CN107412159A - A kind of preparation method and applications of triblock polymer micella - Google Patents
A kind of preparation method and applications of triblock polymer micella Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of triblock polymer micella, including step：(1) chitosan oligosaccharide reacts with dithiodipropionic acid in the presence of activator, obtains the first intermediate；(2) first intermediates react with polyethyleneimine in the presence of activator, obtain the second intermediate；(3) second intermediates react with urocanic acid in the presence of activator, obtain the triblock polymer；(4) it is the triblock polymer obtained in step (3) is soluble in water, obtain the triblock polymer micella.Triblock polymer micella has excellent tissue penetration and enhancing infiltration and retention effect, and so as to possess natural passive target effect, a kind of new candidate's system is provided for the exploitation of nano-carrier.
Description
Technical field
The present invention relates to technical field of polymer, more particularly to the preparation method of a kind of triblock polymer micella and its should
With.
Background technology
At present, the treatment of tumour turns into global problem urgently to be resolved hurrily, and chemotherapy hand is usually taken in clinical treatment
Duan Jinhang is treated, and has different degrees of harmful effect to the health of patient, the adverse reactions such as vomiting, hair loss easily occurs,
Drug resistance is also easy to produce, albumin nano granular listed etc. involves great expense, and tumor-targeting is undesirable, it is impossible to quick and effective
The release medicine or gene that are loaded, and chemotherapy means be also easy to produce multidrug resistance, tumour can not cut off it is clean, tumour fast
The bad phenomenon such as speed invasion and attack and transfer.Polymer micelle is aobvious in various fields such as medicine as a kind of new pharmaceutical carrier
Huge application potential is shown, there is unique advantage, wherein than it is more prominent be higher stability and preferable bio-compatible
Property, the dissolubility of insoluble medicine can be increased, toxic side effect can be reduced as the effective carrier of gene, there is passive and active target
To effect, curative effect of medication is improved.According to tumor microenvironment feature (pH value, glutathione concentrations, active oxygen such as inside and outside tumour cell
Concentration, temperature change etc.), excitant response can occur in tumour cell for some intelligent polymer micelles, so that structure
Destroy, the loaded chemotherapeutics or gene of release, as Genexol-PM has successfully been listed, also there is multiple polymers micella in addition
Chemotheraping preparation is in clinical research.
Most of chemotherapeutics poorly water-soluble, selectivity is lacked to tumor locus, and normal tissue or organ have stronger
Toxic side effect；Nowadays, occurred using siRNA by interference cell it is intragentic expression come the multidrug resistance of reversing tumor,
Phenomena such as accelerating apoptosis, easily shifting, but siRNA molecule amount is larger, negative electrical charge is stronger, is easily lacked by nuclease degraded etc.
Point, therefore, select suitable gene/drug carrier particularly significant.Due to its stability of cationic polymer in non-virus carrier
It is good, maturation is prepared, it is easily-controllable that structure is adjustable, and convenient modification etc. turns into the main carriers of genomic medicine or antineoplastic.Report
The cationic polymerization species in road are a lot, such as polyethyleneimine (PEI), polyamide, poly-D-lysine, chitosan.It is wherein poly-
Aziridine and polyamide surface have abundant amino, can prepare positive charge nano-particle, with negatively charged cell membrane phase interaction
With, intake of the increase tumour cell to carrier, but such polymer generally has higher cytotoxicity, limits to a certain extent
Its application.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide a kind of excellent tissue penetration and retention effect three
Block polymer micelle, and application of the micella in tumor.
The preparation method of the triblock polymer micella of the present invention, comprises the following steps：
(1) chitosan oligosaccharide (CSO) reacts with dithiodipropionic acid (- ss-) in the presence of activator, obtains the first intermediate
Chitosan oligosaccharide-dithiodipropionic acid (CSO-ss-).
(2) the first intermediate obtained in step (1) reacts with polyethyleneimine (PEI) in the presence of activator, obtains
To the second center casing oligosaccharides-dithiodipropionic acid-polyethyleneimine (CSO-ss-PEI).
(3) the second intermediate obtained in step (2) and urocanic acid (UA) are reacted in the presence of activator, obtained
To triblock polymer chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine-urocanic acid (CSO-ss-PEI- of formula (I)
UA)。
Wherein, n=18-32, m=232-233；
(4) it is the triblock polymer obtained in step (3) is soluble in water, obtain the triblock polymer micella.
Wherein, in step (1)：
Reaction temperature is 30-45 DEG C, preferably 40 DEG C.
Reaction carry out in a solvent, solvent be water, DMF, methanol, chloroform and acetone in one or more, preferably water and
DMF。
Reaction time is 10-16h, preferably 12h.
CSO and dithiodipropionic acid mass ratio 1:2-3.
In step (2)：
Reaction temperature is 20-30 DEG C, preferably 20 DEG C.
Reaction is carried out in a solvent, and solvent is one or both of water and phosphate buffer, preferably water, and phosphate delays
The phosphate buffer of fliud flushing preferably 7.4.
Reaction time is 10-16h, preferably 12h.
Chitosan oligosaccharide-dithiodipropionic acid and PEI mass ratio are 1:1.5-3 preferably 1:2.
In step (3)：
Reaction is carried out in a solvent, and reaction temperature is 40-55 DEG C, preferably 50 DEG C.
Solvent is the one or more in water, DMF, methanol, chloroform and acetone, preferably water and DMF.
Reaction time is 10-16h, preferably 12h.
The mass ratio of chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine and urocanic acid is 1.2-1.5:1, preferably
1.3-1.4:1。
In step (4)：
Reaction temperature is 20-30 DEG C.
The concentration of triblock polymer is 0.8-1.2g/ml, preferably 1mg/ml.
The preparation method of triblock polymer micella is specially：
(1) the chitosan oligosaccharide aqueous solution is obtained by chitosan oligosaccharide (CSO) is soluble in water, and adjust pH to 7-8, under agitation will
It is slowly dropped into the mixed solution of activator and dithiodipropionic acid (- ss-) DMF solution, and at 30-45 DEG C (preferably 40 DEG C)
Lower stirring reaction 10-16h (preferably 12h), obtain the first center casing oligosaccharides-dithiodipropionic acid (CSO-ss-).
Wherein, after reaction terminates, dialysed 1-2 days, preferably 24h (MWCO=1000) with distilled water, dialysate filter, filtrate
Freeze-drying, yield is about 95%.
The CSO molecular weight is 3-5kDa.
The concentration of chitosan oligosaccharide is 20-40mg/ml, preferably 25-35mg/ml in the chitosan oligosaccharide aqueous solution, further preferably
30mg/ml。
The dithio dipropyl acid concentration of the dithiodipropionic acid DMF solution is 65-80mg/ml, preferably 70-80mg/ml,
Further preferred 70mg/ml.
The preferred distilled water of water.
PH can add micro NaOH to adjust.
(2) it is the first intermediate obtained in step (1) is soluble in water, obtain the first intermediate water solution, and with activation
Agent, the aqueous solution mixing of polyethyleneimine (PEI), and stirring reaction 10-16h, preferably 12h at 20-30 DEG C, are obtained in second
Mesosome chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine (CSO-ss-PEI).
Wherein, after reaction terminates, dialysed 1-2 days, preferably 2 days, freezed, yield is about using bag filter (MWCO=1000)
90%.
When pH is about 6 in the aqueous solution of the PEI aqueous solution the first intermediate of addition, remaining PEI water is added after about 30min
Solution.
PEI molecular weight is 600g/mol.
First intermediate water solution (chitosan oligosaccharide-dithio dipropyl aqueous acid) concentration is 5-15mg/ml, preferably 8-
12mg/ml, further preferred 10mg/ml.
Polyethyleneimine concentration is 350-450mg/ml, preferably 400-450mg/ml in the aq. polyethyleneimine,
Further preferred 400mg/ml.
The preferred distilled water of water.
(3) the second intermediate (chitosan oligosaccharide-two thio two is obtained by the second intermediate obtained in step (2) is soluble in water
Propionic acid-polyethyleneimine) aqueous solution, and be slowly dropped in the mixed solution of activator and urocanic acid (UA) DMF solution,
Stirring reaction 10-16-h, preferably 12h under 40-55 DEG C (preferably 50 DEG C), obtain the triblock polymer chitosan oligosaccharide-two of formula (I)
Thio-2 acid-polyethyleneimine-urocanic acid (CSO-ss-PEI-UA).
Wherein, after reaction terminates, liquid is placed in bag filter (MW=1000), distilled water dialysis 2-3d, preferably 2d, thoroughly
Liquid filtering is analysed, takes supernatant to be freeze-dried, yield is about 90%.
UA molecular weight is 138.12g/mol.
Chitosan oligosaccharide-dithiodipropionic acid-polyethylene in the chitosan oligosaccharide-dithiodipropionic acid-aq. polyethyleneimine
Imines concentration is 4-6mg/ml, preferably 4-5mg/ml, further preferred 4.8mg/ml.
Urocanic acid concentration is 15-20mg/ml, preferably 17-18mg/ml in the urocanic acid DMF solution, enters one
Walk preferred 17.2mg/ml.
The preferred distilled water of water.
The reaction equation of step (1) to (3) is as follows：
Wherein, n=18-32, m=232-233.
(4) it is at 20-30 DEG C, the triblock polymer obtained in step (3) is soluble in water, by filtering membrane filtration
Afterwards, the triblock polymer micella is obtained.
Further, in step (1), occur after described activated dose of activated carboxyl of dithiodipropionic acid with the chitosan oligosaccharide
Reaction.
In step (2), reacted after described activated dose of activated carboxyl of first intermediate with polyethyleneimine.
In step (3), reacted after described activated dose of activated carboxyl of urocanic acid with second intermediate.
Further, the activator for 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC) hydrochloride,
One or more in N- hydroxysuccinimides (NHS), dicyclohexylcarbodiimide (DCC).
It is preferred that EDC and NHS, both mol ratios are 1：1.
Further, in step (1), the mol ratio of activator and-ss- is 0.8-1.2:1, preferably 1：1, dithio dipropyl
Carboxyl mole in acid is excessive, it is ensured that only reacts a carboxyl.
In step (1), at 30-45 DEG C, preferably 40 DEG C, the dithio dipropyl acid solution carries out carboxyl in activator
Activate about 30min.
Further, in step (2), activator and the first intermediate CSO-ss- mol ratio are 0.8-1.2:1, preferably
1：1.
In step (2), the first intermediate CSO-ss- aqueous solution is mixed with the activator aqueous solution, stirring reaction, carry out carboxylic
Base activates.
Further, in step (3), the mol ratio 0.8-1.2 of activator and urocanic acid:1, preferably 1：1.
In step (3), at 40-55 DEG C, the urocanic acid solution carries out activated carboxylic about in activator
30min。
Further, in step (4), the triblock polymer is added to the water, and formation three is embedding under conditions of ultrasound
Section polymer micelle.
It is it is preferred that ultrasonic in ice bath.
Preferred probes ultrasound 50 times (400w, work 2s stop 2s).
Triblock polymer micella is as tumour medicine and/or the carrier of gene made from the above-mentioned preparation method of the present invention
Application, the triblock polymer micella for being loaded with tumour medicine and/or gene is triblock polymer carrier micelle.
Further, the gene is siRNA, the acceptor gene of Toll-like 4, MDRG, Bcl-2 anti-apoptotic bases
One or more in cause, programmed death ligand gene, Mcl-1 anti-apoptotic genes expressions.
Further, the tumour medicine is adriamycin, Osthole, taxol, harmine, camptothecine, sand difficult to understand
One or more in sharp platinum, Etoposide and Cetuximab.
The preparation method of the triblock polymer carrier micelle of the present invention, comprises the following steps：By drug solution and/or base
Because triblock polymer micellar solution mixes made from solution and the above method, triblock polymer carrier micelle is obtained.
The preparation method of triblock polymer carrier micelle is specially：By drug solution (DMSO solution of such as adriamycin, if
For doxorubicin hydrochloride, its hydrochloric acid need to be neutralized with triethylamine) it is slowly dropped in triblock polymer micellar solution, ultrasonic 20-
40min, preferably 30min, preferably magnetic agitation 1-3h, 2h, are subsequently placed in bag filter the 4h that dialyses, and first water is changed per 1h,
0.45 μm of filtering with microporous membrane of dialyzate, obtains triblock polymer carrier micelle after freeze-drying；Or by gene freeze-dried powder
The aqueous solution (such as siRNA freeze-dried powders 0.5OD adds 62.5 μ L DEPC water to dissolve to form 20 μM of stock solutions, -20 DEG C of preservations) and three block
Polymer micelle solution/triblock polymer polypeptide drug-loaded micelle solution is well mixed, and 20-30 DEG C (room temperature) is incubated 30min, obtains band
The triblock polymer carrier micelle of gene；Wherein, the gene, which is siRNA, programmed death ligand gene, Mcl-1 are anti-withers
Die the one or more in gene；The medicine is adriamycin, Osthole, taxol, harmine, camptothecine, sand difficult to understand
One or more in sharp platinum, Etoposide and Cetuximab.
Further, the N/P ratios of the gene and the triblock polymer micella are 4-20：1, N is that three block polymerize
Amino in thing micella, P are phosphate groups in gene.
Wherein, the mass ratio of the tumour medicine and the triblock polymer micella is 0.1-0.5：1-3.
By such scheme, the present invention at least has advantages below：
CSO of the present invention has good biocompatibility and water solubility, can make hydrophily shell, but individually make
It is extremely unstable to be used as carrier；PEI is widely used in genophore, has a proton sponge effect, but be used alone toxicity compared with
Greatly, therefore in order to solve this problem, two activated carboxylics in dithiodipropionic acid are distinguished chain by the present invention as linking arm
CSO and PEI are met, relative to PEI, the grafting rate that CSO grafting rate is 3.33%, UA is 44.4%, PEI one end connection imidazoles third
Olefin(e) acid (UA) is that hydrophobic inner core can be used to contain insoluble drug, and the other end passes through the disulfide bond (- ss-) in dithiodipropionic acid
One layer of CSO of outsourcing is used for that to maintain whole polymer be positive electricity, while reduces PEI toxicity, polymer can in water self assembly shape
Into with the nano-particle that pH is sensitive and reduction is sensitive, so as to reduce the toxicity of polymer and to embody polymer micro- in tumour
Reduction-sensitive (- ss-) and pH sensitiveness (proton sponge effect) in environment, use PEI to can be used to adsorb siRNA for main chain
(being by EDC and NHS activated carboxyls between each unit, amino is formed amido link with carboxyl reaction), the load formed with this
Body can realize medicine and the gene association treatment of tumour, and a kind of novel carriers are provided for the delivery system of tumour.
The nano particle diameter that the polymer micelle of the present invention is formed is 140nm or so, has excellent tissue penetration
And enhancing infiltration and retention effect, so as to possess natural passive target effect, one kind is provided for the exploitation of nano-carrier
New candidate's system；, can be after particle be formed again by gene and this polymer has the polymer beads of unique three-decker
Compress into PEI layers, be advantageous to control micella particle size and micella particle large-scale production, for structure nano-carrier,
Develop the new drug-loading system based on acid-sensitive sensitive polymeric and chemical constitution reference is provided.
Polymer medicament carrying micelle nano-particle prepared by the present invention, by PEI protonate caused by hydrophobe property change,
And-ss- high concentration glutathione (GSH) effects in tumour cell it is lower can fast fracture, make polymer micelle nano-particle tool
There are certain acid-sensitive characteristic and reduction sensitivity characteristic, conformation is changed under tumour cell sour environment, and release is loaded
Gene and medicine；Available under the conditions of existing for serum rotaring redyeing gene enter target cell and adjust destination protein expression, and
And can simultaneously carrying anti-tumor medicine and gene, so as to combine performance antitumor action.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is CSO (a), CSO-ss- (b), PEI (c), CSO-ss-PEI (d), CSO-ss- in embodiments of the invention 1
PEI-UA's (e)1H-NMR collection of illustrative plates；
Fig. 2 illustrates fluorescence intensity of the triblock polymer in various concentrations in embodiments of the invention 1；
Fig. 3 illustrates in embodiments of the invention 1 triblock polymer in λ1At=372nm and λ3Fluorescence at=383nm
Business (the I of intensity1/I3) ratio with the logarithm of respective concentration；
Fig. 4 illustrates what triblock polymer in embodiments of the invention 1 detected in dynamic light scattering particle size analyzer DLS
Particle size changes；
Fig. 5 is the transmission electron microscope picture of the triblock polymer micella prepared in embodiments of the invention 1；
Fig. 6 is the In-vitro release curves figure of Dox-micelle in embodiments of the invention 1；
Fig. 7 is that siRNA and the electrophoretogram of triblock polymer micella binding ability are determined in embodiments of the invention 1；
Fig. 8 is that the electrophoresis that triblock polymer micella is acted on siRNA enzyme protection is determined in embodiments of the invention 1
Figure；
Fig. 9 is that the serum stability electricity after siRNA is combined with triblock polymer micella is determined in embodiments of the invention 1
Swimming figure；
Figure 10 is the In-vitro release curves figure of siRNA-micelle in embodiments of the invention 1, and wherein A is pH5.3, and B is
PH=5.3+GSH, C pH7.4, D pH=7.4+GSH.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
Embodiment 1
The preparation of triblock polymer, comprises the following steps：
(1) CSO-ss- synthesis
1.578g dithiodipropionic acids, 1.437g EDC and 0.861g NHS are dissolved in 20ml DMF, 40 DEG C of activation
30min is stand-by；Weigh 0.6g CSO to be scattered in 20mL distilled water, it is 7-8 to add micro NaOH and adjust its pH, will under stirring condition
It is slowly dropped into dithio dipropyl acid solution, after 40 DEG C of stirring reaction 12h, with distilled water dialyse 1d (MWCO=1000), thoroughly
Liquid filtering is analysed, filtrate freeze-drying, produces CSO-ss-, yield 95%.
(2) CSO-ss-PEI synthesis
CSO-ss- prepared by 1g steps (1) is dissolved in 100mL distilled water, stirring forms the aqueous solution；By 2.935g
EDC, 1.435g NHS are dissolved in the formation activator aqueous solution in 5ml distilled water, and are added into the CSO-ss- aqueous solution and activate
30min, 2g PEI are dissolved in 5ml distilled water, be added into the CSO-ss- aqueous solution after activation, and as pH to 6 or so
Stop adding, add the remaining PEI aqueous solution, 20 DEG C of stirring reaction 12h, 1000 bag filters dialysis (MWCO=after 30min again
1000) 2 days, dialysate filter, filtrate freeze-drying, CSO-ss-PEI, yield 90% are produced.
(3) CSO-ss-PEI-UA synthesis
0.43g UA, 1.19g EDC and 0.71gNHS are added in the flask containing 25ml DMF, stirring 30min is extremely
It dissolves；The CSO-ss-PEI-UA prepared in 0.6g steps (2) is dissolved in 125mL distilled water, is then slowly added dropwise
Into above-mentioned flask, 12h is reacted at 50 DEG C, the liquid in flask is placed in bag filter (MW=1000) by reaction after terminating, and is steamed
Distilled water dialysis 2d, dialysate filter, takes supernatant to be freeze-dried, obtains CSO-ss-PEI-UA, yield 90%.
(4) synthesis of triblock polymer micella
Polymer CSO-ss-PEI-UA is dissolved in 5ml deionized waters, (400w, work 2s stop for ice bath Probe Ultrasonic Searching 50 times
2s), 1mgmL is made-1Blank triblock polymer micellar solution (blank-micelle).
(5) synthesis of triblock polymer carrier micelle
0.5mg doxorubicin hydrochlorides (Dox) DMSO solution (is added into the hydrochloric acid in triethylamine and in Dox, wherein Dox:Three
Ethamine mol ratio=1:2) it is slowly dropped in the micellar solution in step (4), ultrasonic 30min, magnetic agitation 2h, is subsequently placed in
Dialysed 4h in bag filter, and first water, 0.45 μm of filtering with microporous membrane of dialyzate are changed per 1h, and freeze-drying obtains three block
Polymer medicament carrying micelle (being load adriamycin micella Dox-micelle in the present embodiment).
(6) synthesis of the triblock polymer carrier micelle with gene
SiRNA freeze-dried powders 0.5OD is added into 62.5 μ L pyrocarbonic acid diethyl esters (DEPC), and (DEPC is exactly coke diethylester, DEPC
Water i.e. coke diethylester processing and the water after autoclave sterilization, or DEPC water be changed to sterilized water can also) place
Manage and the water through autoclave sterilization dissolves to form 20 μM of stock solutions, -20 DEG C of preservations；By Dox-micelle, blank-
Micelle respectively with siRNA according to N/P (N be triblock polymer micella in amino, P be gene in phosphate groups, N/P
Can be understood as the mol ratio of carrier and gene) ratio is well mixed for 1-8, and 30min is incubated at room temperature, produces Dox-siRNA-
Micelle and siRNA-micelle, rifle point used passes through DEPC processing with EP pipes in reaction.
The compound in the embodiment is characterized below and the performance measurement of product：
1st, nuclear-magnetism characterizes
CSO, CSO-ss-, PEI, CSO-ss-PEI and CSO-ss-PEI-UA are dissolved in D respectively2In O, in 400MHz nuclear-magnetisms
Tested on resonance instrument,1H-NMR collection of illustrative plates is as shown in Figure 1：δ=4.67 (H1) 3.20-4.00ppm (sugared ring) in CSO, 2.01 (-
CH2)；The specific proton peak of five heterocyclic groups of the signals assignment in dithiodipropionic acid at δ=2.74,2.93ppm, shows
Dithiodipropionic acid is successfully grafted to CSO；Proton peak in CSO-ss-PEI conjugation spectrograms at δ=2.3-2.7ppm confirms
PEI introducing；UA signal is located at δ=8.23 (Ha), δ=7.61 (Hb), δ=7.5 (Hc) and δ=6.51 (H1), shows UA
It is successfully introduced into CSO-ss-PEI；Given CSO-ss-PEI-UA spectrum, distributing to the resultful peak of above-mentioned institute means CSO-
The successful synthesis of ss-PEI-UA copolymers, CSO-ss-PEI-UA polymer are successfully prepared.
2nd, critical micelle concentration (CMC) measure of polymer
The CMC value in pH 7.4 using pyrene as hydrophobic fluorescence probe assay polymer, with each sample solution of measure
Log concentration value (lgC) be abscissa, as shown in Fig. 2 fluorescence intensity of the polymer in various concentrations, with each sample solution
In λ1At=372nm and λ3Ratio (the I of fluorescence intensity at=383nm1/I3) it is ordinate, scatter diagram is drawn, such as Fig. 3 institutes
Show, the horizontal tangent of data point, and mutation curve near tangent, the polymerization corresponding to the intersection point of two tangent lines are made according to each point
Thing concentration, as critical micelle concentration (CMC).
As can be seen from Figure 3, when polymer concentration is relatively low, I1/I3Value keeps constant, shows that polymer does not form micella；When dense
After degree reaches certain value, I1/I3Value drastically declines, and illustrates that polymer initially forms micella in the concentration.With small molecule surface
Activating agent is compared, and the CMC of polymer can reach 7.94 × 10-3mg.mL-1, this explanation is in dilution, the polymer shape
Into micellar phase to stable, have the possibility as pharmaceutical carrier.
3rd, the pH sensitiveness of polymer and reduction-sensitive measure
Polymer nano-particle (2mg/mL) is prepared by probe sonication method, for Study Polymer Melts nano-particle
PH and redox respondent behavior, we with dynamic light scattering particle size analyzer DLS detection particle diameter size variation, such as Fig. 4
It is shown.The polymer nano-particle tested is dissolved under the following conditions in different solution：(i) under pH 7.4,
(ii) under pH 5.3, (iii) uses GSH (100mM) and (iv) in 5.3 times GSH of pH (100mM) 7.4 times in pH.
It is small that polymer nano-particle is incubated 1 with phosphate buffer (PBS) (pH 7.4)+GSH and PBS (pH 5.3) respectively
Shi Hou, 365.3 and 622.6nm are expanded to from 124.6nm, it is bimodal for PBS (pH 5.3)+GSH acquisitions, it is steady which imply micella
It is qualitative destroyed.The change of particle diameter is due to the protonation of imidazole radicals in an acidic solution, and two occur in the presence of GSH
The fracture of sulfide linkage, this causes the fast decoupled of hydrophilic/hydrophobic balance and promotes to discharge in the rapid cellular of medicine and gene.
4th, the transmission electron microscope picture of triblock polymer micella
As shown in figure 5, micellar conformation rounding, consistent with granularmetric analysis result, accumulated using EPR effects in tumor locus
Realize that passive target is treated.
5th, In-vitro release curves of the Dox-micelle in different buffer solutions (PBS)
As shown in fig. 6, Dox releases of the Dox-micelle under pH 7.4PBS is suppressed, the cumulative release in 48 hours
26.71% ± 1.96%.However, being capable of quick medicament release in pH 5.3PBS or glutathione (GSH) (10mM).Having
In the pH 5.3PBS for having GSH (10mM), preparations of the Dox in Dox-micelle is up to 72.92% ± 3.22%.This
Kind fractional release is almost twice in pH7.4PBS.Protonation of the imidazole group in sour environment contributes to fast quick-release
Put, cause the hydrophily of micellar hydrophobic kernel to increase.The fracture of disulfide bond is also to cause hydrophilic/hydrophobic to balance under the reducing conditions
Fast decoupled a factor.As a result show that the polymer micelle has what extracellular relatively stable and cell interior energy quickly released the drug
Characteristic, effective controlled release of medicine is realized, is advantageous to the treatment of tumour.
6th, siRNA and the triblock polymer micella binding ability are determined using agarose gel electrophoresis
0.25g agaroses are weighed, is dissolved in 1 × electrophoretic buffers of 25mL (TAE) buffer solution, is heated in micro-wave oven
2.5 μ L nucleic acid gels dyestuffs (GelRed) are added after dissolving, is well mixed, solution is then poured into glue in glue groove, treated cold
But electrophoresis is prepared to being put into electrophoresis tank after room temperature.
0.025nmolsiRNA is taken to be then respectively adding 17 into 6 EP pipes without ribalgilase (RNase) respectively
Treated μ L DEPC water, 1 μ L triblock polymers micellar solution, 2 μ L triblock polymers micellar solutions, 4 μ L three blocks are gathered
Compound micellar solution, 8 μ L triblock polymers micellar solutions, 16 μ L triblock polymer micellar solutions, and treated with DEPC
Water each group is supplied to 18 μ L.30min is stored at room temperature after solution in each EP pipes is sufficiently mixed, takes 15 μ L to add 3 μ respectively
Loading after 6 × loading of L buffer are mixed, gel imager observation experiment result, such as Fig. 7 are used after 100V, electrophoresis 15min
It is shown, each swimming lane siRNA and polymer N/P ratios respectively 1,2,4,5,6 in figure, as can be seen from Fig., with N/P than increasing
Add, more weakened by carrier Electrostatic Absorption, the fluorescence intensity for the band that dissociates, when N/P ratios are 5, envelop rate is about 90%, and
When N/P ratios are 6, almost all contained, its envelop rate is about 98%.
7th, the triblock polymer micella is determined using agarose gel electrophoresis to act on siRNA enzyme protection
Degradation capabilities of the siRNA to nuclease-resistant is protected using agarose gel electrophoresis experimental examination micella, with free
SiRNA is compared.0.12nmolsiRNA is taken to add 17 μ L DEPC in 1-2 pipes into 6 EP pipes without RNase respectively
Treated water；4 μ L triblock polymers micellar solutions and 13 μ L DEPC water are separately added into 3-6 pipes, will be molten in each EP pipes
Liquid is stored at room temperature 30min after being sufficiently mixed；2-6 pipes are separately added into 1U of ribalgilase RNase I/ μ gsiRNA, respectively room
Temperature is incubated 30min, 30min, 60min, 90min, 120min；3-6 pipes are separately added into liquaemin, are incubated at room temperature 15min, embedding from three
Decomplexing goes out siRNA in section polymer micelle.After taking the μ L of 1-6 pipes sample 15 and 3 μ 6 × loading of L buffer to mix respectively
Loading, gel imaging system observation and analysis result after 100V, electrophoresis 15min, as shown in Figure 8.
As a result show, exposed siRNA and 1U of RNase I/ μ gsiRNA, which are incubated after 30min, occurs degraded (band
2), and almost without degraded (band 3) occurs after siRNA-ACPU micelle and RNase I incubations 30min, it is incubated 120min
Remain to preferably keep siRNA integrality (band 6) afterwards.As a result the micella for showing to prepare can slow down nucleic acid to a certain extent
Degraded of the enzyme to siRNA.
8th, the serum stability after being combined using agarose gel electrophoresis measure siRNA with the triblock polymer micella
Degraded situation after siRNA-micelle is incubated different time in serum is compared using agarose gel electrophoresis,
Compared with free siRNA.0.012nmolsiRNA is taken to add 17 μ in 1-2 pipes into 6 EP pipes without RNase respectively
Water treated L DEPC；4 μ L triblock polymers micellar solutions and 13 μ L DEPC water are separately added into 3-6 pipes, each EP is managed
In solution be sufficiently mixed after be stored at room temperature 30min；2-6 pipes are separately added into 20% hyclone, are incubated at room temperature respectively
30min、30min、60min、90min、120min；3-6 pipes are separately added into liquaemin, are incubated at room temperature 20min, polymerize from three block
Decomplexing goes out siRNA in thing micella.Take respectively the μ L of 1-6 pipes sample 15 and 3 μ 6 × loading of L buffer mix after loading,
Gel imaging system observation and analysis result after 100V, electrophoresis 15min, as shown in Figure 9.
It can be seen that triblock polymer micella combine siRNA after heparin decomplexing, in the electric field by negative pole
Moved to positive pole, dyed on gel slab by GelRed, white bright band occur.Exposed siRNA and 20% sera incubation
Degraded (band 2) occurs after 30min, and remains to preferably protect after siRNA-micelle and 20% sera incubation 120min
Hold siRNA integrality (band 6).Illustrate exposed siRNA and sera incubation, easily degraded by the enzyme in serum, show bleeding
Clear unstability；And after siRNA-ACPU micelle are incubated altogether with serum, siRNA degradeds are reduced, and degradation time extends, explanation
Micella can reduce degraded of the enzyme to siRNA in serum.As a result there is the micella for showing to prepare certain protection to make to siRNA
With.
9th, releases of the SiRNA in micella
PBS (pH=5.3), PBS (pH=5.3)+GSH, PBS (pH=7.4) and PBS (pH=7.4)+GSH are chosen respectively
Release characteristics of the siRNA-micelle under different pH environment are investigated as dissolution medium.
SiRNA-micelle is diluted with different dissolution mediums, is respectively placed in centrifuge tube, every group of parallel three sample
Product, in 37 DEG C, 100rpmmin-1Under the conditions of carry out extracorporeal releasing experiment, be respectively separated 0.5h, 1h, 2h, 6h, 12h, 24h,
48h is sampled, 4 DEG C, 50000rpmmin-145min is centrifuged, takes supernatant, is existed with excitation wavelength 480nm measure prepare liquids
Emission spectrum in the range of 500nm-600nm, it is 10nm to excite with transmite slit bandwidth, as a result as shown in Figure 10.
Under pH 7.4 and pH 7.4+GSH media, siRNA is adsorbed onto PEI layers, and such case effectively prevents siRNA
Discharged from siRNA-micelle；However, micella becomes unstable so as to discharging siRNA under pH 5.3 and pH 5.3+GSH.
These results show that siRNA-micelle can protect siRNA intracellular from the enzyme degraded in physiological environment and enhancing
SiRNA discharges, and the siRNA discharged in cytoplasm is targetted with the transcription of silence target gene and can lower the table of destination protein
Reach.
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of triblock polymer micella, it is characterised in that comprise the following steps：
(1) chitosan oligosaccharide reacts with dithiodipropionic acid in the presence of activator, obtains the first intermediate；
(2) the first intermediate obtained in step (1) reacts with polyethyleneimine in the presence of activator, obtains among second
Body；
(3) the second intermediate obtained in step (2) is reacted in the presence of activator with urocanic acid, obtains formula (I)
Triblock polymer；
Wherein, n=18-32, m=232-233；
(4) it is the triblock polymer obtained in step (3) is soluble in water, obtain the triblock polymer micella.
2. preparation method according to claim 1, it is characterised in that：
In step (1), reacted after described activated dose of activated carboxyl of dithiodipropionic acid with the chitosan oligosaccharide；
In step (2), reacted after described activated dose of activated carboxyl of first intermediate with polyethyleneimine；
In step (3), reacted after described activated dose of activated carboxyl of urocanic acid with second intermediate.
3. preparation method according to claim 2, it is characterised in that：The activator be 1- (3- dimethylamino-propyls)-
One or more in 3- ethyl-carbodiimide hydrochlorides, N- hydroxysuccinimides, dicyclohexylcarbodiimide.
4. preparation method according to claim 1, it is characterised in that：In step (1), the activator and dithio dipropyl
The mol ratio 0.8-1.2 of acid：1.
5. preparation method according to claim 1, it is characterised in that：In step (2), the activator and the first intermediate
The mol ratio 0.8-1.2 of chitosan oligosaccharide-dithiodipropionic acid：1.
6. preparation method according to claim 1, it is characterised in that：In step (4), the triblock polymer adds water
In, triblock polymer micella is formed under ultrasound condition.
7. triblock polymer micella made from the preparation method as described in 1-6 any claims as tumour medicine and/or
The application of the carrier of gene.
8. application according to claim 7, it is characterised in that：The gene is siRNA, the acceptor gene of Toll-like 4, multiple medicine
One or more in drug resistant gene, Bcl-2 anti-apoptotic genes expressions, programmed death ligand gene, Mcl-1 anti-apoptotic genes expressions.
9. application according to claim 7, it is characterised in that：The tumour medicine is adriamycin, Osthole, Japanese yew
One or more in alcohol, harmine, camptothecine, oxaliplatin, Etoposide and Cetuximab.
10. a kind of preparation method of triblock polymer carrier micelle, it is characterised in that comprise the following steps：By tumour medicine
And/or triblock polymer micella made from any one of gene and claim 1-6 mixes, and obtains the triblock polymer
Carrier micelle.
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