CN109589413A - Target polypeptide, the targeted nano particle and its preparation method and application of placenta sample chondroitin sulfate A (CSA) - Google Patents
Target polypeptide, the targeted nano particle and its preparation method and application of placenta sample chondroitin sulfate A (CSA) Download PDFInfo
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- CN109589413A CN109589413A CN201710906587.6A CN201710906587A CN109589413A CN 109589413 A CN109589413 A CN 109589413A CN 201710906587 A CN201710906587 A CN 201710906587A CN 109589413 A CN109589413 A CN 109589413A
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- 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
Abstract
The present invention provides a kind of nano particles for targeting placenta sample chondroitin sulfate A (CSA), including hydrophobic cores, wrap up the single layer lipid molecule layer of the hydrophobic cores and the hydrophily shell of targeting placenta sample chondroitin sulfate A (CSA), the hydrophobic cores include the hydrophobic polymer, the ingredient of the hydrophily shell is polypeptide grafted amphiphilic macromolecular compound, the hydrophobic side of the amphiphilic macromolecular compound is interspersed in the single layer lipid molecule layer, the water-wet side and the polypeptide of the amphiphilic macromolecular compound are keyed by amide, the polypeptide is exposed to outside the single layer lipid molecule layer, wherein, the amino acid sequence of the polypeptide is selected from one of amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:3 or a variety of.The present invention also provides the preparation method and application of the nano particle, and the polypeptide for targeting placenta sample chondroitin sulfate A (CSA).
Description
Technical field
The invention belongs to pharmaceutical carrier technical field, in particular to a kind of placenta sample chondroitin sulfate A (CSA) targeted nano particle
And its preparation method and application.
Background technique
Cancer seriously endangers human health, currently, the treatment of cancer is mainly with operation, based on chemotherapy and radiation, but changes
Treat drug effect be mostly it is non-selective, distribution is wide in vivo, and normal tissue organ toxic side effect is big under therapeutic dose.Design and
The medicine-carried system of specific recognition cancer cell is capable of in building, while reducing the toxic side effect of chemotherapeutics, is risen to the treatment of cancer
Vital effect.
The research of the targeted delivery systems based on nanotechnology brings new opportunity to treatment of cancer in recent years.It can be incited somebody to action
Therapeutic agent is transported to target organ to the maximum extent, and influences very little to non-target organ, to reach the treatment effect of high-efficiency low-toxicity
Fruit, and nano material itself has the advantages such as slow controlled capability, transmucosal, transdermal, is of great importance to treatment of cancer.Wherein,
It can be with target cell (brain microvessel endothelial cells in vitro for constituting blood-brain barrier) specific binding by the connection of drug-loading nanoparticles surface
Ligand, such as antibody, peptide chain make nano particle that Nano medication are delivered to cancer by receptor-mediated transcytosis
Position is more common technology.
Chondroitin sulfate (CS) is to be covalently attached a kind of glycosaminoglycan that proteoglycans is formed on protein.Chondroitin sulfate
Element is distributed widely in the extracellular matrix and cell surface of animal tissue, plays important physiological function.Although chondroitin sulfate
Polysaccharide skeleton it is simple, but degree, sulfate and two species diversity for distribution in chain, are deposited to isomery uronic acid
In biggish difference.The fine structure of chondroitin sulfate decides the specificity of its function, and with multiple proteins molecule
Interaction.
Placenta sample chondroitin sulfate A (CSA) (pl-CSA) belongs to glycosaminoglycan family, be attached to proteoglycans, it is alternate
The straight chain polymer of amino sugar and hexuronic acid residue, glycosylation pattern are different from conventional sulfuric acid chondroitin.Early stage research refers to
Pl-CSA is the reason of causing the red blood cell of Infected With Plasmodium in placenta to be isolated out, and Ali Salanti in 2015 et al. exists
" Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein " middle finger
Out, pl-CSA is expressed in a variety of cancer cells, specifies new target spot for the treatment of cancer.However the specific receptor of pl-CSA
It is receptor, delivery vector etc. that be unknown, can more being specifically bound at present with pl-CSA without document report road.
Summary of the invention
To solve the above problems, the present invention provides the polypeptides and targeted nano of a kind of targeting of placenta sample chondroitin sulfate A (CSA)
Particle, preparation method and application.The stability and targets identification ability of the targeted nano particle are good, improve carrying medicament pair
The targeting effect of cancer cell.
In a first aspect, the present invention provides a kind of polypeptides, wherein the polypeptide is used to target placenta sample chondroitin sulfate A (CSA),
The amino acid sequence of the polypeptide is selected from amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:3.
The polypeptide can be such as SEQ ID NO:1, SEQ ID NO:2 or a kind of sequence as shown in SEQ ID NO:3,
It can also be a variety of in the sequence as shown in SEQ ID NO:1-SEQ ID NO:3.
The polypeptide can be modified in polymer (such as polyethyleneimine, chitosan), liposome, gold nano grain, two
On the common pharmaceutical carrier or genophore such as silica, seralbumin, the delivering of targeting placenta sample chondroitin sulfate A (CSA) is obtained
System.In addition, the polypeptide can also be coupled with small-molecule drug, the targeted delivery system of specially polypeptide drugs conjugate is obtained
System.
Second aspect, the present invention provides a kind of placenta sample chondroitin sulfate A (CSA) targeted nano particle, the targeted nanos
Grain includes hydrophobic cores, the single layer lipid molecule layer of the package hydrophobic cores and targeting placenta sample chondroitin sulfate A (CSA)
Hydrophily shell, the hydrophobic cores include the hydrophobic polymer, and the ingredient of the hydrophily shell is polypeptide grafted
Amphiphilic macromolecular compound, the hydrophobic side of the amphiphilic macromolecular compound is interspersed in the single layer lipid molecule layer
In, the water-wet side and the polypeptide of the amphiphilic macromolecular compound are keyed by amide, and the polypeptide is exposed to described
Outside single layer lipid molecule layer, wherein the amino acid sequence of the polypeptide is selected from ammonia shown in SEQ ID NO:1-SEQ ID NO:3
One of base acid sequence is a variety of.
In the present invention, the polypeptide be can be such as SEQ ID NO:1, SEQ ID NO:2 or as shown in SEQ ID NO:3
Sequence is the one or more of the polypeptide with sequence shown in SEQ ID NO:1-SEQ ID NO:3 respectively.
Preferably, the diameter of the targeted nano particle is 80~150nm.The partial size is using transmission electron microscope
It measures.
Preferably, the hydrophobic polymer, single layer lipid molecule, the amphiphilic macromolecular compound mass ratio be
1:(0.04-0.2): (0.1-0.4).Under the mass ratio, can be formed between each component of the targeted nano particle pattern compared with
The more uniform structure of rule, favorable dispersibility, particle diameter distribution, the stable structure of the targeted nano particle, is not easy by body
Liquid dilution dissolves and disintegrates, and is conducive to the cancer cell that the targeted nano particle is targeted to expression placenta sample chondroitin sulfate A (CSA),
Help to ensure that the enough administration concentrations of targeting moiety.Have with the targeted nano particle package delivering drug in biomedical applications
Greater advantage.
Preferably, the single layer lipid molecule is selected from least one of lecithin and cephalin (phosphatidyl-ethanolamine),
The lecithin is selected from one of soybean lecithin, hydrogenated soy phosphatidyl choline, egg yolk lecithin and phosphatidyl choline or a variety of.
It is further preferred that the single layer lipid molecule is with the hydrophobic part towards the hydrophobic cores and towards the nanometer
Hydrophilic segment outside grain.
Preferably, the mass ratio of the amphiphilic macromolecular compound and the polypeptide is 1:1~4.Under the mass ratio,
Polypeptide is higher to the grafting rate of the amphiphilic macromolecular compound.
As described in the present invention, the polypeptide grafted amphiphilic macromolecular compound layer includes amphiphilic macromolecular chemical combination
Object and polypeptide, the water-wet side that the amphiphilic macromolecular compound has hydrophobic side and connect with the rouge end.In the present invention, institute
The hydrophobic side for stating amphiphilic macromolecular compound can help the amphiphilic macromolecular compound to be inserted into the single layer lipid point
Sublayer, and the water-wet side and the polypeptide grafted branches and the outside for extending in the nano particle.
Preferably, the amphiphilic macromolecular compound is polyglycol derivatization phospholipid, the polyglycol derivatization
Phosphatide is connected to obtain by covalent bond by polyethylene glycol and its derivative with phospholipid substance.At this point, the amphiphilic macromolecular
The hydrophobic side of compound is the phospholipid substance, and the water-wet side is carboxyl or amido modified polyethylene glycol or is band
There is the polyethyleneglycol derivative of other active function groups.
Wherein, the molecular weight of the polyethylene glycol is preferably 200~20000.Specifically, the molecular weight of peg molecule
It can be 200,500,1000,2000,5000,7000,10000,15000 or 20000.The phospholipid substance can be artificial
Phosphatide existing for synthesize or nature, the phospholipid substance can be but be not limited to Distearoyl Phosphatidylethanolamine
(DSPE), distearoylphosphatidylglycerol (DSPG) or cholesterol.
It is further preferred that the amphiphilic macromolecular compound is distearoylphosphatidylethanolamine-polyethylene glycol-carboxylic
Acid copolymerization (DSPE-PEG-COOH, also known as phosphatide-PEG- carboxyl), distearoylphosphatidylethanolamine-polyethylene glycol-amino
It is copolymerized (DSPE-PEG-NH2, also known as phosphatide-PEG- amino) or distearoylphosphatidylethanolamine-polyethylene glycol-Malaysia acyl
Amine.
Preferably, the hydrophobic polymer be selected from poly lactide-glycolide acid (also known as poly (glycolide-co-lactide),
Be abbreviated as PLGA), polylactic acid and polycaprolactone it is one or more, but not limited to this.
It is further preferred that the hydrophobic polymer is poly lactide-glycolide acid (being abbreviated as PLGA), it is described
The molecular weight of PLGA is 7000-17000.Wherein, the copolymerization ratio of monomer lactic acid and hydroxyacetic acid is 50:50.
Preferably, the hydrophobic cores further include that target delivers object, and the target delivers object and is adsorbed on the hydrophobicity
On polymer, it is at least one of anticancer drug, contrast agent and fluorescence tracer that the target, which delivers object,.Target deliver object with
The hydrophobic polymer collectively forms the hydrophobic cores.Hydrophobic polymer can adsorb or wrap up target and deliver object structure
At hydrophobic cores, occurs to assemble before reaching cancer cell or reveal it is possible to prevente effectively from the target loaded delivers object, guarantee
The stability of the delivery object of load.
It is further preferred that the mass ratio that the hydrophobic polymer and the target deliver object is 1:0.25~0.75.
It is further preferred that the target is delivered in object, the anticancer drug and the fluorescence tracer or contrast agent
Mass ratio is 1:1.
Preferably, the anticancer drug be selected from adriamycin, Epi-ADM, taxol, navelbine, etoposide,
One of cis-platinum, methopterin, 5 FU 5 fluorouracil and prodigiosin are a variety of, but not limited to this.
Preferably, the fluorescence tracer is selected from indocyanine-green, Evans blue, isosulfan blue, patent blue, methylenum careuleum, tonka-bean
Element 6, IR780 iodide (chloro- three carbon of 1,1'- diη-propyl -3,3,3', the 3'- tetramethyl -10,12- trimethylene indoles flower of 11-
Green salt compounded of iodine) and one of DiR iodide or a variety of, but not limited to this.
Preferably, the contrast agent is selected from one of iohexol, Iopromide, B-15000, pantopaque and barium sulfate
Or it is a variety of, but not limited to this.
It is prepared by the placenta sample chondroitin sulfate A (CSA) targeted nano particle that second aspect of the present invention provides, single layer lipid molecule
In the process, it can be self-assembled into single layer lipid molecule layer, and wrap up the hydrophobic cores, in the amphiphilic macromolecular compound
Hydrophobic side by physical action with the lipid molecule in the single layer lipid molecule layer in conjunction with to be interspersed in the single layer rouge
In class molecular layer, the water-wet side of the polypeptide and the amphiphilic macromolecular compound is covalently attached and extends in the targeting and receives
The outside of rice grain, the polypeptide grafted amphiphilic macromolecular compound provide outside hydrophily for the targeted nano particle
The receptor of layer and targeting placenta sample chondroitin sulfate A (CSA), therefore, the targeted nano particle has good targeting to cancer cell
Property, can carry target delivery object well enters in cancer cell, helps to ensure that the enough administration concentrations of targeting moiety, raising is controlled
Therapeutic effect.
In addition, the water-wet side (such as PEG) of the amphiphilic macromolecular compound, it can be effective, stable for a long time described
Targeted nano particle hinders identification of the immune system to the targeted nano particle, significantly extends the targeted nano particle
Circulation time in vivo, and then it is enriched to cancer cell position by enhancing infiltration retention effect (EPR effect), finally realize cancer
The passive target of cell.Passive target caused by active targeting and PEG based on aforementioned polypeptides receptor, the targeted nano particle
There is very strong affinity to cancer cell.
The third aspect, the present invention provides a kind of preparation methods of placenta sample chondroitin sulfate A (CSA) targeted nano particle, including
Following steps:
(1) hydrophobic polymer is dissolved in organic solvent, obtains hydrophobic polymer solution；
(2) single layer lipid molecule and amphiphilic macromolecular compound are dissolved in the first solvent, obtain the first mixed solution；
(3) the hydrophobic polymer solution is added in first mixed solution, is ultrasonically treated 4-6min, obtains
Second mixed solution carries out centrifugal treating to second mixed solution, collects supernatant and obtains targeted nano particle precursor body；
(4) take the targeted nano particle precursor body, by it with the targeting polypeptide of placenta sample chondroitin sulfate A (CSA), catalyst,
Dehydrating agent carries out amide reaction in the second solvent, so that on the be grafted to amphiphilic macromolecular compound of the polypeptide；It collects
Reaction solution obtains placenta sample chondroitin sulfate A (CSA) targeted nano particle by the reaction solution after isolating and purifying；
The targeted nano particle includes the single layer lipid molecule layer and target of hydrophobic cores, the package hydrophobic cores
To the hydrophily shell of placenta sample chondroitin sulfate A (CSA), the hydrophobic cores include the hydrophobic polymer, the hydrophily
The ingredient of shell is polypeptide grafted amphiphilic macromolecular compound, and the hydrophobic side of the amphiphilic macromolecular compound is interspersed in
In the single layer lipid molecule layer, the water-wet side and the polypeptide of the amphiphilic macromolecular compound are keyed by amide,
The polypeptide is exposed to outside the single layer lipid molecule layer, wherein the amino acid sequence of the polypeptide is selected from SEQ ID NO:1-
One of amino acid sequence shown in SEQ ID NO:3 is a variety of.
Preferably, in step (1), the organic solvent includes acetonitrile, acetone, ether, chloroform, methylene chloride and just
One of hexane is a variety of, but not limited to this.The organic solvent can preferably dissolve the volatile molten of hydrophobic polymer
Agent.
Wherein, first solvent includes that at least one hydrophilic solvent or water and at least one hydrophilic solvent are formed
Mixed solvent.Wherein, the hydrophilic solvent be selected from ethyl alcohol, methanol, 1- octanol, acetonitrile, acetone, dimethylformamide (DMF) and
Dimethyl sulfoxide (DMSO), but not limited to this.First solvent needs so that amphiphilic macromolecular compound, single layer lipid molecule
Delivering object with target can dissolve.
Preferably, first solvent is the mixed solvent that water and at least one hydrophilic solvent are formed, such as various concentration
Ethanol water, various concentration methanol aqueous solution.It is further preferred that in first solvent, the volume fraction of water is
3-8%.
In an embodiment of the present invention, first solvent is the ethanol water that volume fraction is 4% or volume point
The methanol aqueous solution that number is 4%.
Wherein, first solvent contains water, can when the organic solvent solution in later period and hydrophobic polymer mixes,
Making the solubility of hydrophobic polymer reduces, can be convenient for later period ultrasound, emulsification balling-up.
Preferably, object also is delivered containing target in the targeted nano particle, the target delivers object and is adsorbed on described dredge
On aqueous polymer, it is at least one of anticancer drug, contrast agent and fluorescence tracer that the target, which delivers object,.
Preferably, the target delivers 0.1-0.8 times that the quality of object is the hydrophobic polymer quality.It is further excellent
Selection of land, the quality of the target delivery object are 0.25~0.75 times of the hydrophobic polymer quality.
Specifically, when the target is delivered object when containing hydrophobic combination, containing hydrophobic in the hydrophobic polymer solution
Property target deliver object, that is, the hydrophobic target is delivered into object and is dissolved in the organic solvent.Here hydrophobic combination
Refer to substance hardly compatible with water.When the target delivers object when containing hydrophilic composition, contain in first mixed solution
There is hydrophilic target to deliver object, that is, the hydrophilic target is delivered object and is dissolved in first solvent.It is above-mentioned such
Addition sequence is conducive to improve the load factor that the hydrophobic polymer delivers various forms, parent/hydrophobic target object.
Preferably, in first mixed solution, the concentration of the single layer lipid molecule is 10-300 μ g/mL, described two
Parent's property macromolecular compound concentration is 30-600 μ g/mL.
Preferably, in step (1), the concentration of the hydrophobic polymer solution is 1-4mg/ml.
Preferably, in step (3), the volume ratio of the hydrophobic polymer solution and first mixed solution is 1:3.
Preferably, in step (3), by hydrophobic polymer solution in a manner of being added dropwise with first mixed solution
It mixes.It is further preferred that the rate of addition of the hydrophobic polymer solution is 0.2-0.5mL/min.It can make in this way
Hydrophobic polymer is sufficiently complexed target and delivers object, and wraps up into shell, and cooperating ultrasound that could be formed, structure is more stable, loads
More efficient drug-loading nanoparticles.
Preferably, it is described ultrasound after, by second mixed solution at 40-80 DEG C gentle agitation.Gentle agitation,
Suitable solvent volatilization condition is provided, promotes hydrophobic polymer and single layer lipid molecule, amphipathic molecule to interact, finally
The dispersibility of resulting nano particle presoma is preferable, partial size is more uniform.The nano particle presoma is relative to finally obtaining
Nano particle for, the difference is that only, surface be not decorated to placenta sample chondroitin sulfate A (CSA) targeting polypeptide.
Preferably, in step (4), the amide reaction is to carry out at room temperature.Optionally, the time of the amide reaction
For 15-24h.Preferably 15-20h.
Preferably, when the amphiphilic macromolecular compound has amino, then when carrying out amide reaction, by institute
It states targeted nano particle precursor body to be added in the second solvent, addition catalyst, dehydrating agent carry out activation 0.5-3h, add institute
The polypeptide of targeting placenta chondroitin sulfate is stated, 15-24h is reacted at room temperature and (is preferably 18-24h), obtains reaction solution.
In addition, then in activation, being first added described amphipathic big when the amphiphilic macromolecular compound has carboxyl
Molecular compound, catalyst, dehydrating agent；Nano particle presoma is passed in the addition targeting later, is reacted at 3-10 DEG C
15-24h obtains reaction solution.
Preferably, in step (4), second solvent includes water, 2- (N- morpholine) second sulphur that pH value is 5.5~6.7
Acid buffer (referred to as " MES buffer solution "), phosphate (PBS) buffer that pH value is 7.0~7.9 etc..
In step (4), the method for the amidation process is well known to those skilled in the art.Catalyst can be described as again
Activator is often combined with condensing agent, is used for amidation process.
Preferably, in step (4), the condensing agent includes 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride
Salt (abbreviation EDC).
Preferably, in step (4), the catalyst includes n-hydroxysuccinimide (NHS), N- hydroxy succinyl
Any one in imines sodium salt (Sufo-NHS).
Preferably, in step (4), the condensing agent, catalyst and the amphiphilic macromolecular compound mass ratio be
(0.2-0.4): (0.05-0.3): 1.
It is highly preferred that the mass ratio of described EDC, NHS, DSPE-PEG-COOH are 1:0.4:5.
Preferably, in step (4), it is described isolate and purify for use molecular cut off for the ultra-filtration centrifuge tube of 5-10kDa into
Row ultrafiltration centrifugation collects gained supernatant after centrifugation, obtains placenta targeted nano particle.Preferably, the ultrafiltration centrifugation carries out
2-5 times.In addition to last time ultrafiltration centrifugation, water or PBS washing are all made of after centrifugation every time.
Preferably, in step (3), the centrifugal treating is centrifuged in the ultra-filtration centrifuge tube that molecular cut off is 5-10kDa
2-5 times, using water washing.
Preferably, in step (3), the centrifugal treating is to be centrifuged 3- every time at centrifugal rotational speed 3000-5000rpm
6min。
Preferably, in step (3), the ultrasound is the frequency 80-160W for using ultrasonic cell disruption instrument with 20kHz
Power carries out.
Step (3) hydrophobic polymer, single layer lipid molecule and amphiphilic macromolecular compound pass through self assembly
Journey forms the targeted nano particle precursor body (i.e. without the nano particle of targeting), does not need to be chemically reacted, prepare
Journey is environment-protecting and non-poisonous, and method is simple to operation.
In another embodiment of the invention, can also first will it is described it is polypeptide grafted arrive amphiphilic macromolecular chemical combination
On object, then again by polypeptide grafted amphiphilic macromolecular compound, hydrophobic polymer, single layer lipid molecule according to step
(1) the targeted nano particle is made in-(3) operation.
Fourth aspect, the present invention provides a kind of polypeptide for targeting pl-CSA as described in the first aspect of the invention or
Table of the pl-CSA targeted nano particle of person as described in respect of the second aspect of the invention in preparation prevention, diagnosis or treatment and (pl-CSA)
Application up in the drug of (such as inappropriate expression) relevant disease.
The expression to pl-CSA or the relevant disease of inappropriate expression include tumor disease, arthritis, arthropathy, more
Hair property hardening, the pathologic conditions as caused by neurotrosis (such as healing after neurotrosis), the illness of cartilage and scar tissue
(such as rheumatism, repair of cartilage or wound healing), psoriasis, but not limited to this.
Further, the tumor disease include placental villi cancer, breast cancer, cancer of pancreas, oophoroma, carcinoma of endometrium,
Hepatocellular carcinoma, lung cancer, colon cancer, prostate cancer, cervical carcinoma, carcinoma of testis, basal cell skin cancer, clear cell renal cell carcinoma,
Head and neck squamous cell carcinoma, cutaneous squamous cell carcinoma, vulva angling squamous cell carcinoma and basal cell carcinoma of vulva, neuroendocrine
One of cancer, sarcoma, tumour of hemopoietic system cancer and neuroepithelial tissue are a variety of, but not limited to this.
Wherein, the sarcoma includes but is not limited to fibrosarcoma, dedifferentes cartilage and embryonal-cell lipoma, leiomyosarcoma, rouge
Fat sarcoma, mucus embryonal-cell lipoma, corpus uteri leiomyosarcoma, osteosarcoma, Ewing's sarcoma and rhabdomyosarcoma, synovial sarcoma,
Solitary Fibrous；The hemopoietic system cancer includes but is not limited to that chronic lymphocytic leukemia (CLL), acute lymphoblastic are white
Blood disease (ALL), acute myelocytic leukemia (AML), B cell, T cell and large grained lymthoma；The neuroepithelial tissue
Tumour, including but not limited to astrocytoma (pleomorphic xanthoastrocytoma, fibrillary astrocytoma, modification star
Shape cytoma, glioblastoma multiforme), oligodendroglioma, ependymoma, tumor of choroid plexus, few astrocytoma,
(olfactory nerve is female for atypical hyloma, ganglioglioma, retinoblastoma, nerve-cell tumor, neuroblastoma
Cytoma and neuromere blastoma), medulloblastoma and atypia monster sample Rhabdoid tumor.
Placenta sample chondroitin sulfate A (CSA) (pl-CSA) targeted nano particle provided by the invention, good biocompatibility, Neng Gouti
High institute's loaded article (such as chemotherapeutics, fluorescence tracer and contrast agent, photothermal conversion reagent) improves the targets identification of cancer cell
The stability of loaded article extends circulation time in vivo.
To sum up, beneficial effects of the present invention include the following aspects:
1, provided by the present invention for the polypeptide of targeting pl-CSA, specificity knot can be realized to the pl-CSA on cancer cell
It closes, to mediate the peptide modified substance to enter cancer cell；
2, pl-CSA targeted nano particle provided by the invention is modified with the receptor polypeptides of pl-CSA, Ke Yiyou on surface
Effect improves the nano particle to the targeting and enrichment degree of cancer cell, extends its circulation time in vivo；
3, preparation method is simple for the pl-CSA targeted nano particle, convenient for operation；
4, the polypeptide and the pl-CSA targeted nano particle, have a wide range of application, are used to prepare the drug for the treatment of of cancer.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of placenta sample chondroitin sulfate A (CSA) targeted nano particle prepared by embodiment 1；
Fig. 2 is the transmission electron microscope picture of placenta sample chondroitin sulfate A (CSA) targeted nano particle prepared by embodiment 1；
Fig. 3 is the placenta sample chondroitin sulfate A (CSA) targeted nano particle prepared in the embodiment of the present invention 1 and other experimental groups pair
The ingestion result figure of human placenia cancer cell (JEG3)；
Fig. 4 is that the different cancer cells of the placenta sample chondroitin sulfate A (CSA) targeted nano particle prepared in the embodiment of the present invention 1 are taken the photograph
Take result；
Fig. 5 is the placenta sample chondroitin sulfate A (CSA) targeted nano Granules on Mouse villioma prepared in the embodiment of the present invention 1
Therapeutic effect figure；
Fig. 6 is the diagnosing tumour distribution of the placenta sample chondroitin sulfate A (CSA) targeted nano particle prepared in the embodiment of the present invention 2
Figure.
Specific embodiment
The following is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
In the present invention, the sequence such as SEQ ID NO:1- of the polypeptide for targeting placenta sample chondroitin sulfate A (CSA) (pl-CSA)
Shown in SEQ ID NO:3.
Specifically, LKPSHEKKNDDNGKKLCKAC is as shown in SEQUENCE NO.1.
EDVKDINFDTKEKFLAGCLIVSFHEGKC is as shown in SEQUENCE NO.2.
GKKTQELKNIRTNSELLKEWIIAAFHEGKC is as shown in SEQUENCE NO.3.
The polypeptide is carried out according to conventional polypeptid synthesising process, wherein the left end of each sequence is N-terminal, right end
For the C-terminal of polypeptide, C-terminal or N-terminal can be covalently attached with the amphipathy macromolecule compound, this is according to amphiphilic used
Depending on the property of property high-molecular compound.Wherein, when amphipathy macromolecule compound has-COOH, using carboxylic thereon
Base is reacted with amino (that is, amino on cysteine C) Lai Jinhang amide in the C-terminal of the polypeptide.Work as amphipathy macromolecule
When compound has amino, it can be reacted using amino thereon with the carboxyl on the N-terminal of the polypeptide to carry out amide.
Embodiment 1
A kind of preparation method for the placenta sample chondroitin sulfate A (CSA) targeted nano particle being coated with adriamycin, comprising the following steps:
(1) by poly lactide-glycolide acid, (PLGA, molecular weight 15000, monomer lactic acid and hydroxyacetic acid are total to
Poly- ratio is 50:50) it is dissolved in acetonitrile, obtain the acetonitrile solution of PLGA, concentration 2mg/mL；
(2) by the soybean lecithin of 90 μ g, DSPE-PEG-COOH (molecular weight of PEG is 2000), the 750 μ g of 210 μ g
Adriamycin is dissolved in 4% ethyl alcohol of 3mL, obtains the first mixed solution；
(3) the PLGA acetonitrile solution of 1mL is added dropwise in the first mixed solution of 3mL with the speed of 0.3mL/min,
And ultrasonic cell disruption instrument is used to be ultrasonically treated with the power of the frequency of 20KHz and 130W, ultrasonic time 5min；
Solution after ultrasound is subjected to ultrafiltration centrifugation in the ultra-filtration centrifuge tube that molecular cut off is 10kDa, and is washed with water
It washs, is repeated 4 times, wherein centrifugal rotational speed 4000rpm, be centrifuged 4min every time, collect supernatant and obtain targeted nano particle precursor body；
(4) the targeted nano particle precursor body is soluble in water, it is living that 42 μ g EDC and 17 μ g NHS progress surface is added
Change 2h, the sequence that 0.5mg is added later is the polypeptide of LKPSHEKKNDDNGKKLCKAC (as shown in SEQUENCE NO.1),
Amidation process 16h is carried out at room temperature, obtains reaction solution；
With molecular cut off it is that 10kDa super filter tube carries out ultrafiltration centrifugation, and is washed with water by the reaction solution, is repeated 4 times,
Wherein under centrifugal rotational speed 3500rpm, it is centrifuged 4min every time, collects supernatant and obtains placenta sample chondroitin sulfate A (CSA) targeted nano
Grain.
Fig. 1 is the structural schematic diagram of placenta sample chondroitin sulfate A (CSA) targeted nano particle made from the embodiment of the present invention 1.Institute
Stating targeted nano particle includes hydrophobic cores 1, the single layer lipid molecule layer 2 of the package hydrophobic cores and targeting placenta sample
The hydrophily shell 3 of chondroitin sulfate A (CSA), the ingredient of single layer lipid molecule layer 2 are soybean lecithin, the hydrophily shell 3
Ingredient is the DSPE-PEG31 that polypeptide 32 is grafted, and 11 be hydrophobic polymer PLGA, and 12 deliver object-adriamycin for target, and 1 is
11 and 12 hydrophobic cores constituted；In polypeptide grafted DSPE-PEG, the rouge end DSPE of DSPE-PEG 31 inserts in the soybean
In lecithin layer 22, water-wet side PEG and polypeptide 32 are keyed by amide, and polypeptide 32 is exposed to the single layer lipid molecule layer
Outside.
Fig. 2 is the transmission electron microscope picture of the targeted nano particle, figure it is seen that prepared targeted nano particle
Spherical in shape, dispersibility is preferably.The average grain diameter that the nano target particle is measured with Particle Size Analyzer is 80-100nm.
The encapsulation rate of adriamycin: EN%=(1-Cf/Ct) × 100% is calculated separately out using following formula, wherein Cf is
The amount of free drug, Ct are the total amount of drug, obtain the targeted nano particle to the encapsulation rate EN% of adriamycin be 37.2 ±
1.54%.In addition, measuring the bonding ratio of polypeptide using BCA method are as follows: 47.3 ± 5.1%.
Embodiment 2
A kind of placenta sample chondroitin sulfate A (CSA) targeted nano particle contains the preparation method of adriamycin and indocyanine green altogether, with
Embodiment 1 the difference is that, in the first mixed solution in step (2) containing have plenty of 750 μ g indocyanine green (ICG) and
The adriamycin of 750 μ g.
Embodiment 3
A kind of preparation method of placenta sample chondroitin sulfate A (CSA) targeted nano particle, comprising the following steps:
(1) polycaprolactone (molecular weight 10000) is dissolved in acetone, obtains polycaprolactone solution, concentration 1mg/mL；
(2) by the egg yolk lecithin of 40 μ g, the DSPE-PEG-NH of 100 μ g2(molecular weight of PEG is 3000), 250 μ g
Prodigiosin is dissolved in 4% methanol of 3mL, obtains the first mixed solution；
(3) the polycaprolactone solution of 1mL is added dropwise in the first mixed solution of 3mL with the speed of 0.2mL/min,
And ultrasonic cell disruption instrument is used to be ultrasonically treated with the power of the frequency of 20KHz and 80W, ultrasonic time 6min；
Solution after ultrasound is subjected to ultrafiltration centrifugation in the ultra-filtration centrifuge tube that molecular cut off is 5kDa, and is washed with water
It washs, is repeated 4 times, wherein under centrifugal rotational speed 3000rpm, be centrifuged 6min every time, collect supernatant and obtain targeted nano particle precursor
Body；
(4) the targeted nano particle precursor body is soluble in water, 20 μ g EDC and 5 μ g NHS are added and carry out surface active
2h, the sequence that 0.4mg is added later is the polypeptide of LKPSHEKKNDDNGKKLCKAC (as shown in SEQUENCE NO.1), in room
Temperature is lower to carry out amidation process 15h, obtains reaction solution；
With molecular cut off it is that 5kDa super filter tube carries out ultrafiltration centrifugation, and is washed with water by the reaction solution, is repeated 4 times,
Wherein under centrifugal rotational speed 3000rpm, it is centrifuged 6min every time, collects supernatant and obtains placenta sample chondroitin sulfate A (CSA) targeted nano
Grain.
Embodiment 4
A kind of preparation method of placenta sample chondroitin sulfate A (CSA) targeted nano particle, comprising the following steps:
(1) polylactic acid (molecular weight 21800) is dissolved in methylene chloride, obtains PLA solution, concentration 4mg/mL；
(2) by the cephalin of 800 μ g, the purple of the DSPE-PEG-NH2 (molecular weight of PEG is 2000) of 1600 μ g, 3000 μ g
China fir alcohol is dissolved in 4% methanol of 3mL, obtains the first mixed solution；
(3) PLA solution of 1mL is added dropwise in the first mixed solution of 3mL with the speed of 0.4mL/min, and
Ultrasonic cell disruption instrument is used to be ultrasonically treated with the power of the frequency of 20KHz and 160W, ultrasonic time 4min；
Solution after ultrasound is subjected to ultrafiltration centrifugation in the ultra-filtration centrifuge tube that molecular cut off is 10kDa, and is washed with water
It washs, is repeated 4 times, wherein under centrifugal rotational speed 5000rpm, be centrifuged 3min every time, collect supernatant and obtain targeted nano particle precursor
Body；
(4) the targeted nano particle precursor body is soluble in water, it is living that 640 μ g EDC and 80 μ g NHS progress surface is added
Change 4h, the sequence that 1.6mg is added later is EDVKDINFDTKEKFLAGCLIVSFHEGKC (as shown in SEQUENCE NO.2)
Polypeptide carries out amidation process 18h at room temperature, obtains reaction solution；
With molecular cut off it is that 10kDa super filter tube carries out ultrafiltration centrifugation, and is washed with water by the reaction solution, is repeated 4 times,
Wherein under centrifugal rotational speed 5000rpm, it is centrifuged 3min every time, collects supernatant and obtains placenta sample chondroitin sulfate A (CSA) targeted nano
Grain.
The TEM partial size of the targeted nano particle of embodiment 4 is 120-130nm；The connection of polypeptide is measured using BCA method
Rate are as follows: 52.3 ± 3.2%.
Embodiment 5
A kind of preparation method of placenta sample chondroitin sulfate A (CSA) targeted nano particle, comprising the following steps:
(1) by poly lactide-glycolide acid, (PLGA, molecular weight 10000, monomer lactic acid and hydroxyacetic acid are total to
Poly- ratio is 50:50) it is dissolved in acetonitrile, obtain the acetonitrile solution of PLGA, concentration 2mg/mL；
(2) by the phosphatidyl choline of 120 μ g, the DSPE-PEG-NH of 250 μ g2(molecular weight of PEG is 3000), 800 μ g
Prodigiosin is dissolved in 4% methanol of 3mL, obtains the first mixed solution；
(3) acetonitrile solution of the PLGA of 1mL is added dropwise to the first mixed solution of 3mL with the speed of 0.5mL/min
In, and ultrasonic cell disruption instrument is used to be ultrasonically treated with the power of the frequency of 20KHz and 120W, ultrasonic time is
5min；
Solution after ultrasound is subjected to ultrafiltration centrifugation in the ultra-filtration centrifuge tube that molecular cut off is 5kDa, and is washed with water
It washs, is repeated 4 times, wherein under centrifugal rotational speed 3500rpm, be centrifuged 4min every time, collect supernatant and obtain targeted nano particle precursor
Body；
(4) the targeted nano particle precursor body is soluble in water, it is living that 75 μ g EDC and 50 μ g NHS progress surface is added
Change 3h, the sequence that 0.75mg is added later is GKKTQELKNIRTNSELLKEWIIAAFHEGKC (such as SEQUENCE NO.3 institute
Show) polypeptide, at room temperature carry out amidation process 17h, obtain reaction solution；
With molecular cut off it is that 5kDa super filter tube carries out ultrafiltration centrifugation, and is washed with water by the reaction solution, is repeated 4 times,
Wherein under centrifugal rotational speed 3000rpm, it is centrifuged 3min every time, collects supernatant and obtains placenta sample chondroitin sulfate A (CSA) targeted nano
Grain.
The TEM partial size of the targeted nano particle of embodiment 5 is 90-120nm；The connection of polypeptide is measured using BCA method
Rate is 39.6 ± 2.5%.
Embodiment 6
A kind of preparation method of placenta sample chondroitin sulfate A (CSA) targeted nano particle, comprising the following steps:
(1) polycaprolactone (molecular weight 10000) is dissolved in acetone, obtains polycaprolactone solution, concentration 4mg/
mL；；
(2) by the phosphatidyl choline of 800 μ g, DSPE-PEG-COOH (molecular weight of PEG is 3000), 3000 μ of 1600 μ g
The etoposide of g is dissolved in 4% ethyl alcohol of 3mL, obtains the first mixed solution；
(3) acetonitrile solution of the PLGA of 1mL is added dropwise to the first mixed solution of 3mL with the speed of 0.3mL/min
In, and ultrasonic cell disruption instrument is used to be ultrasonically treated with the power of the frequency of 20KHz and 150W, ultrasonic time is
6min；
Solution after ultrasound is subjected to ultrafiltration centrifugation in the ultra-filtration centrifuge tube that molecular cut off is 10kDa, and is washed with water
It washs, is repeated 4 times, wherein under centrifugal rotational speed 5000rpm, be centrifuged 4min every time, collect supernatant and obtain targeted nano particle precursor
Body；
(4) the targeted nano particle precursor body is soluble in water, 480 μ g EDC and 160 μ g NHS are added and carry out surface
4h is activated, the sequence that 0.8mg is added later is GKKTQELKNIRTNSELLKEWIIAAFHEGKC (such as SEQUENCE NO.3 institute
Show) polypeptide and 0.8mg sequence be EDVKDINFDTKEKFLAGCLIVSFHEGKC (as shown in SEQUENCE NO.2),
Amidation process 20h is carried out at room temperature, obtains reaction solution；
With molecular cut off it is that 10kDa super filter tube carries out ultrafiltration centrifugation, and is washed with water by the reaction solution, is repeated 4 times,
Wherein under centrifugal rotational speed 4000rpm, it is centrifuged 3min every time, collects supernatant, obtains placenta sample chondroitin sulfate A (CSA) targeted nano
Grain.
The TEM partial size of the targeted nano particle of embodiment 6 is 120-150nm.
The cancer cell that Application Example 1 is coated with adriamycin placenta sample chondroitin sulfate A (CSA) targeted nano particle absorbs experiment
Placenta sample chondroitin sulfate A (CSA) targeted nano particle made from the embodiment of the present invention 1 (is abbreviated as
CSA-DNPs the intake experiment of different cancer cells) is carried out, and using free adriamycin (Free DOX) as feminine gender
Polypeptide grafted nano particle (DNPs, i.e. nano particle presoma in the present invention), random ordering are not carried out under control, equal conditions
Polypeptide group (SCR-DNPs) is used as positive control, wherein the sequence for the polypeptide that out-of-order polypeptide group SCR-DNPs is modified are as follows:
PNNKCESDKLAKHKKLGDKC is (such as
Shown in SEQUENCE NO.4), the polypeptide is to placenta sample chondroitin sulfate A (CSA) without targeting.
Specific steps are as follows:
By human placenia cancer cell (JEG3), mouse mastopathy cell (4T1), mouse prostate cancer cell (RM1), people
Breast cancer cell (HCC1937) Proliferation of Human Ovarian Cell (SKOV3) is with 104The density of/well is uniformly inoculated in 12 hole cells respectively
It in culture plate, is cultivated with the DMEM/F12 complete medium containing 10% fetal calf serum and 1% mycillin, when reaching 60%
When fusion, various cells are divided into 4 different processing groups: free adriamycin group (Free DOX), common nanoparticle group
(DNPs), out-of-order polypeptide group (SCR-DNPs), placenta sample chondroitin sulfate A (CSA) target polypeptide group (CSA-DNPs), wherein various thin
The above different disposal drug (5 μ g adriamycin equivalent) of isometric (1mL) is added in 4 DEG C of incubation 1h in born of the same parents, discards original in hole
Culture medium changes DMEM/F12 complete medium into, is put into 37 DEG C of incubators and continues to cultivate 30min, PBS buffer solution is cleaned later
Treated cell 3 times, then fixes 20min with 4% paraformaldehyde, PBS is cleaned 3 times and the DAPI of 50 μ g/mL of addition is molten
Liquid 1mL, and in being placed at room temperature for 20min, last PBS is cleaned cell 4 times, using the glimmering of inverted fluorescence microscope observation group of cells
Light, experimental result are as shown in Figure 3-4.
The result of the cellular uptake experiment of 4 processing groups of human placenia cancer cell (JEG3) is as shown in Figure 3.Wherein,
Free DOX group, DNPs group, SCR-DNPs group are hardly visible the red fluorescence of adriamycin, and the cell handled through CSA-DNPs
Compared with other processing groups, it is able to observe that very strong red fluorescence (the red fluorescence corresponding diagram 3 of adriamycin that adriamycin issues
The shallower part of color in middle secondary series).And it is higher with the registration of the nucleus of DAPI dyeing, due to height on JEG3 cell
It expresses placenta sample chondroitin sulfate A (CSA) (pl-CSA), and the pl-CSA targeted nano particle table of coating adriamycin provided by the invention
Face is modified with the specific receptor (polypeptide LKPSHEKKNDDNGKKLCKAC) of CSA, and the targeted nano particle can be fast
Fast ground (30min) is targeted to JEG3 cancer cell, is absorbed by cancer cell.
Fig. 4 be other cancer cells (4T1, RM1, HCC1937, SKOV3) through CSA-DNPs treated fluorescence imaging as a result,
Wherein first row, secondary series correspond respectively to for DAPI, adriamycin fluorescence channel in the fluorescence photo observed, third is classified as
The overlay chart of first row and secondary series result.From fig. 4, it can be seen that other above cancer cells are handled through CSA-DNPs, equally
It is observed that strong red fluorescence (the shallower part of color in secondary series in red fluorescence corresponding diagram 4), this is further demonstrated
The targeting of the targeted nano particle.
In addition, for pl-CSA targeted nano particle made from 4-6 of the embodiment of the present invention to the cancer cell of expression pl-CSA
Similarly there is targeting, no longer carry out providing their fluorescence targeting photo here.
Application Example 2 is coated with the treatment mouse villioma of the placenta sample chondroitin sulfate A (CSA) targeted nano particle of adriamycin
Effect test
The placenta sample chondroitin sulfate A (CSA) targeted nano particle that adriamycin is coated with made from the embodiment of the present invention 1 (is abbreviated as
CSA-DNPs the effect test of mouse villioma) is carried out, and more with PBS group, free adriamycin group, common nanoparticle group, random ordering
Peptide group compares, wherein the sequence for the polypeptide that out-of-order polypeptide group SCR-DNPs is modified are as follows: PNNKCESDKLAKHKKLGDKC
(as shown in SEQUENCE NO.4).Concrete operations are as follows:
Using 4-6 weeks, female BAl BIc/c nude mice that weight is 15-20g as experimental animal, to mouse bare subcutaneous injection 1 ×
106The villus cancer cell (Fluc-JEG3) of a luciferase label every other day, passed through tail vein injection since the 2nd day
The different pharmaceutical of (0.1mL) and grouping in equal volume: PBS group, free adriamycin group (Free DOX), common nanoparticle group
(DNPs, unmodified polypeptide), out-of-order polypeptide group (SCR-DNPs), the placenta sample chondroitin sulfate A (CSA) targeted nano for containing adriamycin
Particle (CSA-DNPs).In addition to PBS group, the equivalent of adriamycin is 5 μ g when per injection in other groups, while recording tumour body
Product, gross tumor volume calculation formula are as follows: length of tumor × (tumor width)2/2.Experimental period is 18 days, test result such as Fig. 5 institute
Show.
From fig. 5, it can be seen that PBS group is it can be seen that obvious tumour, and surrounding tissue enlargement, illustrate that tumour has been spread
Growth, there is black scab in Free DOX group, DNPs group, SCR-DNPs group tumor locus, but there is no spread.And CSA-DNPs group
Compared with other groups, visible tumour is had no.Test result explanation, placenta sample chondroitin sulfate A (CSA) targeting provided by the invention are passed
The growth for sending system that can significantly inhibit cancer cell has good effect to treating cancer.
These results suggest that placenta sample chondroitin sulfate A (CSA) targeted nano particle provided by the invention is being applied to and CA phase
In the cancer of pass, targeting is preferable, therapeutic effect is also more significant.
The diagnostic test of 3 placenta sample chondroitin sulfate A (CSA) targeted nano Granules on Mouse cancer of Application Example
To the placenta sample chondroitin sulfate A (CSA) targeted nano particle for being coated with ICG and adriamycin made from the embodiment of the present invention 2 altogether
The diagnostic test of (being abbreviated as CSA-IDNPs) progress Murine cancer, comprising the following steps:
Using 4-6 weeks, female BAl BIc/c nude mice that weight is 15-20g as experimental animal, to mouse bare subcutaneous injection 1 ×
106The villus cancer cell (Fluc-JEG3) of a luciferase label, was coated with ICG and Ah mould by tail vein injection after the 5th day altogether
The target of the common nanoparticle (IDNPs, unmodified polypeptide) of element and altogether the placenta sample chondroitin sulfate A (CSA) of coating ICG and adriamycin
To nano particle (CSA-IDNPs), the equivalent of ICG is 10 μ g when injection, respectively in petty action behind 10min, 1 hour, 31 hours
It takes pictures under object living imaging instrument observation, and records the variation of fluorescence signal, test result is as shown in Figure 6.
Fig. 6 left column be in two groups of IDNPs, CSA-IDNPs luciferase mark villus cancer cell (Fluc-JEG3) (i.e.
Tumour) where position；Right column the first row, the second row of right column are ICG in mouse after having injected IDNPs, CSA-IDNPs respectively
Fluorescence distribution can detecte the red fluorescence of ICG by small animal imaging instrument.
From fig. 6, it can be seen that may determine that the tumour cell of DNPs group and CSA-IDNPs group from luminous Fluc-JEG3
It is all predominantly located at right side of mice the part between the ribs and the hips nest position, after injecting IDNPs10min, ICG fluorescence signal can be at the entire back of mouse
And neck can detect, but the fluorescence signal of ICG can not be overlapped well with the tumour cell at the part between the ribs and the hips nest position, this explanation
IDNPs does not reach tumor locus；And after injecting CSA-DINPs, right side of mice the part between the ribs and the hips nest tumor locus is able to detect that very strong
ICG fluorescence signal, illustrate that CSA-DINPs has arrived at tumor locus, and the fluorescence signal still can be seen after 31h
It observes, this explanation, being coated with the placenta sample chondroitin sulfate A (CSA) targeted nano particle of ICG, can quickly and to be enduringly targeted to cancer thin
Born of the same parents, therefore can be used as a kind of effective means of diagnosis cancer happening part and development.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
SEQUENCE LISTING
<110>Shenzhen Institutes of Advanced Technology, Chinese Academy of Science
<120>a kind of polypeptide for targeting placenta sample chondroitin sulfate A (CSA), targeted nano particle and its preparation method and application
<130> 2017
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> PRT
<213>artificial sequence
<400> 1
Leu Lys Pro Ser His Glu Lys Lys Asn Asp Asp Asn Gly Lys Lys Leu
1 5 10 15
Cys Lys Ala Cys
20
<210> 2
<211> 28
<212> PRT
<213>artificial sequence
<400> 2
Glu Asp Val Lys Asp Ile Asn Phe Asp Thr Lys Glu Lys Phe Leu Ala
1 5 10 15
Gly Cys Leu Ile Val Ser Phe His Glu Gly Lys Cys
20 25
<210> 3
<211> 30
<212> PRT
<213>artificial sequence
<400> 3
Gly Lys Lys Thr Gln Glu Leu Lys Asn Ile Arg Thr Asn Ser Glu Leu
1 5 10 15
Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly Lys Cys
20 25 30
<210> 4
<211> 20
<212> PRT
<213>artificial sequence
<400> 4
Pro Asn Asn Lys Cys Glu Ser Asp Lys Leu Ala Lys His Lys Lys Leu
1 5 10 15
Gly Asp Lys Cys
20
Claims (10)
1. a kind of polypeptide, which is characterized in that the polypeptide is for targeting placenta sample chondroitin sulfate A (CSA), the amino acid of the polypeptide
Sequence is selected from one of amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:3 or a variety of.
2. a kind of placenta sample chondroitin sulfate A (CSA) targeted nano particle, which is characterized in that the targeted nano particle includes hydrophobicity
The hydrophily shell of kernel, the single layer lipid molecule layer of the package hydrophobic cores and targeting placenta sample chondroitin sulfate A (CSA), institute
Stating hydrophobic cores includes the hydrophobic polymer, and the ingredient of the hydrophily shell is polypeptide grafted amphiphilic macromolecular
Compound, the hydrophobic side of the amphiphilic macromolecular compound are interspersed in the single layer lipid molecule layer, described amphipathic big
The water-wet side of molecular compound and the polypeptide are keyed by amide, and the polypeptide is exposed to the single layer lipid molecule layer
Outside, wherein the amino acid sequence of the polypeptide in the amino acid sequence shown in the SEQ ID NO:1-SEQ ID NO:3 one
Kind is a variety of.
3. targeted nano particle as claimed in claim 2, which is characterized in that the diameter of the targeted nano particle be 80~
150nm。
4. targeted nano particle as claimed in claim 2, which is characterized in that the hydrophobic cores further include that target is delivered
Object, the target are delivered object and are adsorbed on the hydrophobic polymer, and it is anticancer drug, contrast agent and glimmering that the target, which delivers object,
At least one of light tracer.
5. targeted nano particle as claimed in claim 4, which is characterized in that the hydrophobic polymer and the target are delivered
The mass ratio of object is 1:(0.25~0.75).
6. targeted nano particle as claimed in claim 2, which is characterized in that the hydrophobic polymer, single layer lipid molecule
Mass ratio with the amphiphilic macromolecular compound is 1:(0.04-0.2): (0.1-0.4).
7. targeted nano particle as claimed in claim 2, which is characterized in that the amphiphilic macromolecular compound and described more
The mass ratio of peptide is 1:1~4.
8. targeted nano particle as claimed in claim 2, which is characterized in that the amphiphilic macromolecular compound is poly- second two
Alcohol derivatization phospholipid, the polyglycol derivatization phospholipid pass through covalent bond and phospholipid substance by polyethylene glycol and its derivative
It is connected and obtains.
9. a kind of preparation method of placenta sample chondroitin sulfate A (CSA) targeted nano particle, which comprises the following steps:
(1) hydrophobic polymer is dissolved in organic solvent, obtains hydrophobic polymer solution；
(2) single layer lipid molecule and amphiphilic macromolecular compound are dissolved in the first solvent, obtain the first mixed solution；
(3) the hydrophobic polymer solution is added dropwise to first mixed solution with the speed of 0.2-0.5mL/min
In, it is ultrasonically treated 4-6min, obtains the second mixed solution, centrifugal treating is carried out to second mixed solution, collects supernatant
Obtain targeted nano particle precursor body；
(4) take the targeted nano particle precursor body that the second solvent is added, addition catalyst, dehydrating agent are activated, added
Polypeptide carries out amidation process 15-20h at room temperature, obtains reaction solution, by the reaction solution after isolating and purifying, obtain tire
Disk sample chondroitin sulfate A (CSA) targeted nano particle, the targeted nano particle include hydrophobic cores, the package hydrophobic cores
Single layer lipid molecule layer and targeting placenta sample chondroitin sulfate A (CSA) hydrophily shell, the hydrophobic cores include it is described dredge
Aqueous polymer, the ingredient of the hydrophily shell are polypeptide grafted amphiphilic macromolecular compound, described amphipathic big point
The hydrophobic side of sub- compound is interspersed in the single layer lipid molecule layer, the water-wet side of the amphiphilic macromolecular compound and institute
It states polypeptide to be keyed by amide, the polypeptide is exposed to outside the single layer lipid molecule layer, wherein the amino acid of the polypeptide
Sequence is selected from one of amino acid sequence shown in SEQ ID NO:1-SEQ ID NO:3 or a variety of.
10. polypeptide as described in claim 1 or placenta sample chondroitin sulfate A (CSA) targeted nano particle as claimed in claim 2
Application in the drug of preparation prevention, diagnosis or treatment disease relevant to the expression of placenta sample chondroitin sulfate A (CSA).
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| PCT/CN2017/108646 WO2019061648A1 (en) | 2017-09-28 | 2017-10-31 | Polypeptide targeting placental chondroitin sulfate a, targeted delivery system, and preparation method and application thereof |
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| CN117159736A (en) * | 2023-08-25 | 2023-12-05 | 香港大学深圳医院 | Lipid nanoparticle for targeted delivery of nucleic acid and preparation method and application thereof |
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| CN103040757A (en) * | 2012-12-26 | 2013-04-17 | 深圳先进技术研究院 | Core-shell drug nano-particles, as well as preparation method and application thereof |
| CN103099784A (en) * | 2012-10-29 | 2013-05-15 | 深圳先进技术研究院 | Nanometer medicine particle, preparation method and application thereof |
| CN103099782A (en) * | 2012-08-10 | 2013-05-15 | 深圳先进技术研究院 | Core-shell type nano medical granule, preparation method and application thereof |
| CN104136041A (en) * | 2012-02-09 | 2014-11-05 | Var2制药有限公司 | Targeting of chondroitin sulfate glycans |
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|---|---|---|---|---|
| CN104136041A (en) * | 2012-02-09 | 2014-11-05 | Var2制药有限公司 | Targeting of chondroitin sulfate glycans |
| CN103099782A (en) * | 2012-08-10 | 2013-05-15 | 深圳先进技术研究院 | Core-shell type nano medical granule, preparation method and application thereof |
| CN103099784A (en) * | 2012-10-29 | 2013-05-15 | 深圳先进技术研究院 | Nanometer medicine particle, preparation method and application thereof |
| CN103040757A (en) * | 2012-12-26 | 2013-04-17 | 深圳先进技术研究院 | Core-shell drug nano-particles, as well as preparation method and application thereof |
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| CN117159736A (en) * | 2023-08-25 | 2023-12-05 | 香港大学深圳医院 | Lipid nanoparticle for targeted delivery of nucleic acid and preparation method and application thereof |
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