CN104546711A - SiRNA-PLGA/CSO conjugate micelle and preparation method thereof - Google Patents
SiRNA-PLGA/CSO conjugate micelle and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of siRNA-PLGA/CSO conjugate micelle. The preparation method comprises the following steps: (1) preparing PLGA-PDPH from PLGA, DCC, NHS and PDPH; (2) preparing siRNA-PLGA from siRNA and the PLGA-PDPH; (3) preparing the siRNA-PLGA/CSO conjugate micelle from the siRNA-PLGA and CSO. According to the preparation method of the siRNA-PLGA/CSO conjugate micelle, reaction conditions are mild, and the operation is simple and convenient; the sources of the utilized PLGA and CSO are wide, and the utilized PLGA and CSO have the excellent characteristics of low toxicity and biodegradability. The invention further discloses the siRNA-PLGA/CSO conjugate micelle. The siRNA-PLGA/CSO conjugate micelle improves the stability of the siRNA in a delivery process, prevents the siRNA from being degraded by nuclease and lysosome, and increases the cell uptake rate of the siRNA.
Description
Technical field
The present invention relates to gene drug carriers field, particularly relate to a kind of siRNA-PLGA/CSO conjugate micelle and preparation method thereof.
Background technology
SiRNA is the intermediate product that RNA disturbs (RNA interference, RNAi) approach, is also the effector molecule that RNAi plays a role.Since RNA perturbation technique finds, RNAi has become cytogenetic crucial actuator and has illustrated the powerful of body growth, pathogeny and aging.Whether effectively siRNA can be delivered to target cell position, be combined with target mRNA, become the key factor of the application of restriction siRNA.In addition, after siRNA is by cellular uptake, be delivered to by lysosomal degradation in lysosome, greatly reduce the therapeutic activity of siRNA.Therefore the siRNA stability of sending is improved also very important.Only have effective cellular uptake and endocytosis activity and keep the integrity of double-strand siRNA that siRNA just can be made finally to play therapeutic effect.The physicochemical property of siRNA self is as electronegativity, and molecular weight is large, and the features such as volume is large make it not by the cell membrane of Passive diffusion by feminine gender.Therefore, the key of gene therapy is to search out needs modification or the genes of interest suppressed and has desirable carrier.
Non-virus carrier safety is high, and is easy to a large amount of acquisition, in gene therapy, be therefore more subject to the favor of vast researcher than viral vectors.Non-virus carrier is transported to target cell through blood barrier in vivo; with target cell specificity combine; enter Cytoplasm; discharge siRNA, need multiple combination obstacle be overcome, protection and the picked-up of siRNA therefore during application vector, should be conducive to; it is made lessly to be degraded born of the same parents are outer; again after arrival site of action, target cell can be imported efficiently, and effectively discharge in kytoplasm.In the transmission of siRNA, the most often use cation carrier, and with liposome and microgranule in the majority.
The most frequently used cationic-liposome is polymer polyethylenimine (PEI), at (the Developments in Non-Viral of siRNA, Wang Xipei etc., clinical medicine research and development forward position, the world, 29th volume the 1st phase, 2008), in a literary composition, anti-vegf siRNA and Polyethylene Glycol (PEG) by disulfide formation complex, then interact with cationic polymer polymine (PEI) and form polyelectrolyte micelle by the people such as Kim.Find that siRNA can discharge in good condition from complex in prostatic cell experiment.The advantage of this method is that disulfide bond can keep stable in course of conveying, not easy fracture in the outer high oxidation state environment relatively of born of the same parents, and after arrival tumor cell, hypersensitivity disulfide bonds under micro-glutathion (GSH) triggering in kytoplasm, thus reduction release siRNA.In addition, PEI molecule exists a lot of amino, have in the endocytosis of what is called " proton sponge effect, " after entering born of the same parents and lyase process and play cushioning effect, destroy and escape from lysosomal degraded, realizing the high expression of gene.But because PEI has higher cytotoxicity and limited biodegradable degree, cause the toxicity problem of activation to self immune system and Formulations for systemic administration.
Poly(D,L-lactide-co-glycolide (PLGA) is that a kind of research is ripe, and biodegradable, has the polymer of biocompatibility, applies decades at pharmaceutical industry.PLGA is by one of support copolymer as medicine controlled releasing of the most extensively studying.It comparatively cationic-liposome compare with cationic polymer there is more hypotoxicity, PLGA has been widely used in embedding DNA, antisense oligonucleotide and siRNA to improve medicine stability, and realizes sustained release.But PLGA is a kind of degradable macromolecule, produce acidic oligomer or lactic acid, glycolic acid monomers after degraded, the sour environment produced may cause the inactivation of medicine.PLGA is the site lacking specific binding with cell tissue as another shortcoming of pharmaceutical carrier, lacks the functional group of activation, thus can not be combined with active ligand molecule.
Chitosan (CS) is a kind of alkaline polysaccharide, because it has hypotoxicity, reduced immunogenicity and the advantage such as outstanding biological degradability and biocompatibility, primary amino radical in chitosan structure is active reactive group, amino can with the bioactive ligand cou of body.Therefore chitosan has become the common used material forming siRNA nanoparticle.
Molecular weight affects chitosan (CS) physicochemical properties and bioactive key factor, and molecular weight can change by induced conformational, the molecular conformation of low-molecular weight chitoglycan relative to the extensibility of high molecular weight chitosan and rigidity better.The ionic strength increasing solution can make molecule more shrink with curling.Oligochitosan (CSO) is the water-soluble aminosaccharide compound produced after hydrolysis by chitin (chitin) and chitosan.The degree of polymerization of CSO is 2-20, and good water solubility, is easily absorbed and used, and biological activity ratio CS is stronger.Particularly the degree of polymerization is the oligochitosan of about 6, has more physiologically active and the functional character of many uniquenesses.
Based on the character of PLGA and CSO, oligochitosan being modified PLGA surface is a kind of effective means strengthening and improve its performance.
Chinese patent 201410212594.2 discloses a kind of PLGA Nano medication body be made up of PLGA Nano microsphere kernel and vanillin cross-linked chitosan shell, this invention uses chitosan to carry out finishing to PLGA microsphere, use vanillin as cross-linking agent afterwards, PLGA Nano medication body is prepared in amino and the vanillin generation chemical crosslinking of chitosan.This inventive method vanillin used, by synthetic, has toxicity, is unfavorable for the release of medicine.The amino of chitosan and vanillin generation chemical crosslinking, the experiment condition adopted is harsh.
Summary of the invention
The invention provides a kind of preparation method of siRNA-PLGA/CSO conjugate micelle.SiRNA-PLGA and CSO is formed siRNA-PLGA/CSO conjugate micelle by electrostatic interaction by preparation method of the present invention, and reaction condition is gentle, easy and simple to handle.PLGA and the CSO wide material sources that the present invention adopts, have low toxicity and biodegradable good characteristic.
A preparation method for siRNA-PLGA/CSO conjugate micelle, comprises the following steps:
(1) PLGA, N, N '-dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS) react in solvent first, obtained PLGA-NHS ester after process; PLGA-NHS ester and 3-(2-thiopyridine) propionyl hydrazine (PDPH) react in solvent second, obtained PLGA-PDPH grafting after process;
(2) positive-sense strand 3 ' end or antisense strand 5 ' hold the siRNA of modified with mercapto group to be dissolved in phosphate buffer (PBS), PLGA-PDPH grafting is dissolved in dimethyl sulfoxide (DMSO), by reaction after two kinds of solution mixing, treated obtained siRNA-PLGA conjugate micelle;
(3) siRNA-PLGA conjugate micelle and oligochitosan (CSO) processed in water at pyrophosphoric acid diethylester (DEPC) and hatch, obtained skin is the siRNA-PLGA/CSO conjugate micelle of CSO.
PLGA of the present invention is end carboxyl PLGA, also referred to as carboxylated PLGA.
The physical propertys such as the dissolubility of the molecular weight effects PLGA of PLGA and viscosity; The speed that in PLGA, two kinds of monomer Acetic acid, hydroxy-, bimol. cyclic esters (PLA) are degraded with the scale effect PLGA of lactide (PGA), two kinds of monomer ratio are more close, and the speed of PLGA degraded is faster.
Preferably, PLGA molecular weight is two kinds of monomer Acetic acid, hydroxy-, bimol. cyclic esters (PLA) of 4000-50000, PLGA is 50 ~ 90:50 ~ 10 with the ratio of lactide (PGA).
The PLGA that molecular weight is large is unfavorable for the chemical reaction with other materials, cannot form micelle.
Further preferably, PLGA molecular weight is 8000-20000, and the ratio of two kinds of monomer Acetic acid, hydroxy-, bimol. cyclic esters and lactide is 50 ~ 75:50 ~ 25.
Step (1) DCC, as dehydrant and catalyst, accelerates PLGA with NHS and becomes ester to react, and PLGA and the PDPH after DCC, NHS activation is obtained by reacting PLGA-PDPH, containing disulfide bond in PLGA-PDPH.
Preferably, step (1) PLGA:DCC and NHS mol ratio is 1:2 ~ 5:2 ~ 5, and excessive NHS ensures PLGA complete reaction.
Preferably, the mol ratio of step (1) PLGA-NHS ester and PDPH is 1:1 ~ 3, and excessive PDPH ensures PLGA-NHS ester complete reaction.
The selection principle of step (1) solvent is abundant solubilizing reaction thing.
Preferably, described solvent first is at least one in dichloromethane, dimethyl formamide, oxolane and N-Methyl pyrrolidone.
The reaction temperature of step (1) PLGA, DCC and NHS is 10 DEG C-40 DEG C, and the response time is 5h-8h.
Solvent second described in step (1) is at least one in dichloromethane, dimethyl formamide, oxolane, N-Methyl pyrrolidone and ethyl acetate.
Step (1) PLGA-NHS ester and PDPH reaction temperature are 10 DEG C-40 DEG C, and the response time is 5h-8h.
Step (2) positive-sense strand 3 ' end or antisense strand 5 ' hold the siRNA of modified with mercapto group, form siRNA-PLGA with the disulfide bond on PLGA-PDPH by the displacement reaction of disulfide bond.
Step (2) described siRNA comprises 19 ~ 30 nucleotide, and molecular weight is 10000-30000.
Mol ratio 1:20 ~ 50 of step (2) siRNA and PLGA-PDPH.
Step (2) PBS and DMSO volume ratio is 1:8 ~ 12, and PBS amount too much can affect the dissolving of PLGA-PDPH.
Step (2) reaction temperature is 10 DEG C ~ 40 DEG C, and the response time is 16h ~ 26h.
The particle diameter of the obtained siRNA-PLGA conjugate micelle of step (2) is 200nm ~ 246nm, and surface potential is-40mV ~-25mV.
The siRNA-PLGA conjugate micelle that step (3) is electronegative and positively charged CSO form compound polyelectrolyte micelle by electrostatic interaction, and the charging property of siRNA-PLGA turns negative number to positive number.
Preferably, oligochitosan (CSO) molecular weight described in step (3) is 1000 ~ 3000, and the degree of polymerization is 6-8.
Preferred incubation conditions is: siRNA-PLGA conjugate micelle is hatched under N/P is than the condition for 1-80:1 with CSO, and incubation temperature is 10 DEG C ~ 40 DEG C, incubation time 15min ~ 30min.
Described N/P is than the ratio representing N element and P element.
Preferably, siRNA-PLGA conjugate micelle is hatched under N/P is than the condition for 10-80:1 with CSO.At preferred N/P than under condition, it is positively charged that obtained siRNA-PLGA conjugate micelle is hatched in the present invention, can be combined with electronegative cell membrane well, increases the cellular uptake rate of siRNA.
Present invention also offers the siRNA-PLGA/CSO conjugate micelle that described preparation method is obtained.
Described siRNA-PLGA/CSO conjugate micelle improves the stability of siRNA in delivery process, makes siRNA from nuclease and lysosomal degraded, increases the cellular uptake rate of siRNA.
The particle diameter of described siRNA-PLGA/CSO conjugate micelle is 150nm-200nm, and surface potential is-18mV ~ 40mV.SiRNA-PLGA/CSO conjugate micelle is along with the increase of N/P ratio, and its surface potential turns negative number to positive number.Positively charged siRNA-PLGA/CSO conjugate micelle is combined with electronegative cell membrane well, increases the cellular uptake rate of siRNA.
Compared with prior art, the present invention has following beneficial effect:
(1) preparation method reaction condition of the present invention is gentle, and easy and simple to handle, PLGA, CSO wide material sources of employing, have low toxicity and biodegradable good characteristic.
(2) siRNA-PLGA and CSO is formed siRNA-PLGA/CSO conjugate micelle by electrostatic interaction by preparation method of the present invention, and this micelle is combined with electronegative cell membrane well, increases the cellular uptake rate of siRNA.
(3) the siRNA-PLGA/CSO conjugate micelle that prepared by the present invention improves the stability of siRNA in delivery process, makes siRNA from nuclease and lysosomal degraded, increases the cellular uptake rate of siRNA.
(4) the present invention with the born of the same parents' evagination solving gene vector system release with born of the same parents in be difficult to be released to impetus, reduce prominent outside born of the same parents of siRNA by chemical bond mode and polyelectrolyte effect and to release and by nuclease degradation; In reduction-state environment, trigger fracture through GSH by disulfide bond again, promote that it is in arrival Cytoplasm, disengages siRNA.
Accompanying drawing explanation
Fig. 1 is synthesis and the cell traffic process schematic thereof of siRNA-PLGA/CSO conjugate micelle.
Fig. 2 is the form transmission electron microscope picture of siRNA-PLGA conjugate.
Fig. 3 is the form transmission electron microscope picture of siRNA-PLGA/CSO conjugate micelle.
Fig. 4 measures different N/P than siRNA-PLGA/CSO conjugate micelle particle diameter and potentiometric analysis figure under condition by dynamic light scattering (DLS) method.
Fig. 5 evaluates stability to nuclease, respectively with naked siRNA and siRNA-PLGA conjugate micelle, and the electrophoresis photographs of siRNA palliating degradation degree in time under nuclease existence condition.
Fig. 6 is the critical micelle concentration figure of siRNA-PLGA conjugate micelle.
Fig. 7 is hydroxyl fluorescein (FAM) the siRNA/CSO conjugate of 20,40 and 80, the picked-up result flow cytometer showed figure after FAM siRNA-PLGA/CSO micelle and lipofectamine 2000 transfected with human ovarian cancer SKOV3 cell with N/P ratio respectively.
Fig. 8 uses negative control (N.C) siRNA-PLGA/CSO micelle respectively, after intracellular signaling and activating transcription factor 3 (STAT3) siRNA/CSO conjugate, STAT3 siRNA-PLGA/CSO conjugate micelle, lipofectamine 2000 transfected with human ovarian cancer SKOV3 cell, to the qRT-PCR analysis chart of STAT3 mRNA downward effect in SKOV3.
Fig. 9 is after using N.C siRNA-PLGA/CSO micelle, STAT3 siRNA/CSO conjugate STAT3 siRNA-PLGA/CSO conjugate micelle transfected with human ovarian cancer SKOV3 cell respectively, to the western blot analysis figure of STAT3 albumen downward effect in SKOV3.
Figure 10 is after using N.C siRNA-PLGA/CSO micelle, STAT3 siRNA/CSO conjugate STAT3 siRNA-PLGA/CSO conjugate micelle transfected with human ovarian cancer SKOV3 cell respectively, the Cell proliferation results analysis chart obtained is detected with 2-(2-methoxyl group-4-nitrobenzophenone)-3-(4-nitrobenzophenone)-5-(2,4-disulfonic acid benzene)-2H-tetrazolium monosodium salt (CCK-8).
Figure 11 is after using N.C siRNA-PLGA/CSO micelle, STAT3 siRNA/CSO conjugate, STAT3 siRNA-PLGA/CSO conjugate micelle transfected with human ovarian cancer SKOV3 cell respectively, the apoptosis interpretation of result figure that flow cytometer obtains.
Detailed description of the invention
Embodiment 1
(molecular weight is 10000 to PLGA, two kinds of monomer ratio are 50:50), N, N '-dicyclohexylcarbodiimide (DCC) and N-hydroxy-succinamide (NHS) react 6h by mol ratio room temperature (25 DEG C) in 5mL dichloromethane of 1:3:3,0.22um filtering with microporous membrane, filtrate is concentrated into after 0.5mL ~ 1mL through rotary evaporation and dropwise joins in 35mL cold diethyl ether, 4500rpm, 4 DEG C, centrifugal 30min, by PLGA-NHS ester obtained after precipitate lyophilization.PLGA-NHS ester and 3-(2-thiopyridine) propionyl hydrazine (PDPH) react 0.22um filtering with microporous membrane after 6 hours by mol ratio room temperature (25 DEG C) in 5mL dichloromethane of 1:2, filtrate joins in the cold ethanol of 35mL after rotary evaporation is concentrated, 4500rpm, 4 DEG C, centrifugal 30min, by PLGA-PDPH obtained after precipitate lyophilization.PLGA-PDPH is after 20mmol/L dithiothreitol, DTT (DTT) process, and ultraviolet spectrophotometry records grafting rate and reaches more than 90%.
Of short duration for the common siRNA (purchased from Shanghai JiMa pharmacy Technology Co., Ltd) of 10nmol positive-sense strand 3 ' end modified with mercapto group centrifugal rear 50ul 10mmol/LPBS (pH7.4) is dissolved, 400nmolPLGA-PDPH 500ul DMSO dissolves, in solution, (siRNA and PLGA-PDPH mol ratio is 1:40, PBS and DMSO volume ratio is 1:10), first to dialyse 12h with DMSO after both 30 DEG C of reaction 24h, obtain siRNA-PLGA conjugate micelle after dialysing 24 hours with the distilled water of pH7.4 again, see accompanying drawing 1.Conjugate micelle is through 3000rpm, and 4 DEG C of centrifugal 10min, repeat 3 times, remove excessive PLGA-PDPH, and record obtains micelle volume.
For evaluating the grafting rate of siRNA-PLGA, by siRNA-PLGA micellar solution 12000rpm, 90min, 4 DEG C centrifugal, the amount of siRNA in filtrate is measured with nucleic acid determination instrument, the theoretical siRNA amount that drops into deducts siRNA amount in filtrate and drops into theoretical the grafting rate that ratio that siRNA measures is siRNA and PLGA, and result obtains grafting rate and is about 87%.
In order to by siRNA-PLGA conjugate energy Successful delivery in cell, siRNA-PLGA conjugate and low-molecular-weight cationic substance oligochitosan are formed compound polyelectrolyte micelle.500pmol siRNA-PLGA conjugate is dissolved in 500ul DEPC water, adds isopyknic oligochitosan solution, hatches under N/P ratio is 80:1 condition, and incubation temperature is 25 DEG C, and incubation time is 30min, obtains siRNA-PLGA/CSO conjugate micelle.DLS methods analyst is used to measure current potential and the particle diameter of siRNA-PLGA and siRNA-PLGA/CSO conjugate micelle.Record the mean diameter 200nm of siRNA-PLGA, average potential is the mean diameter of-40mV, siRNA-PLGA/CSO conjugate micelle is 156nm, and average potential is+40mV, uses the form of transmission electron microscope analysis micelle.
Record siRNA-PLGA conjugate with transmission electron microscope, see accompanying drawing 2.
The form of siRNA-PLGA/CSO polyelectrolyte conjugate micelle is spherical, sees accompanying drawing 3.
Embodiment 2
With embodiment 1, but in PLGA, Acetic acid, hydroxy-, bimol. cyclic ester (PLA) is 75:25 with the ratio of lactide (PGA), and the siRNA-PLGA conjugate micelle mean diameter obtained is about 246nm, and average potential is-29mV.
Embodiment 3
With embodiment 2, but siRNA and PLGA-PDPH reaction mol ratio is 1:20, and the siRNA-PLGA conjugate micelle mean diameter obtained is about 200nm, and average potential is-30mV.Obtain siRNA and PLGA-PDPH grafting rate about 60%.
Execute example 4
With embodiment 1, but siRNA and PLGA-PDPH reaction mol ratio is 1:4, obtains siRNA and PLGA-PDPH grafting rate about 10%.And the siRNA-PLGA conjugate micelle DLS obtained measures, and because particle concentration is too low, cannot measure particle diameter and current potential.
Embodiment 5
With embodiment 1, but the weight average molecular weight of PLGA to be 50000, PLGA viscosity too high, the product after DCC, NHS and PDPH activation cannot be dissolved completely in DMSO, and PLGA and PDPH grafting rate about 50%.
The synthesis of embodiment 6FAM siRNA-PLGA/CSO conjugate micelle
With embodiment 1, but siRNA is the FAM siRNA (purchased from Shanghai JiMa pharmacy Technology Co., Ltd) that positive-sense strand 3 ' holds modified with mercapto group, obtains the FAM siRNA-PLGA/CSO conjugate micelle being with green fluorescence.
The synthesis of embodiment 7STATS siRNA-PLGA/CSO conjugate micelle
With embodiment 1, but siRNA is the STAT3 siRNA of positive-sense strand 3 '-sulfydryl modification, obtains the STAT3 siRNA-PLGA/CSO conjugate micelle for ovarian cancer STAT3 gene.
Embodiment 8
With embodiment 1, but siRNA-PLGA and CSO is hatched under for 1:1 condition at N/P, and the mean diameter obtaining siRNA-PLGA/CSO conjugate micelle is 194nm, and average potential is-18mV.
Embodiment 9
With embodiment 1, but siRNA-PLGA and CSO is hatched under for 10:1 condition at N/P, and the mean diameter obtaining siRNA-PLGA/CSO conjugate micelle is 179nm, and average potential is 16mV.
Embodiment 10
With embodiment 1, but siRNA-PLGA and CSO is hatched under for 20:1 condition at N/P, and the mean diameter obtaining siRNA-PLGA/CSO conjugate micelle is 188nm, and average potential is 25mV.
Embodiment 11
With embodiment 1, but siRNA-PLGA and CSO is hatched under for 40:1 condition at N/P, and the mean diameter obtaining siRNA-PLGA/CSO conjugate micelle is 185nm, and average potential is 28mV.
Embodiment 12
With embodiment 1, but siRNA-PLGA and CSO is hatched under for 60:1 condition at N/P, and the mean diameter obtaining siRNA-PLGA/CSO conjugate micelle is 173nm, and average potential is 32mV.
Different N/P is shown in accompanying drawing 4 than lower current potential and granularmetric analysis figure.
The test result of siRNA-PLGA conjugate micelle prepared by the present invention and siRNA-PLGA/CSO conjugate micelle is as follows:
1. under nuclease existence condition based on the stability of the siRNA-PLGA conjugate of disulfide bond
Naked siRNA and the siRNA-PLGA conjugate for preparing are being cultivated 0,1,2,3,4,5 hour containing 0.1mg/mLRNase solution A 37 DEG C respectively, use 2% agarose gel electrophoresis analyze both stability.
Found that: after 5h, the good stability of siRNA-PLGA conjugate, and naked siRNA deposits in case there being nuclease, degrades soon, specifically sees accompanying drawing 5.
The mensuration of 2.siRNA-PLGA conjugate critical micelle concentration (CMC)
The siRNA-PLGA conjugate DEPC water of synthesis in embodiment 1 is mixed with the solution of 0.1 ~ 12ug/mL, adds the ultimate density 6.0 × 10 that pyrene solution makes pyrene
7m.30 DEG C of lucifuge jolting 5h.With the emission spectra that fluorescent spectrophotometer assay excitation wavelength is 337nm place pyrene.The critical micelle concentration that result obtains siRNA-PLGA conjugate is about 3mg/L, sees accompanying drawing 6.
Result shows that the siRNA-PLGA conjugate micelle prepared has good thermodynamic stability and good anti-dilution capacity.
3. cellular uptake analysis
By human ovarian cancer SKOV3 cell by 2 × 10
5cells/well is inoculated in 6 orifice plates, transfection is carried out with FAM-siRNA/CSO and FAM-siRNA-PLGA/CSO with green fluorescence that N/P ratio is respectively 20,40,80 after overnight incubation, lipofectamine 2000 as positive control, without the cell of any process as blank (Blank).
Found that: the cellular uptake rate of alone siRNA/CSO electrostatical binding thing is very low, and the cellular uptake rate of siRNA-PLGA/CSO conjugate micelle increases along with the increase of N/P ratio.When N/P ratio is 80:1, its uptake ratio and cationic-liposome lipofectamine 2000 positive controls there was no significant difference, uptake ratio, more than 90%, is shown in accompanying drawing 7.
The qRT-PCR quantitative analysis that in 4.SKOV3 cell, STAT3 mRNA measures
By human ovarian cancer SKOV3 cell by 2 × 10
5cells/well is inoculated in 6 orifice plates, after overnight incubation respectively with N/P than the N.C-siRNA-PLGA/CSO conjugate micelle being 80, STAT3 siRNA/CSO conjugate, the transfection of STAT3 siRNA-PLGA/CSO conjugate micelle, the cell be left intact is as blank (Blank), and lipofectamine 2000 transfection group is as positive control.Analyze the level of SKOV3 cell STAT3 mrna expression after different reagent transfection by qRT-PCR analytic process after cultivating 48h.
Found that: SKOV3 cell is through N.C-siRNA-PLGA/CSO conjugate micelle, STAT3 mrna expression there was no significant difference (P > 0.05) compared with blank group after the transfection of STAT3 siRNA/CSO conjugate, and STAT3 siRNA-PLGA/CSO conjugate micelle transfection group is compared with blank group, STAT3 mrna expression obviously declines, there is significant difference (P < 0.001), but see accompanying drawing 8 with lipofectamine 2000 positive controls without explicitly difference (P > 0.05).
The Western blot quantitative analysis of STAT3 protein content in 5.SKOV3 cell
By SKOV3 cell by 2 × 10
5cells/well is inoculated in 6 orifice plates, respectively with the N.C-siRNA-PLGA/CSO conjugate micelle that N/P value is 80 after overnight incubation, STAT3 siRNA/CSO conjugate, the transfection of STAT3 siRNA-PLGA/CSO conjugate micelle, with the cell be left intact for blank (Blank).Analyze the level of SKOV3 cell STAT3 protein expression after different reagent transfection by Western blot analytic process after cultivating 72h.
Found that: SKOV3 cell is through N.C-siRNA-PLGA/CSO conjugate micelle, after the transfection of STAT3 siRNA/CSO conjugate STAT3 protein band compared with blank group without explicitly difference, and STAT3 siRNA-PLGA/CSO conjugate micelle transfection group is compared with blank group, STAT3 protein band is obviously thin out, and result has significant difference and sees accompanying drawing 9.
6.STAT3 siRNA-PLGA/CSO conjugate micelle is to the inhibitory action of SKOV3 Growth of Cells
CCK-8 test kit is used to analyze STAT3 siRNA-PLGA/CSO conjugate micelle to the growth inhibited effect of SKOV3 cell.SKOV3 cell is inoculated in 96 orifice plates by 6000 cells/well, 37 DEG C, 5%CO
2use N.C-siRNA-PLGA/CSO conjugate micelle after incubator overnight incubation respectively, STAT3 siRNA/CSO conjugate, the transfection of STAT3 siRNA-PLGA/CSO conjugate micelle, with the cell be left intact for blank (Blank).After cultivation 24h, 48h, 72h, add 10ul CCK-8 reagent in each hole respectively, cell culture incubator hatches 1h, measures the absorbance at 450nm place by microplate reader.
Found that: SKOV3 cell is through N.C-siRNA-PLGA/CSO conjugate micelle, after the transfection of STAT3 siRNA/CSO conjugate compared with blank group, Growth of Cells is not all significantly suppressed, and STAT3 siRNA-PLGA/CSO conjugate micelle transfection group is compared with blank group, the growth of cell is suppressed significantly, especially, after having arrived 72h, cell proliferation is obviously subject to weakening sees accompanying drawing 10.
7.STAT3 siRNA-PLGA/CSO conjugate micelle is to the apoptotic facilitation of SKOV3.
Utilize apoptosis kit to carry out cell apoptosis assay.By SKOV3 cell by 2 × 10
5cells/well is inoculated in 6 orifice plates, N.C-siRNA-PLGA/CSO conjugate micelle is used respectively after incubator overnight incubation, STAT3 siRNA/CSO conjugate, the transfection of STAT3 siRNA-PLGA/CSO conjugate micelle, with the cell be left intact for blank (Blank).
Found that: SKOV3 cell is through N.C-siRNA-PLGA/CSO conjugate micelle, SKOV3 apoptosis rate (early apoptosis+late apoptic) there was no significant difference (P > 0.05) compared with blank group after the transfection of STAT3 siRNA/CSO conjugate, and STAT3 siRNA-PLGA/CSO conjugate micelle transfection group is compared with blank group, apoptosis rate obviously declines, and has significant difference (P < 0.01) and sees that (abscissa FITC is Fluorescein isothiocyanate to accompanying drawing 11; Vertical coordinate PI is propidium iodide).
Claims (10)
1. a preparation method for siRNA-PLGA/CSO conjugate micelle, comprises the following steps:
(1) PLGA, DCC and NHS react in solvent first, obtained PLGA-NHS ester after process; PLGA-NHS ester and PDPH react in solvent second, obtained PLGA-PDPH grafting after process;
(2) positive-sense strand 3 ' end or antisense strand 5 ' hold the siRNA of modified with mercapto group to be dissolved in PBS, and PLGA-PDPH grafting is dissolved in DMSO, reaction after two kinds of solution mixing, treated obtained siRNA-PLGA conjugate micelle;
(3) siRNA-PLGA conjugate micelle and CSO are hatched in pyrocarbonic acid diethyl ester process water, obtained siRNA-PLGA/CSO conjugate micelle.
2. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, it is characterized in that, PLGA molecular weight described in step (1) is the ratio of two kinds of monomer Acetic acid, hydroxy-, bimol. cyclic esters and lactide in 8000 ~ 20000, PLGA is 50 ~ 75:50 ~ 25.
3. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, is characterized in that, the mol ratio of step (1) PLGA, DCC and NHS is 1:2 ~ 5:2 ~ 5, and the mol ratio of PLGA-NHS ester and PDPH is 1:1 ~ 3.
4. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, it is characterized in that, solvent first described in step (1) is at least one in dichloromethane, dimethyl formamide, oxolane and N-Methyl pyrrolidone, and described solvent second is at least one in dichloromethane, dimethyl formamide, oxolane, N-Methyl pyrrolidone and ethyl acetate.
5. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, it is characterized in that, the described positive-sense strand 3 ' of step (2) is held or antisense strand 5 ' holds the siRNA of modified with mercapto group to comprise 19-30 nucleotide, and molecular weight is 10000-30000.
6. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, is characterized in that, the mol ratio of siRNA and the PLGA-PDPH described in step (2) is 1:20 ~ 50, and PBS and DMSO volume ratio is 1:8 ~ 12.
7. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, is characterized in that, the particle diameter of the siRNA-PLGA conjugate micelle described in step (2) is 200nm ~ 246nm, and surface potential is-40mV ~-25mV.
8. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, is characterized in that, the CSO molecular weight described in step (3) is 1000 ~ 3000, and the degree of polymerization is 6-8.
9. the preparation method of siRNA-PLGA/CSO conjugate micelle according to claim 1, it is characterized in that, step (3) siRNA-PLGA conjugate micelle is hatched under N/P is than the condition being 1 ~ 80:1 with oligochitosan, incubation temperature is 10 DEG C ~ 40 DEG C, and incubation time is 15min ~ 30min.
10. the siRNA-PLGA/CSO conjugate micelle obtained according to the preparation method of the arbitrary described siRNA-PLGA/CSO conjugate micelle of claim 1-9, particle diameter is 150nm-200nm, and surface potential is-18mV ~ 40mV.
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| CN101926775A (en) * | 2010-09-07 | 2010-12-29 | 上海交通大学 | Preparation and application methods of difunctional naonparticle preparation entrapping vincristine sulphate |
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| FU-QIANG HU ET AL.: "A novel chitosan oligosaccharide-stearic acid micelles for gene delivery:Properties and in vitro transfection studies", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》 * |
| SOO HYEON LEE ET AL.: "Self-assembled siRNA-PLGA conjugate micells for gene silencing", 《JOURNAL OF CONTROLLED RELEASE》 * |
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| CN113633613A (en) * | 2021-07-20 | 2021-11-12 | 河南大学 | siRNA micelle, preparation method, composition and application thereof |
| CN113633613B (en) * | 2021-07-20 | 2023-04-25 | 河南大学 | siRNA micelle, preparation method, composition and application thereof |
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