CN107233332A - Preparation, activity and the application of GO PLL RGDS/VEGF siRNA target gene medicines - Google Patents
Preparation, activity and the application of GO PLL RGDS/VEGF siRNA target gene medicines Download PDFInfo
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Abstract
The invention discloses the delivery system GO PLL RGDS/VEGF siRNA for delivering VEGF siRNA, disclose the preparation method of delivery vector and delivery system, disclose its nanostructured, disclose the cellular uptake situation of delivery system, disclose the effect of delivery system silence VEGF genes, disclose the efficiency that it lowers vegf expression, disclose it and suppress the effect of tumor cell proliferation, and further disclose it and suppress the effect of lotus S180 sarcoma mice tumors grews and the cancer target effect of delivery system.Therefore application of the genes delivery system in Antioncogene medicine is illustrated.
Description
Technical field
The present invention relates to the delivery system GO-PLL-RGDS/VEGF-siRN A for delivering VEGF-siRNA, it is related to it
Preparation method, be related to its nanostructured, be related to its cellular uptake situation, be related to the effect of its silence VEGF gene, relate to
And it lowers the efficiency of vegf expression, it is related to it and suppresses the effect of tumor cell proliferation, and it is small to be related to its suppression lotus S180 sarcoma
The effect of mouse tumour growth and the cancer target effect of delivery system, thus it is related to it as non-virus carrier in antitumor base
Because of the application in medicine.Belong to biologic pharmacological science field.
Background technology
In recent years, research finds that in the gene therapy of tumour, non-virus carrier is more safe than viral vector, it is to avoid
The viral vectors such as adenovirus cause the possibility of immunogenic response, so that the focus studied as gene delivery.Report at present
The non-virus carrier in road is broadly divided into cationic polymerization species (such as polyethyleneimine PEI), liposome class, inorganic nano material
(such as Nano diamond, Jenner's grain of rice), but the problem of still suffering from some urgent need to resolve is studied mostly.For example, PEI cell
Toxicity is larger;The load efficiency of liposome is low;Inorganic nano material is mostly hydrophobicity, stability and biocompatibility compared with
Difference.In addition, the tumor-targeting difference of non-virus carrier be also cause that gene delivery efficiency is low, internal toxic side effect is big it is crucial because
Element.
Since graphene self-discovery, because its unique monoatomic layer architectural feature and physicochemical property cause great concern,
Except the application in the fields such as electricity, optics cause it is widely studied in addition to, its increasing medical application value is gradually excavated out
Come.Graphene oxide (Graphene Oxide, GO) is the oxide of graphene, is to prepare graphene with oxidation-reduction method
When obtained presoma, there are a variety of oxygen-containing functional groups such as carboxyl, hydroxyl, epoxy radicals in its surface, biocompatibility preferably and
Be conducive to carrying out functional modification to it.Further, since its unique two-dimensional nanostructure, the specific surface area ratio of graphene oxide
General nano-carrier is big, and upper and lower surface can be carried out modification.Polylysine (PLL) is a kind of basic amino acid polymerization
Thing, due to containing multiple free amino, so dissociating in physiological conditions, with positive charge.In addition, PLL is
A kind of stronger compound of hydrophily, biodegradable in vivo, monomer is essential amino acid lysine, so raw
Thing compatibility is preferable.RGD (arginyl-glycyl-aspartic acid) sequence peptide is extracellular matrix (extracellular
Matrix, ECM) in glycoprotein components fibronectin (fibronectin, FN) conservative sequence, to fibronectin and cell
Recognition reaction is played in interaction between surface receptor.Acceptor-integrin of RGD sequence peptide, is a class membrane glycoprotein man
Race.There are some researches show the integrin receptor height expression on some solid tumor cell films.RGDS (argininyl-glycyls-asparagus fern
Aminoacyl-serine) it is a kind of linear RGD sequence peptide, research shows integrin receptor that can be with high expression on tumor cell membrane
Specific binding, therefore can be modified as targeting sequence pair medicine or genophore, realize that the targeting of medicine or gene is passed
Send.
The present invention carries out electropositive and Hydrophilic modification using polylysine PLL to graphene oxide, then utilizes target
Targeting sex modification is carried out to sequence peptide RGDS, is connected by covalent bond, obtains gene VEGF-siRNA delivery vector GO-
PLL-RGDS.The present invention is improves VEGF-siRNA load capacity, from the larger graphene oxide of specific surface area as carrier,
And GO-PLL-RGDS is made by being covalently attached with PLL and Targeting sequences peptide RGDS, then supported V EGF-siRNA, as a result
It was found that 1mg GO-PLL-RGDS can load 100 μ g VEGF-siRNA, gene is applied to be greatly improved in efficiency, and passes through medicine point
Cloth studies the tumor-targeting for confirming delivery system.According to this discovery, the present invention is inventors herein proposed.
The content of the invention
It is an object of the invention to provide a kind of genes delivery system with antitumor activity:GO-PLL-RG DS/
VEGF-siRNA。
Genes delivery system of the present invention, by Antioncogene medicine VEGF-siRNA and targeted delivery vector
GO-PLL-RGDS is prepared from.
Genes delivery system of the present invention, GO-PLL-RGDS and VEGF-siRNA mass ratio is:10:1.
Genes delivery system of the present invention, is nanostructured.
Genes delivery system of the present invention, targeted delivery vector GO-PLL-RGDS is made up of following component:GO、
PLL、RGDS。
It is another object of the present invention to provide the preparation method of genes delivery system.
Preparation method of the present invention:By GO-PLL-RGDS and VEGF-siRNA according to mass ratio 10:1 mixing, room temperature
Stand, produce GO-PLL-RGDS/VEGF-siRNA.
Wherein, targeted delivery vector GO-PLL-RGDS preparation:Graphene oxide GO passes through ring-opening reaction, condensation reaction
It is sequentially connected PLL (polylysine) and RGDS and is deprotected and obtains GO-PLL-RGDS.It is preferred that, gene of the present invention
The preparation method of delivery system, comprises the following steps:
1) GO-PLL is prepared
2.0mg GO ultrasonic disperses are weighed in 4mL ultra-pure waters, are sonicator ultrasound 4h (2 with Probe Ultrasonic Searching instrument
Second batch (-type)).The KOH aqueous solution (pH9) is added to 8mL.10.0mg PLL are added, now there is sediment generation, KOH water is used in continuation
It is 9 that solution (pH11), which is adjusted to pH value,.Reaction bulb is placed in 70 DEG C of oil baths and stirs 24h.Stop heating, centrifugation after 24h
(12000g, 20min), discards supernatant, washes three times, washes unreacted PLL off.It is lyophilized standby.
2) Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OH is prepared
Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OBzl is prepared according to standard method.By Boc-Arg (Tos)-
Gly-Asp (OMe)-Ser-OBzl 430mg are added in 20mL methanol, are added 64.5mg palladium carbons, are passed through hydrogen, are reacted at room temperature,
TLC monitoring reactions.React complete after 5h, filter out palladium carbon, rotation removes methanol, obtains Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-
OH。
3) GO-PLL-RGDS is prepared
10mg Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OH is weighed, is dissolved in 10mL DMF, is added
30mg EDCHCl, stir-activating 15min, form reaction solution A.By 2mg GO-PLL ultrasonic disperses in 10mL DMF, then
It is mixed with the reaction solution A of above-mentioned activation, 30mg HOBt is added, is stirred at room temperature.After 24h, reaction solution is centrifuged
(12000g, 20min), collects sediment, is centrifuged three times with methanol and milli-Q water, removes uncombined Boc-Arg
(Tos)-Gly-Asp (OMe)-Ser-OH and unreacted EDCHCl and HOBt.It is lyophilized to obtain GO-PLL-Ser-Asp
(OMe)-Gly-Arg(Tos)-Boc.It is dispersed in 10mL methanol solutions, ultrasonic 30min, under condition of ice bath, uses NaOH
(2N) solution adjusts its pH value equal to 12, reacts 6h, after question response terminates, and centrifuges 10min with 10000g rotating speed, uses ultra-pure water
Rinsing 3 times, then rinsed three times with absolute ethyl alcohol, 15min is centrifuged with 12000g rotating speeds, ventilation is placed and spontaneously dries.Under ice bath,
The product of above-mentioned drying is dispersed in (TFA in 5mL mix acid liquors:TfOH=4:1, v/v) 1h, is reacted.Transfer them to centrifugation
Guan Zhong, centrifugation (7000g, 15min) discards supernatant.Three times are rinsed with absolute ether rinsing and ultra-pure water obtain target production respectively
Thing GO-PLL-Ser-Asp-Gly-Arg (GO-PLL-RGDS).5mL ultra-pure waters are eventually adding, ultrasonic disperse finds that product disperses
Property is good.Dispersion liquid is loaded into bag filter (molecular cut off:8000), dialysis 24h removes impurity.Will be scattered after dialysis purification
Liquid is freezed, and determines infrared spectrum.
4) GO-PLL-RGDS/VEGF-siRNA is prepared
2mg GO-PLL-RGDS are weighed, are dispersed in 2mL DEPC water, ultrasonic 40min, 1000 μ g/mL are configured to respectively
GO-PLL-RGDS storing solutions, then 1.0OD VEGF-siRNA (33 μ g) are dissolved in 250 μ L DEPC water.By GO-PLL-
RGDS is mixed with VEGF-siRNA according to certain N/P ratios, is stored at room temperature 30min, is produced GO-PLL-RGDS/VEGF-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 evaluate genes delivery system GO-PLL-RGDS/VEGF-siRNA in gene and
Protein level lowers the efficiency of vegf expression.
It is another object of the present invention to evaluate genes delivery system GO-PLL-RGDS/VEGF-siRNA to suppress tumour
The effect of cell propagation.
It is another object of the present invention to evaluate genes delivery system GO-PLL-RGDS/VEGF-siRNA to suppress lotus
The effect of S180 sarcoma mice tumors grews.
It is another object of the present invention to evaluate genes delivery system GO-PLL-RGDS/VEGF-siRNA targeting lotuses
The targeting effect of S180 sarcoma mouse tumors.
The following word occurred in specification is further explained:
GO:Graphene Oxide, graphene oxide
PLL:Polylysine
RGDS:Arg-Gly-Asp-Ser, Arg-Gly-Asp-serine
The english abbreviation that other positions occur in specification, please be set out to make further explanation:
VEGF:VEGF
PEI:Polyethyleneimine
TLC:TLC
DMF:N,N-dimethylformamide
EDC·HCl:1- (3- dimethylaminopropyls) -3- ethyl-carbodiimide hydrochlorides
HOBt:I-hydroxybenzotriazole
TFA:Trifluoroacetic acid
TfOH:Trifluoromethanesulfonic acid
OD values:OD value
DEPC:Pyrocarbonic acid diethyl ester
N/P ratios:Nitrogen/phosphorus ratio, refers to the ratio of genophore and gene
NC:Non-homogeneous-siRNA
DMEM:Improve Du Shi Eagle's mediums
FBS:Hyclone
FAM:Hydroxyl fluorescein
MTT:3- (4,5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromides
DMSO:Dimethyl sulfoxide (DMSO)
NS:Physiological saline
Cy3:Cyanine dye fluorescence labeling
ELISA:Enzyme linked immunosorbent assay (ELISA)
RT-qPCR:Real-time quantitative reverse transcriptase chain reaction
Brief description of the drawings:
Fig. 1 .GO-PLL-RGDS/VEGF-siRNA syntheti c route.
Note:Reaction condition:I:KOH,70℃.II:Boc-Arg(Tos)-Gly-Asp(OMe)-Ser-OH/EDC;NaOH/
CH3OH;Trifluoroacetic acid/trifluoromethanesulfonic acid III:Incubation at room temperature stands 30min.
Fig. 2 Ago-Gel retardation experiment result figures.
Fig. 3 .GO, GO-PLL-RGDS and GO-PLL-RGDS/VEGF-siRNA transmission electron microscope picture.
Note:GO (A), GO-PLL-RGDS (B), GO-PLL-RGDS/VEGF-siRNA (C) transmission electron micrograph
Fig. 4 blank water, GO, GO-PLL-RGDS and GO-PLL-RGDS/VEGF-siRNA AFM.
Note:Water (A), GO (B), GO-PLL-RGDS (C), GO-PLL-RGDS/VEGF-siRNA (D) AFM
Picture.
The result figure that Fig. 5 .HeLa cells are absorbed to GO-PLL-RGDS/VEGF-siRNA.
A:Blank control group
B:Naked FAM-VEGF-siRNA groups
C:GO-PLL-RGDS/VEGF-siRNA groups
D:Lipo/FAM-VEGF-siRNA groups
The measurement result of Fig. 6 .GO-PLL-RGDS/VEGF-siRNA real time RT quantitative polyase chain reaction.(n=
3)
Note:GPR is GO-PLL-RGDS reference literary style;NC is non-homogeneous negative control siRNA.
Fig. 7 .GO-PLL-RGDS/VEGF-siRNA enzyme-linked immunosorbent assay result figure.(n=3)
Note:GPR is GO-PLL-RGDS reference literary style;NC is non-homogeneous negative control siRNA.
Fig. 8 .MTT methods survey GO-PLL-RGDS cytotoxicity result.(n=3)
Fig. 9 .GO-PLL-RGDS/VEGF-siRNA suppress HeLa cel l proliferation results.(n=3)
Note:GPR is GO-PLL-RGDS reference literary style;NC is non-homogeneous negative control siRNA.
Figure 10 .GO-PLL-RGDS/VEGF-siRNA internal antitumor activity result.(n=10)
Note:Blank Control are blank control physiological saline group;Negative Control are VEGF-siRNA negative
Control group;Positive Control are adriamycin group.GPR is GO-PLL-RGDS reference literary style.
Dosage:Blank control physiological saline group is 0.2mL/, and negative control group VEGF-siRNA dosages are
GO-PLL-RGDS dosage is 3mg/kg in 0.3mg/kg, GPR/VEGF-siRNA group, and VEGF-siRNA dosages are 0.3mg/
Kg, positive control doxorubicin dosages are 2 μm of ol/kg.
A is each group tumour photo, and B is tumor weight, and C is tumour inhibiting rate.
Figure 11 .GO-PLL-RGDS/VEGF-siRNA cancer target effect.
A:Blank control group
B:Naked Cy3-VEGF-siRNA groups
C:GO-PLL-RGDS/Cy3-VEGF-siRNA groups
Embodiment:
In order to which the present invention is expanded on further, a series of embodiments are given below.These embodiments be entirely it is illustrative, it
Only be used for the present invention is specifically described, be not construed as limitation of the present invention.
Embodiment 1 prepares GO-PLL-RGDS/VEGF-siRNA
Prepare GO-PLL
2.0mg GO ultrasonic disperses are weighed in 4mL ultra-pure waters, are sonicator ultrasound 4h (2 with Probe Ultrasonic Searching instrument
Second batch (-type)).The KOH aqueous solution (pH9) is added to 8mL.10.0mg PLL are added, now there is sediment generation, KOH water is used in continuation
It is 9 that solution (pH11), which is adjusted to pH value,.Reaction bulb is placed in 70 DEG C of oil baths and stirs 24h.Stop heating, centrifugation after 24h
(12000g, 20min), discards supernatant, washes three times, washes unreacted PLL off.It is lyophilized standby.Determine infrared spectrum.Fourier
Leaf conversion infrared spectrum (FTIR, Thermo ScientificTM NicoletTM iSTM5, cm-1):3242.74,2935.15,
1635.09,1520.51,1340.47,1037.13,601.35.
Prepare Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OH
Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OBzl is prepared according to standard method.By Boc-Arg (Tos)-
Gly-Asp (OMe)-Ser-OBzl 430mg are added in 20mL methanol, are added 64.5mg palladium carbons, are passed through hydrogen, are reacted at room temperature,
TLC monitoring reactions.React complete after 5h, filter out palladium carbon, rotation removes methanol, obtains Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-
OH。
Determine nuclear magnetic resonance,1H-NMR(500MHz,DMSO-d6):δ/ppm=δ/ppm=12.656 (s, 1H), 8.169
(d, J=7.50Hz, 1H), 8.047 (t, J=8.00Hz, 1H), 7.924 (d, J=7.50Hz, 1H), 7.630 (d, J=
8.00Hz, 2H), 7.283 (d, J=8.50Hz, 2H), 7.016 (m, 1H), 6.915 (d, J=8.00Hz, 1H), 6.614 (m,
1H),4.725(dd,J1=13.50, J2=7.50Hz, 1H), 4.209 (m, 1H), 3.890 (m, 1H), 3.782 (d, J=
5.50Hz,2H),3.711(dd,J1=11.00Hz, J2=5.00Hz, 1H), 3.619 (dd, J1=11.00Hz, J2=
5.00Hz,1H),3.595(s,3H),3.308(s,2H),3.029(s,2H),2.746(dd,J1=16.00Hz, J2=
5.50Hz,1H),2.531(dd,J1=16.00Hz, J2=8.00Hz, 1H), 2.343 (s, 3H), 1.603 (s, 1H), 1.450
(m,2H),1.379(s,9H).
13C-NMR(125MHz,DMSO-d6):δ/ppm=172.87,172.15,170.96,170.63,169.18,
157.10,155.89,142.26,141.49,129.50,126.04,78.68,61.71,55.33,54.43,51.98,
49.55,42.47,36.67,29.63,28.67,21.35.
ESI-MS(m/z):[M-1]-:700.47;[2M-1]-:1401.70.
Prepare GO-PLL-RGDS
10mg Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OH is weighed, is dissolved in 10mL DMF, is added
30mg EDCHCl, stir-activating 15min, form reaction solution A.By 2mg GO-PLL ultrasonic disperses in 10mL DMF, then
It is mixed with the reaction solution A of above-mentioned activation, 30mg HOBt is added, is stirred at room temperature.After 24h, reaction solution is centrifuged
(12000g, 20min), collects sediment, is centrifuged three times with methanol and milli-Q water, removes uncombined Boc-Arg
(Tos)-Gly-Asp (OMe)-Ser-OH and unreacted EDCHCl and HOBt.It is lyophilized to obtain GO-PLL-Ser-Asp
(OMe)-Gly-Arg(Tos)-Boc.It is dispersed in 10mL methanol solutions, ultrasonic 30min, under condition of ice bath, uses NaOH
(2N) solution adjusts its pH value equal to 12, reacts 6h, after question response terminates, and centrifuges 10min with 10000g rotating speed, uses ultra-pure water
Rinsing 3 times, then rinsed three times with absolute ethyl alcohol, 15min is centrifuged with 12000g rotating speeds, ventilation is placed and spontaneously dries.Under ice bath,
The product of above-mentioned drying is dispersed in (TFA in 5mL mix acid liquors:TfOH=4:1, v/v) 1h, is reacted.Transfer them to centrifugation
Guan Zhong, centrifugation (7000g, 15min) discards supernatant.Three times are rinsed with absolute ether drift and ultra-pure water obtain target product respectively
GO-PLL-Ser-Asp-Gly-Arg(GO-PLL-RGDS).5mL ultra-pure waters are eventually adding, ultrasonic disperse finds product dispersiveness
Well.Dispersion liquid is loaded into bag filter (molecular cut off:8000), dialysis 24h removes impurity.By the dispersion liquid after dialysis purification
It is lyophilized, determine infrared spectrum.Fourier transform infrared spectrum (FTIR, Thermo ScientificTM NicoletTM
iSTM5,cm-1):3253.22,2922.98,1643.00,1518.86,1056.93,666.97.
Prepare GO-PLL-RGDS/VEGF-siRNA
2mg GO-PLL-RGDS are weighed, are dispersed in 2mL DEPC water, ultrasonic 40min, 1000 μ g/mL are configured to respectively
GO-PLL-RGDS storing solutions, then 1.0OD VEGF-siRNA (33 μ g) are dissolved in 250 μ L DEPC water.By GO-PLL-
RGDS is mixed with VEGF-siRNA according to certain N/P ratios, is stored at room temperature 30min, you can obtain transfection composite GO-PLL-
RGDS/VEGF-siRNA。
Determine the load capacity of VEGF-siRNA in GO-PLL-RGDS/VEGF-siRNA
Prepare sample and prepare GO-PLL-RGDS with RNA dilution buffers:VEGF-siRNA (mass ratio) is 2:1,8:1,
10:1,20:1,30:Isosorbide-5-Nitrae 0:1,50:The 1 μ L of mixing suspension 100, shake up, are stored at room temperature 30min.
Experimental procedure weighs 0.5g agaroses in 100mL beakers, adds 5mL TBE (10 ×) and 45mL deionized waters,
High fire heating 2min dissolves agarose in being placed in micro-wave oven.10 μ L ethidium bromide solutions (10mg/mL) are added after slightly cooling down,
Stir evenly, pour into mould and (clean and dried with absolute ethyl alcohol rinse in advance), insert comb, it is standby after solidification.Take 30mL TBE
(10 ×) and 270mL ultra-pure waters, are poured into electrophoresis tank as electrophoresis liquid after being well mixed.The Ago-Gel after solidification is taken out,
It is placed in electrophoresis tank, 20 μ L is loaded per hole.Turn on the power, voltage is arranged to 120V, set of time is 20min.From electrophoresis tank
Middle taking-up Ago-Gel, is placed in gel imager and observes and take pictures.
Experimental result as shown in Fig. 2 different quality than GO-PLL-RGDS/VEGF-siRNA fluorescent bands than trip
It is weak from siRNA groups (control), illustrate that VEGF-siRNA is arrested in loading hole to some extent, show VEGF-siRNA successes
By partially or completely steady load on GO-PLL-RGDS surfaces.When mass ratio is 10:When 1, fluorescent bands are wholly absent, explanation
Now VEGF-siRNA is arrested in loading hole completely, i.e. VEGF-siRNA is supported on GO-PLL-RGDS surfaces completely.Institute
So that 1mg GO-PLL-RGDS can load 100 μ g VEGF-siRNA.
Embodiment 2 determines GO-PLL-RGDS and GO-PLL-RGDS/VEGF-siRNA nanostructured
GO, GO-PLL-RGDS aqueous dispersions that experimental method takes 100 μ L concentration to be 1000 μ g/mL are diluted to pure water
2mL.According to mass ratio 10:1 concentration for preparing GO-PLL-RGDS/VEGF-siRNA aqueous dispersions, i.e. GO-PLL-RGDS is 50 μ
G/mL, VEGF-siRNA concentration are 5 μ g/mL.Respectively take 20 μ L to be added drop-wise to carbon to support on film (copper mesh), be placed in 37 DEG C of baking ovens and do
Dry 24h.Each sample is placed in and is observed under transmission electron microscope (TEM, JEM-1230, JEOL, Japan) and obtains photo.20 μ L are respectively taken again
It is added drop-wise on mica sheet, is placed in room temperature natural air drying.Each sample is placed in AFM (AFM, Veeco
Instruments, Inc, Plainview, USA) under observe and obtain photo.Using water as blank background.
Experimental result from the images of transmissive electron microscope in Fig. 3 we can see that GO has typical fold flake nano structure,
Particle size is uneven, in the range of 100-350nm.And GO-PLL-RGDS particle size is significantly smaller, 60-180nm it
Between,.This is due to hydrophilic compounds PLL and RGDS modification, and the water dispersible for making GO-PLL-RGDS more preferably, is not susceptible to
Aggregation.And VEGF-siRNA GO-PLL-RGDS/VEGF-siRNA particle diameter increase has been loaded, partly reach more than 200nm.
In addition, the atomic force microscope images in Fig. 4 are shown, GO-PLL-RGDS thickness is increased by unmodified 1.7nm or so (GO)
It is added to 3.0nm.Compared with GO-PLL-RGDS, GO-PLL-RGDS/VEGF-siRNA particle diameter and thickness have increase, and thickness increases
It is added to 4.4nm or so.
Embodiment 3 determines GO-PLL-RGDS and GO-PLL-RGDS/VEGF-siRNA Zeta potential and hydration particle diameter
The aqueous dispersions that experimental method prepares 100 μ g/mL GO and GO-PLL-RGDS are standby.Prepare 100 μ g/mL (i.e.
The concentration that GO-PLL-RGDS concentration is 100 μ g/mL, VEGF-siRNA is 10 μ g/mL) GO-PLL-RGDS/VEGF-
SiRNA aqueous dispersions.With the particle diameter distribution and Zeta potential of laser particle size analyzer determination sample.
Experimental result GO, GO-PLL-RGDS and GO-PLL-RGDS/VEGF-siRNA Zeta potential value is respectively-
40.59±2.23mV、25.87±0.47mV、15.36±2.62mV.GO, GO-PLL-RGDS and GO-PLL-RGDS/VEGF-
SiRNA hydration particle diameter is respectively 275.8 ± 2.7nm, 162.9 ± 4.5nm, 196.6 ± 2.7nm.Zeta potential value is by negative value
It is changed on the occasion of also further illustrating GO-PLL-RGDS successful modification.After supported V EGF-siRNA, compound GO-PLL-
RGDS/VEGF-siRNA still shows certain electropositive, and this is significant to cellular uptake.
The laser confocal microscope of embodiment 4 investigates HeLa cells and GO-PLL-RGDS/VEGF-siRNA cell is taken the photograph
Take situation
Experimental method
1) medicine is prepared:By GO-PLL-RGDS ultrasonic disperses in DEPC water, the deposit that concentration is 1000 μ g/mL is made into
Liquid.(thickness is no more than 1cm), ultraviolet irradiation 24h are placed it in sterile sampling bottle.By 1.0OD (2.5nmol, 33 μ g)
FAM-VEGF-siRNA is dissolved in 250 μ L DEPC water, is made into 10nmol/mL storing solution.Lipo's (LipoRNAiMAX)
Concentration recommended amounts to specifications.100nmol/L is made into respectively according to following component ratio (by siRNA densimeter)
Naked FAM-VEGF-siRNA, GO-PLL-RGDS/FAM-VEGF-siRNA, Lipo/FAM-VEGF-siRNA.
1. naked FAM-VEGF-siRNA (negative control group):24 μ L FAM-VEGF-siRNA storing solutions are taken to be added to 2376 μ
Mixed in L serum free mediums standby.
2.GO-PLL-RGDS/FAM-VEGF-siRNA (experimental group):Take 24 μ L FAM-VEGF-siRNA and 34 μ L GO-
PLL-RGDS storing solutions are added in 2342 μ L serum free mediums and mixed, and are stored at room temperature 30min standby.
3.Lipo/FAM-VEGF-siRNA (positive controls):Take 24 μ L FAM-VEGF-siRNA and 20 μ L Lipo
(LipoRNAiMAX) storing solution is added in 2356 μ L serum free mediums and mixed, and is stored at room temperature 30min standby.
2) cell recovery and culture:
1. opening water-bath, preheat (37 DEG C).
2. taking out the HeLa cells frozen from liquid nitrogen container, it is put into rapidly in 37 DEG C of water-baths, is dissolved in 1min.
3. the cell of dissolving is moved rapidly into centrifuge tube in super-clean bench, 2mL complete medium is added.Centrifugation
(1000g, 5min).
4. abandoning supernatant, 1mL culture mediums are added, piping and druming is uniform, moves into 25cm2In blake bottle, 4mL culture mediums are added, 37 DEG C,
5%CO2Incubator in cultivate.
3) passage:Former culture medium is discarded, 2mL PBS carefully purging 2 times is added, adds 1mL pancreatin, piping and druming is equal
It is even, it is put into after incubator, 4min, is observed under inverted microscope, cell retracts into spherical suspending.3mL is added to train completely
Base is supported, digestion is terminated.Careful piping and druming cell, and be transferred in centrifuge tube, centrifuge (1000g, 5min), carefully topple over supernatant
Liquid, adds the piping and druming of 4mL complete mediums uniform.Each 2mL cell suspensions of drawing are in 25cm2In blake bottle.3mL trainings are each added again
Nutrient solution, piping and druming is uniform, is put into incubator, 37 DEG C, 5%CO2Culture.
4) laser confocal microscope is determined:By 3 × 105Individual (2.0 × 105/ ml) HeLa cells kind in internal diameter 10mm swash
In light copolymerization Jiao's capsule, 37 DEG C, 5%CO224h is cultivated, (is free of after changing the pastille culture medium for preparing after cell attachment into
FBS).Blank control group (adding isometric blank cultures), positive controls, negative control group, experimental group are set respectively.Turn
Contaminate after 4h, discard pastille culture medium, washed with PBS three times, the working solution 1mL of Hoechst 33342 are added per ware, be incubated after 15min
Discard, washed with PBS three times, a small amount of PBS is finally reserved in every ware prevents dry plate.It is placed under laser confocal microscope and observes simultaneously
Take pictures.
Experimental result FAM is a kind of fluoresceins labelled reagent of carboxylated, excitation wavelength 492nm, launch wavelength
518nm.As shown in figure 5, A groups are blank control groups, Hoechst33342 is by nuclei dyeing au bleu.It can only be observed under B group mirrors
To the nucleus of blueness, it is known that, free FAM-VEGF-siRNA can not be by cellular uptake.In C groups, in the observation of nucleus external enwergy
To green fluorescence, it is observed that green fluorescence integrated distribution is in kytoplasm under Merge passages, it is known that, FAM-VEGF-
SiRNA enters cell by GO-PLL-RGDS Successful deliveries.D groups can also be observed green as positive control around nucleus
Color fluorescence.Therefore, it can draw to draw a conclusion from result above:Transfection composite GO-PLL-RGDS/FAM-VEGF-siRNA into
Work(is positioned in cytoplasm by cellular uptake.
The real time RT quantitative polyase chain reaction of embodiment 5 evaluates GO-PLL-RGDS/VEGF-siRNA silences VEGF
The effect of encoding gene
Experimental method
1) medicine is prepared:By GO-PLL-RGDS ultrasonic disperses in DEPC water, concentration is made into for 1000 μ g/mL (storing solutions
1) storing solution.(thickness is no more than 1cm) is individually positioned in sterile sampling bottle, and ultraviolet irradiation 24h is standby.By 1.0OD
(2.5nmol, 33 μ g) homo-VEGF-siRNA is dissolved in 250 μ L DEPC water, is made into 10nmol/mL storing solution.
1.0OD (2.5nmol, 33 μ g) non-homogeneous-siRNA (NC) is dissolved in 250 μ L DEPC water, is made into 10nmol/mL deposit
Liquid.Lipo (LipoRNAiMAX) concentration recommended amounts to specifications.
According to the form below prepares each 6mL of pastille culture medium (concentration is 100nM):
The medicine configuration proportion of table 1.
2) cell transfecting:Take the logarithm phase HeLa cell, counting, adjustment cell concentration to 1.6 × 105Individual/mL (cultures used
Base is the DMEM culture mediums without antibiotic but containing 10%FBS).2mL cell suspension inoculations are drawn per hole into six orifice plates, are made every
Hole number of cells is 3.2 × 105It is individual, 37 DEG C, 5%CO2Culture.After after cell attachment after 12h, culture medium is carefully suctioned out.Per hole
Each 2mL of pastille culture medium prepared is added, every group sets 3 multiple holes, and blank control group adds isometric DMEM.37 DEG C, 5%
CO2Under the conditions of continue cultivate 6h.After 6h, pastille culture medium is carefully suctioned out, complete medium is changed into, continues to cultivate 42h.
When carrying out cell transfecting, it has been found that if giving the use of the cell before transfection reagent Lipo (LipoRNAiMAX)
Containing dual anti-complete medium culture, find that cell shape in 30min becomes round by fusiformis after transfection reagent Lipo is added
Shape, floating is in the medium.And GO-PLL-RGDS/VEGF-siRNA is then without this phenomenon.Afterwards, with without dual anti-culture medium
Then culture is given transfection reagent and is transfected to adherent, and this phenomenon disappears.
3) the full RNA of cell extraction:Work top is wiped with RNase scavenger before operation, using without enzyme centrifuge tube and rifle
Head.
1. six orifice plates for taking transfection to terminate, discard culture medium, 1mL Trizol lysate (Trizol lysates are added per hole
Consumption calculated according to hole area, 10cm2Plus 1mL Trizol lysates), blown and beaten with liquid-transfering gun it is uniform, be transferred to after 3min from
In heart pipe, 5min is stood so that nucleic acid-protein compound is kept completely separate.
2. often pipe adds 0.2mL chloroforms, 15s is acutely shaken, 3min is stored at room temperature, centrifuged (4 DEG C, 12000g, 15min).
3. mixed liquor is divided into three layers after centrifugation, here is red phenol chloroform layer, is aqueous phase above, and RNA has aqueous phase
In, about of the total volume 50%.
4. precipitate RNA:Upper strata aqueous phase is slowly carefully transferred in another centrifuge tube, 0.5mL isopropanols are added, mixed
It is even, 10min is stored at room temperature, is then centrifuged for (4 DEG C, 12000g, 15min), it is seen that white precipitate.
5. wash RNA:Supernatant is abandoned, the 75% ethanol (V that 1mL has been configured is addedAlcohol/VDEPC water=3:1).Wash RNA precipitate,
Concussion shakes up, and centrifuges (4 DEG C, 12000g, 15min), repeated washing three times.Supernatant is abandoned, dries and (notes:It need not be completely dried, it is no
Then greatly reduce its solubility).
6.RNA is redissolved:RNA is resuspended with DEPC water (the often μ L of pipe 60), piping and druming several times, makes RNA complete with low power ultrasound
Dissolving, 55 DEG C of water-baths are incubated 10min, are cooled to room temperature, -80 DEG C of preservations after packing.
7. purity and concentration mensuration:After DEPC water correction Nanodrop-1000, measurement is dropped in the 2 μ L RNA aqueous solution
On arm, selection nucleic acid RNA-measure measures OD260/OD280 values and concentration value.
4) reverse transcription:
1. preparation of samples:According to the RNA concentration of measure, the RNA volumes (sample needs 2 μ g) needed for calculating configure sample.
2. each reacting hole adds 10 μ 2 × RT of L Buffer, 1 μ L RT EnzymeMix and the RNA solution of respective volume,
DEPC water supplies 20 μ L, is added to MicroAmpTMIn Optical 96-Well Reaction Plate, sealed with sealed membrane,
4 DEG C, 1000g centrifuges 3min, removes bubble.
3. by MicroAmpTMOptical 96-Well Reaction Plate are put into Real Time PCR System
In 7500, condition, 94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 25s, 72 DEG C of 7min, 4 DEG C of ∞ are set.Totally 30 circulations.Obtain
CDNA is obtained, its concentration is surveyed.
5) polymerase chain reaction:
1.cDNA applied sample amount is 50ng, and the volume for each group cDNA samples that need to be added is calculated according to its concentration.According to
Lower each group partial volume prepares mixed liquor:Each reacting hole mixeding liquid volume is 50 μ L, including 25 μ L Gene
ExpressionMaster Mix, 2.5 μ L probes mark primer (VEGF or GAPDH), the cDNA samples of respective volume and
DEPC water.
2. the mixed liquor prepared is added into MicroAmpTMIn Optical 96-Well Reaction Plate, per hole 50
μ L, centrifuge (4 DEG C, 1000g, 3min), mixed liquor is sunken to reacting hole bottom and discharge bubble,
3. 96 orifice plates are put into Real-Time PCR System7500, condition, Hold are set:50 DEG C of 2min, 95 DEG C
10min, Cycle (40cycles), 95 DEG C of 15s, 60 DEG C of 1min.Start amplification.
4. data processing.Using GAPDH as internal reference, with 2-△△CtEach mRNA of method relative quantification amount.Above-mentioned experiment repeats three
It is secondary.
Experimental result
The VEGF-mRNA of HeLa cells under GO-PLL-RGDS/VEGF-siRNA effects of table 2. relative quantity (mean value ±
SD, n=3)
As can be seen from Figure 6:The negative control group free VEGF-siRNA's and non-homogeneous siRNA of GO-PLL-RGDS/
MRNA relative expression quantities are compared with blank control group, there was no significant difference (P>0.05), i.e., both of which can not effectively lower mRNA
Expression.And experimental group GO-PLL-RGDS/VEGF-siRNA mRNA relative expression quantity compared with blank control group have significantly
Sex differernce (P<0.01), GO-PLL-RGDS/VEGF-siRNA can effectively lower mRNA expression, and downward rate is 40.86 ±
4.09%, it is suitable with positive controls Lipo/VEGF-siRNA.
The enzyme linked immunosorbent assay of embodiment 6 evaluates the effect that GO-PLL-RGDS/VEGF-siRNA lowers vegf expression
Experimental method
1) medicine is prepared:By GO-PLL-RGDS ultrasonic disperses in DEPC water, concentration is made into for 1000 μ g/mL (storing solutions
1) storing solution.(thickness is no more than 1cm) is individually positioned in sterile sampling bottle, and ultraviolet irradiation 24h is standby.By 1.0OD
(2.5nmol, 33 μ g) homo-VEGF-siRNA is dissolved in 250 μ LDEPC water, is made into 10nmol/mL storing solution.1.0OD
(2.5nmol, 33 μ g) non-homogeneous-siRNA (NC) is dissolved in 250 μ LDEPC water, is made into 10nmol/mL storing solution.Lipo
(LipoRNAiMAX) concentration recommended amounts to specifications.Distinguish compounding pharmaceutical according to the method in 2.1.3.
According to the form below prepares each 6mL of pastille culture medium (concentration is 100nM):
The medicine configuration proportion of table 1.
2) cell transfecting is taken the logarithm phase HeLa cell, counting, adjustment cell concentration to 1.6 × 105Individual/mL (cultures used
Base is the DMEM culture mediums without antibiotic but containing 10%FBS).2mL cell suspension inoculations are drawn per hole into six orifice plates, are made every
Hole number of cells is 3.2 × 105It is individual, 37 DEG C, 5%CO2Culture.After after cell attachment after 12h, culture medium is carefully suctioned out.Per hole
The pastille culture medium 2mL prepared is added, every group sets 3 multiple holes, and blank control group adds isometric DMEM.37 DEG C, 5%CO2
Under the conditions of continue cultivate 6h.After 6h, pastille culture medium is carefully suctioned out, complete medium is changed into, continues to cultivate 42h.Due to VEGF
It is secreted protein, it is that can detect its content directly to collect cell culture medium supernatant.Specific method is:Draw the culture medium per hole
In centrifuge tube, centrifuge (4 DEG C, 8000g, 10min), take supernatant, then centrifuge (4 DEG C, 12000g, 10min), take supernatant.
3) BCA Protein Assay Kit determine total protein concentration in nutrient solution supernatant
With BCA protein quantification kits, standard curve is described according to explanation, the concentration per histone sample is determined (by egg
White sample stoste dilutes 5 times of measure).
4) in Human VEGF ELISA kits determination sample albumen VEGF content
1. the loading volume of each sample is calculated (based on each μ g of sample loading 130 according to the concentration per histone sample
Calculate) 50 μ L sample liquid is made into ultra-pure water.
2. dilute Wash Buffer (25 ×), Streptavidin-HRP (100 ×) according to thinner ratio.
3. all holes add 50 μ L Incubation Buffer per hole in addition to Chromogen blanks.In standard sample wells
Add 100 μ L Standard Diluent Buffer, sample well add 50 μ L Standard Diluent Buffer and
50 μ L sample liquid, and three multiple holes are set.Covered with sealed membrane, 2h is placed at room temperature.
4. carefully absorbing liquid, 4 times are washed with Wash Buffer (1 ×) per hole, every time 100 μ L/ holes.Except Chromogen
100 μ L Hu VEGF Biotin Conjugate are added outside blanks per hole.Covered with sealed membrane, room temperature places 1h.
5. carefully absorbing liquid, Wash Buffer (1 ×) are washed 4 times.100 μ are added per hole in addition to Chromogen blanks
L Streptavidin-HRP, are covered with sealed membrane, and 30min is placed at room temperature.
6. carefully absorbing liquid, Wash Buffer (1 ×) are washed 4 times, and 100 μ L Stabilized are added per hole
Chromogen, another 3 Chromogen blanks also add 100 μ L Stabilized Chromogen hole.Cover sealed membrane,
Lucifuge, is stored at room temperature after 30min, and 100 μ L Stop Solution are added per hole, are rapped, to complete discoloration.Survey at 450 nm
Determine OD values.
Above-mentioned experiment is in triplicate.
Experimental result
Relative quantity (the mean value ± of the vegf protein of HeLa cells under GO-PLL-RGDS/VEGF-siRNA effects of table 2.
SD%, n=3)
It can be seen from figure 7 that the negative control group free VEGF-siRNA and non-homogeneous siRNA of GO-PLL-RGDS/
VEGF relative expression quantities are compared with blank control group, there was no significant difference (P>0.05), i.e., both of which can not effectively lower VEGF
Expression.And experimental group GO-PLL-RGDS/VEGF-siRNA VEGF relative expression quantity compared with blank control group have significantly
Sex differernce (P<0.01), GO-PLL-RGDS/VEGF-siRNA is capable of the expression of effective down-regulation protein, and downward rate is 51.71 ±
4.54%.
Embodiment 7 evaluates GO-PLL-RGDS/VEGF-siRNA cytotoxicity and suppresses the effect of tumor cell proliferation
Experimental method
1) preparation of reagents
Complete medium:The hyclone 50mL (10%) of inactivation, penicillin are added in DMEM culture mediums (450mL)
100U/mL, the μ g/mL of streptomysin 100.
Serum free medium:DMEM culture mediums.
The inactivation of hyclone:Finished product hyclone is incubated 30min in 56 DEG C of water-baths, 50mL/ parts, -20 are packed as
DEG C preserve.
PBS:PBS powder is illustratively configured to pH7.4 with ultra-pure water, concentration is 0.01M PBS, and high pressure is gone out
Bacterium.4 DEG C of preservations.
Pancreatin:Finished product pancreatin is dispensed into 10mL/ parts, 4 DEG C of preservations.
MTT working solutions:50mg MTT powder is weighed, is dissolved in 10mL PBS, ultrasound makes it fully molten
Solution, is configured to 5mg/mL working solution, with 0.22 μm of filter membrane filtration sterilization.
2) medicine is prepared
By intermediate product GO-PLL ultrasonic disperses in DEPC water, the storing solution 1 that concentration is 1000 μ g/mL is made into.
By GO-PLL-RGDS ultrasonic disperses in DEPC water, concentration is made into for 1000 μ g/mL (storing solution 2) and 500 μ g/
ML (storing solution 3) storing solution.(thickness is no more than 1cm) is individually positioned in sterile sampling bottle, and ultraviolet irradiation 24h is standby
With.1.0OD (2.5nmol, 33 μ g) homo-VEGF-siRNA is dissolved in 500 μ LDEPC water, 5nmol/mL deposit is made into
Liquid.1.0OD (2.5nmol, 33 μ g) non-homogeneous-siRNA (NC) is dissolved in 500 μ LDEPC water, is made into 5nmol/mL deposit
Liquid 4.Lipo (LipoRNAiMAX) concentration recommended amounts to specifications.
1.GO-PLL-RGDS Cytotoxic evaluation
GO-PLL and GO-PLL-RGDS:GO-PLL and GO-PLL-RGDS storing solutions are diluted into concentration respectively is respectively
50,100,200,300,400,500,750 μ g/mL GO-PLL mother liquors and GO-PLL-RGDS mother liquors.
The effect of 2.GO-PLL-RGDS/VEGF-siRNA suppression HeLa cells propagation
Blank background group:Blank cultures are only added, without cell.
Blank control group:Without processing, the cell of the culture medium of same volume is only added.
Negative control group:Non-homogeneous-the siRNA of GO-PLL-RGDS, VEGF-siRNA, GO-PLL-RGDS/, Lipo/ are non-same
Source-siRNA
Positive controls:Lipo/VEGF-siRNA
Experimental group:GO-PLL-RGDS/VEGF-siRNA, GO-PLL-RGDS, VEGF-siRNA, GO-PLL-RGDS/ are non-same
Non-homogeneous-the siRNA of source-siRNA, Lipo/, Lipo/VEGF-siRNA and GO-PLL-RGDS/VEGF-siRNA concentration is
10nM, 40nM, 80nM, 120nM.
3) mtt assay evaluates GO-PLL-RGDS cytotoxicity
Take the logarithm phase HeLa cell, counting, adjustment cell concentration to 4 × 104Individual/mL.96 orifice plates PBS fluid-tights, per hole
Take 100 μ L cell suspension kind plates, 37 DEG C, 5%CO2Culture.After after cell attachment after 12h, it is administered.Above-mentioned prepare is added per hole
GO-PLL mother liquors or the μ L of GO-PLL-RGDS mother liquors 25, the final concentration for making the GO-PLL-RGDS or GO-PLL of addition is 10,20,
40,60,80,100,150 μ g/mL, each concentration sets 6 multiple holes, and blank background group and blank control control group add 25 μ L
DEPC water.37 DEG C, 5%CO2Under the conditions of continue cultivate 48h.After 48h, add 25 μ LMTT working solutions per hole, left-right and front-back rocks
It is even, 37 DEG C of incubation 4h.4000g is centrifuged 10 minutes after 4h.It is careful to suction out per hole culture medium.The μ L of DMSO 150 are added per hole, with micro-
Type oscillator shakes (500r, 10min).On ELIASA, absorbance (OD) value is determined by 570nm of Detection wavelength, calculates thin
Born of the same parents' survival rate.Cell survival rate (%)=(experimental group OD values-blank group OD values)/(control group OD values-blank group OD values) ×
100%.Above-mentioned experiment is in triplicate.
4) GO-PLL-RGDS/VEGF-siRNA suppresses the effect of HeLa cells propagation
Take the logarithm phase HeLa cell, counting adjusts cell concentration to 4 × 10 with complete medium4Individual/mL.96 orifice plates are used
PBS fluid-tights, 100 μ L cell suspension kind plates, 37 DEG C, 5%CO are taken per hole2Culture.After 12h, cell attachment is treated, it is careful to suction out completely
Culture medium, 100 μ L serum free mediums are added per hole.Then, the GO-PLL- that 25 μ L prepare various concentrations is separately added into per hole
Non-homogeneous-the siRNA of RGDS, VEGF-siRNA, GO-PLL-RGDS/, the non-homogeneous-siRNA of Lipo/, Lipo/VEGF-siRNA and
GO-PLL-RGDS/VEGF-siRNA is transfected, and each group final concentration is respectively 10,40,80,120nM (based on siRNA).Transfection
After 6h, each hole changes isometric complete medium into, continues to cultivate 42h.Add 25 μ L MTT working solutions per hole, left-right and front-back rocks
Uniformly, 37 DEG C of incubation 4h.4000g is centrifuged 10 minutes after 4h.It is careful to suction out per hole culture medium.The μ L of DMSO 150 are added per hole, are used
Microoscillator shakes 500r, 15min.On ELIASA, absorbance (OD) value is determined by 570nm of Detection wavelength, calculates thin
Born of the same parents' survival rate.Above-mentioned experiment is in triplicate.
Experimental result
1.GO-PLL-RGDS Cytotoxic evaluation
Preferable genophore is not only effective, it is also important that hypotoxicity.By mtt assay to GO-PLL-RGDS and
Intermediate GO-PLL cytotoxicity is evaluated.As a result as shown in figure 8, when GO-PLL-RGDS concentration increases to 150 μ g/
During mL, its cell survival rate is thrown away more than 85%, to show obvious cytotoxicity.And it is treated with the GO-PLL of concentration
Cell, survival rate is lower than GO-PLL-RGDS groups, and when concentration increases to 60,80,100,150 μ g/mL, has therebetween
Significant difference (P<0.05), i.e., GO-PLL cytotoxicity is more than GO-PLL-RGDS.There are document report, electropositive nanometer
Carrier can interact with the electronegative glycoprotein of cell membrane surface, so as to show cytotoxicity;And by modification
With the electropositive of nano-carrier, its cytotoxicity can be reduced.In above-mentioned chapters and sections, we have measured GO-PLL-RGDS's
Zeta potential value (25.87 ± 0.47mV) is significantly less than GO-PLL Zeta potential value (40.20 ± 4.89mV), i.e., by RGDS
Modification, reduce the amino on PLL, neutralized the positive charge of part on GO-PLL.
The effect of 2.GO-PLL-RGDS/VEGF-siRNA suppression HeLa cells propagation
As can be drawn from Figure 9:
1. the VEGF-siRNA of various concentrations is compared with blank control group, cell survival rate does not show significant difference,
I.e. the unrestraint of VEGF-siRNA cell proliferations is acted on.
2. GO-PLL-RGDS, GO-PLL-RGDS/ of various concentrations non-homogeneous-siRNA and Lipo (LipoRNAiMAX)/
Non-homogeneous-siRNA is compared with blank control, although show difference (P<0.05), but cell survival rate is more than 80%,
Cell proliferation is without obvious inhibiting effect.
3. positive controls Lipo (LipoRNAiMAX)/VEGF-siRNA has significant difference compared with blank control group
(P<0.01) the obvious effect for suppressing cell propagation, is shown.Demonstrate the correctness of experimental method.
4. 10nM GO-PLL-RGDS/VEGF-siRNA is compared with blank control group, cell survival rate is shown significantly
Sex differernce (P<0.05), and with the increase of concentration, its cell survival rate is gradually reduced, i.e. suppressing cell reproduction effect is presented one
Determine the dose dependent of scope.
Embodiment 8 evaluates the effect that GO-PLL-RGDS/VEGF-siRNA suppresses lotus S180 sarcoma mice tumors grews
1) Tumor growth inhibition is acted on
Experimental method
1. dosage
Blank control physiological saline (NS) group:0.2mL/ is only;
The free VEGF-siRNA groups of negative control:0.3mg/kg;
Positive control adriamycin (DOX) group:2μmol/kg;
Experimental group GO-PLL-RGDS/VEGF-siRNA:0.3mg/kg (based on VEGF-siRNA);3.0mg/kg (presses GO-
PLL-RGDS is counted).
2. administering mode
Tail vein injection, once every other day, is administered 5 times altogether.
3. the foundation of lotus S180 sarcoma mouse models
It is 1 × 10 by 0.2mL concentration7Individual/mL S180 cell suspension inoculations are subcutaneous in the left armpit of every mouse, set up lotus
S180 sarcoma mouse models.Mouse feeder environment is neatly hygienic, 5 cages, and temperature is 18-22 DEG C, and humidity is 50-60%, drink
Food is sufficient and free.The knurl volume of mouse is measured every other day, is raised 7 days, now the knurl volume of mouse is (long × wide2/ 2) be about
350mm3.Experimental animal ICR mouse male, 20 ± 2g of body weight (x ± SD) ties up the limited public affairs of the magnificent animal experiment technique of tonneau by Beijing
Department provides.
4. evaluate
Successful mouse will be modeled and be randomly divided into four groups, every group 10, weighed in every other day and knurl volume.And by above-mentioned dose
Amount and method give physiological saline (NS), free VEGF-siRNA, adriamycin, GO-PLL-RGDS/VEGF-siRNA respectively.
The next day after terminating is administered in last time, weighs first, then carries out de- cervical vertebra and puts to death mouse, eyeball takes blood 2mL/ only, every group
Take 6 mouse.1h is stored at room temperature, (4 DEG C, 4000g, 10min) is centrifuged after serum is separated out, takes supernatant to obtain blood serum sample, -80 DEG C
Preserve.Taking internal organ (knurl, the heart, liver,spleen,kidney, brain), claims its weight, calculates tumour inhibiting rate and dirty body ratio, and keep a record.Internal organs are placed in-
80 DEG C of preservations.
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.
Experimental result each group knurl weight and tumour inhibiting rate result are the transplanting taken out after each group mouse is dissected in A as shown in Figure 10
Knurl picture, directly perceived can must find out the sarcoma volume ratio blank control life of the mouse of experimental group GO-PLL-RGDS/VEGF-siRNA groups
Reason salt solution group is small, the sarcoma small volume for VEGF-siRNA groups of dissociating than negative control group.It is the average knurl weight (g) of each group, C in B
It is the tumour inhibiting rate of each group.As a result show that the average knurl weight of free VEGF-siRNA groups is compared with blank control group, no conspicuousness is poor
Different (P>0.05).The average knurl weight of GO-PLL-RGDS/VEGF-siRNA groups is swum with blank control physiological saline group and negative control
Compare from VEGF-siRNA groups, there is significant difference (P<0.01), tumour inhibiting rate is 51.74%, and its tumour inhibiting rate is slightly less than chemotherapeutic
DOX (56.23%).The above results illustrate that free VEGF-siRNA can not effectively suppress the growth of tumour, and GO-PLL-
RGDS/VEGF-siRNA then can effectively suppress tumour growth, show internal antitumor activity.
2) in each group tumor-bearing mice serum VEGF expression
Experimental method takes the mice serum sample of above-mentioned acquisition, and room temperature is melted, and is retouched by Mus-VEGF-ELISA kit explanations
Paint standard curve and determine the vegf protein content in serum.
Experimental result prepares a series of standard protein liquid of concentration according to explanation, determines its absorption value, draws standard curve
It is as follows:Regression equation is y=0.0096x+0.1278 (R2=0.999).VEGF absolute content in each group tumor-bearing mice serum
As a result it is as shown in the table:VEGF contents in the mice serum of GO-PLL-RGDS/VEGF-siRNA groups are 61.44 ± 2.62pg/
ML, hence it is evident that less than blank control group and free VEGF-siRNA groups.As a result illustrate, GO-PLL-RGDS/VEGF-siRNA can be with
The expression quantity of the vegf protein in Mice Body is effectively lowered, i.e., VEGF-siRNA is played gene silencing effect.This and body
Outer result matches with discussion.
VEGF contents (mean value ± SD, pg/mL) in each group mice serum of table 1.
Tumor-targeting effect-fluorescence imaging method that embodiment 9 evaluates GO-PLL-RGDS/VEGF-siRNA determines GO-
PLL-RGDS/VEGF-siRNA Tissue distribution
Experimental method:
1. the foundation of lotus S180 sarcoma mouse models
It is 1 × 10 by 0.2mL concentration7Individual/mL S180 cell suspension inoculations are subcutaneous in the left armpit of every mouse, set up lotus
S180 sarcoma mouse models.Mouse feeder environment is neatly hygienic, 5 cages, and temperature is 18-22 DEG C, and humidity is 50-60%, drink
Food is sufficient and free.
2. administration
It is random that the successful mouse of modeling is divided into 3 groups, every group 3.It is administered, gives according to following dosage tail vein injection respectively
Medicine is once.
Physiological saline group:0.2mL/ is only
Cy3-VEGF-siRNA groups:0.3mg/kg
GO-PLL-RGDS/Cy3-VEGF-siRNA groups:0.3mg/kg (based on Cy3-VEGF-siRNA);3.0mg/kg (is pressed
GO-PLL-RGDS is counted), compound method is as described in 2.1.3.
3. fluorescence imaging is observed
It is administered after 24h, mouse is put to death, takes out each internal organs, note lucifuge.It is placed in observation and quantitative scoring under phosphorimager
Calculate the fluorescence intensity level of each internal organs.Excitation wavelength is 550nm, and launch wavelength is 570nm.Note:6% chloraldurate is first used during 12h
0.2mL/ is only injected intraperitoneally mouse anesthesia, and then living imaging, observes fluorescence.As a result due to chaeta influence, cause can not be into
Picture, the chaeta of its belly is shaved off, is placed under phosphorimager, as a result still can't see fluoroscopic image afterwards.Analyze reason:May
Because Cy3 fluorescence intensity is weaker, mouse skin and tissue are thicker.Observed so taking and being directly taken out after execution mouse after internal organs
This method.Note:Cy3 fluorescent dyes are a kind of water-soluble dyes for being usually used in biomolecular labeling.
As shown in Figure 11 A, each organ of physiological saline group does not observe fluorescence, free Cy3-VEGF- to experimental result
In siRNA groups, weaker fluorescence, and the fluorescence intensity of tumor locus can be observed in tumour, liver, lung, kidney and brain
Fluorescence intensity unlike other organs is strong.This is due to that free Cy3-VEGF-siRNA is easily inactivated by enzymolysis, and comparatively fast
Ground is excreted.And in GO-PLL-RGDS/Cy3-VEGF-siRNA groups, it is dirty that the fluorescence intensity of tumor locus is significantly stronger than other
Device, also, not it was observed that fluorescence in brain.In addition, the fluorescence intensity of kidney is strong compared with the fluorescence intensity of other organs, GO-PLL-
RGDS/Cy3-VEGF-siRNA may be by kidney excretion, so as to have accumulation in kidney.What the fluorescence area of lung relatively dissociated
Cy3-VEGF-siRNA groups lung fluorescence area is big, can be deposited on lung with partial oxidation of graphite alkene relevant.It is in B, fluorescence is strong
Angle value quantifies, it can be deduced that same result.Fluorescence intensity levels of the GO-PLL-RGDS/Cy3-VEGF-siRNA in tumor locus
It is 2.34 times of kidney.To sum up after explanation administration 24h, GO-PLL-RGDS/Cy3-VEGF-siRNA is most of in tumor tissues point
Cloth, next to that in kidney distribution, may be relevant with Metabolism Excretion approach;Also there is a small amount of distribution in liver and lung in addition.This
As a result demonstrating GO-PLL-RGDS/Cy3-VEGF-siRNA has tumor-targeting.
Claims (9)
1. a kind of genes delivery system with antitumor activity:GO-PLL-RGDS/VEGF-siRNA.
2. the genes delivery system described in claim 1, it is characterised in that the genes delivery system is by Antioncogene curative
Thing VEGF-siRNA and targeted delivery vector GO-PLL-RGDS are prepared from.
3. the genes delivery system described in claim 1, it is characterised in that GO-PLL-RGDS and VEGF-siRNA weight ratio
For:10:1.
4. the genes delivery system described in claim 1, it is characterised in that the genes delivery system is nanostructured.
5. the genes delivery system described in claim 1, it is characterised in that targeted delivery vector GO-PLL-RGDS by below into
Divide and be made:GO、PLL、RGDS.
6. the preparation method of the genes delivery system described in claim 1, it is characterised in that by GO-PLL-RGDS and VEGF-
SiRNA is according to 10:1 mass ratio is mixed, and is stored at room temperature, is produced GO-PLL-RGDS/VEGF-siRNA.
7. the preparation method of the genes delivery system described in claim 1, it is characterised in that targeted delivery vector GO-PLL-
RGDS preparation:Graphene oxide GO is sequentially connected PLL (polylysine) and RGDS by ring-opening reaction, condensation reaction and taken off
Protection obtains GO-PLL-RGDS.
8. the preparation method of the genes delivery system described in claim 1, it is characterised in that comprise the following steps:
1) GO-PLL is prepared
2.0mg GO ultrasonic disperses are weighed in 4mL ultra-pure waters, with Probe Ultrasonic Searching instrument are sonicator ultrasound 4h (between 2 seconds
Have a rest formula).The KOH aqueous solution (pH9) is added to 8mL.10.0mg PLL are added, now there is sediment generation, the KOH aqueous solution is used in continuation
(pH11) it is 9 to be adjusted to pH value.Reaction bulb is placed in 70 DEG C of oil baths and stirs 24h.Stop heating after 24h, centrifuge (12000g,
20min), supernatant is discarded, washes three times, washes unreacted PLL off.It is lyophilized standby.
2) Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OH is prepared
Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OBzl is prepared according to standard method.By Boc-Arg (Tos)-Gly-Asp
(OMe)-Ser-OBzl 430mg are added in 20mL methanol, are added 64.5mg palladium carbons, are passed through hydrogen, are reacted at room temperature, and TLC monitorings are anti-
Should.React complete after 5h, filter out palladium carbon, rotation removes methanol, obtains Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OH.
3) GO-PLL-RGDS is prepared
10mg Boc-Arg (Tos)-Gly-Asp (OMe)-Ser-OH is weighed, is dissolved in 10mL DMF, 30mg is added
EDCHCl, stir-activating 15min, form reaction solution A.By 2mg GO-PLL ultrasonic disperses in 10mL DMF, then by it
Mixed with the reaction solution A of above-mentioned activation, add 30mg HOBt, be stirred at room temperature.After 24h, reaction solution is centrifuged (12000g,
20min), sediment is collected, is centrifuged three times with methanol and milli-Q water, removes uncombined Boc-Arg (Tos)-Gly-Asp
(OMe)-Ser-OH and unreacted EDCHCl and HOBt.It is lyophilized to obtain GO-PLL-Ser-Asp (OMe)-Gly-Arg
(Tos)-Boc.It is dispersed in 10mL methanol solutions, ultrasonic 30min, under condition of ice bath, it is adjusted with NaOH (2N) solution
PH value is equal to 12, reacts 6h, after question response terminates, and 10min is centrifuged with 10000g rotating speed, is rinsed 3 times with ultra-pure water, then with nothing
Water-ethanol is rinsed three times, and 15min is centrifuged with 12000g rotating speeds, is placed ventilation and is spontaneously dried.Under ice bath, by the production of above-mentioned drying
Thing is dispersed in (TFA in 5mL mix acid liquors:TfOH=4:1, v/v) 1h, is reacted.Transfer them in centrifuge tube, centrifuge
(7000g, 15min) discards supernatant.Three times are rinsed with absolute ether drift and ultra-pure water obtain target product GO-PLL- respectively
Ser-Asp-Gly-Arg(GO-PLL-RGDS).5mL ultra-pure waters are eventually adding, ultrasonic disperse finds product favorable dispersibility.Will
Dispersion liquid loads bag filter (molecular cut off:8000), dialysis 24h removes impurity.Dispersion liquid after dialysis purification is freezed, surveyed
Determine infrared spectrum.
4) GO-PLL-RGDS/VEGF-siRNA is prepared
2mg GO-PLL-RGDS are weighed, are dispersed in 2mL DEPC water, ultrasonic 40min, 1000 μ g/mL GO- is configured to respectively
PLL-RGDS storing solutions, then 1.0OD VEGF-siRNA (33 μ g) are dissolved in 250 μ L DEPC water.By GO-PLL-RGDS with
VEGF-siRNA, than mixing, is stored at room temperature 30min, produces GO-PLL-RGDS/VEGF-siRNA according to certain N/P.
9. application of the genes delivery system in the medicine for suppressing tumour is prepared described in claim 1.
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CN110180029A (en) * | 2019-05-16 | 2019-08-30 | 暨南大学 | A kind of preparation method and application with induced osteogenesis differentiation and the degradation material of bone regeneration function |
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