CN108948152A - A kind of amphipathic cell-penetrating peptide key compound, preparation method and the usage - Google Patents
A kind of amphipathic cell-penetrating peptide key compound, preparation method and the usage Download PDFInfo
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- CN108948152A CN108948152A CN201710352646.XA CN201710352646A CN108948152A CN 108948152 A CN108948152 A CN 108948152A CN 201710352646 A CN201710352646 A CN 201710352646A CN 108948152 A CN108948152 A CN 108948152A
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- cell
- penetrating peptide
- amphipathic
- key compound
- self
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229940097275 indigo Drugs 0.000 description 1
- COHYTHOBJLSHDF-UHFFFAOYSA-N indigo powder Natural products N1C2=CC=CC=C2C(=O)C1=C1C(=O)C2=CC=CC=C2N1 COHYTHOBJLSHDF-UHFFFAOYSA-N 0.000 description 1
- 229960004657 indocyanine green Drugs 0.000 description 1
- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 description 1
- ICIWUVCWSCSTAQ-UHFFFAOYSA-M iodate Chemical compound [O-]I(=O)=O ICIWUVCWSCSTAQ-UHFFFAOYSA-M 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- BZCKRPHEZOHHBK-UHFFFAOYSA-N methyl 2-phenoxyacetate Chemical compound COC(=O)COC1=CC=CC=C1 BZCKRPHEZOHHBK-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000011368 organic material Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 238000007626 photothermal therapy Methods 0.000 description 1
- QWYZFXLSWMXLDM-UHFFFAOYSA-M pinacyanol iodide Chemical class [I-].C1=CC2=CC=CC=C2N(CC)C1=CC=CC1=CC=C(C=CC=C2)C2=[N+]1CC QWYZFXLSWMXLDM-UHFFFAOYSA-M 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000002601 radiography Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000008684 selective degradation Effects 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000004073 vulcanization Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0039—Coumarin dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0076—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion
- A61K49/0082—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form dispersion, suspension, e.g. particles in a liquid, colloid, emulsion micelle, e.g. phospholipidic micelle and polymeric micelle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Abstract
The present invention relates to a kind of amphipathic cell-penetrating peptide key compounds, it includes hydrophobic patches and cell-penetrating peptide segment, wherein the hydrophobic patches and the cell-penetrating peptide segment are bonded by the functional group reactions that they are included, bonding pattern includes, but it is not limited to, amido bond, ester bond, ehter bond or acyl sulfide linkage.The present invention also provides the purposes of the preparation method of the amphipathic cell-penetrating peptide key compound, its micella being self-assembly of in water or buffer and the micella in terms of diagnosing tumor imaging and oncotherapy.The amphipathic cell-penetrating peptide key compound can be self-assembly of nano-micelle in water, can be used as carrier to convey drug or contrast agent.
Description
Technical field
The invention belongs to biomedical engineering fields, in particular to a kind of amphipathic cell-penetrating peptide key compound, its preparation
Method, its micella being self-assembly of in water or buffer and the micella are used as the purposes of transport vehicle, can be used for passing
The contrast agent that defeated diagnosing tumor is imaged and the drug for oncotherapy.
Background technique
Nano medication can change by the pharmacokinetics behavior of improvement drug and overcome tumour resistance to mechanism such as cytosis modes
Medicine.The realization of the antitumor drug resistance function of Nano medication depend greatly on Nano medication and cell interaction and
Its pharmacokinetics behavior, and this is closely related with the physicochemical property of nano particle.Wherein, the pattern of nano particle and particle is thin
Born of the same parents' intake and body-internal-circulation ability have a significant impact, and the surface characteristic of nano particle enters endochylema or nucleus with it,
The ability for delivering drugs into its target spot has direct relation, thus the pattern of nano particle and surface characteristic fight tumor drug resistance
Effect has important influence.
At present overwhelming majority organic materials by conventional method nano-carrier obtained based on spheric granules.Although passing through
Nanocrystal, mechanical stretching, inscription rubbing, microfluid and particles self assemble technology can prepare different-shape particle, but by above-mentioned
The particle major part size that technology is prepared is unfavorable for transporting in vivo more than several hundred nanometers even micron.Nanocrystal
Although method can prepare the particle of small size, it is typically limited to the preparation of inorganic nanoparticles, and nearest research is sent out
Existing difference of the nano particle in flexibility even meeting reverse both prompts nothing to the intake preference of two kinds of different-shape nano particles
Possible limitation and the necessity of pharmaceutical carrier as research object is used when machine nano particle is as cellular uptake model particle
Property.Spherical or cylindrical micellar and vesica, but more points of polymer can be prepared by the composition and molecular weight that adjust block copolymer
Scattered characteristic would generally have adverse effect on carrier pattern, obtain highly homogeneous block copolymer for vector construction and be still
Crucial and technological difficulties therein.Nano-carrier, especially different-shape are constructed by self-assembling technique using uniform molecule
Nano-carrier, disclose nano particle and organism interacts basic law to further, realize that drug is thin to drug-resistant tumor
Effective conveying of born of the same parents all has important meaning.
The functional modification on Nano medication surface is to improve its effective means accumulated in cells of resistant tumors.With swollen
The cell-penetrating peptide of oncocyte film penetration capacity answering in Nano medication surface modification and in terms of overcoming Tumor Heterogeneity and multidrug resistance
With increasingly extensive.Nano-carrier etc. is conveyed altogether for example, modifying cell-penetrating peptide to albumin, inorganic nano-particle, micella, liposome
Grain can dramatically increase accumulation of the package-contained therapeutic agent in cells of resistant tumors behind surface, significantly improve to drug-resistant tumor
Therapeutic effect.Meanwhile the development of cell-penetrating peptide can be activated to improve its selectivity, keep cell-penetrating peptide big in the application in Nano medication field
To expand.Although its antitumous effect can be significantly improved by way of carrying out cell-penetrating peptide modification to Nano medication, its shape
Looks determine that essentially spherical particle, granule-morphology enter cells of resistant tumors ability to cell-penetrating peptide by being modified nano particle
Further investigation is not yet received in influence.Design synthesizes completely new cell-penetrating peptide derivative, constructs novel different characteristics (such as not similar shape
Looks) cell-penetrating peptide modify nano-carrier, to overcoming tumor multi-medicine drug-resistant to have great importance.
Based on cell-penetrating peptide in the great potential of molecule transportation aspect, the present inventor is hydrophobic to cell-penetrating peptide progress to be repaired
Decorations construct amphipathic cell-penetrating peptide, and prepare the novel nano carrier based on cell-penetrating peptide by self assembly means.It is worn with amphipathic
The building of film peptide is prepared into nanoparticle as carrier material based on cell-penetrating peptide, can adjust its pattern by adjusting molecular structure, retain
And the passive target advantage of nano-delivery system is strengthened, and increase hydrophobic drug drugloading rate, while can also be to amphipathic
Cell-penetrating peptide one of carbon tip is extended, and addition can be stimulated responsiveness connection molecule, as MMP degrade oligopeptide sequence, and then introduce
Polyethylene glycol.Toxic side effect can be reduced the tumor locus selective degradation the characteristics of according to it, improve curative effect, and can be used
In the diagnosis of tumour.
Summary of the invention
Based on background above, it is an object of the present invention to provide a new class of amphipathic cell-penetrating peptide key compound, described two
Parent's property cell-penetrating peptide key compound can be self-assembly of nano-micelle in water, can be used as carrier to transmit drug or radiography
Agent.
It is a further object of the present invention to provide the nano-micelles that the amphipathic cell-penetrating peptide key compound is formed.
Another object of the present invention is to provide the preparation method of the amphipathic cell-penetrating peptide key compound.
It is yet another object of the invention to provide the purposes of the amphipathic cell-penetrating peptide key compound.
According to an aspect of the invention, there is provided a kind of amphipathic cell-penetrating peptide key compound, it includes hydrophobic patches and
Cell-penetrating peptide segment, wherein the hydrophobic patches and the cell-penetrating peptide segment are the functional group reactions key for being included by them
It closes, bonding pattern includes, but are not limited to amido bond, ester bond, ehter bond and acyl sulfide linkage.Preferably, in each amphipathic cell-penetrating peptide
Include one or more hydrophobic patches and a cell-penetrating peptide segment in key compound molecule.
Preferably, the hydrophobic patches and the cell-penetrating peptide segment are the function positioned at end for being included by them
Group reacts and is bonded, it is, the hydrophobic patches and the cell-penetrating peptide segment are that respective end is bonded.
Preferably, the hydrophobic patches are only bonded one end of the cell-penetrating peptide segment.
If it is necessary, the amphipathic cell-penetrating peptide bonding can be further included positioned at the hydrophobic patches and described be worn
Amino acid or small peptide between film peptide fragment are used to for one or more hydrophobic patches to be connected to the cell-penetrating peptide segment
On；
Wherein, the hydrophobic patches can be derived from, but be not limited to, and C9~C18 saturated fatty acid, C9~C18 are unsaturated
Fatty acid, fluorine-containing C9~C18 saturated fatty acid, fluorine-containing C9~C18 unsaturated fatty acid, vitamin E, cholic acid or signal point
Sub (such as fluorescent dye) etc.；
According to specific technical requirements, it is preferable that the hydrophobic patches can be derived from, but be not limited to, palmitinic acid, 2H,
2H, 3H, 3H- perfluoro-pelargonic acid or camptothecine fluorescent molecule etc..
The amino acid number of the cell-penetrating peptide segment is 5~30, and the cell-penetrating peptide fragment derivitization certainly, but is not limited to, HIV
Source CPP (GRKKRRQRRRPPQ), oligomerization arginine, MPG (Ac-GALFLGFLGAAGSTMGAWSQPKKKRKVC), EB1
(LIKLWSHLIHIWFQNRRLKWKKK) etc..
Preferably, the cell-penetrating peptide segment is GRKKRRQRRRPPQ or GRKKRRQRRR.
The cell-penetrating peptide segment and the molar ratio of the hydrophobic patches are 1:1~1:8, preferably 1:2 and 1:4.
Preferably, hydrophilic fractions are connected in the other end of the cell-penetrating peptide segment.The hydrophilic fractions are hydrophilic
Macromolecule group (polyethylene glycol, polysaccharide etc.), targeting ligand (folic acid, small peptide, antibody etc.), or both conjugate.
It is directly connected between the hydrophilic fractions and the cell-penetrating peptide or is connected by stimulating responsive linking group, institute
State stimulating responsive linking group refer to can the physiological and pathologicals such as enzyme, acid and glutathione stimulation under recurring structure or conformation change
Chemical group, the stimulating responsive linking group be selected from MMP degradation oligopeptide sequence (GGGYIPVSLRSGYC), or comprising
The chemical group of disulfide bond (such as cystamine), ester bond (such as aldehyde radical methyl phenoxyacetate) or hydrazone bond (such as hydrazides) etc..
Preferably, the amphipathic cell-penetrating peptide key compound molecular weight ranges are in 1000~20000Da.
According to another aspect of the present invention, a kind of amphipathic cell-penetrating peptide key compound self-assembled nano micelle is additionally provided,
It is self-assembly of in water or buffer by the amphipathic cell-penetrating peptide key compound.Specifically the preparation method is as follows: weighing amphiphilic
Property cell-penetrating peptide key compound, be directly dissolved in water or buffer, wherein the concentration of amphipathic cell-penetrating peptide key compound be 1nM-
50mM.In course of dissolution, amphipathic cell-penetrating peptide key compound can be kept completely molten by means such as vortex, water bath sonicator, heating
Solution.
Obtained amphipathic cell-penetrating peptide key compound self-assembled nano micelle can be characterized as follows: by institute
The concentration for obtaining amphipathic cell-penetrating peptide key compound is adjusted to 1mM, draws a drop in the groove of drop reaction porcelain plate with a fine droplets pipe
It is interior, and carbon-sprayed copper net is put on test solution (film surface is downward), copper mesh is taken out after 1~2 minute, is inhaled with filter paper small pieces from copper mesh edge
Abandon surplus liquid；In same way as described above, which is placed on about 30s on 1% (w/v) acetic acid uranium dyeing liquor, with filter paper small pieces
Extra dye liquor is blotted from copper mesh edge, then is washed with distilled water 1~2 time, drying after abandoning moisture content is inhaled with filter paper, under transmission electron microscope
Observe the mode of appearance and distribution character of amphipathic cell-penetrating peptide key compound self-assembled nano micelle.By the knot for controlling hydrophobic part
Structure and quantity, the pattern for being self-assembly of nano-micelle of the invention include spherical shape, stub shape, sheet, tubulose etc..
Amphipathic cell-penetrating peptide key compound self-assembled nano micelle of the present invention with by amphipathy macromolecule or amphipathic
There were significant differences for the nano-micelle that beta sheet peptide is formed, the pattern of nano-micelle of the present invention can variform (ball, stick,
Pipe, piece etc.) between by control hydrophobic part structure and quantity adjust, the preparation method of nano-micelle of the present invention is simple,
It only needs to add water or buffer solution that can prepare, and due to being self-assembly of, dimensional homogeneity is high.
According to another aspect of the invention, the preparation method of the amphipathic cell-penetrating peptide key compound is provided, including with
Lower step:
Cell-penetrating peptide is mixed with hydrophobic compound, condensing agent, reacts to obtain amphipathic cell-penetrating peptide bonding in a solvent
Object.Preferably, the reaction is amidation process, esterification, acyl vulcanization reaction, etherification reaction etc..
For example, the condensing agent is in the case where the bonding pattern of the amphipathic cell-penetrating peptide key compound is amido bond
Benzotriazole-N, N, N ', N '-tetramethylurea hexafluorophosphate (HBTU) and diisopropylethylamine (DIEA) and solvent are two
Methylformamide (DMF).
Preferably, the molar ratio of the cell-penetrating peptide, hydrophobic compound, HBTU and DIEA are 1:4:4:6.
Preferably, the reaction time of the amidation process is 3-12 hours.
Wherein, the cell-penetrating peptide can use 9-fluorenylmethyloxycarbonyl (Fmoc) Solid phase peptide synthssis technology according to required sequence
Synthesis.Cell-penetrating peptide is sequentially synthesized on solid-phase resin particle, during peptide chain extension, amino protecting group Fmoc uses 4- first
The DMF solution reaction of phenylpiperidines is sloughed, and is coupled first that side-chain amino group and amino is protected amino acid derived when subsequent amino-acid
Object and HBTU and DIEA activated carboxyl, then with polypeptides reactive on resin；In the synthesis process, all reactive amino acid side chains are equal
It is protected by blocking group, avoids polypeptide protracting and occur branched side products in the process；It, can be further at it after Peptide systhesis
Amino terminal performs the derivatization, to meet different cell-penetrating peptide preparations；Be coupled to polypeptide on resin by with trifluoroacetic acid, water
It reacts and recycles with tri isopropyl silane；All blocking groups are cut from peptide side chain, are purified by precipitating, drying and preparation, can
Obtain high-purity cell-penetrating peptide.Preferably, the volume ratio of trifluoroacetic acid, water and tri isopropyl silane is 95:2.5:2.5.
According to another aspect of the invention, the amphipathic cell-penetrating peptide key compound self-assembled nano micelle is provided as defeated
Send the purposes of carrier.
The delivery vehicles can be used to convey contrast agent or treat the drug of tumour.
Amphipathic cell-penetrating peptide key compound of the present invention can combine contrast agent in such a way that covalent coupling or physics contain
Or the drug for the treatment of tumour.
The contrast agent is selected from fluorine-containing hydrophobic molecule, fluorescent molecule and light absorption molecule etc..
The drug of the treatment tumour is selected from small point of hydrophobicitys of taxanes, camptothecin, CHROMATOGRAPHIC FRACTIONATION AND MASS, rapamycin etc.
Sub- anti-tumor drug, including such as indocyanine green of the small molecule with photo-thermal effect (ICG), iodate indigo carbocyanine derivative (DiI)
Deng including such as chlorin e 6 of the small molecule with photodynamic effect (Ce6) etc..
Wherein, the step of drug covalent coupling and physics of contrast agent or treatment tumour contain is distinguished as follows:
(1) covalent coupling: selection is with contrast agent of functional groups or swollen for treating such as carboxyl, hydroxyl, amino or sulfydryls
The drug of tumor is coupled by chemical bond and the amphipathic cell-penetrating peptide key compound, and resulting coupling has contrast agent or treats tumour
The amphipathic cell-penetrating peptide key compound of drug prepares nano-micelle method preparation load contrast agent nano-micelle, the chemical bond by aforementioned
Include, but are not limited to amido bond, ester bond, ehter bond and acyl sulfide linkage.
(2) physics contains: by amphipathic cell-penetrating peptide and contrast agent or being used to treat the drug co-dissolve of tumour in organic
In solvent (such as hexafluoroisopropanol), after removing organic solvent after being thoroughly mixed uniformly, water or buffer is added by aforementioned
Nano-micelle (film aquation embrane method) preparation carries contrast agent nano-micelle.
Can according to ultrasonic imaging,19F-NMR imaging, photoacoustic imaging, fluorescence imaging, the requirement selection of photothermal imaging are appropriate
Contrast agent.
The present invention provides the amphipathic cell-penetrating peptide key compound self-assembled nano micelles in diagnosing tumor imaging and tumour
Purposes in treating.
The tumour includes breast cancer, lung cancer, oophoroma, prostate cancer, cancer of pancreas, liver cancer, head-neck carcinoma or gastric cancer.
According to the characteristic of entrained drug, obtained amphipathic cell-penetrating peptide key compound can be used for of self-assembled nano micelle
Treatment, photo-thermal therapy, optical dynamic therapy etc..
Amphipathic cell-penetrating peptide key compound of the present invention, which is self-assembly of nano-micelle, can utilize its regulatable shape
Looks and surface characteristic are accumulated by passive and active targeting mode in tumor locus, the exposure cell-penetrating peptide under particular stimulation, significantly
The accumulation of nano-micelle and its entrained drug in tumour cell is improved, is had in terms of antitumor especially multidrug resistance of tumor
There is significant advantage.
Detailed description of the invention
Fig. 1 is the molecular structure and TOF MS ESI mass spectrogram of dC16-CPP made from embodiment 1 (1)；
Fig. 2 is the molecular structure and TOF MS ESI mass spectrogram of dC16-CPP-MCL made from embodiment 1 (2)；
Fig. 3 is dC16-CPP-PEG made from embodiment 1 (3)5000Molecular structure and TOF MS ESI mass spectrogram；
Fig. 4 is the molecular structure and TOF MS ESI mass spectrogram of qC16-CPP made from embodiment 1 (4)；
Fig. 5 is the molecular structure and TOF MS ESI mass spectrogram of qCF9-CPP made from embodiment 1 (5)；
Fig. 6 is the molecular structure and TOF MS ESI mass spectrogram of qCPT-CPP made from embodiment 1 (7)；
Fig. 7 is the TEM figure for several amphipathic cell-penetrating peptide key compound self-assembled nano micelles that embodiment 2 obtains；Wherein:
(a) the TEM figure of dC16-CPP self-assembled nano micelle；(b) the TEM figure of qC16-CPP self-assembled nano micelle；(c)qCPT-CPP
The TEM of self-assembled nano micelle schemes；(d) the TEM figure of qC9F-CPP self-assembled nano micelle；
Fig. 8 is that dC16-CPP self-assembled nano micelle made from embodiment 2 contains light irradiation figure after coumarin 6；Wherein:
(a) dC16-CPP contains under photo (b) white light of coumarin 6 that dC16-CPP is contained under photo (c) green light of distilled water under white light
The fluorescence photo of coumarin 6；(d) under green light distilled water photo
Fig. 9 is fluorescence imaging of the qCPT-CPP self-assembled nano micelle at 365nm made from embodiment 2, in which: (a)
Distilled water；(b)qCPT-CPP；
Figure 10 is that amphipathic cell-penetrating peptide key compound dC16-CPP made from embodiment 4 contains taxol self-assembled nano micelle
To paclitaxel-sensitive A549 cytotoxicity experiment result histogram；Wherein: (a) dC16-CPP contains taxol self-assembled nanometer glue
Beam is in the chart to the MTT toxicity test data in paclitaxel-sensitive lung cell A549；(b) dC16-CPP contains taxol certainly
The chart of MTT toxicity test data of the assemble nanometer micella in lung cancer taxol resistance strain A549/T；DC16-CPP in figure
Represent the test group that amphipathic cell-penetrating peptide key compound dC16-CPP contains the taxol self-assembled nano micelle of 10% molar ratio；
PTX is represented without the free PTX group contained.
Specific embodiment
General exemplary description is carried out to the present invention now, this can be more easily to understand in conjunction with the following examples
Invention, these embodiments and examples are used only to understand certain aspects of the present disclosure and embodiment, rather than to this hair
Bright spirit and scope progress limits in all senses, and protection scope of the present invention is limited by the following claims and their equivalents
It is fixed.
Reagent
Taxol (PTX, Shanghai Sunve Pharmaceutical Co., Ltd.)；Methyl thiazoly tetrazolium assay (MTT, Sigma)；RPMI-1640
Dehydrated medium (Gibco)；0.25% trypsase 0.02%EDTA digestive juice (Gibco)；Fetal calf serum (FBS, Gibco)；
Ampicillin, strepto- (Sigma)；1,1,1,3,3,3- chlordene -2- acetone (HFIP, Beijing lark prestige Science and Technology Ltd.)；
Coumarin 6 (Beijing lark prestige Science and Technology Ltd.).In the present specification, unless otherwise specified, remaining agents useful for same and solvent
It is purchased from Chinese medicines group (Shanghai) chemical reagent Co., Ltd.
Biomaterial
Paclitaxel-sensitive strain A549 (ATCC, U.S. Manassas), lung cancer taxol resistance strain A549/T (ATCC, the U.S.
Manassas)。
Instrument
BP190S type electronic balance (German Sartorius company)；(the U.S. Millipore pure water meter ZLXS5003
Millipore company)；B ü chi R-114 type Rotary Evaporators (B ü ch company of Switzerland)；Waters2695 (Alliance system)
Liquid chromatograph (Waters, US)；Waters2489 UV detector (Waters, US)；UV-2450 is visible-
Ultraviolet specrophotometer (Shimadzu, Japan)；XW-80A turbula shaker (kylin medical apparatus factory of Jiangsu Haimen City)；
The desk-top air bath shaking table of HZ-9100k (Jiangsu Hua Lida experimental facilities Co., Ltd)；JEM-1230 transmission electron microscope (day
This JEOL Electronics Co., Ltd)；DZF-6090 vacuum oven (Shanghai Yi Heng laboratory apparatus Co., Ltd)；CO2Incubator (beauty
Thermo-FisherScientific company of state)；VS-840-2 super-clean bench (Shanghai Bo Xun Industrial Co., Ltd. Medical Devices
Factory)；Tecan Infinit F200 type multi-function microplate reader (Australian Salzburg company)；PB-10pH meter (Germany
Sartorius company)；YXQ-LS-50SII vertical pressure steam sterilizer (Shanghai Bo Xun Industrial Co., Ltd. Medical Devices
Factory).
Embodiment 1, different structure amphipathic cell-penetrating peptide key compound preparation
The present embodiment is the demonstration to the amphipathic cell-penetrating peptide key compound of different structure is constructed in summary of the invention of the invention, just
In the understanding to its structure and adjustability of structure.
(1) it is bonded the preparation of the amphipathic cell-penetrating peptide key compound dC16-CPP of two palmitinic acid chains
0.3g Rink Amide-MBHA Resin resin is weighed, using 9-fluorenylmethyloxycarbonyl (Fmoc) Solid phase peptide synthssis
Technology (W.C.Chan and P.D.Whhite, the Oxford Fmoc Solid Phase Peptide Synthesis publishing house 2000
Year), cell-penetrating peptide CPP (GRKKRRQRRRPPQ) is sequentially synthesized on solid-phase resin particle, wherein select in the Peptide systhesis
One amino side chain by Fmoc radical protection, side-chain amino group by the lysine of Boc radical protection, the 4- methyl piperidine of 20wt%
After DMF solution reacts the blocking group (Fmoc) for sloughing glycine, using two amino side chains by the bad ammonia of Fmoc blocking group
Acid is added 0.3755g HBTU, 0.25ml DIEA, 0.2954g lysine solution the reaction 3h for being dissolved in 15ml DMF, uses
The blocking group (Fmoc) of lysine is sloughed in the DMF solution reaction of the 4- methyl piperidine of 20wt%, introduces 2 amino, is added
Overnight, reaction solution 15ml is added in the polypeptide being bonded on resin for 0.751g HBTU, 0.5ml DIEA, the reaction of 0.64g palmitinic acid
2 hours recycling product dC16-CPP of (trifluoroacetic acid: water: tri isopropyl silane=95:2.5:2.5, volume ratio) concussion reaction are produced
Light yellow solid is obtained after product vacuum drying.Amphipathic cell-penetrating peptide key compound (dC16-CPP) structure of two fat chains of the bonding with
Mass spectrum is as shown in Figure 1, mass-to-charge ratio is consistent with the molecular weight being fitted with ChemDraw Professional 15.0, it was demonstrated that successfully makes
It is standby to go out to be bonded the amphipathic cell-penetrating peptide key compound of two fat chains.
(2) preparation of the amphipathic cell-penetrating peptide key compound dC16-CPP-MCL of the oligopeptide sequence of the degradation containing MMP
0.3gRink Amide-MBHA Resin resin is weighed, using 9-fluorenylmethyloxycarbonyl (Fmoc) Solid phase peptide synthssis
Technology sequentially synthesizes the cell-penetrating peptide (GRKKRRQRRRGGGYIPV of the oligopeptide sequence of the degradation containing MMP on solid-phase resin particle
SLRSGYC), wherein the lysine of a Fmoc radical protection is selected when the Peptide systhesis, the 4- methyl piperidine of 20wt%
After DMF solution reacts the blocking group (Fmoc) for sloughing glycine, using containing there are two the lysine of Fmoc blocking group, additions
It is dissolved in 0.3755g HBTU, 0.25ml DIEA, 0.2954g lysine solution the reaction 3h of 15ml DMF, with the 4- of 20wt%
The blocking group (Fmoc) of lysine is sloughed in the DMF solution reaction of methyl piperidine, introduces 2 amino, be added 0.751g HBTU,
Overnight, reaction solution (trifluoroacetic acid: water: three is added in the polypeptide being bonded on resin for 0.5ml DIEA, the reaction of 0.64g palmitinic acid
Isopropyl base silane=95:2.5:2.5, volume ratio) it recycles within concussion reaction 2 hours, light yellow solid is obtained after product vacuum drying.
Obtain the amphipathic cell-penetrating peptide key compound (dC16-CPP-MCL) of the oligopeptide sequence of the degradation containing MMP of two fat chains of bonding, structure
With mass spectrum as shown in Fig. 2, mass-to-charge ratio is consistent with the molecular weight being fitted with ChemDraw Professional 15.0, it was demonstrated that success
Prepare the amphipathic cell-penetrating peptide key compound of the oligopeptide sequence of the degradation containing MMP.
(3) the key compound dC16-CPP-PEG of the cell-penetrating peptide of the oligopeptide sequence of the degradation containing MMP and polyethylene glycol5000Preparation
Weigh amphipathic cell-penetrating peptide key compound dC16-CPP-MCL 100mg, the HS-PEG prepared in embodiment 1 (2)5000-
CH3500mg is dissolved in 20ml DMF, is stirred to react 48h under room temperature, to end of reaction, selects ether precipitating, will precipitate vacuum
After drying, it is dissolved in acetonitrile: in water (v:v)=1:1 solution, being purified with preparation liquid phase, obtain amphipathic cell-penetrating peptide key compound
dC16-CPP-PEG5000.Structure is as shown in Figure 3 with mass spectrum.
(4) it is bonded the preparation of the amphipathic cell-penetrating peptide key compound qC16-CPP of four fat chains
0.3g Rink Amide-MBHA Resin resin is weighed, using 9-fluorenylmethyloxycarbonyl (Fmoc) Solid phase peptide synthssis
Technology sequentially synthesizes cell-penetrating peptide (GRKKRRQRRRPPQ) on solid-phase resin particle, wherein selects a Fmoc radical protection
Alpha-amido Boc protects the lysine of side-chain amino group, and the protection of glycine is sloughed in the DMF solution reaction of the 4- methyl piperidine of 20wt%
After group (Fmoc), using containing there are two the lysine of Fmoc blocking group, addition is dissolved in the 0.3755g of 15ml DMF
HBTU, 0.25ml DIEA, 0.2954g lysine solution react 3h, react de- with the DMF solution of the 4- methyl piperidine of 20wt%
The blocking group (Fmoc) for removing lysine is added containing there are two the lysine of Fmoc blocking group, and addition is dissolved in 15ml
The 0.3755g HBTU of DMF, 0.25ml DIEA, 0.2954g lysine solution react 3h, with the 4- methyl piperidine of 20wt%
DMF solution, which reacts, sloughs the blocking group (Fmoc) of lysine, introduces 4 amino, be added 1.502g HBTU, 1ml DIEA,
The reaction of 1.28g palmitinic acid overnight, be bonded to polypeptide on resin be added reaction solution (trifluoroacetic acid: water: tri isopropyl silane=
95:2.5:2.5, volume ratio) it recycles within concussion reaction 2 hours, light yellow solid is obtained after product vacuum drying.Four rouge of the bonding
Amphipathic cell-penetrating peptide key compound (qC16-CPP) structure of chain and mass spectrum are as shown in figure 4, mass-to-charge ratio and use ChemDraw
The molecular weight that Professional 15.0 is fitted is consistent, it was demonstrated that successfully prepares the amphipathic cell-penetrating peptide key of four fat chains of bonding
Close object.
(5) it is bonded the preparation of the amphipathic cell-penetrating peptide key compound qC9F-CPP of four rouge derivatives
0.3g Rink Amide-MBHA Resin resin is weighed, using 9-fluorenylmethyloxycarbonyl (Fmoc) Solid phase peptide synthssis
Technology sequentially synthesizes cell-penetrating peptide (GRKKRRQRRRPPQ) on solid-phase resin particle, wherein a Fmoc is selected to protect α-ammonia
Base Boc protects the lysine of side-chain amino group, and the blocking group of glycine is sloughed in the DMF solution reaction of 20% 4- methyl piperidine
(Fmoc) after, using containing there are two amino by fmoc-protected lysine, the 0.3755g for being dissolved in 15ml DMF is added
HBTU, 0.25ml DIEA, 0.2954g lysine solution react 3h, are sloughed with the DMF solution reaction of 20% 4- methyl piperidine
The blocking group (Fmoc) of lysine is added containing there are two the lysine of Fmoc blocking group, and addition is dissolved in 15ml DMF
0.3755g HBTU, 0.25ml DIEA, 0.2954g lysine solution react 3h, the DMF with 20% 4- methyl piperidine is molten
Liquid reacts the blocking group (Fmoc) for sloughing lysine, and after introducing 4 amino, 1.502g HBTU, 1ml DIEA, 1.6g is added
The reaction of 2H, 2H, 3H, 3H- perfluoro-pelargonic acid is stayed overnight, the amphipathic cell-penetrating peptide key compound (qC9F- of four rouge derivative chains of the bonding
CPP) structure with mass spectrum as shown in figure 5, mass-to-charge ratio with ChemDraw Professional 15.0 be fitted molecular weight it is consistent,
Prove the amphipathic cell-penetrating peptide key compound for successfully preparing four rouge derivatives of bonding.
(6) preparation for the camptothecine fluorescent molecule CPT-S-S-Pyr that sulfydryl is protected
Weigh 2.0g 4- bromo-butyric acid, 1.0g thio urea is dissolved in 50ml ethyl alcohol and reacts 4h, addition 5g NaOH continuation
Back flow reaction 16h, residue are extracted with ether, and water layer is adjusted to PH5 with the hydrochloric acid of 3mol/L, use Na2SO4It is molten with 5ml methanol after drying
Solution is added dropwise two pyridine of 4g 2- sulphur dissolved with 5ml methanol, is stirred to react 3h, after vacuum drying, resulting product is added
Enter 200mg camptothecine fluorescent molecule, 44mg 4- dimethylamino pyridine, 436 μ l N, N'- diisopropylcarbodiimide to be stirred to react
36h, with using Na after 60ml chloroform desalination2SO4It is dry, obtain the camptothecine fluorescent molecule (CPT-S-S- that sulfydryl is protected
Pyr)。
(7) it is bonded the preparation of the amphipathic cell-penetrating peptide key compound qCPT-CPP of four hydrophobic fluorescence molecules containing sulfydryl
0.3g Rink Amide-MBHA Resin resin is weighed, using 9-fluorenylmethyloxycarbonyl (Fmoc) Solid phase peptide synthssis
Technology sequentially synthesizes cell-penetrating peptide (GRKKRRQRRRPPQ) on solid-phase resin particle, wherein a Fmoc is selected to protect α-ammonia
Base Boc protects the lysine of side-chain amino group, and the blocking group of glycine is sloughed in the DMF solution reaction of 20% 4- methyl piperidine
(Fmoc) after, using containing being fmoc-protected lysine there are two amino, the 0.3755g for being dissolved in 15ml DMF is added
HBTU, 0.25ml DIEA, 0.2954g lysine solution react 3h, are sloughed with the DMF solution reaction of 20% 4- methyl piperidine
The blocking group (Fmoc) of lysine is added containing there are two the lysine of Fmoc blocking group, and addition is dissolved in 15ml DMF
0.3755g HBTU, 0.25ml DIEA, 0.2954g lysine solution react 3h, the DMF with 20% 4- methyl piperidine is molten
Liquid, which reacts, sloughs the blocking group (Fmoc) of lysine, after introducing 4 amino, be added 1.502g HBTU, 1ml DIEA,
The reaction of 1.1892g cysteine overnight, after dry, is dissolved in 20ml methanol, and obtained 300mg is reacted in rear be added embodiment 1 (6)
CPT-S-S-Pyr reaction overnight, obtains the amphipathic cell-penetrating peptide key compound (qCPT-CPP) of four rouge derivative chains of bonding, knot
Structure is with mass spectrum as shown in fig. 6, mass-to-charge ratio is consistent with the molecular weight being fitted with ChemDraw Professional 15.0, it was demonstrated that at
Function prepares the amphipathic cell-penetrating peptide key compound of four hydrophobic drugs containing sulfydryl of bonding.
By Fig. 1~6 it is found that cell-penetrating peptide by be bonded different hydrophobic structures can effectively construct structure it is controllable, it is quality controllable,
The amphipathic cell-penetrating peptide key compound of monodisperse, uniform molecular weight.
Embodiment 2, the preparation of amphipathic cell-penetrating peptide key compound self-assembled nano micelle and its pattern
The present embodiment is the demonstration that different-shape nano micella can be constructed to the present invention, to understand the nano-micelle system
Standby simplification and its pattern adjustability.
Dissolve embodiment 1 (1), (4), (5), each amphipathic cell-penetrating peptide key compound obtained in (7), ultrasound respectively with pure water
Amphipathic cell-penetrating peptide key compound self-assembled nano micelle is prepared, dC16-CPP self-assembled nano micelle, qC16-CPP are respectively obtained
Self-assembled nano micelle, qCF9-CPP self-assembled nano micelle, qCPT-CPP self-assembled nano micelle.
Self-assembled nano micelle is diluted with water to 2mM in right amount, draws a drop in drop reaction porcelain plate with a fine droplets pipe
Groove in, and carbon-sprayed copper net is put on test solution (film surface is downward), takes out copper mesh after 1~2min, with filter paper small pieces from copper mesh
Edge, which is inhaled, abandons surplus liquid；In same way as described above, which is placed on about 30s on 1% (w/v) acetic acid uranium dyeing liquor, with filter
Paper small pieces blot extra dye liquor from copper mesh edge, then are washed with distilled water 1~2 time, and drying after abandoning moisture content is inhaled with filter paper, is being transmitted
The mode of appearance and distribution character of electric cell-penetrating peptide CPP key compound self-assembled nano micelle amphipathic under the microscope.
As seen from Figure 7, the cell-penetrating peptide hydrophobic structure different by bonding, can form the nano-micelle of different-shape,
Realize the morphology controllable of nano-micelle.
The fluorescence imaging of embodiment 3, amphipathic cell-penetrating peptide key compound self-assembled nano micelle
The present embodiment is the demonstration to amphipathic cell-penetrating peptide key compound self-assembled nano micelle imaging capability, to understand it
Application in terms of diagnosing tumor imaging.
(1) dC16-CPP contains coumarin 6 fluorescence imaging
Precision weighs dC16-CPP made from embodiment 1 (1) (10mg) dissolution 2mL HFIP in 10mL round-bottomed flask, adds
The HFIP solution (coumarin 6 is dissolved in HFIP with 1mg/mL) for entering 2 μ L dissolution coumarin 6, is protected from light mediation 30s, in reduced pressure
Lower rotation evaporating organic solvent makes it form the film of layer of transparent in bottle wall, and the distillation of 1mL preheating is then added
Water, jog aquation contains fluorescent molecule coumarin 6 to get amphipathic cell-penetrating peptide key compound dC16-CPP physics under the conditions of 37 DEG C
Self-assembled nano micelle.The nano-micelle of preparation is excited under 488nm fluorescence (Fig. 8).
(2) fluorescence imaging of qCPT-CPP
Precision weigh qCPT-CPP made from embodiment 1 (7) (5mg) be dissolved in 1mL preheating distilled water, ultrasonic 30s, i.e.,
Obtain the self-assembled nano micelle of amphipathic cell-penetrating peptide key compound qCPT-CPP.The nano-micelle of preparation is excited under 365nm glimmering
Light (Fig. 9).
The investigation of embodiment 4, taxol self-assembled nano micelle encapsulation rate and drugloading rate
The present embodiment is the demonstration that hydrophobic molecule is contained to the self-assembled nano micelle physics, to understand the nanometer
Application of the micella in drug conveying.
Precision weighs dC16-CPP made from embodiment 1 (1) (25mg), PTX (1mg), i.e. dC16-CPP:PTX=25:1
(w:w), ultrasonic dissolution is added in 10mL round-bottomed flask in 2mL HFIP, rotates evaporating organic solvent under reduced pressure, make
Its film that layer of transparent is formed in bottle wall, the distilled water that then addition 1mL is preheated, jog aquation under the conditions of 37 DEG C, i.e.,
Obtain the taxol self-assembled nano micelle that amphipathic cell-penetrating peptide key compound dC16-CPP contains 10% molar ratio.Draw it is a certain amount of not
It is centrifuged stoste, to measure PTX content through HPLC after dilution in acetonitrile, is denoted as WAlways.The nano-micelle of preparation is centrifuged in 4 DEG C
(5000rmp, 5min).Centrifugation gained filtrate is drawn to measure PTX content through HPLC after dilution in acetonitrile, is denoted as WTrip, count as the following formula
Calculate dC16-CPP encapsulation rate and drugloading rate:
Encapsulation rate (EE, %)=[(WAlways-WTrip)/WAlways] × 100%
Drugloading rate (DL, %)=[(WAlways-WTrip)/WCarrier] × 100%
Wherein,
WTripFor the drug weight dissociated in filtrate
WAlwaysFor total drug weight
WCarrierFor carrier (dC16-CPP) weight
The average encapsulation rate for measuring dC16-CPP is 87.59%, drugloading rate 6.05%.
The vitro cytotoxicity experiment of embodiment 5, amphipathic cell-penetrating peptide key compound nano-micelle
The present embodiment is the demonstration applied in oncotherapy to the nano-micelle, to understand that the nano-micelle exists
Purposes in antitumor especially multidrug resistance of tumor.
(1) cell culture
The cell frozen is taken out from liquid nitrogen container, is thawed in 37 DEG C of warm water rapidly, and cell suspension is centrifuged in 15mL
Complete 1640 culture medium of 5mL is added in Guan Zhong, gently blows and beats to being uniformly mixed, is centrifuged (1500rmp, 1.5min), discards supernatant
Liquid.Complete 1640 culture medium of 2mL is added, gently blows and beats to being uniformly mixed, which is added in culture dish, is added
Complete 1640 culture medium of 8mL mixes.Culture dish is placed in 5%CO2, cultivate in 37 DEG C of incubators.
A549/T cell is with containing 200ngmL-1The complete 1640 culture medium culture of taxol, complete 1640 training of A549
Support base culture.
(2) cell-penetrating peptide key compound (dC16-CPP material) contains PTX cytotoxicity experiment
A549, A549/T cell are inoculated into 96 well culture plates with the density in 3000/hole, after culture for 24 hours, more renewed
Fresh complete 1640 culture medium, and PTX dimethyl sulfoxide solution (PTX-DMSO) conduct of final concentration of 2.5,5,10 μm of ol/L is added
Free PTX group, final concentration of 50 μm of ol/L dC16-CPP that the preparation of 4 method of the embodiment of the present invention is added contain 0,2.5,5,10 μ
Mol/L PTX is as test group.In 5%CO after cell dosing2, be incubated for for 24 hours in 37 DEG C of incubators, inhale the drug containing culture abandoned in hole
Base is washed twice with culture medium, and fresh complete 1640 culture medium, 200 μ L is added, and after continuing culture for 24 hours, inhales the culture abandoned in hole
Base is washed three times with PBS, 180 μ L of fresh complete 1640 culture is added, while 20 μ L MTT solution (5mgmL are added in every hole-1), continue in 5%CO2, cultivate 4h in 37 DEG C of incubators, terminate culture.Careful inhale abandons supernatant, and 100 μ L are added in every hole
DMSO is protected from light concussion 10min, so that purple crystal thing sufficiently dissolves mixing.The light absorption value (A) at 570nm is measured with microplate reader,
According to: cells survival (survival) rate % (Cell viability, %)=experimental group average A-value/blank control group average A-value ×
100%, it calculates cell survival rate and draws a diagram.
With mtt assay detect cell survival rate the result shows that, in A549/T cell strain, with the increase of PTX concentration, toxicity
Become larger, in the concentration of 10 μm of ol/L, cell survival rate is i.e. less than 50%；And in sensitive cells strain, due to cell itself pair
Taxol is just more sensitive, so containing the difference of PTX and free PTX cellular control unit survival rate not in drug-resistant cell strain
Significantly.
The results are shown in Figure 10, cytotoxicity power be respectively contain the cell-penetrating peptide key compound self-assembled nano micelle of PTX >
The amphipathic cell-penetrating peptide key compound nano micellar solution of free PTX > do not contain PTX.
Claims (10)
1. a kind of amphipathic cell-penetrating peptide key compound, it is characterised in that: include hydrophobic patches and cell-penetrating peptide segment, wherein described dredge
Aqueous segment and the cell-penetrating peptide segment are bonded by the functional group reactions that they are included, and bonding pattern is selected from amide
Key, ester bond, ehter bond or acyl sulfide linkage.
2. amphipathic cell-penetrating peptide key compound as described in claim 1, it is characterised in that: the amphipathic cell-penetrating peptide key compound into
One step includes the amino acid or small peptide between the hydrophobic patches and the cell-penetrating peptide segment, is used for one or more
A hydrophobic patches are connected in cell-penetrating peptide segment.
3. amphipathic cell-penetrating peptide key compound as claimed in claim 1 or 2, it is characterised in that: the hydrophobic patches are derived from
C9~C18 saturated fatty acid, C9~C18 unsaturated fatty acid, fluorine-containing C9~C18 saturated fatty acid, fluorine-containing C9~C18 are not
Saturated fatty acid, vitamin E, cholic acid or signaling molecule；Preferably, the hydrophobic patches are derived from palmitinic acid, 2H, 2H, 3H,
3H- perfluoro-pelargonic acid or camptothecine fluorescent molecule.
4. amphipathic cell-penetrating peptide key compound as claimed in claim 1 or 2, it is characterised in that: the cell-penetrating peptide fragment derivitization is certainly
The source HIV CPP, oligomerization arginine, MPG or EB1；Preferably, the cell-penetrating peptide segment be GRKKRRQRRRPPQ or
GRKKRRQRRR。
5. amphipathic cell-penetrating peptide key compound as described in claim 1, it is characterised in that: described in the hydrophobic patches are only bonded
One end of cell-penetrating peptide segment is connected with hydrophilic fractions in the other end of the cell-penetrating peptide segment；The hydrophilic fractions are parent
Water macromolecule group, targeting ligand, or both conjugate；Preferably, the hydrophilic high mol group is polyethylene glycol or more
Sugar；The targeting ligand is folic acid, small peptide or antibody.
6. amphipathic cell-penetrating peptide key compound as claimed in claim 5, it is characterised in that: the hydrophilic fractions wear film with described
It is directly connected between peptide or is connected by stimulating responsive linking group.
7. a kind of preparation method of amphipathic cell-penetrating peptide key compound described in claim 1, which is characterized in that including following step
Rapid: cell-penetrating peptide is mixed with hydrophobic compound, condensing agent, reacts to obtain amphipathic cell-penetrating peptide key compound in a solvent.
8. a kind of amphipathic cell-penetrating peptide key compound self-assembled nano micelle, amphipathic as described in any one of claim 1~6
Cell-penetrating peptide key compound is self-assembly of in water or buffer.
9. purposes of the self-assembled nano micelle according to any one of claims 8 as delivery vehicles；Preferably, the carrier is for conveying
Contrast agent or the drug for treating tumour.
10. purposes according to claim 9, wherein the contrast agent is selected from fluorine-containing hydrophobic molecule, fluorescent molecule and light and inhales
Contracture；The drug of the treatment tumour is selected from taxanes, camptothecin, CHROMATOGRAPHIC FRACTIONATION AND MASS, rapamycin hydrophobic small molecules
Anti-tumor drug.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3682899A1 (en) * | 2019-01-15 | 2020-07-22 | Tianjin University | Compound amphiphilic peptide nanomicelle, preparation and use thereof |
| CN113209020A (en) * | 2021-05-18 | 2021-08-06 | 中南民族大学 | Enzyme-sensitive polypeptide micelle diagnosis and treatment agent and application thereof in preparation of antitumor drugs |
| WO2022013306A1 (en) * | 2020-07-14 | 2022-01-20 | Universität Heidelberg | Oral pharmaceutical compositions comprising lipid conjugates |
| WO2022077815A1 (en) * | 2020-10-16 | 2022-04-21 | 上海交通大学 | Affibody-cytotoxin conjugate used for active tumor targeting therapy, and nanoparticles, preparation method, and application thereof |
| CN115198015A (en) * | 2022-05-19 | 2022-10-18 | 广州臻美生物科技研究有限公司 | Tumor-associated protein and application thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007104422A2 (en) * | 2006-03-14 | 2007-09-20 | Lts Lohmann Therapie-Systeme Ag | Agent-enriched nanoparticles based on hydrophilic proteins |
| CN102002096A (en) * | 2010-09-10 | 2011-04-06 | 中国药科大学 | Amphipathic compound using somatostatin analogue as target radical and pharmaceutics application thereof |
| CN103690961A (en) * | 2013-12-10 | 2014-04-02 | 深圳先进技术研究院 | Intelligent amphiphilic polymer nano micelle and preparation method and application thereof |
| CN104497147A (en) * | 2014-12-23 | 2015-04-08 | 四川大学 | Double receptor identified serial cell-penetrating peptide modified tumor targeted nano drug delivery system |
| CN104940949A (en) * | 2015-06-16 | 2015-09-30 | 国家纳米科学中心 | Antineoplastic polypeptide nanometer drug, preparation method and application thereof |
| CN104940950A (en) * | 2015-07-09 | 2015-09-30 | 武汉大学 | Tumor targeted polypeptide photosensitizer bonding object |
| CN105412011A (en) * | 2016-01-11 | 2016-03-23 | 四川大学 | Amphiphilic micelle of carrier with anti-tumor and anti-metastasis activity |
| CN106389384A (en) * | 2016-03-14 | 2017-02-15 | 四川大学 | Preparation method and application of multi-stage liver-targeted intelligent nano drug delivery system |
-
2017
- 2017-05-18 CN CN201710352646.XA patent/CN108948152A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007104422A2 (en) * | 2006-03-14 | 2007-09-20 | Lts Lohmann Therapie-Systeme Ag | Agent-enriched nanoparticles based on hydrophilic proteins |
| CN102002096A (en) * | 2010-09-10 | 2011-04-06 | 中国药科大学 | Amphipathic compound using somatostatin analogue as target radical and pharmaceutics application thereof |
| CN103690961A (en) * | 2013-12-10 | 2014-04-02 | 深圳先进技术研究院 | Intelligent amphiphilic polymer nano micelle and preparation method and application thereof |
| CN104497147A (en) * | 2014-12-23 | 2015-04-08 | 四川大学 | Double receptor identified serial cell-penetrating peptide modified tumor targeted nano drug delivery system |
| CN104940949A (en) * | 2015-06-16 | 2015-09-30 | 国家纳米科学中心 | Antineoplastic polypeptide nanometer drug, preparation method and application thereof |
| CN104940950A (en) * | 2015-07-09 | 2015-09-30 | 武汉大学 | Tumor targeted polypeptide photosensitizer bonding object |
| CN105412011A (en) * | 2016-01-11 | 2016-03-23 | 四川大学 | Amphiphilic micelle of carrier with anti-tumor and anti-metastasis activity |
| CN106389384A (en) * | 2016-03-14 | 2017-02-15 | 四川大学 | Preparation method and application of multi-stage liver-targeted intelligent nano drug delivery system |
Non-Patent Citations (7)
| Title |
|---|
| AMIR NASROLAHI SHIRAZI等: "Peptide amphiphile containing arginine and fatty acyl chains as molecular transporters", 《MOL PHARM》 * |
| AMIR NASROLAHI SHIRAZI等: "Peptide amphiphile containing arginine and fatty acyl chains as molecular transporters", 《MOL PHARM》, vol. 10, no. 12, 12 November 2013 (2013-11-12), pages 4717 - 4727 * |
| YUNFEI SUN等: "New amphiphilic N-phosphoryl oligopeptides designed for gene delivery", 《INT J PHARM》 * |
| YUNFEI SUN等: "New amphiphilic N-phosphoryl oligopeptides designed for gene delivery", 《INT J PHARM》, vol. 468, no. 1, 5 April 2014 (2014-04-05), pages 83 - 90, XP029029798, DOI: 10.1016/j.ijpharm.2014.04.007 * |
| 刘亚杰等: "微纳米粒子的形貌调控及其对药物/基因传递体系的影响", 《化学进展》, vol. 28, no. 01, 31 December 2016 (2016-12-31), pages 67 - 74 * |
| 张霞等: "负载紫杉醇的双配体纳米药物输送系统的制备", 《数理医药学杂志》 * |
| 张霞等: "负载紫杉醇的双配体纳米药物输送系统的制备", 《数理医药学杂志》, no. 04, 15 August 2013 (2013-08-15), pages 450 - 453 * |
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| WO2022013306A1 (en) * | 2020-07-14 | 2022-01-20 | Universität Heidelberg | Oral pharmaceutical compositions comprising lipid conjugates |
| WO2022077815A1 (en) * | 2020-10-16 | 2022-04-21 | 上海交通大学 | Affibody-cytotoxin conjugate used for active tumor targeting therapy, and nanoparticles, preparation method, and application thereof |
| CN113209020A (en) * | 2021-05-18 | 2021-08-06 | 中南民族大学 | Enzyme-sensitive polypeptide micelle diagnosis and treatment agent and application thereof in preparation of antitumor drugs |
| CN113209020B (en) * | 2021-05-18 | 2022-05-03 | 中南民族大学 | Enzyme-sensitive polypeptide micelle diagnosis and treatment agent and application thereof in preparation of antitumor drugs |
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