction tube, adds 50% trifluoracetic acid (TFA)/CH to get resin 110mg 2Cl 2Solution, stirring reaction remove tryptophan Boc protecting group (3ml, 15min, 2 times), use CH successively 2Cl 2With DMF agitator treating resin (CH 2Cl 2: 3ml, 1min, 5 times; DMF:3ml, 1min, 4 times).After the ninhydrin solution inspection reacts completely, get the Boc-D-Leu-OH and the HOBt of 4 times of moles of resin, the DIPEA of 8 times of moles is dissolved among the 4ml DMF, behind the activation 30min, adds in the reaction tube, and stirring reaction 30min uses DMF and CH successively 2Cl 2Agitator treating resin (DMF:3ml, 1min, 4 times; CH 2Cl 2: 3ml, 1min, 5 times).Add 50% trifluoracetic acid (TFA)/CH again 2Cl 2After solution removes the Boc protecting group, insert follow-up aminoacid successively according to sequence.After aminoacid all connects, add 20% piperidines/DMF solution, stirring reaction (3ml, 10min, 2 times), CH 2Cl 2Agitator treating resin (3ml, 1min, 6 times).Resin is changed in the HF reaction tube, add 10ml liquid HF and an amount of p-cresol, stirring reaction 1h under the ice bath, negative pressure is removed HF, adds ice ether sedimentation and cyclic washing polypeptide, adds 50% acetonitrile/water (0.1%TFA) dissolving cyclic peptide.Partly prepare purification, lyophilization, both white polypeptide powder.
The half polypeptide 20mg of preparation behind the purification that learn from else's experience places round-bottomed flask, adds the PyBOP of 5 times of moles and the DIPEA of 10 times of moles, 20ml DMF dissolving, the about 20h of stirring at room reaction.Reactant liquor partly prepares purification after adding 20ml water dilution, lyophilization, both white cyclic peptide powder.Product yield 30～40%, purity height＞95%.
Embodiment 2
Polypeptide and cyclic peptide partly prepare purification
Chromatographic condition: Ecilpse XDB-C18 post, 9.4 μ m * 250mm; Mobile phase A: 0.1%TFA/H 2O, Mobile phase B: 0.1%TFA/ acetonitrile; Gradient condition (polypeptide): 0～3min 30%B, 3～4min 30%B → 50%B, 4～6min 50%B, 6～26min 50%B → 60%B, 26～30min 60%B → 30%B; Gradient condition (cyclic peptide): 0～5min 30%B, 5～7min 30%B → 50%B, 7～10min 50%B, 10～30min 50%B → 60%B, 30～35min60%B, 35～40min 60%B → 30%B; Detect wavelength: 280nm; Flow velocity: 4ml/min; Column temperature: 25 ℃; Sample size: 1ml.
Synthetic cyclic decapeptide cyclo[ LW LW LW LW LQ] through HPLC, LC/MS, FT-IR, UV, 1HNMR characterizes, and has verified its structure.
Embodiment 3
The preparation of Cyclopeptide nanotube and sign
In solvent, Cyclopeptide nanotube self assembly behavior is observed.Cyclic decapeptide 1mg places the 1.5ml centrifuge tube, uses 0.5ml 1%TFA/CHCl 3Dissolving is left standstill the observation crystallization and is separated out situation.After placing a few hours or more than ten hour, observing gradually has the crystallization of hour hand shape to separate out in the solution.When crystallization was separated out degree and reached maximum, centrifugal (10000rpm 10min), removed organic solvent, and the gained precipitate adds the appropriate amount of purified water suspendible, does further to analyze.The Cyclopeptide nanotube that forms carries out optical microscope and transmission electron microscope observing, shown in accompanying drawing 3A and accompanying drawing 3B.Viewed Cyclopeptide nanotube is pencil and assembles, for long bar-shaped.
Embodiment 4
Cyclopeptide nanotube mediation medicine transmembrane transport is investigated on the liposome phospholipid bilayer model
Liposome adopts the reverse phase evaporation preparation, and medicine is a 5-fluorouracil.Preparation process is as follows: get phospholipid (purity＞80%) 60mg, cholesterol 15mg, be dissolved in 6ml CHCl 3, add 2ml 5mg/ml medicine/PBS solution; Adopt ultrasonic probe to carry out ultrasonic emulsification, ultrasound parameter: super 3s, to stop 1s, 40 times, power 150W; The W/O Emulsion of the stable homogeneous that makes is changed in the round-bottomed flask, and rotary evaporation is removed CHCl 3Make gel, parameter: 30 ± 2 ℃ of bath temperatures, vacuum 0.07～0.08MPa; Add 1.5～2ml PBS solution and carry out aquation, make the medicinal liposome suspension, parameter: 30 ± 2 ℃ of bath temperatures, vacuum 0.09～0.10MPa, time 40～60min; Cross 1.2 μ m microporous filter membrane; Dialysis or mistake sephadex column are removed non-encapsulated free drug, both get the pastille liposome.With the pastille liposome of method for preparing, wherein the envelop rate of 5-fluorouracil, cisplatin, ftorafur, cytosine arabinoside is respectively: 14.43%, 8.38%, 12.13%, 16.25%; Particle diameter is respectively 794,603,470,454,371nm.
Hydrion (H +) the transhipment investigation:
Utilize the sensitivity of 5 (6)-carboxymethyl fluoresceins, Cyclopeptide nanotube mediation H to pH +Transmembrane transport changes the pH value of water in the liposome, thereby causes the variation of system fluorescence intensity.Get 5 (6)-carboxymethyl fluorescein liposomees, 100 μ l (interior water pH7.3), be suspended among the 2.5ml pH4.0 PBS, cyclic peptide/DMF solution 25 μ l (the cyclic peptide concentration: 0mg/ml that add variable concentrations, 1mg/ml, 2mg/ml), place the variation of measuring fluorescence intensity on the spectrofluorophotometer, every 3min measures once, and each cyclic peptide concentration is made 6 multiple pipes.
Figure G2009101452490D0000101
Wherein, I 0: 0 moment fluorescence intensity, I t: t is fluorescence intensity constantly, I ∞: the fluorescence intensity behind the breakdown of emulsion.I nTo time t mapping, promptly obtain Cyclopeptide nanotube mediation hydrion (H +) stride artificial lipid film transhipment situation, as shown in Figure 4.Along with the increase of cyclic peptide concentration, hydrionic transport speed is accelerated.
5-fluorouracil (5-FU) transhipment is investigated:
Get 5-FU liposome 1ml, in the bag filter of packing into (3.5kD), add cyclic peptide/DMF solution 25 μ l (the cyclic peptide concentration: 0mg/ml of variable concentrations, 1mg/ml, 2mg/ml 4mg/ml), places the conical flask that 30mlPBS (pH7.3) buffer is housed, agitator 100rpm, 37 ℃ of water-baths discharge 90min, and every 3min sampling once, each 40 μ l, every batch is repeated to investigate 6 samples.Chromatographic condition: Agilent C18 post, 4.6 μ m * 150mm; Mobile phase: 10%CH 3OH/H 2O; Detect wavelength: 265nm; Flow velocity: 0.8ml/min; Column temperature: 30 ℃; Sample size: 20ul.
Figure G2009101452490D0000111
Wherein, C 0: 0 moment drug level, C t: t is drug level constantly, C ∞: the drug level behind the breakdown of emulsion.Discharge percentage rate to time t mapping, promptly obtain Cyclopeptide nanotube mediation 5-FU and stride artificial lipid film transhipment situation, as shown in Figure 5.Do not adding under the situation of cyclic peptide, the transmembrane transport speed of fluorouracil is extremely low, and along with the increase of cyclic peptide amount, transport speed is accelerated.
Cisplatin (DDP) transhipment is investigated:
Get cisplatin liposome 1ml, in the bag filter of packing into (3.5kD), add cyclic peptide/DMF solution 25 μ l (the cyclic peptide concentration: 0mg/ml of variable concentrations, 1mg/ml, 2mg/ml, 4mg/ml), place the stripping rotor that 50ml 0.2%NaCl is housed, little oar method stirs, 100rpm, 37 ℃ of water-baths, discharge 90min, every 5min takes a sample once, each 0.5ml, and every batch is repeated to investigate 5 samples.Sample adopts GFAAS to measure.Discharge percentage rate to time t mapping, promptly obtain Cyclopeptide nanotube mediation cisplatin and stride artificial lipid film transhipment situation, as shown in Figure 6.Do not adding under the situation of cyclic peptide, the transmembrane transport speed of cisplatin is lower, and along with the increase of cyclic peptide amount, transport speed is accelerated.
The ftorafur transhipment is investigated:
Get ftorafur liposome 1ml, in the bag filter of packing into (3.5kD), add cyclic peptide/DMF solution 25 μ l (the cyclic peptide concentration: 0mg/ml of variable concentrations, 1mg/ml, 2mg/ml, 4mg/ml), place the stripping rotor that 50mlPBS (pH7.3) buffer is housed, little oar method stirs, 100rpm, 37 ℃ of water-baths, discharge 90min, every 5min takes a sample once, each 0.5ml, and every batch is repeated to investigate 5 samples.Chromatographic condition: Agilent C18 post, 4.6 μ m * 150mm; Mobile phase: 15%CH 3OH/H 2O; Detect wavelength: 282nm; Flow velocity: 1.0ml/min; Column temperature: 30 ℃; Sample size: 20ul.Discharge percentage rate to time t mapping, promptly obtain Cyclopeptide nanotube mediation ftorafur and stride artificial lipid film transhipment situation, as shown in Figure 7.
Cytosine arabinoside (Ara-C) transhipment is investigated:
Get Ara-C liposome 1ml, in the bag filter of packing into (3.5kD), add cyclic peptide/DMF solution 25ul (the cyclic peptide concentration: 0mg/ml of variable concentrations, 1mg/ml, 2mg/ml 4mg/ml), places the conical flask that 30mlPBS (pH7.3) buffer is housed, agitator 100rpm, 37 ℃ of water-baths discharge 90min, and every 3min sampling once, each 40 μ l, every batch is repeated to investigate 6 samples.Chromatographic condition: Diamonsil C18 post, 4.6 μ m * 250mm; Mobile phase: CH 3OH/0.01mol/l PBS (pH4.0,0.01mol/l SDS) (50: 50); Detect wavelength: 270nm; Flow velocity: 1ml/min; Column temperature: 30 ℃; Sample size: 20ul.Discharge percentage rate to time t mapping, promptly obtain Cyclopeptide nanotube mediation Ara-C and stride artificial lipid film transhipment situation, as shown in Figure 8.The molecular size of Ara-C is 1.11nm, greater than cyclic decapeptide cyclo[ LW LW LW LW LQ] model Dehua internal diameter (1nm), this medicine can not mediate transmembrane transport by Cyclopeptide nanotube in theory.The result shows cisplatin under the condition of not adding and adding cyclic peptide, all can not penetrate the bimolecular tunic preferably.
Embodiment 5
Cyclopeptide nanotube is to the investigation of tumor cell effect
Tumor cell line: human hepatocellular carcinoma BEL-7402 cell's strain.
After investigating Cyclopeptide nanotube and acting on tumor cell membrane, to the change situation of the sensitivity of 5-FU.
Experimental implementation: tumor cell line is inoculated on 96 orifice plates, 3000/hole, adds the culture fluid effect 48h that contains finite concentration CP and 5-FU, the cytotoxicity of mtt assay detection of drugs behind the 24h.
The result shows that after cyclic peptide (64 μ g/ml) acted on human hepatocellular carcinoma BEL-7402 cell's strain, 5-FU was reduced to 25.02 μ g/ml to the IC50 of this cell from 47.93 μ g/ml.This explanation cyclic peptide can be on tumor cell membrane self assembly form Cyclopeptide nanotube, and the outside small-molecule drug 5-FU of mediation enters fast by this passage and produces cytotoxicity in the tumor cell.Because it is fast that 5-FU strides film diffusion path by the transport velocity ratio of this passage, thereby can kill tumor cell faster.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; can also make some improvement and replenish, these improvement and replenish and also should be considered as protection scope of the present invention.
SEQUENCE?LISTING
＜110〉Fudan University
＜120〉a kind of Cyclopeptide nanotube medicinal composition and application thereof
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<170>PatentIn?version?3.1
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1 5 10

Claims (10)

1. the application of Cyclopeptide nanotube medicinal composition in preparation treatment tumor disease, bacterial infective diseases or virus infection medicine, described Cyclopeptide nanotube medicinal composition is made up of Cyclopeptide nanotube and medicine.
2. application according to claim 1, it is characterized in that: described Cyclopeptide nanotube Van der Waals internal diameter is 0.75-1.3nm, pipe range is 10-100nm, Cyclopeptide nanotube basic composition unit is a cyclic peptide, cyclic peptide is formed by 8,10,12 aminoacid cyclizations, and the diameter of described drug molecule is less than Cyclopeptide nanotube Van der Waals internal diameter.
3. application according to claim 2 is characterized in that: the cyclic peptide in the described Cyclopeptide nanotube replaces covalent bond by the D type of equal number and L type aminoacid and forms, and described drug molecule is a hydrophilic medicament.
4. application according to claim 3, it is characterized in that: the aminoacid sequence of cyclic peptide such as SEQ ID NO:1 in the described Cyclopeptide nanotube, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, SEQ ID NO:21, SEQ ID NO:22, shown in SEQ ID NO:23 or the SEQ ID NO:24, described medicine is selected from 5-fluorouracil, flucytosine, ftorafur, salicylic acid, para-aminosalicylic acid, cisplatin, carboplatin, chlormethine, cyclophosphamide, ifosfamide, formylmerphalan, glyforfin, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, Seninon, ethambutol, ribavirin, zidovudine or metronidazole.
5. application according to claim 4 is characterized in that: the aminoacid sequence of cyclic peptide is shown in SEQ ID NO:23 or SEQ ID NO:24 in the described Cyclopeptide nanotube, and described medicine is selected from 5-fluorouracil, ftorafur, cisplatin or carboplatin.
6. Cyclopeptide nanotube medicinal composition, it is characterized in that: form by Cyclopeptide nanotube and medicine, described Cyclopeptide nanotube Van der Waals internal diameter is 0.75-1.3nm, pipe range is 10-100nm, Cyclopeptide nanotube basic composition unit is a cyclic peptide, cyclic peptide is formed by 8,10,12 aminoacid cyclizations, and the diameter of described drug molecule is less than Cyclopeptide nanotube Van der Waals internal diameter.
7. pharmaceutical composition according to claim 6 is characterized in that: the cyclic peptide in the described Cyclopeptide nanotube replaces covalent bond by the D type of equal number and L type aminoacid and forms, and described drug molecule is a hydrophilic medicament.
8. pharmaceutical composition according to claim 6 is characterized in that: the aminoacid sequence of cyclic peptide such as SEQ ID NO:1 in the described Cyclopeptide nanotube, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQID NO:20, SEQ ID NO:21, SEQ ID NO:22, shown in SEQ ID NO:23 or the SEQ ID NO:24.
9. pharmaceutical composition according to claim 6 is characterized in that: described medicine is selected from 5-fluorouracil, flucytosine, ftorafur, salicylic acid, para-aminosalicylic acid, cisplatin, carboplatin, chlormethine, cyclophosphamide, ifosfamide, formylmerphalan, glyforfin, melphalan, carmustine, lomustine, semustine, nimustine, dopamine, isoniazid, Seninon, ethambutol, ribavirin, zidovudine or metronidazole.
10. pharmaceutical composition according to claim 6, it is characterized in that: the aminoacid sequence of cyclic peptide is shown in SEQ ID NO:23 or SEQ ID NO:24 in the described Cyclopeptide nanotube, described medicine is selected from 5-fluorouracil, ftorafur, cisplatin or carboplatin, the Cyclopeptide nanotube pipe range is 20-50nm, described Cyclopeptide nanotube Van der Waals internal diameter is 1.0nm, and the diameter of described drug molecule is less than 1.0nm.
CN200910145249A 2008-11-10 2009-05-22 Cyclopeptide nanotube medicinal composition and application thereof Pending CN101732699A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106188228A (en) * 2016-07-06 2016-12-07 厦门大学 A kind of method stablizing ternary cyclic peptide based on phenyl tetrafluoride dimethoxy nitrile structure
CN107406486A (en) * 2015-03-31 2017-11-28 新加坡科技研究局 The ultrashort aliphatic cyclic peptide of self assembly for biomedical applications
WO2024041535A1 (en) * 2022-08-22 2024-02-29 赣州和美药业股份有限公司 Nano-composition, preparation method therefor, and use thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107406486A (en) * 2015-03-31 2017-11-28 新加坡科技研究局 The ultrashort aliphatic cyclic peptide of self assembly for biomedical applications
CN106188228A (en) * 2016-07-06 2016-12-07 厦门大学 A kind of method stablizing ternary cyclic peptide based on phenyl tetrafluoride dimethoxy nitrile structure
CN106188228B (en) * 2016-07-06 2019-03-22 厦门大学 A method of it is constructed based on tetrafluoro benzene dicarbonitrile and stablizes ternary cyclic peptide
WO2024041535A1 (en) * 2022-08-22 2024-02-29 赣州和美药业股份有限公司 Nano-composition, preparation method therefor, and use thereof

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