CN110269944A - A kind of intelligent response type genophore transport system and its preparation method and application - Google Patents
A kind of intelligent response type genophore transport system and its preparation method and application Download PDFInfo
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- CN110269944A CN110269944A CN201910308135.7A CN201910308135A CN110269944A CN 110269944 A CN110269944 A CN 110269944A CN 201910308135 A CN201910308135 A CN 201910308135A CN 110269944 A CN110269944 A CN 110269944A
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- thioketal
- transport system
- genophore
- glucan
- response type
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- 239000003814 drug Substances 0.000 claims description 20
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 16
- 229940079593 drug Drugs 0.000 claims description 16
- 239000002246 antineoplastic agent Substances 0.000 claims description 15
- 229940041181 antineoplastic drug Drugs 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
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- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical class NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 6
- 229920002873 Polyethylenimine Polymers 0.000 claims description 5
- 108010039918 Polylysine Proteins 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- 229920000656 polylysine Polymers 0.000 claims description 5
- 229920001661 Chitosan Polymers 0.000 claims description 4
- 239000004475 Arginine Substances 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
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- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
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- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- KFNRNFXZFIRNEO-NSHDSACASA-N (2s)-5-(diaminomethylideneamino)-2-[(4-methylphenyl)sulfonylamino]pentanoic acid Chemical compound CC1=CC=C(S(=O)(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C=C1 KFNRNFXZFIRNEO-NSHDSACASA-N 0.000 description 2
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102100028999 High mobility group protein HMGI-C Human genes 0.000 description 2
- 101000986379 Homo sapiens High mobility group protein HMGI-C Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
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- 230000003612 virological effect Effects 0.000 description 2
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- -1 Methyl benzenesulfonyl Chemical group 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 239000003054 catalyst Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
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- 239000007789 gas Substances 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
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- 239000005457 ice water Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 125000005323 thioketone group Chemical group 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
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Classifications
-
- 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
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- 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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
Abstract
The present invention provides a kind of intelligent response type genophore transport system and its preparation method and application, intelligent response type genophore transport system provided by the invention includes kernel and the shell that is coated on kernel；Wherein, the glucan by selecting the shell modified for thioketal；And in the glucan for keeping the thioketal modified thioketal and glucan molar ratio (1~1200): 1；The kernel is polycation；As a result, it has been found that genophore transport system provided by the invention has lower cytotoxicity and excellent H2O2Responsiveness, and there is efficient transfection efficiency in tumor locus, the genophore transport system mediated therapy gene of building has significant antitumous effect.Genophore transport system i.e. provided by the invention has broad application prospects in the antitumor field of gene therapy.
Description
Technical field
The present invention relates to bio-medical new material technology fields more particularly to a kind of intelligent response type genophore to transmit
System and its preparation method and application.
Background technique
Cancer threatens the life and health of the mankind as a kind of malignant disease.In the treatment means of cancer, gene is controlled
It treats as a kind of completely new therapeutic modality, obtains the extensive concern of people.The key of gene therapy is efficient genophore.
Genophore is broadly divided into virus gene carrier and non-viral genoid carrier at present.Virus gene carrier presence is higher
Immunogenicity, therefore clinically application is limited.In non-viral genoid carrier, polycation gene carrier is only due to it
Special performance obtains the very big concern of people.Generally, electropositive polycation and electronegative nucleic acid are mutual by electrostatic
Effect forms composite particles, carries out supporting and transmit [referring to Fang, H.P. for gene；Guo, Z.P.；Lin, L.；Chen,
J.；Sun, P.J.Wu, J.Y.；Caina Xu；Tian, H.Y.；Chen, X.S.Molecular Strings
Significantly Improved the Gene Transfection Efficiency of
Polycations.J.Am.Chem.Soc. 2018,140,11992-12000.Fang, H.P.；Lin, L.；Chen, J.；Wu,
J.Y.；Tian, H.Y.；Chen, X.S.Zinc ion coordination significantly improved the
transfection efficiency of low molecular weight
Polyethylenimine.Biomater.Sci., 2019, C9BM00039A].But for the body of polycation gene carrier
For interior application, due to, containing there are many electronegative albumen, it is mutual that electrostatic easily occurring with polycation gene carrier in blood
Effect, causes carrier/nucleic acid complexes particle to be reunited, and then influences the transmission performance of carrier.
For horizontal polycation gene carrier in vivo, it is intended that it is protected in transmission process in vivo
Lower positive charge density is held, is conducive to carrier/nucleic acid complexes in this way and stablizes in transmission process in vivo, and it is thin in tumour
It is intracellular to gulp down the stage, it is intended that its positive charge density with higher, it in this way can be preferably by tumour cell endocytosis.According to
The difference of tumor microenvironment and normal physiological environment, the polycation gene carrier that people construct different intelligent response pass
System is passed [referring to Guan, X.W.；Guo, Z. P., Lin, L.；Chen, J.；Tian, H.Y.；Chen,
X.S.Ultrasensitive pH Triggered Charge/Size Dual-Rebound Gene Delivery
System.Nano Lett.2016,16,6823-6831.Tian, H.Y；Tang, Z.H.；Zhuang, X.L.；Chen, X.S.；
Jing, X.B. Biodegradable synthetic polymers:Preparation, functionalization and
Biomedical application.Prog.Polym.Sci.2012,37,237-280].Currently, building drug carrying ability is preferable
Polycation gene carrier transport system be still everybody study hot spot.
Summary of the invention
In view of this, technical problem to be solved by the present invention lies in provide a kind of intelligent response type genophore transmitting
System and its preparation method and application, a kind of intelligent response type genophore shading system provided by the invention, which has, tells somebody what one's real intentions are
Cytotoxicity, and it is high with high transfection efficiency in tumour.
The present invention provides a kind of intelligent response type genophore transport systems, comprising: kernel and is coated on kernel
Shell；
Wherein, the shell is the modified glucan of thioketal；
Thioketal and the molar ratio of glucan are (1~1200) in the modified glucan of the thioketal: 1；
The kernel is polycation.
Preferably, the number-average molecular weight of the glucan in the modified glucan of the thioketal is 2000~200000.
Preferably, thioketal and the molar ratio of glucan are (30~1000) in the modified glucan of the thioketal: 1,
Preferably (60~500): 1.
Preferably, the modified glucan of the thioketal has structure shown in formula (I),
Wherein, m is 1~1200；
N is 12~1200；
A is 0~15；
B is 0~15.
Preferably, the modified glucan of the thioketal is prepared in accordance with the following methods:
Mercaptoalkyl acid is reacted under chlorination hydrogen atmosphere with acetone, obtains thioketal；
Then thioketal is reacted with glucan, obtains the modified glucan of thioketal.
Preferably, the polycation be the modified poly- α-lysine of p-toluenesulfonyl arginine, polyethyleneimine,
One or more of polylysine, chitosan and polyamide-amide.
The present invention also provides a kind of anti-tumor drugs, including carrier and the drug being supported on carrier；
Wherein, the carrier is intelligent response type genophore transport system of the present invention, and the drug is DNA
And/or RNA.
Preferably, the mass ratio of the polycation in the intelligent response type genophore transport system and drug is (2
~10): 1.
Preferably, the tumour cell in the anti-tumor drug be 4T1, MCF-7, HeLa, B16F10, CT26, SKBR3,
A549, HepG-2, PC3, A2780, SK-OV-3 or Huh-7.
The present invention also provides a kind of preparation methods of anti-tumor drug of the present invention, comprising:
1) by intelligent response type genophore transport system polycation and drug it is compound, obtain polycation medicine
Object compound；
2) the modified glucan of thioketal is mixed with polycation medicinal composition, obtains anti-tumor drug.
Compared with prior art, the present invention provides a kind of intelligent response type genophore transport system and its preparation sides
Method and application, intelligent response type genophore transport system provided by the invention include kernel and are coated on outer on kernel
Shell；Wherein, the glucan by selecting the shell modified for thioketal；And it contracts in the glucan for keeping the thioketal modified
The molar ratio of thioketones and glucan is (1~1200): 1；The kernel is polycation；By the experimental results showed that, the present invention
The genophore transport system of offer has lower cytotoxicity and excellent H2O2Responsiveness, and have in tumor locus
The genophore transport system mediated therapy gene of efficient transfection efficiency, building has significant antitumous effect.That is this hair
The genophore transport system of bright offer has broad application prospects in the antitumor field of gene therapy.
Specific embodiment
The present invention provides a kind of intelligent response type genophore transport systems, comprising: kernel and is coated on kernel
Shell；
Wherein, the shell is the modified glucan of thioketal；
Thioketal and the molar ratio of glucan are (1~1200) in the modified glucan of the thioketal: 1；
The kernel is polycation.
According to the present invention, the number-average molecular weight of glucan (DSDPA-Dex) is preferred in the modified glucan of the thioketal
Be 2000~200000, more preferably 10000~180000, more preferably 20000~150000, more preferably 50000~
100000；The modified glucan of the thioketal has structure shown in formula (I),
Wherein, m is 1~1200, preferably 10~1000, more preferably 50~800, most preferably 100~500；
N is 12~1200, preferably 20~1000, more preferably 50~800, most preferably 100~600；
The a is 0~15, preferably 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15；The b be 0~
15, preferably 0,1,2,3,4,5,6,7,8,9,10,11,12,13,14 or 15.Contracting sulphur in the modified glucan of the thioketal
The molar ratio of ketone and glucan is (30~1000): 1, more preferably (60~500): 1, more preferably (90~300): 1, more
Preferably (120~200): 1.
In the present invention, the source of the present invention glucan modified to the thioketal does not have a particular/special requirement, preferably can be by
It is prepared according to following methods:
Mercaptoalkyl acid is reacted under chlorination hydrogen atmosphere with acetone first, obtains thioketal；Wherein, the sulfydryl alkane
The molar ratio of base acid and acetone is preferably 1: (2~4), more preferably 1: 3；The temperature of the reaction is preferably 20~40 DEG C, more
Preferably 30~35 DEG C；The reaction time is preferably 6~for 24 hours, more preferably 10~20 hours；After completion of the reaction, preferably will
Reaction solution crystallisation by cooling under the conditions of salt ice obtains thioketal (DSDPA).
Then thioketal is reacted with glucan (Dex), obtains the modified glucan of thioketal；Wherein, thioketal and Portugal
The molar ratio of glycan is (30~1000): 1, more preferably (60~500): 1, more preferably (90~300): 1, more preferably
(120~200): 1；The catalyst of the reaction is preferably C8H17N3HCl and 4-dimethylaminopyridine (DMAP)；It is described
C8H17N3The molar ratio of HCl, DMAP and DSDPA are preferably (1~5): (1~5): 1, more preferably (2~4): (2~4):
1, more preferably (2.5~3): (2.5~3): 1；The solvent of the reaction is preferably DMSO；The Dex is in the dense of DMSO solution
Degree is preferably 0.01~0.5mg/mL, more preferably 0.1~0.4mg/mL, more preferably 0.2~0.3mg/mL；The reaction
Temperature be preferably 20~40 DEG C, more preferably 30~35 DEG C, the reaction time is preferably 60~80h, more preferably 70~75h；
In the present invention, thioketal and glucan (Dex) after completion of the reaction, also dialyse obtained reaction solution, obtain what thioketal was modified
Then glucan is lyophilized；Wherein, the molecular weight of the dialysis bag filter is 1000~7000；In the step thoroughly
The time of analysis is 60~80h；The freeze temperature is set as -50~-80 DEG C.
More specifically, the reaction process of the modified glucan of the thioketal with formula (I) structure is as follows:
The compound of the compound of formula (II) structure, formula (III) is reacted under hydrogen chloride gas atmosphere with acetone, is obtained
To the compound of formula (IV) structure；
The compound of obtained formula (IV) structure is reacted with glucan, obtains the modified Portugal of the thioketal of formula (I) structure
Glycan.
According to the present invention, the polycation is preferably the modified poly- α-lysine (PLL- of tosyl arginine
RT), polyethyleneimine (PEI), polylysine (PLL), chitosan (CS) or polyamide-amide (PAMAM), more preferably toluene
The modified poly- α-lysine of sulfonyl arginine, wherein in the modified poly- α-lysine of the tosyl arginine
Poly- α-lysine and the arginic molar ratio of tosyl are preferably 1: (10~200), more preferably 1: (30~150),
Most preferably be 1: (60~120) are most preferably 1: (70~90).
According to the present invention, in intelligent response type genophore transport system of the present invention, kernel and it is coated on kernel
On shell between realize cladding by electrostatic interaction, and the Portugal that the thioketal of the polycation of kernel and shell is modified
The mass ratio of glycan is preferably 1: (0.8~1.2), more preferably 1: 1.
The present invention also provides a kind of anti-tumor drugs, including carrier and the drug being supported on carrier；
Wherein, the carrier is intelligent response type genophore transport system of the present invention, and the drug is DNA
And/or RNA, the polycation of the intelligent response type genophore transport system and the mass ratio of drug are (2~10): 1,
More preferably (2.5~8): 1, most preferably (3~7): 1, most preferably (4~6): 1, most preferably (5~5.5): 1；This hair
The bright tumour cell in the anti-tumor drug does not have a particular/special requirement, all tumour cells, preferably 4T1, MCF-7,
HeLa, B16F10, CT26, SKBR3, A549, HepG-2, PC3, A2780, SK-OV-3 or Huh-7.
The present invention also provides a kind of preparation methods of anti-tumor drug of the present invention, comprising:
1) by intelligent response type genophore transport system polycation and drug it is compound, obtain polycation medicine
Object compound；
2) the modified glucan of thioketal is mixed with polycation medicinal composition, obtains anti-tumor drug.
According to the present invention, the present invention is first by the polycation and drug in intelligent response type genophore transport system
It is compound, obtain polycation medicinal composition；Then the modified glucan of thioketal is mixed with polycation medicinal composition
It closes, obtains anti-tumor drug；Wherein, the present invention does not have particular/special requirement, those skilled in the art to compound or mixed method
Suitable compound or hybrid mode can be selected according to general knowledge known in this field；Wherein, the intelligent response type genophore passes
The mass ratio of the polycation and drug of passing system is (2~10): 1, more preferably (2.5~8): 1, most preferably (3~7):
1, most preferably (4~6): 1, most preferably (5~5.5): 1.
The present invention provides a kind of intelligent response type genophore transport system and its preparation method and application, the present invention
The intelligent response type genophore transport system of offer includes kernel and the shell that is coated on kernel；Wherein, pass through selection
The shell is the modified glucan of thioketal；And thioketal and glucan rub in the glucan for keeping the thioketal modified
You are than being (1~1200): 1；The kernel is polycation；So that genophore transport system provided by the invention have compared with
Low cytotoxicity and excellent H2O2Responsiveness, and there is efficient transfection efficiency in tumor locus, the gene of building carries
Body transport system mediated therapy gene has significant antitumous effect.Genophore transport system i.e. provided by the invention exists
The antitumor field of gene therapy has broad application prospects.
It is clearly and completely described below in conjunction with the technical solution of the embodiment of the present invention, it is clear that described reality
Applying example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field
Those of ordinary skill's every other embodiment obtained without making creative work, belongs to guarantor of the present invention
The range of shield.
Embodiment 1
1, the synthesis of DSDPA-Dex
By taking the Dex of molecular weight 20000 as an example, by the hydrogen chloride of anhydrous mercaptopropionic acid and anhydrous propanone mixture drying
It is full of, is reacted at room temperature, it is cooling under the conditions of salt ice until crystallization is complete after completion of the reaction.By what is obtained
Crystal filtering, is washed with n-hexane and ice water, product obtained is lyophilized, DSDPA is obtained.
Then C is being added8H17N3Under the conditions of HCl and DMAP, according to the molar ratio of DSDPA and Dex in table 1 respectively into
Row mixing, the DSDPA and Dex for obtaining different proportion obtain mixture, said mixture are dissolved in DMSO solution respectively, into
Row reaction.Then by the mixture deionized water dialysis after reaction.Finally the product after dialysis is lyophilized, obtains thioketal
Modified glucan (DSDPA-Dex).
Table 1 is the corresponding feed ratio of shell DSDPA-Dex of genophore transport system
Wherein, we are on the Dex of molecular weight 20000 by being grafted different number of DSDPA, the throwing provided according to table 1
Expect that ratio, DSDPA-Dex-1, DSDPA-Dex-2, DSDPA-Dex-3 and DSDPA-Dex-4 indicate the number difference of grafting DSDPA
It is 30,60,90,120.
2, using DSDPA-Dex as the shielding layer of genophore system, polycation gene carrier as kernel, with
(it is formed is shown in Table 2 to PLL-RT, and table 2 is different number of to Methyl benzenesulfonyl base essence ammonia to be grafted on the PLL of molecular weight 20000
The result of acid) for, as the genophore transport system of DNA, apply to kinds of tumor cells system (for example, 4T1, MCF-7,
HeLa, B16F10, CT26, SKBR3, A549, HepG-2, Huh-7 etc.).
Table 2 is the corresponding feed ratio of kernel PLL-RT for synthesizing genophore transport system
Wherein, according to the different feed ratios of such as table 2, PLL20k-RT1, PLL20k-RT-2, PLL20k-RT-3 and
PLL20k-RT-4 indicates that the number of grafting RT is respectively 30,60,90,120,150.
3, DSDPA-Dex/PLL-RT is as green fluorescent protein plasmid (pEGFPN1) and luciferase plasmids (pGL3)
The usage of carrier
Step and condition are as follows:
(1) cell culture
Cell is cultivated in the culture solution of volume fraction containing fetal calf serum 10%, the temperature of cell incubator is set as
37 DEG C, the volume fraction of carbon dioxide be set as 5%, carry out constant temperature incubation.
(2) cell transfecting
The cell of logarithmic growth phase is carried out after pancreatin digestion with the culture solution of volume fraction containing fetal calf serum 10% dilute
It releases, according to every hole 1 × 104Cell bed board in 96 porocyte culture plates, being placed in cultivation temperature is 37 DEG C, containing CO2Volume fraction
It is cultivated in 5% constant incubator, until cell confluency degree is up to 80~90%.When carrying out transfection experiment, by PLL-RT/
After the compound 20min of pDNA, DSDPA-Dex is then added thereto again compound 20min.96 are added to according to 0.2 hole μ gpDNA/
In porocyte plates, continue to cultivate 48h.For needing H2O2The experimental group of effect, before DSDPA-Dex is added, by H2O2Add
Enter into DSDPA-Dex solution, so that H after being added2O2Substance withdrawl syndrome is 100uM in the solution, carries out reaction 12h, so
Treated DSDPA-Dex is added in PLL-RT/pDNA afterwards and is transfected.
(3) measurement of cell transfecting efficiency
A) luciferase assays
After cultivating 48h in cell incubator, tissue culture plate is taken out, cell culture fluid is sucked out, washs 2 with PBS
Secondary, 50uL cell pyrolysis liquid is added in every hole, is put into 30min in -80 DEG C of refrigerators.Then the glimmering of equivalent is added in basis in every hole
Light element zymolyte detects transfection efficiency by photometric quantification, the results are shown in Table 3.
Table 3 is that the genophore transport system of building mediates the transfection efficiency in vitro of luciferase plasmids
As can be seen from the table, we are by determining luciferase of the genophore system of building in 4T1 cell
The transfection efficiency of plasmid is found in PLL20k-RT1, PLL20k-RT2, PLL20k-RT3, PLL20k-RT4 and PLL20k-
In RT5, PLL20k-RT2/pDNA obtains optimal transfection efficiency in mass ratio 2.5/1.Then we select PLL20k-
RT2 is as kernel, and DSDPA-Dex-1, DSDPA-Dex-2, DSDPA-Dex-3, DSDPA-Dex-4 are respectively as shell, measurement
Transfection efficiency in 4T1 cell.Highest transfection efficiency is shown when we have found that DSDPA-Dex-2 is as shell, and
In H2O2Under effect, outer shell is detached from, and transfection efficiency significantly improves.
B) the expression of green fluorescent protein (GFP)
After cultivating 48h in constant temperature cell incubator, tissue culture plate is taken out, places it in fluorescence microscopy under the microscope
The green fluorescent protein signal of cell expression.The cell for generating green florescent signal is the positive cell transfected, without producing
The cell of raw green florescent signal is the negative cells not transfected.Flow cytometer (Flow cytometry) can be used
Percentage shared by the positive cell transfected is detected, the results are shown in Table 4.
Table 4 is that the genophore transport system of building mediates the transfection efficiency in vitro of green fluorescent protein plasmid
From table 4, it can be seen that we are by measuring green fluorescence egg of the genophore system of building in 4T1 cell
The transfection efficiency in vitro of white matter grain, discovery in PLL20k-RT1, PLL20k-RT2, PLL20k-RT3, PLL20k-RT4 and
In PLL20k-RT5, PLL20k-RT2/pDNA obtains optimal transfection efficiency in mass ratio 2.5/1.Then we select
PLL20k-RT2 is as kernel, and DSDPA-Dex-1, DSDPA-Dex-2, DSDPA-Dex-3, DSDPA-Dex-4 is respectively as outer
Shell, measurement turn seven efficiency in 4T1 cell.Highest transfection is shown when we have found that DSDPA-Dex-2 is as shell
Efficiency, and in H2O2Under effect, outer shell is detached from, and transfection efficiency significantly improves.
(4) measurement (MTT) of cytotoxicity
The evaluation of its biocompatibility of genophore transport system is carried out using MTT method.Logarithmic growth phase
Cell is diluted, according to every hole 1 × 10 with the culture solution of volume fraction containing fetal calf serum 10% after pancreatin digestion4Cell
Bed board is carried out in 96 porocyte culture plates.Placing it in cultivation temperature is 37 DEG C, contains CO2The constant temperature incubation of volume fraction 5%
Culture is carried out in case until cell confluency degree is up to 80~90%.The carrier of different quality ratio/pDNA compound is added to 96 holes
Culture 48h is carried out in the cell of tissue culture plate, and the thiazolyl blue solution that 20 μ L mass fractions are 5% is then added in every hole,
4h is cultivated under 37 DEG C of constant temperature, culture solution is sucked out, and then 200 μ L DMSO are added in every hole, detect absorption value, choosing with microplate reader
It is 490nm with wavelength.Cell survival rate is calculated according to following formula:
Cell survival rate (%)=(Asample/Acontrol)×100
AsampleIt is the absorption of material group sample well, AcontrolIt is the absorption that the control wells of material are not added, every group of experiment weight
Again three times, survival rate (being shown in Table 5) is calculated；
Table 5 is that the genophore transport system of building mediates the cell survival rate of pDNA
As can be seen from the table, cytotoxicity of the pDNA in 4T1 cell is supported by the genophore of measurement building.
We have found that PLL20k-RT2/DNA has negligible cytotoxicity in best transfection efficiency.In addition DSDPA-Dex-
2/PLL20k-RT-2/DNA is in best transfection efficiency, whether there is or not H2O2Under the conditions of show negligible cytotoxicity.
4. using DSDPA-Dex as the shielding layer of genophore system, polycation gene carrier as kernel, with
For PLL-RT, as the genophore transport system of RNA, apply at present various cell lines (for example, 4T1, MCF-7,
HeLa, B16F10, CT26, SKBR3, A549, HepG-2, Huh-7 etc.), step and correlated condition are as follows:
(1) we synthesize siRNA by conventional method, and the sequence of the siRNA is 5 '-CUUACGCU
GAGUACUUCGAdTdT-3 ', being capable of silencing luciferase.
(2) culture of cell
Cell is placed in the culture solution of volume fraction containing fetal calf serum 10%, in 37 DEG C of lower body fractions of cultivation temperature
For 5% CO2Insulating box in continuously cultivate.
(3) cell transfecting
Before transfection for 24 hours, the cell of logarithmic growth phase, with the tire ox blood containing volume fraction 10% after pancreatin digestion
Clear culture solution is diluted, according to 1 × 104The density of cells/well is plated in 96 porocyte culture plates, is placed in cultivation temperature
For 37 DEG C, contain CO2Culture in the constant incubator of volume fraction 5% until cell confluency degree up to 80~90%.When transfection,
After the compound 20min of PLL-RT/siRNA compound, compound 20min then is added in DSDPA-Dex, according to O.2 μ g
The hole siRNA/ is added in 96 porocyte plates, continues to cultivate 48h.For needing H2O2DSDPA- is being added in the experimental group of effect
Before Dex, by H2O2It is added in DSDPA-Dex solution, so that H after being added2O2Substance withdrawl syndrome is in the solution
100uM carries out reaction 12h, and then treated DSDPA-Dex is added in PLL-RT/siRNA and transfects.
(4) measurement of cell transfecting efficiency
Tissue culture plate is taken out from constant incubator, cell culture fluid is sucked out, is washed 2 times with PBS, and 50 μ are added in every hole
L cell pyrolysis liquid, which is put into -80 DEG C of refrigerators, cracks 20min, tissue culture plate is then taken out placement at room temperature, wait crack
After liquid melts, the luciferase substrate of equivalent is added in every hole, is measured cell transfecting efficiency by photometric quantification and is shown in Table 6.
Table 6 is that the genophore transport system of building mediates the external silence efficiency of gene siRNA of silencing luciferase
As can be seen from Table 6, we are by measuring the genophore system constructed in the 4T1- of permanent expressing luciferase
The silence efficiency of mediation silencing luciferase gene siRNA in Luc cell, finds in PLL20k-RT1, PLL20k-RT2,
In PLL20k-RT3, PLL20k-RT4 and PLL20k-RT5, PLL20k-RT2/pDNA is obtained most preferably in mass ratio 2.5/1
Silence efficiency.Then we select PLL20k-RT2 as kernel, DSDPA-Dex-1, DSDPA-Dex-2, DSDPA-Dex-
3, DSDPA-Dex-4 mediate silencing fluorescein respectively as shell, measurement in the 4T1 Luc cell of permanent expressing luciferase
The silence efficiency of enzyme gene siRNA.Show highest silence efficiency when we have found that DSDPA-Dex-2 is as shell, and
H2O2Under effect, outer shell is detached from, and silence efficiency significantly improves.
(5) measurement (MTT) of cytotoxicity
The evaluation of its biocompatibility of genophore transport system is carried out using MTT method.Logarithmic growth phase
Cell is diluted, according to every hole 1 × 10 with the culture solution of volume fraction containing fetal calf serum 10% after pancreatin digestion4Cell
Bed board is carried out in 96 porocyte culture plates.Placing it in cultivation temperature is 37 DEG C, contains CO2The constant temperature incubation of volume fraction 5%
Culture is carried out in case until cell confluency degree is up to 80~90%.The carrier of different quality ratio/siRNA compound is added to 96
Culture 48h is carried out in the cell of porocyte culture plates, and the thiazolyl blue solution that 20 μ L mass fractions are 5% is then added in every hole,
4h is cultivated under 37 DEG C of constant temperature, and culture solution is sucked out, then 200 μ L DMSO are added in every hole, absorption value is detected with microplate reader,
Selection wavelength is 490nm.Cell survival rate is calculated according to following formula:
Cell survival rate (%)=(Asample/Acontrol)×100
AsampleIt is the absorption of material group sample well, AcontrolIt is the absorption that the control wells of material are not added, every group of experiment weight
Again three times, it calculates survival rate and is shown in Table 7.
Table 7 is that the genophore transport system of building mediates the cell survival rate of siRNA compound
As can be seen from the table, we support siRNA in 4T1-Luc cell by the genophore of measurement building
Cytotoxicity finds that PLL20k-RT2/DNA has negligible cytotoxicity in best transfection efficiency.In addition DSDPA-
Dex-2/PLL20k-RT-2/DNA is in best transfection efficiency, whether there is or not H2O2Under the conditions of show negligible cell
Toxicity.
5. present invention building genophore transport system mediates the application of pDNA in vivo, it is accomplished in that
(1) culture of 4T1 cell
4T1 cell is placed in the culture solution of volume fraction containing fetal calf serum 10%, is containing CO2Volume fraction 5%, training
It supports and is cultivated in the insulating box that temperature is 37 DEG C.
(2) tumor inoculation
The BALB/c mouse of the weight 20g of purchase week old 5-6 weeks or so, before tumor inoculation, logarithmic growth phase
4T1 cell is digested through pancreatin, and cell culture fluid is then added and is diluted, and 1 × 103Rpm is centrifuged 5min, and PBS is washed three times,
Then PBS suspension cell is used.According to every mouse 3 × 106Cell inoculation is in mouse oxter.Tumour to tumor-bearing mice is average
Volume reaches 200mm3When, carry out the intracorporal transfection experiment of pDNA.
(3) transfection in vivo
It will be carried using the pDNA of expressing luciferase as internal rotaring redyeing gene according to the 20 μ g of pDNA dosage of every mouse
In 200 μ L Tail Vein injection Mouse body of body/pDNA compound normal saline solution, 48h is transfected.
(4) measurement of transfection efficiency efficiency in vivo
After carrying out the internal transfection of 48h, BABL/c mouse is put to death, takes out each group tumour, PBS is washed 2 times, with group
It knits lysate to be cracked, be homogenized, luciferase substrate is then added, measure transfection efficiency, the results are shown in Table 8.
Table 8 is that the genophore transport system of building mediates the transfection of luciferase plasmids in vivo
As can be seen from the table, we carry out tail vein note by the way that the genophore of building is supported luciferase plasmids
It is mapped in lotus 4T1 tumor mouse, the expression of luciferase in tumor tissues is detected after 48h.It was found that in PLL20k-RT1, PLL20k-
In RT2, PLL20k-RT3, PLL20k-RT4 and PLL20k-RT5, PLL20k-RT2/pDNA is in mass ratio 2.5/1 in tumour
Optimal transfection efficiency is obtained in tissue.Then we select PLL20k-RT2 as kernel, DSDPA-Dex-1, DSDPA-
Dex-2, DSDPA-Dex-3, DSDPA-Dex-4 carry out tail vein injection into lotus 4T1 tumor mouse respectively as shell.I
Find to show highest transfection efficiency in tumor tissues when DSDPA-Dex-2 is as shell after 48h, this is because
DSDPA-Dex-2 can reach tumor tissues by blood circulation after tail vein injection as shielding layer, and due to tumour
Organize H2O2Content is high, can promote the disengaging of shielding layer, is conducive to tumour cell endocytosis, and transfection efficiency acquisition mentions significantly
It is high.
6. the application in siRNA transfection in vivo of present invention building genophore transport system, real in the following way
It is existing:
(1) culture of 4T1 cell
4T1 cell is placed in the culture solution containing fetal calf serum 10%, in the CO for being 5% containing volume fraction2, temperature be
It is cultivated in 37 DEG C of constant incubator.
(2) tumor inoculation
The BABL/c mouse of weight 20g or so of purchase week old 5-6 weeks, before tumor inoculation, the 4T1 of logarithmic growth phase
Cell, with trypsin digestion, then with cell culture fluid mixing trypsase, 1 × 103Rpm is centrifuged 5min, PBS washing 3
It is secondary, with PBS suspension cell.According to every mouse 3 × 106Cell inoculation is in mouse oxter.Tumour to tumor-bearing mice is averaged body
Product reaches 100mm3When, carry out the interior therapeutic of siRNA.
(3) interior therapeutic
Gene selects shVEGF plasmid, according to the shVEGF dosage 20ug of every mouse, carrier/shVEGF compound
In 200 μ L Tail Vein injection Mouse body of normal saline solution volume, a medicine is every other day given, is administered 14 days.
(4) measurement of mice tumors grew
In mouse, administration starts for the first time, measures the variation of mouse tumor size and weight daily.Table 9 gives pair
Tumor-bearing mice carries out the volume of tumour after treatment 14 days.
Table 9 is that the genophore transport system of building mediates the tumor size after shVEGF treatment
As can be seen from Table 9, we carry out tail vein note by the way that the genophore of building is supported therapeutic gene shVEGF
It is mapped in lotus 4T1 tumor mouse, gave a medicine every 1 day, be administered 6 times in total, observe tumour growth situation, discovery PLL20k makees
For kernel, DSDPA-Dex-2 can inhibit the growth of tumour significantly, show as shell mediated therapy gene shVEGF
Optimal antitumous effect.This is because DSDPA-Dex-2 as shielding layer, can be followed after tail vein injection by blood
Ring reaches tumor tissues, and due to tumor tissues H2O2Content is high, can promote the disengaging of shielding layer, be conducive to tumour cell
Endocytosis, have significant antitumous effect.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims (10)
1. a kind of intelligent response type genophore transport system, comprising: kernel and the shell being coated on kernel；
Wherein, the shell is the modified glucan of thioketal；
Thioketal and the molar ratio of glucan are (1~1200) in the modified glucan of the thioketal: 1；
The kernel is polycation.
2. intelligent response type genophore transport system according to claim 1, which is characterized in that the thioketal is modified
Glucan in glucan number-average molecular weight be 2000~200000.
3. intelligent response type genophore transport system according to claim 1, which is characterized in that the thioketal is modified
Glucan in thioketal and the molar ratio of glucan be (30~1000): 1, preferably (60~500): 1.
4. intelligent response type genophore transport system according to claim 1, which is characterized in that the thioketal is modified
Glucan have formula (I) shown in structure,
Wherein, m is 1~1200；
N is 12~1200；
A is 0~15；
B is 0~15.
5. intelligent response type genophore transport system according to claim 1, which is characterized in that the thioketal is modified
Glucan be prepared in accordance with the following methods:
Mercaptoalkyl acid is reacted under chlorination hydrogen atmosphere with acetone, obtains thioketal；
Then thioketal is reacted with glucan, obtains the modified glucan of thioketal.
6. intelligent response type genophore transport system according to claim 1, which is characterized in that the polycation is
In the modified poly- α-lysine of p-toluenesulfonyl arginine, polyethyleneimine, polylysine, chitosan and polyamide-amide
It is one or more of.
7. a kind of anti-tumor drug, including carrier and the drug being supported on carrier；
Wherein, the carrier is intelligent response type genophore transport system described in claim 1~7 any one, described
Drug is DNA and/or RNA.
8. anti-tumor drug according to claim 7, which is characterized in that the intelligent response type genophore transport system
In polycation and drug mass ratio be (2~10): 1.
9. anti-tumor drug according to claim 7, which is characterized in that the tumour cell in the anti-tumor drug is
4T1, MCF-7, HeLa, B16F10, CT26, SKBR3, A549, HepG-2, PC3, A2780, SK-OV-3 or Huh-7.
10. a kind of preparation method of anti-tumor drug as claimed in claim 7, comprising:
1) by intelligent response type genophore transport system polycation and drug it is compound, it is compound to obtain polycation drug
Object；
2) the modified glucan of thioketal is mixed with polycation medicinal composition, obtains anti-tumor drug.
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