CN107137355A - A kind of genes delivery system - Google Patents
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- CN107137355A CN107137355A CN201710382838.5A CN201710382838A CN107137355A CN 107137355 A CN107137355 A CN 107137355A CN 201710382838 A CN201710382838 A CN 201710382838A CN 107137355 A CN107137355 A CN 107137355A
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Classifications
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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- A—HUMAN NECESSITIES
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Abstract
The invention discloses the acyl RGDV (IRV) of gene nano liposome vectors Isoquinoline 3 and IRV/STAT3 siRNA genes delivery systems preparation method, IRV/STAT3 siRNA genes delivery systems are made up of in proportion liposome vectors IRV and STAT3 siRNA, nucleoprotamine, calf thymus DNA in invention, with sustained release and targeting；The present invention with lung cancer A549 cell strain for model evaluation the cell transfecting efficiency of compound, anti tumor activity in vitro, and gene silencing efficiency and cytosis mechanism in mRNA level in-site and protein level, as a result show that IRV/STAT3 siRNA genes delivery systems have more excellent antitumor activity and gene silencing efficiency than each control group.Present invention antitumor activity of IRV/STAT3 siRNA genes delivery systems using lotus S180 ascites tumors mouse as model evaluation, as a result shows 100%IRV/STAT3 siRNA groups with antitumous effect.
Description
Technical field
The present invention relates to the novel amphiphilic that the isoquinolin -3- carboxylic acids by lipophilic and hydrophilic RGDV sequence peptides are combined to form
The preparation of liposome vectors, and IRV/STAT3-siRNA genes delivery systems structure, and it has been carried out inside and outside resist
The evaluation of tumor promotion and gene silencing efficiency.This patent mainly strengthens antitumor activity by isoquinoline structure, with reference to
RGDV sequences peptide strengthens the targeting of liposome, makes it in the concentration increase of target site, so as to reduce side effect, improves treatment
Index.
Background technology
Gene therapy refers to the gene of performance therapeutic action importing target cell by certain way, to correct gene defect
Or silence related gene expression, so as to play antineoplastic purpose.STAT genes are signal transducer and transcription activator, are
The new Intracellular signals transmission and gene expression regulation by the polypeptide ligand activation such as cell factor, growth factor of one class because
Sub-family.Wherein STAT3 is the important composition of this family, the wide participation responsing reaction of cytokine profiles.Research hair
It is existing, STAT3 for the normal cells, tissue to most of adults it is not necessary to, but the STAT3 of activation is to tumour cell
The process such as formation, growth, apoptosis but play important regulating and controlling effect, be the medium played an important role in metastases.Together
When STAT3 abnormal activation also result in the activation of downstream gene, including VEGF and survivin genes.
RNA perturbation techniques are to cause purpose mRNA selective degradations by siRNA, so that silence after genetic transcription
A kind of means.The specific expressed of STAT3 genes is blocked to turn into important anticancer research using STAT3siRNA.But it is naked
SiRNA is easily degraded by RNase enzymes in vivo, and half-life short, transfection efficiency is low, and these shortcomings limit siRNA activity, because
And can stablize parcel siRNA reach and be transferred to cancer cell carrier it is very necessary.
Phosphatide is substituted using the amphipathic isoquinolin -3- acyl groups-RGDV of synthesis, blank liposomes are combined to form with cholesterol
Body.Liposome-polycation-DNA composite technologies are recycled, positively charged nucleoprotamine can adsorb siRNA and small
Bovine chest gland DNA, forms a stable electronegative compound, and can compress siRNA to greatest extent, this compound again with sun from
Sub- liposome is compounded to form liposome/siRNA compounds, forms a kind of novel siRNA delivery system, improves the transfection of gene
Efficiency.Wherein isoquinolin is to have been demonstrated the structure with the antitumor isoreactivity of antithrombotic, RGD be can with it is whole in tumour cell
The sequence peptide that plain receptor-specific is combined is closed, therefore the carrier also has targeting and certain antitumor activity.
RGDV is a kind of polypeptide for containing " arginine-glycine-aspartic acid " amino acid sequence, can recognize that tumour cell table
The integrin of face specifically expressing, and specifically bind to carry out the targeted therapy of mediate tumor with it.Repaiied by RGDV sequence peptides
The antineoplastic and its delivery system of decorations, can increase the active targeting of medicine, so as to reach the purpose of specific treatment.
The content of the invention
It is an object of the invention to provide a kind of gene liposome carrier with targeting:Isoquinoline-3-
acyl-RGDV。
The preparation method of gene liposome carrier of the present invention, comprises the following steps：Chemical synthesis is obtained into IQ-
COOH, is produced with RGDV condensations.
It is another object of the present invention to provide a kind of genes delivery system, by Antioncogene medicine STAT3-
SiRNA, and gene liposome carrier Isoquinoline-3-acyl-RGDV described in claim 1 are prepared from.
In genes delivery system of the present invention, Antioncogene medicine STAT3-siRNA (100nM) and gene
Liposome vectors Isoquinoline-3-acyl-RGDV (100%) amount ratio is 1:1.1(μL:μL).
Genes delivery system of the present invention, nanostructured.
It is another object of the present invention to provide the preparation method of genes delivery system.
Preparation method of the present invention, comprises the following steps：Gene liposome carrier Isoquinoline-3-acyl-
RGDV is mixed at room temperature with nucleoprotamine, is stood, standby, takes calf thymus DNA and Antioncogene medicine STAT3-
SiRNA is mixed at room temperature, stand for standby use, after above two solution is mixed, produces genes delivery system.
Wherein, the preparation of gene liposome carrier：
IRV synthesis：PS condensation reactions occur under HCl catalysis and obtain tetrahydroisoquinoline carboxylic for phenylalanine and formaldehyde
Acid, piptonychia ester obtains isoquinolin -3- carboxylic acids after esterification is reoxidized.IQ-COOH and H-Arg (Tos)-Gly-Asp
(OBzl)-Val-OBzl is reacted in THF by DCC/HOBt, then takes off blocking group formation Isoquinoline-3-acyl-
RGDV(IRV)。
It is preferred that, the preparation of gene liposome carrier of the present invention：
Under HCl catalysis, with formaldehyde PS reactions, condensation generation tetrahydrochysene occur under 80~90 DEG C of oil baths for 5g phenylalanines
Isoquinolinecarboxylic acid (4H IQ-COOH)；By 4H IQ-COOH under SOCl2 catalysis, occur esterification with methanol, obtain 4H
IQ-OMe；Solvent is made with THF, is IQ-OMe with potassium permanganate catalysis oxidation 4H IQ-OMe；Using methanol as solvent, in 2N NaOH
Catalysis under, IQ-OMe sloughs methyl esters, obtains IQ-COOH.
H-Arg (Tos)-Gly-Asp (OBzl)-Val-OBzl synthesis
Take 6g Boc-Arg (Tos)-Gly-Asp (OBzl)-Val-OBzl to be placed in eggplant bottle, be slowly added under condition of ice bath
50mL hydrochloric ethyl acetates, bottleneck connects thin-layered chromatography monitoring reaction process to reaction after drying tube and is fully completed, under tepidarium
Drain liquid with water pump, and with dry ethyl acetate and absolute ether respectively wear away 3 times after drain more than 8h.
Isoquinoline-3-acyl-RGDV synthesis
Take compound Isoquinoline-3-carboxylic acid 646mg to be dissolved in a small amount of anhydrous THF, add
HoBt 605mg are stirred to dissolving, added under condition of ice bath added after DCC 923mg, activation 30min 3.49g H-Arg (Tos)-
Gly-Asp (OBzl)-Val-OBzl, adjusts PH to 8-9 with NMM, removes and reaction process is monitored after ice bath.Reaction is revolved after terminating to remove
The yellow liquid being filtrated to get, is cleaned 18 times, anhydrous Na 2SO4 is done totally with saturation alkali, salt, acid respectively after being dissolved with CH2Cl2
Rotation removes filtrate after the dry filtrate 4h being filtrated to get, and obtains faint yellow solid Isoquinoline-3-acyl--Arg (Tos)-Gly-
Asp(OBzl)-Val-OBzl.Column chromatography carries out Structural Identification after purification.Take 300mg Isoquinoline-3-acyl--Arg
(Tos)-Gly-Asp (OBzl)-Val-OBzl is placed in eggplant bottle, and TFA 4.8ml, TfOH are slowly added under condition of ice bath
Ice absolute ether terminating reaction is added after 1.2ml, 30min, centrifuges (3000rpm, 3min), is taken out after wearing away 5 times with absolute ether
Dry to obtain yellowish crude product, column chromatography freezes obtain clear crystal after purification.
The preparation of gene nano carrier IRV blank liposomes of the present invention, comprises the following steps：
100%IRV membrane materials, IRV and CH weight ratio are 15:1,1% (g/100ml) Tween-80 solution, Probe Ultrasonic Searching
20min (130W, 90%, super 2s, stop 2s).It is prominent to compare the superiority of 100%IRV membrane materials, at the same be prepared for 75%IRV,
The blank liposome membrane material of 50%IRV and 25%IRV contents is contrasted.
It is preferred that, the preparation of gene nano carrier IRV blank liposomes of the present invention comprises the following steps：
60mg IRV accurately are weighed, 10mL volumetric flasks is transferred to after being dissolved with a small amount of methanol, is settled to chloroform after 10mL
Ultrasonic dissolution.0.4g Tween-80 are weighed in 50ml beakers, water ultrasonic dissolution is handled with 40mL DE PC.Accurately weigh
38.7mg CH, are transferred to 10mL volumetric flasks after being dissolved with a small amount of chloroform, 10mL are settled to chloroform.75mg PE accurately are weighed,
10ml volumetric flasks are transferred to after being dissolved with a small amount of chloroform, 10mL is settled to chloroform,
Take 0.906mL IRV and 0.094mL CH to be added in 25mL eggplants bottle, mend chloroform to 2mL.Rotary evaporation in vacuo
Condition be 37 DEG C of water-baths, 120rpm, 250mbar, after 30min per 5min reduction 50mbar until 1mbar, obtains homogeneous thin
Film.Popped one's head in after the appropriate rehydration solution i.e. Tween-80 of various concentrations, jog aquation ultrasound 10min are added after vacuum drying
The blank liposomes liquid solution containing 100%IRV is obtained after ultrasound, cooling.
The preparation method of genes delivery system of the present invention, comprises the following steps：
10 μ L STAT3-siRNA are drawn first, add 6.5 μ L calf thymus DNA, 10min is stood at room temperature；Draw 1
μ L IRV adds 5 μ L nucleoprotamine, and 10min is stood at room temperature；After two kinds of compounds are mixed, 15min is stood at room temperature.
The DEPC water for adding 967.5 μ L obtains 100nM 100%IRV/STAT3-siRNA.10 μ L STAT3-siRNA are drawn, plus
Enter 6.5 μ L calf thymus DNA, 10min is stood at room temperature；The IRV for drawing 14 μ L adds 15 μ L nucleoprotamine, quiet at room temperature
Put 10min；After two kinds of compounds are mixed, 15min is stood at room temperature.The DEPC water for adding 954.5 μ L obtains 100nM's
75%IRV/STAT3-siRNA.10 μ L STAT3-siRNA are drawn, 6.5 μ L calf thymus DNA is added, stands at room temperature
10min；The IRV for drawing 20 μ L adds 20 μ L nucleoprotamine, and 10min is stood at room temperature；After two kinds of compounds are mixed, room temperature
Lower standing 15min.The DEPC water for adding 943.5 μ L obtains 100nM 50%IRV/STAT3-siRNA.Draw 10 μ L
STAT3-siRNA, adds 6.5 μ L calf thymus DNA, 10min is stood at room temperature；The IRV for drawing 38 μ L adds 22.5 μ L's
Nucleoprotamine, stands 10min at room temperature；After two kinds of compounds are mixed, 15min is stood at room temperature.Add 923 μ L DEPC water
Obtain 100nM 25%IRV/STAT3-siRNA.
It is another object of the present invention to provide application of the genes delivery system in the medicine for suppressing tumour is prepared.
It is another object of the present invention to be related to lung cancer A549 cell strain for model, genophore is evaluated with mtt assay
IRV to the cytotoxicity of A549 cells and STAT3-siRNA/IRV anti tumor activity in vitro, as a result show 100%IRV and
75%IRV goes out weaker antitumor activity to A549 cells shows, and 50%IRV and 25%IRV have cytotoxicity in higher concentrations,
And 100%IRV/STAT3-siRNA has good anti tumor activity in vitro.
It is double with AnnexinV-FITC/PI it is another object of the present invention to be related to lung cancer A549 cell strain for model
Dye method investigates IRV/STAT3-siRNA and induces the apoptosis rate of lung cancer A549 cell, and has investigated IRV/ by PI decoration methods
Influences of the STAT3-siRNA to the lung cancer A549 cell cycle.The double dye method results of AnnexinV-FITC/PI show IRV/STAT3-
The A549 cells of siRNA (concentration is 100nM) effects are 43.8% in 48h apoptosis rate；Cycle experimental result shows 100%
IRV/STAT3-siRNA has cyclin dependent, will be in mitotic A549 cell blocks in the G2/M phases.
It is another object of the present invention to be related to lung cancer A549 cell strain for model, albumen water is determined by ELISA
Flat gene silencing efficiency, the gene silencing efficiency of mRNA level in-site is determined by RT-PCR.In mRNA level in-site, 100%IRV/
STAT3-siRNA can lower the expression of STAT3-siRNA corresponding mRNAs, and the inhibiting rate that concentration is 100nM is 49.5 ±
2.8%, while STAT3-siRNA downward causes the reduction that downstream gene VEGF and SURVIVIN corresponding mRNA are expressed, VEGF
Inhibiting rate be 66.8 ± 4.7%, SURVIVIN inhibiting rate be 51.9 ± 11.9%；In protein level, 100%IRV/
STAT3-siRNA can lower the expression of stat protein, and the inhibiting rate that concentration is 100nM is 37.82 ± 2.98%.
It is another object of the present invention to be related to comment with the internal antitumor activity of lotus S180 ascites tumors mouse model progress
STAT3-siRNA dosage is 0.3mg/kg in valency, IRV/STAT3-siRNA compounds, and its tumour inhibiting rate is 52.73%；
And by determining FT-MS after carrying out homogenized to Organs of Mice, as a result it is shown in tumor locus and is able to detect that RGDV
Molecule, illustrates that IRV/STAT3-siRNA has tumor-targeting.
The following word occurred in specification is further explained：
RGDV：Arg-Gly-Asp-Val, Arg-Gly-Asp-valine
STAT：Signal transducer and transcription activator
DMF：N,N-dimethylformamide
DCC：Dicyclohexylcarbodiimide
HOBt：I-hydroxybenzotriazole
TFA：Trifluoroacetic acid
TfOH：Trifluoromethanesulfonic acid
OD values：OD value
DEPC：Pyrocarbonic acid diethyl ester
NC：Non-homogeneous-siRNA
FBS：Hyclone
FAM：Hydroxyl fluorescein
MTT:3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides
DMSO：Dimethyl sulfoxide (DMSO)
NS：Physiological saline
ELISA：Enzyme linked immunosorbent assay (ELISA)
RT-PCR：Reverse transcriptase chain reaction
Brief description of the drawings
Accompanying drawing 1 is transfection figures of the IRV/STAT3-siRNA in A549 cells.
Note：IRV is the abbreviation of " Isoquinoline-3-acyl-RGDV ", and lipo2000 is " LipofectamineTM
2000 " abbreviation.
Accompanying drawing 2 is cytotoxicities of the IRV to A549 cells.
Accompanying drawing 3 is IRV/STAT3-siRNA anti tumor activity in vitro.
Note：IRV is the abbreviation of " Isoquinoline-3-acyl-RGDV ", and lipo is " LipofectamineTM 2000”
Abbreviation；▲ the P ＜ 0.05 compared with STAT3-siRNA/100%IRV groups are represented, there is significant difference.
Accompanying drawing 4 is apoptotic effects of the 100%IRV/STAT3-siRNA to A549 cells.
A:To the apoptotic effect of A549 cells in blank control group 24h
B:To the apoptotic effect of A549 cells in naked siRNA control groups 24h
C:To the apoptotic effect of A549 cells in 100%IRV/STAT3-siRNA groups 24h
D:To the apoptotic effect of A549 cells in blank control group 48h
E:To the apoptotic effect of A549 cells in naked siRNA control groups 24h
F:To the apoptotic effect of A549 cells in 100%IRV/STAT3-siRNA groups 24h
Accompanying drawing 5 is influences of the 100%IRV/STAT3-siRNA to the A549 cell cycles.
A:The influence of each group medicine cell cycle in 24h
B:The influence of each group medicine cell cycle in 48h
Accompanying drawing 6 is the content of each group STAT3 albumen.
Note：IRV is the abbreviation of " Isoquinoline-3-acyl-RGDV ", and lipo2000 is
The abbreviation of " LipofectamineTM2000 ".
Accompanying drawing 7 is each group STAT3-mRNA content.
Note：IRV is the abbreviation of " Isoquinoline-3-acyl-RGDV ", and lipo2000 is
The abbreviation of " LipofectamineTM2000 ".
Accompanying drawing 8 is each group VEGF-mRNA and SURVIVIN-mRNA content.
Note：IRV is the abbreviation of " Isoquinoline-3-acyl-RGDV ".
Accompanying drawing 9 is the dirty body ratio of NS, 100%IRV/STAT3-siRNA, STAT3-siRNA, 100%IRV and DOX group.
Accompanying drawing 10 is the knurl weight of NS, 100%IRV/STAT3-siRNA, STAT3-siRNA, 100%IRV and DOX group.
Accompanying drawing 11 is the FT-MS figures of NS groups and 100%IRV/STAT3-siRNA group internal organs homogenates.
A:The FT-MS figures of 100%IRV/siRNA group tumor tissues liquid
B:The FT-MS figures of 100%IRV/siRNA group heart tissue liquid
C:The FT-MS figures of 100%IRV/siRNA group renal tissue liquid
D:The FT-MS figures of 100%IRV/siRNA group spleen tissue liquid
E:The FT-MS figures of 100%IRV/siRNA group brain tissue liquid
F:The FT-MS figures of 100%IRV/siRNA group liver organization liquid
G:The FT-MS figures of physiological saline group tumor tissues liquid
H:The FT-MS figures of physiological saline group heart tissue liquid
I:The FT-MS figures of physiological saline group renal tissue liquid
J:The FT-MS figures of physiological saline group spleen tissue liquid
K:The FT-MS figures of physiological saline group brain tissue liquid
L:The FT-MS figures of physiological saline group liver organization liquid
Embodiment
Embodiment 1.IRV/STAT3-siRNA preparation
Isoquinoline-3-carboxylic acid synthesis
Under HCl catalysis, with formaldehyde PS reactions, condensation generation tetrahydrochysene occur under 80~90 DEG C of oil baths for 5g phenylalanines
Isoquinolinecarboxylic acid (4H IQ-COOH)；By 4H IQ-COOH in SOCl2Catalysis under, with methanol occur esterification, obtain 4H
IQ-OMe；Solvent is made with THF, is IQ-OMe with potassium permanganate catalysis oxidation 4H IQ-OMe；Using methanol as solvent, in 2N NaOH
Catalysis under, IQ-OMe sloughs methyl esters, obtains IQ-COOH.
H-Arg (Tos)-Gly-Asp (OBzl)-Val-OBzl synthesis
Take 6g Boc-Arg (Tos)-Gly-Asp (OBzl)-Val-OBzl to be placed in eggplant bottle, be slowly added under condition of ice bath
50mL hydrochloric ethyl acetates, bottleneck connects thin-layered chromatography monitoring reaction process to reaction after drying tube and is fully completed, under tepidarium
Drain liquid with water pump, and with dry ethyl acetate and absolute ether respectively wear away 3 times after drain more than 8h.
Isoquinoline-3-acyl-RGDV synthesis
Take compound Isoquinoline-3-carboxylic acid 646mg to be dissolved in a small amount of anhydrous THF, add
HoBt 605mg are stirred to dissolving, added under condition of ice bath added after DCC 923mg, activation 30min 3.49g H-Arg (Tos)-
Gly-Asp (OBzl)-Val-OBzl, adjusts PH to 8-9 with NMM, removes and reaction process is monitored after ice bath.Reaction is revolved after terminating to remove
The yellow liquid being filtrated to get, uses CH2Cl2Respectively with saturation alkali, salt, acid cleaning totally ten eight times, anhydrous Na after dissolving2SO4Dry
Rotation removes filtrate after the filtrate 4h being filtrated to get, and obtains faint yellow solid Isoquinoline-3-acyl--Arg (Tos)-Gly-
Asp(OBzl)-Val-OBzl.Column chromatography carries out Structural Identification after purification.Take 300mg Isoquinoline-3-acyl--Arg
(Tos)-Gly-Asp (OBzl)-Val-OBzl is placed in eggplant bottle, and TFA 4.8ml, TfOH are slowly added under condition of ice bath
Ice absolute ether terminating reaction is added after 1.2ml, 30min, centrifuges (3000rpm, 3min), is taken out after wearing away 5 times with absolute ether
Dry to obtain yellowish crude product, column chromatography freezes obtain clear crystal after purification.
Structural Identification result：[M-H]+=599.51, [M+H]+=601.13；
1H NMR(500MHz,DMSO-d6) δ/ppm=10.430 (s, 1H), 9.391 (s, 1H), 9.129 (d, J=8Hz,
1H), 8.915 (t, 1H), 8.847 (d, J=8Hz, 1H), 8.554 (s, 1H), 8.247 (d, J=8Hz, 1H), 8.186 (d, J=
8Hz, 1H), 7.874 (m, 1H), 7.807 (m, 1H), 7.241 (s, 2H), 7.014 (d, J=8.5Hz, 1H), 4.704 (m, 1H),
4.337 (m, 1H), 4.112 (dd, J=10Hz, J=8Hz, 1H), 3.934 (dd, J=8Hz,
J=4.5Hz, 1H), 3.516 (dd, J=17Hz, J=5Hz, 1H), 3.194 (m, 1H), 2.974 (m, 1H),
2.621 (dd, J=16Hz, J=3.5Hz, 1H), 2.252 (dd, J=11Hz, J=5.5Hz, 1H), 2.195 (m, 1H), 2.028
(m, 1H), 1.911 (m, 1H), 1.583 (m, 2H), 0.812 (d, J=3.5Hz, 6H)；
13C NMR(125MHz,DMSO-d6) δ/ppm=175.90,174.07,172.43,170.62,168.80,
164.03,158.09,152.20,143.59,135.83,131.91,129.78,129.72,128.48,128.36,120.27,
58.61,52.55,50.67,42.91,41.07,38.02,31.67,31.36,24.72,19.72,18.26；
UV detects a length of 275.5nm of maximum absorption wave of compound；
HPLC detections, purity=98.08 ± 1.2% are carried out under 275.5nm wavelength, separation condition is chromatographic column 4.6mm
× 5.0um × 250mm Symmetry C18 5um 4.6 × 250mm Column, methanol：Water (0.1% glacial acetic acid)=45:
55, flow velocity 1mL/min, time 20min；
IR：νN-H 3293cm-1,3204cm-1,δN-H 1625cm-1；νC=O 1650cm-1；ν=C-H 3064cm-1；νC- H2967cm-1,2933cm-1,2831cm-1,δC-H 1468cm-1,1390cm-1；νC=C 1514cm-1,1468cm-1,δC=C 746cm-1。
The preparation of IRV blank liposomes
60mg IRV accurately are weighed, 10mL volumetric flasks is transferred to after being dissolved with a small amount of methanol, is settled to chloroform after 10mL
Ultrasonic dissolution.0.4g Tween-80 are weighed in 50ml beakers, water ultrasonic dissolution is handled with 40mL DEPC.Accurately weigh
38.7mg CH, are transferred to 10mL volumetric flasks after being dissolved with a small amount of chloroform, 10mL are settled to chloroform.75mg PE accurately are weighed,
10ml volumetric flasks are transferred to after being dissolved with a small amount of chloroform, 10mL is settled to chloroform.
Take 0.906mL IRV and 0.094mL CH to be added in 25mL eggplants bottle, mend chloroform to 2mL.Rotary evaporation in vacuo
Condition be 37 DEG C of water-baths, 120rpm, 250mbar, after 30min per 5min reduction 50mbar until 1mbar, obtains homogeneous thin
Film.Popped one's head in after the appropriate rehydration solution i.e. Tween-80 of various concentrations, jog aquation ultrasound 10min are added after vacuum drying
The blank liposomes liquid solution containing 100%IRV is obtained after ultrasound, cooling.Prepare 75%IRV with same method, 50%IRV and
25%IRV.
IRV/STAT3-siRNA preparation
According to electrophoresis the selection result, 10 μ L STAT3-siRNA are drawn first, add 6.5 μ L calf thymus DNA, room temperature
Lower standing 10min；The IRV for drawing 1 μ L adds 5 μ L nucleoprotamine, and 10min is stood at room temperature；After two kinds of compounds are mixed,
15min is stood at room temperature.The DEPC water for adding 967.5 μ L obtains 100nM 100%IRV/STAT3-siRNA.Draw 10 μ L
STAT3-siRNA, adds 6.5 μ L calf thymus DNA, 10min is stood at room temperature；The IRV for drawing 14 μ L adds 15 μ L fish
Protamine, stands 10min at room temperature；After two kinds of compounds are mixed, 15min is stood at room temperature.Add 954.5 μ L DEPC water
Obtain 100nM 75%IRV/STAT3-siRNA.10 μ L STAT3-siRNA are drawn, 6.5 μ L calf thymus DNA is added,
10min is stood at room temperature；The IRV for drawing 20 μ L adds 20 μ L nucleoprotamine, and 10min is stood at room temperature；By two kinds of compounds
After mixing, 15min is stood at room temperature.The DEPC water for adding 943.5 μ L obtains 100nM 50%IRV/STAT3-siRNA.Inhale
10 μ L STAT3-siRNA are taken, 6.5 μ L calf thymus DNA is added, 10min is stood at room temperature；The IRV for drawing 38 μ L is added
22.5 μ L nucleoprotamine, stands 10min at room temperature；After two kinds of compounds are mixed, 15min is stood at room temperature.Add 923 μ L
DEPC water be to obtain 100nM's
25%IRV/STAT3-siRNA.
Embodiment 2.IRV/STAT3-siRNA anti tumor activity in vitro evaluation
Laser confocal microscope investigates siRNA transfection
Human lung carcinoma cell lines' A549 cells are the cells of adherent growth, are inoculated in appropriate concentration in blake bottle, are added
Containing 10% hyclone, 100U/mL penicillin, 1640 complete culture solutions of 100 μ g/mL streptomysin, be placed in 37 DEG C of constant temperature,
5%CO2Cultivated in the incubator of saturated humidity.Take the logarithm growth period A549 cell, first washed with PBS three times, plus 1mL pancreatin,
Digest after 1min30s, centrifuge (2000rpm, 3min), cell concentration is adjusted extremely with 1640 culture mediums containing 10% hyclone
1.5×1052mL cell liquid, totally six groups, 37 DEG C of constant temperature, 5%CO are added in individual/mL, each laser co-focusing culture capsule2Culture
Overnight.Supernatant carefully is discarded, PBS is washed once, be separately added into 100%IRV/STAT3-siRNA, 75% that 2mL contains 100nM
IRV/STAT3-siRNA, 50%IRV/STAT3-siRNA, 25%IRV/STAT3-siRNA and naked STAT3-siRNA without blood
Clear 1640 culture medium, and 2mL contain 100nM IRV/LipofectamineTM2000 culture medium of serum-free 1640, transfection
4h.Supernatant is sucked, 1mL PBS are washed 3 times, each culture dish adds 1mL Hochest dye liquors, and (PBS dilutes, and working concentration is 4 μ g/
), mL 15min is dyed.Supernatant is sucked, is washed with 1mL PBS 3 times, is eventually adding 1mL PBS.It is placed in laser confocal microscope
Lower observation.
Experimental result
In this experiment, the siRNA that IRV is contained be FAM mark STAT3-siRNA, from Fig. 1 we can see that
100%IRV/STAT3-siRNA groups and Lipo 2000/STAT3-siRNA nucleus (dye indigo plant by Hochest 33342
Color) around there is fraction green fluorescence around most of green fluorescence (FAM), 75%IRV/STAT3-siRNA group nucleus,
Have a small amount of or almost glimmering without green around 50%IRV/STAT3-siRNA groups, 25%IRV/STAT3-siRNA group nucleus
Light, and do not have viridescent fluorescence around naked STAT3-siRNA groups nucleus, it can be deduced that conclusion：STAT3-siRNA is by 100%
It is intracellular that IRV, 75%IRV and the commercially available Successful deliveries of transfection carrier reagent Lipo 2000 enter A549, and is distributed in nucleus
Around.
IRV CDCC
A549 cells are inoculated in blake bottle with appropriate concentration, adds and contains 10% hyclone, 100U/mL mould
Element, 1640 complete culture solutions of 100 μ g/mL streptomysin, are placed in 37 DEG C of constant temperature, 5%CO2Trained in the incubator of saturated humidity
Support.Take the logarithm growth period A549 cell, first washed with PBS three times, plus 1mL pancreatin, after digestion 1min30s, centrifugation (2000rpm,
3min), cell concentration is adjusted to 1.5 × 10 with 1640 culture mediums containing 10% hyclone5Individual/ml.96 well culture plates are taken,
PBS fluid-tights, if blank cultures group, remaining adds 100 μ L cell suspensions per hole, in 37 DEG C, 5%CO2Incubator in cultivate
Overnight.It is careful to discard nutrient solution, the μ L of 1640 culture medium 100 containing 100%IRV, 75%IRV, 50%IRV, 25%IRV are added,
Concentration is followed successively by 40nM, 60nM, 80nM, 100nM, 120nM.Each concentration sets five multiple holes.It is placed in culture in incubator
24h.The concentration that 20 μ L Fresh are added per hole is 5mg/mL MTT, 37 DEG C, 5%CO2Incubator continues to cultivate 4h.Centrifugation
Careful abandoning supernatant, and add 150 μ L DMSO afterwards, 700r, 10min are shaken with microoscillator.After mixing, in ELIASA
On by 490nm of Detection wavelength determine absorbance (OD) value, calculate cell survival rate.Cell survival rate (%)=[(experimental group OD
Value-blank group OD values)/(control group OD values-blank group OD values)] × 100%.Above-mentioned experiment is in triplicate.
Experimental result
We can see that with the increase of concentration from Fig. 2, compared with blank group, 100%IRV and 75%IRV groups
Cell survival rate does not have obvious sex differernce (P>0.05) antitumor activity, is produced weaker.50%IRV and 25%IRV are in high concentration
It is lower to produce stronger cytotoxicity, illustrate that IRV has antitumor action.
IRV/STAT3-siRNA anti tumor activity in vitro
Take the logarithm the A549 cells in growth period, after pancreatin digestion, adjust thin with 1640 culture mediums containing 10% hyclone
Born of the same parents' concentration is to 1.5 × 105Individual/ml.Per the μ l cell suspensions of hole kind 100 in 96 well culture plates, 37 DEG C, 5%CO2Overnight incubation.It is small
The heart discards nutrient solution, adds 100 μ L and contains 40nM, 60nM, 80nM and 100nM, 120nM STAT3-siRNA, 100%IRV/NC,
The Lipo 2000/STAT3-siRNA culture medium of serum-free 1640, and 100%IRV/STAT3-siRNA, 75%IRV/
STAT3-siRNA, 50%IRV/STAT3-siRNA and the 25%IRV/STAT3-siRNA culture medium of serum-free 1640.Every group every
Individual concentration sets five multiple holes.4h is transfected, supernatant is discarded, 1640 culture mediums of the 100 μ L containing 10%FBS are added per hole, then
Continue to cultivate 20h.MTT, 37 DEG C, 5%CO that the concentration that 20 μ L of addition are newly prepared per hole is 5mg/mL2Continue to cultivate 4h.Centrifugation
Careful abandoning supernatant, and add 150 μ L DMSO afterwards, 700r, 10min are shaken with microoscillator.After mixing, in ELIASA
On by 490nm of Detection wavelength determine absorbance (OD) value, calculate cell survival rate.Cell survival rate (%)=[(experimental group OD
Value-blank group OD values)/(control group OD values-blank group OD values)] × 100%.Above-mentioned experiment is in triplicate.
Experimental result
As can be drawn from Figure 3 to draw a conclusion：
(1) blank group is compared, STAT3-siRNA and each equal no difference of science of statistics of concentration group of 100%IRV/NC, STAT3-
Also illustrate that NC-siRNA does not have cytotoxicity without significant difference between siRNA and NC-siRNA each concentration group, it is free
STAT3-siRNA can not enter cell and produce cytotoxicity.
(2) Lipo 2000/STAT3-siRNA are compared with 100%IRV/NC groups, 40,60, P ＜ between 80nM concentration groups
0.05,100, P ＜ 0.01 between 120nM concentration group, have significant difference, illustrate that STAT3-siRNA is commercially available transfection reagent
It is successfully loaded cell and produces cytotoxicity.
(3) 100%IRV/STAT3-siRNA is compared with 100%IRV/NC groups, and P ＜ 0.01 between each concentration group have aobvious
Work property significant difference, illustrates that siRNA groups effectively can be taken in cell and produce cytotoxicity by 100%IRV/STAT3-siRNA.
100%IRV/STAT3-siRNA is compared with Lipo 2000/STAT3-siRNA groups, and P ＞ 0.05, are not counted between 120nM groups
Difference is learned, illustrates that 2000 groups of effects of this concentration and Lipo are identical.40th, P ＜ 0.05 between 60,80nM groups, there is significant difference,
Illustrate that the cytotoxicity that these concentration groups are produced is better than 2000 groups of Lipo.Compare, P ＜ 0.01, have significantly between 100nM groups
Property significant difference, illustrate that the cytotoxicity that carrier at this concentration is loaded into siRNA and produced is most strong, be significantly better than Lipo
2000 groups.
(4) 75%IRV/STAT3-siRNA is compared with 100%IRV/NC groups, 40,60,80, P ＜ between 100nM groups
0.05,120nM group P ＜ 0.01, there is significant difference, illustrate that this group also has cytotoxicity.50%IRV/STAT3-siRNA with
NC/100%IRV groups are compared, and P ＜ 0.01 between P ＜ 0.05,40,100,120nM groups, have statistics poor between 60,80nM groups
It is different.25%IRV/STAT3-siRNA is compared with NC/100%IRV groups, and P ＞ 0.05, poor without statistics between 40,60nM groups
It is different, 80,100, P ＜ 0.01 have notable statistics difference between 120nM groups.
According to Fig. 1, Fig. 2 and Fig. 3 result, the cytotoxicity of 100%IRV/STAT3-siRNA groups is largely sunk by gene
Silent to cause, transfection efficiency is best.75%IRV/STAT3-siRNA group effects are taken second place.50%IRV/STAT3-siRNA groups and 25%
The cytotoxicity of IRV/STAT3-siRNA groups is largely that carrier toxicity is caused.
Embodiment 3.100%IRV/STAT3-siRNA study on mechanism
The double dye methods of AnnexinV-FITC/PI investigate the apoptosis that 100%IRV/STAT3-siRNA induces lung cell A549
Rate
Cell culture
Take the logarithm the A549 cells in growth period, first washed with PBS three times, plus 1mL pancreatin, after digestion 1min30s, 3000rpm
Centrifuge 3min.Cell concentration is adjusted to 2 × 10 with 1640 culture mediums containing 10% hyclone5Individual/ml, adds 2mL in six per hole
In orifice plate, if three multiple holes, after culture 24h, culture medium is discarded, 2mL is separately added into and contains 100nM 100%IRV/STAT3-
SiRNA and STAT3-siRNA 1640 culture mediums without serum, blank group adds 2mL 1640 culture mediums without serum,
4h is transfected, culture medium is discarded, adds the fresh culture that 2mL contains 10%FBS per hole.Continue in 37 DEG C, 5%CO2Under the conditions of train
Support 20h or 44h.
Dyeing
Eluted and merged with the suspension cell of culture medium supernatant with the pancreatin digestion without EDTA, 2000rpm centrifugation 3min,
Then washed 2 times with the PBS of precooling, the cell mass of centrifugation is suspended in 200 μ L Binding Buffer (1 ×) completely
In, it is then diluted to 5 × 105Individual/mL, at least 50 μ L, add 5 μ L AnnexinV-FITC and 1 μ L PI, lucifuge, incubation at room temperature
15min.Upper machine check.
Experimental result
As shown in Figure 4, apoptosis rates and blank group and STAT3-siRNA of the 100%IRV/STAT3-siRNA to A549 cells
Group compares, and has significant difference.In 48h, the apoptosis rate of 100%IRV/STAT3-siRNA groups can reach 43.8% (early
Adjust：23.8%；Evening adjusts：20.0%).We may safely draw the conclusion：100%IRV/STAT3-siRNA can be successfully by STAT3-
SiRNA is delivered to that A549 is intracellular, and can induce the apoptosis of A549 cells.
PI decoration methods investigate the influence of 100%IRV/STAT3-siRNA cell cycles
Cell culture
Take the logarithm the A549 cells in growth period, first washed with PBS three times, plus 1mL pancreatin, after digestion 1min30s, 2000rpm
Centrifuge 3min.Cell concentration is adjusted to 2 × 10 with 1640 culture mediums containing 10% hyclone5Individual/ml, adds 2mL in six per hole
In orifice plate, if three multiple holes, cultivate 24h, discard culture medium, add 2mL contain 100nM 100%IRV/STAT3-siRNA and
STAT3-siRNA 1640 culture mediums without serum, blank group adds 2mL 1640 culture mediums without serum, transfects 4h,
Culture medium is discarded, adds the fresh culture that 2mL contains 10%FBS per hole.Continue in 37 DEG C, 5%CO2Under the conditions of cultivate.
The fixation of cell
Respectively at 12h, 24h, 48h collects cell.Eluted with the pancreatin digestion without EDTA and outstanding with culture medium supernatant
Floating cell merges, 2000rpm centrifugation 3min, is then washed three times with the PBS of precooling, centrifuge cell agglomerate is suspended in completely
In 0.5ml PBS, it is gradually dropped in the ethanol solution for the 9.5mL 80% being ready for, 4 DEG C of preservations are stand-by.
Dyeing
The cell centrifugation (8000rpm) for each period that previous step is collected into, abandoning supernatant, and with cold
PBS is washed three times, is dispersed in 500 μ L PBS and (contains the PI dye liquors of 5 μ L RNase stostes and concentration for 1mg/ml) cell, 37 DEG C
Lucifuge dyes 30min, upper machine testing.
Experimental result
From Fig. 5 it will be seen that compared with blank group and naked STAT3-siRNA groups, 100%IRV/STAT3-
SiRNA groups increase with the time, G2The cell number of/M phases increases, and G1The cell number of phase is reduced, and shows G2/ M the phases block existing
As.It can be seen that STAT3-siRNA enters cell by IRV Successful deliveries, by cell block in G2/ M the phases, suppress cell propagation,
And then inducing cell apoptosis.
The 100%IRV/STAT3-siRNA of embodiment 4 protein level silence efficiency is evaluated
Cell culture
Take the logarithm and grow A549 cells, after pancreatin digestion, adjust cell with 1640 culture mediums containing 10% hyclone dense
Spend to 2 × 105Individual/ml, adds 2mL in 24 orifice plates per hole, if three multiple holes, 24h is cultivated, when cell density length to 80% or so
When, abandoning supernatant adds 2mL and contains 100nM STAT3-siRNA, 100%IRV/NC, 100%IRV/STAT3-siRNA,
Lipo 2000/STAT3-siRNA and Lipo the 2000/NC culture medium of serum-free 1640, blank group add 2mL serum-frees 1640
Fresh 1640 culture mediums containing 10%FBS are changed into after culture medium, transfection 4h, continue to cultivate 44h.
Extract albumen
Operate on ice：Supernatant is discarded, is then washed per hole with PBS 3 times, abandoning supernatant, 500 μ L protein cleavages are added
30min is cracked in liquid, ice bath, is extracted in centrifuge tube, centrifugation (10000rpm, 10min) takes supernatant.
Protein concentration in BCAProtein Assay Kit quantitative cleavage liquid
The protein lysate that previous step is extracted dilutes ten times, three multiple holes of every group of setting, according to BCA work liquid kits
Operating procedure, determines per histone concentration, does lower record, seek every group of sample total protein concentration average value.
STAT3 content in Human STAT3 ELISA kit determination sample albumen
Albumen applied sample amount is 20 μ g, according to protein concentration, adds the protein extract of respective volume and with DEPC water constant volumes
To 50 μ L, OD values are finally determined according to kit step operation at 450 nm.Above-mentioned experiment is in triplicate.
Experimental result
Protein quantification result
Draw standard curve
The OD values of the standard protein of various concentrations
Normal equation is y=0.01x+0.215.R2=0.992
Total protein concentration in each group protein sample
ELISA
The relative amount of stat protein in each group protein sample
It will be seen that naked STAT3-siRNA groups are compared with blank group from Fig. 6, without obvious sex differernce (P>
0.05), i.e., naked STAT3-siRNA can not influence the expression of A549 cell stat proteins；100%IRV/NC and Lipo 2000/NC
Compared with blank group, without obvious sex differernce (P>0.05), show that expression of the carrier material on stat protein does not influence；And
100%IRV/STAT3-siRNA has obvious sex differernce (P compared with blank group<0.01), with Lipo 2000/STAT3-siRNA
Positive controls are compared, without obvious sex differernce (P>0.05), illustrate that STAT3-siRNA is deliverrf into cell simultaneously by 100%IRV
The expression of stat protein is lowered in success.
Embodiment 5.100%IRV/STAT3-siRNA mRNA level in-site silence efficiency is evaluated
Cell culture
Take the logarithm growth period A549 cell, when cell density reaches 80% or so, washed with PBS three times, add 1mL pancreatin
Digest 1min 30s, centrifuge (2000rpm, 3min), with 1640 culture mediums containing 10% hyclone adjust cell concentration to 2 ×
105Individual/ml, adds 2mL in six orifice plates per hole, cultivates 24h, if three multiple holes, supernatant discarding, adds 2mL and contains 100nM
STAT3-siRNA, 100%IRV/NC, 100%IRV/STAT3-siRNA and Lipo 2000/STAT3-siRNA serum-free
1640 culture mediums, transfect 4h, change fresh 1640 culture mediums containing serum into, continue to cultivate 44h.
RNA extraction
(1) supernatant of six orifice plates is discarded first, is then washed three times with 1mL PBS, and 1mL Trizol are added per hole and are split
Liquid is solved (per 10cm2Add 1mL Trizol).The uniform simultaneously lysis at room temperature 10min of piping and druming, no RNase EP are transferred to by lysate
Pipe, stands 5min.
(2) it is separated.0.2mL chloroforms are added in every 1mL Trizol, then acutely vibration, room temperature is placed 3 minutes, centrifugation
(4 DEG C, 12000rpm, 15minutes).Liquid is divided into three layers, and the red of lower floor is phenol chloroform phase, and upper strata is colourless water
Phase.RNA is present in aqueous phase, aqueous phase about of the total volume 50%.
(3) RNA is precipitated.Carefully upper strata aqueous phase liquid is transferred to it is another clean without in RNase EP pipes, then often
Pipe adds 0.5mL isopropanols, mixes, is stored at room temperature 10min, is then centrifuged for (4 DEG C, 12000rpm, 10min).In pipe after centrifugation
Bottom cotton-shaped semi-transparent gelatinoid RNA precipitate can be seen.
(4) RNA is washed.Careful supernatant discarding, adds 75% ethanol (preparation of DEPC water) that 1mL is prepared, and washing RNA sinks
Form sediment, oscillator is mixed, centrifuge (4 DEG C, 12000rpm, 10min), careful supernatant discarding.
(5) RNA redissolution.RNA precipitate is placed into 10min, treats that RNA precipitate is dried.It is noted that can not be complete by RNA precipitate
White drying, because this can greatly reduce its solubility.RNA precipitate is resuspended with DEPC water, and is blown and beaten repeatedly with pipette tips several
It is secondary, 10 minutes are stood, 55 DEG C of water-bath 10-15min.Determine immediately.
(6) RNA concentration is surveyed：After DEPC water correction Nanodrop-1000, the RNA solution sample after 2 μ L mixings, drop are taken
It is added in measuring arm, selects RNA, clicks on measure measurements.Measure all RNA solution samples successively with method.
Reverse transcription
(1) preparation of samples：Reverse transcription RNA applied sample amounts are 2 μ g, and the RNA concentration measured according to previous step calculates our institutes
Need RNA volumes.
(2) MicroAmp is takenTMOptical 96-Well Reaction Plate, each reacting hole adds 10 2 × RT of μ L
Buffer, 1 μ L 20 × RT EnzymeMix, add the RNA solution of respective volume, 20 μ L are supplied with DEPC water.Use Optical
Adhesive film seal plank.Centrifuge (4 DEG C, 1000rpm, 1min), discharge is gathered in the bubble of reacting hole bottom.
(3) it is put into PCR System 9700, reaction condition is set：94℃-5min,94℃-30s,55℃-30s,72
DEG C -25s, 72 DEG C of -7min, 4 DEG C of ∞, totally 30 circulations.
(4) concentration is surveyed：After reaction terminates, first with DEPC water correction Nanodrop-1000, the cDNA after 2 μ L mixings is taken
Solution example, is added drop-wise in measuring arm, covers measuring arm, selects DNA-50, clicks on measure measurements.Measure institute successively with method
There are cDNA solution examples, and record the result of measurement.It is diluted, is surveyed after dilution with same method according to applied sample amount
It is fixed.Preserved at -20 DEG C.
RT-PCR
CDNA applied sample amount is 50ng, the volume of each cDNA samples added needed for calculating accordingly.Each reacting hole is added
25μL Gene ExpressionMaster Mix, 2.5 μ L probes mark primer (STAT3 or GAPDH or VEGF
Or SURVIVIN), the cDNA samples of respective volume, with the μ L of DEPC water polishing 50, shake up.Centrifuge (4 DEG C, 1000rmp, 1min),
Make liquid accumulation in bottom and discharge bubble；It is put into Applied Biosystems 7500Real-Time PCR System,
Setting condition.Hold:50 DEG C of 2min, 95 DEG C of 10min, Cycle (40cycles), 95 DEG C of 15s, 60 DEG C of 1min.Data processing,
According to Ct values using GAPDH as internal reference, with each experimental group mRNA of 2^- △ △ Ct method relative quantifications amount.Above-mentioned experiment repeats three
It is secondary.
Experimental result
The each group RNA concentration of extraction
The each group RNA concentration of extraction
The cDNA of each group concentration after reverse transcription
The cDNA of each group concentration after reverse transcription
RT-PCR
Each group 2^- △ △ Ct values are as shown in the table after RT-PCR：
Gene silencing efficiency on STAT3mRNAlevel
As can be seen from Figure 7：
(1) naked STAT3-siRNA groups are compared with blank group, without significant difference (P>0.05) naked STAT3, is illustrated
Transcription of the group on STAT3-mRNA does not influence；
(2) 100%IRV/NC groups group is compared with blank group, without significant difference (P>0.05) carrier material pair, is illustrated
STAT3-mRNA transcription does not influence；
(3) 100%IRV/STAT3-siRNA groups have explicitly poor respectively compared with blank group and 100%IRV/NC
Different (P<0.01), and compared with Lipo 2000/STAT3-siRNA groups, there is no X significant differences (P>0.05), it can be deduced that
100%IRV/STAT3-siRNA can lower STAT3-mRNA transcriptional level, and efficiency and positive controls are suitable.
As can be seen from Figure 8,100%IRV/STAT3-siRNA can lower the same of STAT3-mRNA transcriptional level
When, downstream gene is impacted, the expression of VEGF and SURVIVIN genes is also lowered.
Embodiment 6.100%IRV/STAT3-siRNA internal antitumor activity
Experiment packet
Experiment is divided into 5 groups, is physiological saline group (NS), 100%IRV/STAT3-siRNA groups, naked STAT3- respectively
SiRNA groups, 100%IRV groups and doxorubicin hydrochloride group (DOX).Every group of 10 mouse, six groups totally 60.
Dosage
(1) 100%IRV/STAT3-siRNA groups：STAT3-siRNA dosage is 0.3mg/kg, proportionally configures 100%
IRV/STAT3-siRNA compounds.
(2) STAT3-siRNA groups：Dosage is 0.3mg/kg
(3) 100%IRV groups：Dosage is suitable in dosage and 100%IRV/STAT3-siRNA groups
(4) DOX groups：Dosage is 2 μm of ol/kg
(5) NS groups：Isometric physiological saline, i.e., every 0.2mL
Administering mode：Tail vein injection
Experimental animal：ICR mouse, male, 20 ± 2g of body weight (x ± SD), tieing up the magnificent animal experiment technique of tonneau by Beijing has
Limit company provides.
Experimental program
Set up lotus S180 ascites tumor mouse models：Take 1 × 107Individual/mL S180 cell suspensions are subcutaneous in healthy mice armpit
Inoculation, 0.2mL/ only, after mouse inoculation knurl liquid supports 7 days (long × wide to knurl volume2/ 2) be 150mm3Left and right, by whole mouse with
Machine is grouped, and is divided into 5 groups, and number.Body weight is measured, then starts administration, 100%IRV/STAT3-siRNA, STAT3-siRNA
Group and 100%IRV groups are administered once by the daily tail vein injection of dosage, each 0.2mL/, successive administration 5 days；It is negative
Compare and be administered with isometric physiological saline tail vein injection, is administered once a day, 0.2mL/ is only, continuous to give 5 days；Positive control
DOX groups are administered with tail vein injection, are administered once a day by dosage, 0.2mL/, successive administration 5 days, daily by number
Measure mouse weight.
The measure of solid tumor tumour inhibiting rate and organ index
Every group of successive administration is after 5 days, and in the 6th day, de- cervical vertebra put to death mouse, body weight was weighed before execution, then by mouse solution
Cut open, take out knurl, brain, the heart, liver,spleen,kidney, and weigh, tumour inhibiting rate and organ index is calculated as follows.
Tumour inhibiting rate %=[(the average knurl weight of the average knurl weight-administration group of physiological saline group) the average knurl weight of/physiological saline group] ×
100%, body weight before organ index (%)=internal organs weight/execution.Data statistics is examined and variance analysis using t, with (mean ±
SD g) represent.
From Fig. 9 it will be seen that each group such as 100%IRV/STAT3-siRNA groups to the heart of animal, liver, spleen, lung,
Kidney, brain are all without toxicity.It can be seen from fig. 10 that naked STAT3-siRNA groups are compared with blank group, without significant difference (P
>0.05), illustrate that growth unrestraints of the naked STAT3-siRNA to tumour is acted on；100%IRV/STAT3-siRNA and blank group
Compare, there is significant difference (P<0.01), compared with DOX groups, without significant difference (P>0.05) 100%IRV/, is illustrated
STAT3-siRNA substantially suppresses the growth of tumour, and tumour inhibiting rate is close to positive drug DOX.100%IRV groups have statistics with blank group
Learn difference (P<0.05), but compared with 100%IRV/STAT3-siRNA groups, there is significant difference (P<0.05) carrier material, is illustrated
Material has weaker antitumor action.
100%IRV/STAT3-siRNA targeting Journal of Sex Research
Knurl that previous step is taken out, brain, the heart, liver,spleen,kidney are respectively organized, and precise weighing is first washed one time with PBS, is washed till no blood
Water.Then certain volume (m is added：V=1:3) PBS (pH7.4), homogenate centrifuges (6000rpm, 10min), takes upper strata to centrifuge
The μ L of liquid 200, add 400 μ L methanol, are vortexed and mix, and centrifuge (10000rpm, 10min), take the μ L of supernatant 600, are concentrated and dried, and add
200 μ L chromatograms methanol redissolve, and determine FT-MS.
[M+1]+ RGDVTheoretical value 446.23632, by Figure 11 we can see that in the tissue extract of blank group
RGDV is not found, and RGDV [M+1] is found in the tumor tissues extract solution of 100%IRV/STAT3-siRNA groups+Peak
(446.23250).Thus we show that 100%IRV/STAT3-siRNA has targeting, can be targeted to tumor locus performance
Antitumor action.
Claims (10)
1. a kind of gene liposome carrier with targeting:Isoquinoline-3-acyl-RGDV.
2. the preparation method of the gene liposome carrier described in claim 1, comprises the following steps：Chemical synthesis is obtained into IQ-
COOH, is produced with RGDV condensations.
3. a kind of genes delivery system, it is characterised in that by Antioncogene medicine STAT3-siRNA, and claim 1
Described gene liposome carrier Isoquinoline-3-acyl-RGDV is prepared from.
4. the genes delivery system described in claim 3, it is characterised in that Antioncogene medicine STAT3-siRNA, and
Gene liposome carrier Isoquinoline-3-acyl-RGDV amount ratio is 1:1.1.
5. the genes delivery system described in claim 3, nanostructured.
6. the preparation method of the genes delivery system described in claim 3, it is characterised in that comprise the following steps：Gene lipid
Body carrier Isoquinoline-3-acyl-RGDV is mixed at room temperature with nucleoprotamine, stand, it is standby, take calf thymus DNA with
Antioncogene medicine STAT3-siRNA is mixed at room temperature, stand for standby use, after above two solution is mixed, produces base
Because of delivery system.
7. the preparation method of the genes delivery system described in claim 3, it is characterised in that the preparation of gene liposome carrier：
Under HCl catalysis, with formaldehyde PS reactions, condensation generation Tetrahydroisoquinoli- occur under 80~90 DEG C of oil baths for 5g phenylalanines
Dicarboxylic acid moiety (4H IQ-COOH)；By 4H IQ-COOH under SOCl2 catalysis, occur esterification with methanol, obtain 4H IQ-
OMe；Solvent is made with THF, is IQ-OMe with potassium permanganate catalysis oxidation 4H IQ-OMe；Using methanol as solvent, 2N NaOH's
Under catalysis, IQ-OMe sloughs methyl esters, obtains IQ-COOH.
H-Arg (Tos)-Gly-Asp (OBzl)-Val-OBzl synthesis
Take 6g Boc-Arg (Tos)-Gly-Asp (OBzl)-Val-OBzl to be placed in eggplant bottle, 50mL is slowly added under condition of ice bath
Hydrochloric ethyl acetate, bottleneck connects thin-layered chromatography monitoring reaction process to reaction after drying tube and is fully completed, and water is used under tepidarium
Pumping dry liquids, and drain more than 8h with drying after ethyl acetate and absolute ether respectively wear away 3 times.
Isoquinoline-3-acyl-RGDV synthesis
Take compound Isoquinoline-3-carboxylic acid 646mg to be dissolved in a small amount of anhydrous THF, add HoBt
605mg is stirred to dissolving, and 3.49g H-Arg (Tos)-Gly- is added after DCC 923mg, activation 30min are added under condition of ice bath
Asp (OBzl)-Val-OBzl, adjusts PH to 8-9 with NMM, removes and reaction process is monitored after ice bath.Rotation is except filtering after reaction terminates
Obtained yellow liquid, is cleaned 18 times, anhydrous Na 2SO4 is dried totally with saturation alkali, salt, acid respectively after being dissolved with CH2Cl2
Filter rotation after obtained filtrate 4h and remove filtrate, obtain faint yellow solid Isoquinoline-3-acyl--Arg (Tos)-Gly-Asp
(OBzl)-Val-OBzl.Column chromatography carries out Structural Identification after purification.Take 300mg Isoquinoline-3-acyl--Arg
(Tos)-Gly-Asp (OBzl)-Val-OBzl is placed in eggplant bottle, and TFA 4.8ml, TfOH are slowly added under condition of ice bath
Ice absolute ether terminating reaction is added after 1.2ml, 30min, centrifuges (3000rpm, 3min), is taken out after wearing away 5 times with absolute ether
Dry to obtain yellowish crude product, column chromatography freezes obtain clear crystal after purification.
8. the preparation method of the genes delivery system described in claim 3, it is characterised in that the preparation of blank liposome：Accurately
60mg IRV are weighed, 10mL volumetric flasks is transferred to after being dissolved with a small amount of methanol, ultrasonic dissolution after 10mL is settled to chloroform.Weigh
0.4g Tween-80 handle water ultrasonic dissolution in 50ml beakers with 40mLDEPC.38.7mg CH accurately are weighed, a small amount of chlorine is used
10mL volumetric flasks are transferred to after imitative dissolving, 10mL is settled to chloroform.75mg PE accurately are weighed, are turned after being dissolved with a small amount of chloroform
10ml volumetric flasks are moved to, 10mL is settled to chloroform,
Take 0.906mL IRV and 0.094mL CH to be added in 25mL eggplants bottle, mend chloroform to 2mL.The bar of rotary evaporation in vacuo
Part is 37 DEG C of water-baths, 120rpm, 250mbar, after 30min per 5min reduction 50mbar until 1mbar, obtains homogeneous film.Very
Sky carries out Probe Ultrasonic Searching after adding the appropriate rehydration solution i.e. Tween-80 of various concentrations, jog aquation ultrasound 10min after drying,
The blank liposomes liquid solution containing 100%IRV is obtained after cooling.
9. the preparation method of the genes delivery system described in claim 3, it is characterised in that comprise the following steps：
10 μ L STAT3-siRNA are drawn first, add 6.5 μ L calf thymus DNA, 10min is stood at room temperature；Draw 1 μ L's
IRV adds 5 μ L nucleoprotamine, and 10min is stood at room temperature；After two kinds of compounds are mixed, 15min is stood at room temperature.Add
967.5 μ L DEPC water is the 100%IRV/STAT3-siRNA for obtaining 100nM.10 μ L STAT3-siRNA are drawn, 6.5 are added
μ L calf thymus DNA, stands 10min at room temperature；The IRV for drawing 14 μ L adds 15 μ L nucleoprotamine, stands at room temperature
10min；After two kinds of compounds are mixed, 15min is stood at room temperature.The DEPC water for adding 954.5 μ L obtains the 75% of 100nM
IRV/STAT3-siRNA.10 μ L STAT3-siRNA are drawn, 6.5 μ L calf thymus DNA is added, 10min is stood at room temperature；
The IRV for drawing 20 μ L adds 20 μ L nucleoprotamine, and 10min is stood at room temperature；After two kinds of compounds are mixed, stand at room temperature
15min.The DEPC water for adding 943.5 μ L obtains 100nM 50%IRV/STAT3-siRNA.Draw 10 μ L STAT3-
SiRNA, adds 6.5 μ L calf thymus DNA, 10min is stood at room temperature；The IRV for drawing 38 μ L adds 22.5 μ L milt egg
In vain, 10min is stood at room temperature；After two kinds of compounds are mixed, 15min is stood at room temperature.The DEPC water for adding 923 μ L is obtained
100nM 25%IRV/STAT3-siRNA.
10. application of the genes delivery system in the medicine for suppressing tumour is prepared described in claim 3.
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| CN (1) | CN107137355A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113825523A (en) * | 2019-03-13 | 2021-12-21 | 益生生物科技有限公司 | Plant-derived Extracellular Vesicle (EV) composition and use thereof |
| CN116832051A (en) * | 2023-07-08 | 2023-10-03 | 首都医科大学 | Novel antioxidant anti-inflammatory and lipid metabolism promoting synergistic nano drug delivery system |
-
2017
- 2017-05-26 CN CN201710382838.5A patent/CN107137355A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113825523A (en) * | 2019-03-13 | 2021-12-21 | 益生生物科技有限公司 | Plant-derived Extracellular Vesicle (EV) composition and use thereof |
| CN116832051A (en) * | 2023-07-08 | 2023-10-03 | 首都医科大学 | Novel antioxidant anti-inflammatory and lipid metabolism promoting synergistic nano drug delivery system |
| CN116832051B (en) * | 2023-07-08 | 2024-02-09 | 首都医科大学 | Antioxidant anti-inflammatory and lipid metabolism promoting synergistic nano drug delivery system |
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Application publication date: 20170908 |