CN101474408A - Polyethylene glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer and siRNA medicament carrier prepared thereby - Google Patents
Polyethylene glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer and siRNA medicament carrier prepared thereby Download PDFInfo
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- CN101474408A CN101474408A CNA2008100559458A CN200810055945A CN101474408A CN 101474408 A CN101474408 A CN 101474408A CN A2008100559458 A CNA2008100559458 A CN A2008100559458A CN 200810055945 A CN200810055945 A CN 200810055945A CN 101474408 A CN101474408 A CN 101474408A
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Abstract
The invention discloses a siRNA drug carrier. An active component of the siRNA drug carrier is a nano-particle with positive charge formed by polymer which can be polyethylene glycol monomethyl ether-polycaprolactone-polyphosphate ester tri-block copolymer concretely. The invention also provides a polyethylene glycol monomethyl ether-polycaprolactone-polyphosphate ester copolymer and a method for synthesizing the copolymer. The biocompatible tri-block copolymer provided by the invention forms the nano-particles by self-assembly in aqueous solution and has good stability, simple preparation method and high repeatability. The polymer nano-particle is used as a carrier which can protect siRNA from degrading and can also combine with the self size effect of the nano-particle. Nano-micelle has good biocompatibility, and macromolecular micelle has the characteristics of very good stability, convenient preparation and the like, thereby ensuring the biodegradable nano-micelle has good application prospect in siRNA delivery and disease treatment based on RNA interference.
Description
Technical field
The present invention relates to the siRNA pharmaceutical carrier of a kind of poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer and preparation thereof.
Background technology
It is the gene silent technology that a kind of double-chain small molecule RNA interfering of being made up of about more than 20 nucleotide (siRNA) mediates that RNA disturbs, and it has sequence-specific and occurs in after the transcribing of hereditary information.Since the RNA perturbation technique have specificity suppress ability that Disease-causing gene expresses with and efficient, diversified feature, people are constantly excavating the potentiality of siRNA in human disease treatment, up to the present a large amount of zoopery even clinical experiments have been carried out at disease treatment, this is comprising to express VEGF (VEGF), the P glycoprotein, range genes such as telomerase are the treatment of cancer of target gene, at HIV-1, B-mode and hepatitis C, the experiment of virus such as influenza and SARS, and at the research of some neurodegenerative disease etc.Nearest research also shows and utilizes siRNA also may treat type ii diabetes and obesity, and the siRNA therapy of the old maculopathy that the retinal degeneration pathological changes causes has entered clinical experimental stage especially.The Acuity drugmaker in U.S. Philadelphia also announces can " close " the VEGF gene based on the Bevasiranib medicine of RNAi in ASGT's annual meeting in 2006, and the II clinical trial phase that is used for treating treating senile maculopathy has been obtained initial success.Have RNAi expert to point out, the achievement of this test is a milestone of RNA therapy development, but also prophesy RNAi just may be used for clinical treatment in the near future.
Although at present in most cells cultivation and external applied research, siRNA demonstrates the good potential quality of suppressor gene, the transmission result of external siRNA is also satisfied, but is faced with huge challenge aspect the siRNA transmission in the body that with the treatment human diseases is purpose.Because the ability extreme difference of the permeates cell membranes of siRNA molecule own does not possess target function yet, and extremely unstable in physiological environment, so a transmission system that key is siRNA of siRNA drug development.How to strengthen in vivo stability and the ability of permeates cell membranes, and to strengthen the cell of disease treatment and tissue target tropism etc. all be the problem that the siRNA transmission system need solve in a hurry.Therefore except seeking and screening the efficient specific siRNA drug molecule, at the transmission system of too busy to get away targeting particular organization of life system performance RNAi therapeutic efficiency or target cell, therefore transmission system also becomes another key point that develops and implement RNA interference therapy in the body of siRNA.Although the vein fluid power of zoopery mesohigh large volume injection (hydrodynamic injection) can be transported to the siRNA molecule euangiotic tissues such as liver, spleen, lung, kidney, pancreas, and very effective, but in practical clinical, be restricted; Some physical methods such as electroporation (Electroporation), and siRNA directly delivered to target site such as eyes, lung or central nervous system, this class locality carrying method has certain potential, still need to seek the new method of more convenient suitable clinical practice, the method that particularly is administered systemically.Transmission is a problem that is similar to gene therapy in the body of carrier mediated siRNA expression plasmid, can use some physical methods under study for action, and the transmission system of virus or non-virus.Yet although virus has high efficiency to its transmission, potential safety issue is restricting its extensive clinical practice; But not the good characteristics of safety of the cationic-liposome of virus type and cation high molecular carrier make them become the carrier that has potentiality.Existing typical case is non-, and viral siRNA transferring material comprises polymine (PEI), refining collagen " atelocollagen " and some cationic-liposomes of end peptide have been removed, the siRNA-SNALP (stable nucleic-acid-lipid particle) that comprises nearest report, carry by antibody-mediated cell-specific siRNA, and neutral fat plastid DOPC, also useful specificity based on the adaptive son of oligonucleotide (Aptamer) is carried the report of siRNA to cell, still faces the challenge but development efficiently has the interior transmission system of the siRNA body of certain targeting and safety.
The research and development of siRNA drug administration carrier is puzzlement various countries scientist's a key issue always.People are studying the suitable carriers material always can be transported to target cell and position with the siRNA drug molecule effectively, and keeps its effect one period reasonable time.Currently used carrier system is cationic-liposome or water miscible cationic polymer mostly, these carrier molecules and siRNA solution are mixed the complex that obtains the two mutually, thereby realize transmitting the purpose of siRNA, the deficiency of these class methods is exactly to be not easy amplification scale and repeated relatively poor.The Direct RNAi of Alnylam drugmaker development
TMTechnology mainly is the direct administration at lesions position, is used for the treatment of ophthalmic diseases, central nervous system disease and respiratory tract disease; And the Systemic RNAi of the said firm
TMTechnology is used for the treatment of numerous diseases such as comprising tumor, digestive system disease and autoimmune disease.Intradigm company utilizes TargeTran
TMSiRNA technology, this technology utilize cationic block macromolecular to form the kernel of nano-particle by electrostatic interaction and siRNA, and cover the shell that one deck has protection and strengthens siRNA stability in the blood, and the targeting group.
Be widely used in the research of the transmission from micromolecule to the biologically active dna molecule based on high molecular nano-particle, obtained high achievement, wherein a lot of achievements have been used for clinical or clinical trial.The particulate transmission system of high molecular nanometer good biocompatibility, the potential with targeted delivery has better stability, can effectively protect the toleration of bioactive molecule to physiological environment, and strengthens cell to being transmitted the absorption of molecule.It is worthy of note that especially the nanoparticle of macromolecule self assembly has EPR effect (Enhanced permeability andretention), can promote the enrichment of medicine at tumor tissues, and can be absorbed by the tumor locus cell effectively, enter Cytoplasm and various organelle, so the high molecular nanometer grain is the candidate that has the siRNA transmission system of potential.And has the degradable macromolecule carrier of good biocompatibility; the effectively combination and protection siRNA molecule of degradable cationic type macromolecule carrier that particularly has quick deprotonation ability; siRNA is discharged rapidly in Cytoplasm; realize the purpose that blocking gene is expressed, this will make this class carrier have better advantage and application prospect in little RNA administration.Traditional mode all adopts water miscible cationic polymer, these carrier molecules and siRNA are mixed complex or the nano-particle that obtains the two mutually, thereby realize transmitting the purpose of siRNA, the obvious deficiency of these class methods is that technology is difficult to amplify, and is repeatable relatively poor.
Summary of the invention
An object of the present invention is to provide the pharmaceutical carrier of a kind of siRNA, its active component is the positively charged nano-particle of polymer formation.
Described nano-particle can carry out chemical modification or ligand modified.
Described polymer can be poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer.
In above-mentioned poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer, described polycaprolactone is a poly-epsilon-caprolactone, and described poly phosphate is polymerized by the five-membered cyclic phosphate ester monomer of structural formula suc as formula (I):
In above-mentioned poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer, mid-block is described polycaprolactone, and an end-blocks is a poly glycol monomethyl ether, and another end-blocks is described poly phosphate; The degree of polymerization of described poly glycol monomethyl ether block is 45, and corresponding number-average molecular weight is 2,000g/mol; The degree of polymerization of described polycaprolactone block is 45-100, and corresponding number-average molecular weight is 5,130-11,400g/mol; The degree of polymerization of described poly phosphate block is 7-12, and corresponding number-average molecular weight is 1,420-2,430g/mol.
Above-mentioned triblock copolymer can be mPEG
45-PCL
45-PPEEA
7Or mPEG
45-PCL
100-PPEEA
12
The diameter of described nano-particle can be 30-121nm.
The present invention also provides a kind of poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer.
Above-mentioned polycaprolactone is a poly-epsilon-caprolactone, and above-mentioned poly phosphate is polymerized by the five-membered cyclic phosphate ester monomer of structural formula suc as formula (I).
In the above-mentioned triblock copolymer, mid-block is described polycaprolactone, and an end-blocks is a poly glycol monomethyl ether, and another end-blocks is described poly phosphate; The degree of polymerization of described poly glycol monomethyl ether block is 45, and corresponding number-average molecular weight is 2000g/mol; The degree of polymerization of described polycaprolactone block is 45-100, and corresponding number-average molecular weight is 5,130-11,400g/mol; The degree of polymerization of described poly phosphate block is 7-12, and corresponding number-average molecular weight is 1,420-2,430g/mol.
The present invention also provides the method for a kind of synthesizing polyethylene glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer.
The method of described synthesizing polyethylene glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer is to be macromole evocating agent with terminal hydroxy group poly glycol monomethyl ether-polycaprolactone, with the stannous iso caprylate is catalyst, obtains triblock copolymer by the monomeric ring-opening polymerization of annular phosphate.
Above-mentioned terminal hydroxy group poly glycol monomethyl ether-polycaprolactone can poly glycol monomethyl ether be an initiator, is catalyst with the stannous iso caprylate, causes caprolactone monomer and be polymerized under bulk conditions.
Above-mentioned annular phosphate monomer can pass through 2-chloro-2-oxygen-1,3, and 2-dioxaphospholane and N-tertbutyloxycarbonyl amido ethanol synthesis obtain.
Above-mentioned 2-chloro-2-oxygen-1,3, the 2-dioxaphospholane can obtain by ethylene glycol and Phosphorous chloride. reaction.
Above-mentioned N-tertbutyloxycarbonyl amido ethanol (EABoc) can obtain by ethanolamine and Bis(tert-butoxycarbonyl)oxide reaction.
Behind the monomer polymerization of said structure formula suc as formula (I), just can obtain the poly phosphate polymer that side group is an amido after sloughing tertbutyloxycarbonyl (Boc) blocking group.
The triblock copolymer that the present invention obtains has excellent biological compatibility and degradability, and its physics, chemical property can be regulated by the composition of telomerized polymer.The invention provides the triblock copolymer of biocompatibility, self assembly forms nano-particle in aqueous solution, has good stable; preparation method is simple; repeatable high, can protect siRNA to avoid degraded as carrier, scale effect that again can combining nano granule itself.This copolymer mainly is applicable to nano-medicament carrier, gene therapy vector, macromolecule precursor medicament, fields such as biomaterial surface modification.
Poly glycol monomethyl ether-polycaprolactone provided by the invention-poly phosphate block copolymer can form the nano-particle that has positive charge, and the particle diameter of this nano-particle is about 100nm, and surface charge is about 40mV.Suitable positive and negative charge than (N/P than) situation under, nano-particle can combine the nano-particle of the stable load siRNA molecule of formation fully with siRNA.
The present invention utilizes the siRNA of mPEG-PCL-PPEEA nano-particle delivery needle to the GFP gene, and and Lipofectamine 2000 more reticent GFP in the expression of HEK293 cell, proved that nano-particle can be transported to siRNA in the cell and brings into play the effect of silent gene expression.The utilization of the present invention ability that further quantitative analysis has checked nano-particle to carry siRNA at the proteic siRNA of Luciferase, mPEG-PCL-PPEEA nano-particle transfection 150pmol siRNA compares with Lipofectamine 2000 transfection 20pmol siRNA, the expression of Luciferase is suitable in the cell, increase along with siRNA dosage, the Luciferasede expression reduces in the cell, along with the increase of N/P ratio, the expression of Luciferase descends.
The present invention proves that by cytotoxicity experiment this nano-micelle has excellent biological compatibility, macromolecule micelle has characteristics such as good stability and convenient preparation simultaneously, makes the biodegradable nano-micelle of this class have a good application prospect in the interferential disease treatment in the siRNA conveying with based on RNA.
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.
Description of drawings
Fig. 1 is the synthetic route chart of mPEG-PCL-PPEEABoc.
Fig. 2 is the nuclear magnetic resoance spectrum of EABoc and PEEABoc.
Fig. 3 is the GPC spectrum of mPEG-PCL macromole evocating agent and mPEG-PCL-PPEEABoc.
Fig. 4 be the mPEG-PCL macromole evocating agent
1H NMR spectrum.
Fig. 5 is mPEG-PCL-PPEEABoc and mPEG-PCL-PPEEA's
1H NMR spectrum.
Fig. 6 is the excitation spectrum of pyrene in variable concentrations mPEG-PCL-PPEEA polymer solution.
Fig. 7 is the sign of the formed nano-particle of mPEG-PCL-PPEEA copolymer.
Fig. 8 is the cytotoxicity of the formed nano-particle of mPEG-PCL-PPEEA copolymer.
Fig. 9 is that the mPEG-PCL-PPEEA-siRNA complex is in the variation of different N/P than particle diameter under the condition and surface potential.
Figure 10 is the painted mPEG of PKH26
45-PCL
100-PPEEA
12The laser confocal microscope photo that the complex that the siRNA of nano-particle and FAM labelling forms distributes after entering cell.
Figure 11 is the painted mPEG of PKH26
45-PCL
45-PPEEA
7The laser confocal microscope photo that the complex that the siRNA of nano-particle and FAM labelling forms distributes after entering cell.
Figure 12 enters the fluorescence microscope photo of the reticent green fluorescent protein GFP of cell expression effect for nano-particle transportation green fluorescent protein GFP siRNA.
The comparison that Figure 13 enters the reticent Luciferase expression effect of cell for nano-particle transportation various dose LUC Photinus pyralis LUC Photinus pyralis FL (Luciferase) siRNA.
The specific embodiment
The invention provides the pharmaceutical carrier of a kind of siRNA, its active component is the positively charged nano-particle of polymer formation.
Above-mentioned polymer is ABC type poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer, comprise poly glycol monomethyl ether (A block), polycaprolactone (B block) and poly phosphate (C block), described B block is poly-epsilon-caprolactone (PCL), described C block is formed by the monomer polymerization of structural formula suc as formula (I), and this poly glycol monomethyl ether-polycaprolactone-polyphosphate ester copolymer is expressed as mPEG-PCL-PPEEA.
Use the mid-block structure of polycaprolactone, have following advantage and effect as triblock copolymer of the present invention: 1. relative hydrophobicity, therefore hydrophobic-the hydrophobic interaction between polymer chain promotes the copolymer self assembly; 2. biodegradable; 3. bio-compatible; 4. synthetic method simple controllable; 5. raw material is comparatively cheap, saves cost.
Use the hydrophilic segment of hydrophilic poly phosphate as triblock copolymer of the present invention, mainly utilize the easily functionalized advantage of poly phosphate to obtain the copolymer that side group has amidine functional group, siRNA lays the foundation for transmission.
Following embodiment provides by stannous iso caprylate catalysis, the synthetic method of the synthesizing amphipathic triblock copolymer that the macromole polyester causes.This method is: the terminal hydroxy group poly glycol monomethyl ether-polycaprolactone of synthetic different molecular weight; With terminal hydroxy group poly glycol monomethyl ether-polycaprolactone is macromole evocating agent, and stannous iso caprylate is a catalyst, obtains triblock copolymer by the monomeric ring-opening polymerization of annular phosphate.
Poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer has amphipathic feature, self assembly forms the granule of nanoscale in aqueous solution, this type nano granular has hydrophobic polycaprolactone kernel and hydrophilic poly glycol monomethyl ether and poly phosphate shell, and particulate size is relevant with the composition of copolymer, can regulate and control.More meaningfully, among the present invention the poly phosphate part of synthetic amphipathic copolymer all have can be protonated amido, the nano grain surface that forms in omiting sour environment has considerable positive charge, can play the effect of transmission siRNA in conjunction with the siRNA that has negative electricity by electrostatic interaction.Adopt this nano-particle to realize the endocytosis of carrier/siRNA complex, and reach the effect of gene silencing as carrier.
Can triblock copolymer be prepared into nano-particle in water by conventional method, such as solvent evaporation method and dialysis.
Solvent evaporation method: triblock copolymer is dissolved in the oxolane, under stirring, splashes into ultra-pure water and obtain corresponding final concentration, stir after two hours, under decompression, remove organic solvent, be settled to suitable volume again.
Dialysis: triblock copolymer is dissolved in good solvent such as the dimethyl sulfoxide (DMSO), splashes into ultra-pure water and obtain corresponding final concentration under stirring, stir after two hours, organic solvent is removed in dialysis in bag filter.
Following embodiment also provides nano-particle and the siRNA solution that will prepare to mix nano-particle and the siRNA complex for preparing mutually.
Raw materials used source and processing method among the embodiment:
(CL, Acros), purity 〉=99% is at N for 6-caprolactone
2Use CaH under the atmosphere
2Backflow 24h, decompression steams before using.
Poly glycol monomethyl ether (Mn=2000g/mol, mPEG
45), purchase company in Aldrich, purity 〉=99.5% dewaters with methylbenzene azeotropic before using.
Stannous iso caprylate (Chemical Reagent Co., Ltd., Sinopharm Group), earlier with xylol azeotropic twice, and then distilling under reduced pressure, (20～40Pa) fraction is used for polyreaction to collect 152 ℃.
Phosphorous chloride., ethylene glycol heavily steam before the use.
Dichloromethane with the phosphorus pentoxide 24h that refluxes, steams before the use.
Triethylamine is successively used phthalic anhydride, NaOH and CaH
2The 24h that refluxes respectively steams before the use.
Oxolane refluxes with Na-K alloy, steams before the use.
Ether, toluene reflux dry with sodium sand, all steam before use.
Unexplained other reagent directly use.
Experimental technique among the following embodiment if no special instructions, is conventional method.
Synthetic and the sign of embodiment 1, mPEG-PCL-PPEEA
One, 2-(N-tertbutyloxycarbonyl amido) ethyoxyl-2-oxygen-1,3, the monomeric synthetic and sign of 2-dioxaphospholane (PEEABoc) annular phosphate
(1) 2-chloro-2-oxygen-1,3,2-dioxaphospholane (COP) synthetic
The COP synthetic route is shown in Fig. 1 (A).The concrete steps of synthetic COP are: in the dichloromethane solution of 300mL 3.26mol/L Phosphorous chloride., slowly add the dichloromethane solution of 301mL 3.25mol/L ethylene glycol.After waiting to drip off, continue reaction 0.5 hour, the pressure reducing and steaming solvent.After double again decompression steams product, be dissolved in the benzene logical 3 days O
2Up to reacting completely, 72 ℃ fraction is collected in distilling under reduced pressure (20Pa), obtains COP.
(2) the synthetic and sign of N-tertbutyloxycarbonyl amido ethanol (EABoc)
Synthetic route is shown in Fig. 1 (B).The concrete steps of synthetic EABoc are: add 6.1 gram (0.10mol) ethanolamine, 100mL oxolane, 100mL ultra-pure water in 250 milliliters of there-necked flasks successively, stirring adds 8.4g (0.1mol) sodium bicarbonate and 21.8g (0.1mol) Bis(tert-butoxycarbonyl)oxide after treating that it dissolves fully continuously.Naturally rise to room temperature reaction and spend the night after 30 minutes in reaction under 0 ℃.Reactant liquor 100mL extracted with diethyl ether 2 times, the organic facies anhydrous sodium sulfate drying.Decompression removes down and desolvates, and obtains EABoc, and productive rate is 95%.
To EABoc carry out proton nmr spectra (
1H NMR) analyze,
1H NMR spectrum is seen Fig. 2 (A).
By Fig. 2 (A) as seen, each kind of proton all obtains respective home, and the integration ratio is coincide.
(3) 2-(N-tertbutyloxycarbonyl amido) ethyoxyl-2-oxygen-1,3, the synthetic and sign of 2-dioxaphospholane (PEEABoc)
Synthetic route is shown in Fig. 1 (C).The concrete steps of synthetic PEEABoc are: under-5 ℃ low-temp reactions are bathed, with the COP (14.25g that makes, 0.1mol) oxolane (30ml) drips of solution be added to EABoc (16.10g, 0.1mol) and triethylamine (10.12g, 0.1mol) oxolane (120ml) solution in, reaction is spent the night.Then at N
2Protection is filtered above solution down, after filtrate is concentrated, with the exsiccant cold diethyl ether precipitation of 400mL, removes supernatant, and precipitate is drained with oil pump, is PEEABoc.
To PEEABoc carry out proton nmr spectra (
1H NMR) and carbon-13 nmr spectra (
13C-NMR) analyze,
1H NMR spectrum is seen Fig. 2 (B),
13The C-NMR spectrum is seen Fig. 2 (C).
Can observe from Fig. 2 (B), the unimodal of 1.43ppm is 9 protons of the tert-butyl group, and the triplet of 3.36ppm and 4.12ppm is respectively-OCH
2C
H 2NH-and-OC
H 2CH
22 protons on the NH-fragment, the multiplet of 4.36ppm is phosphate ester ring-OC
H 2C
H 24 protons on the O-.In Fig. 2 (C), the chemical shift of various carbon is indicated in the drawings.Above nuclear magnetic resoance spectrum has proved monomeric structure.
Two, the synthetic and sign of poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer (mPEG-PCL-PPEEA)
(1) the synthetic and sign of poly glycol monomethyl ether-polycaprolactone (mPEG-PCL) macromole evocating agent
Terminal hydroxy group poly glycol monomethyl ether-polycaprolactone the macromole evocating agent of various molecular weight is to be initiator with the poly glycol monomethyl ether, causes caprolactone monomer and be polymerized under bulk conditions.By the molecular weight of adjusting poly glycol monomethyl ether and the rate of charge of caprolactone and poly glycol monomethyl ether, can obtain different molecular weight polyethylene glycol monomethyl ether-polycaprolactone polymer.Stannous iso caprylate is because of its high catalytic efficiency and avirulence, by the lactone of extensive use and the catalyst of lactide cyclic monomer ring-opening polymerization, by drugs approved by FDA as food additive.
With mPEG
45Be initiator, stannous iso caprylate (Sn (Oct)
2) be the Preparation of Catalyst polymer, polyreaction is carried out in glove box, and the synthetic concrete experimental procedure of mPEG-PCL is as follows:
1) after the processing of the round-bottomed flask that will react and inflated with nitrogen dry, puts into glove box through repeatedly evacuation flame.
2) proportioning by table 1 feeds intake: add mPEG in flask
45, CL monomer and Sn (Oct)
2, under 120 ℃ of stirrings, react.
3) reaction was shifted out glove box with product after 24 hours, used a spot of CH
2Cl
2Dissolving, with solution precipitation in cold ether, repeatedly twice, the collecting precipitation thing is drained till the constant weight with oil pump, promptly gets product.
Different rate of charges can obtain different products, have so obtained a series of mPEG-PCL copolymers, see Table 1.
The different rate of charges of table 1 (mol ratio) synthesize mPEG-PCL
aThe digitized representation polymer basis in the polymer lower right corner
1The degree of polymerization that H NMR obtains
To above-mentioned mPEG-PCL copolymer carry out proton nmr spectra (
1H NMR) analyzes, measure its degree of polymerization and number-average molecular weight.With gel permeation chromatography (GPC) method is the number-average molecular weight and the molecular weight distribution PDI (molecular weight distribution breadth index) of standard analysis mPEG-PCL copolymer with the polystyrene.The degree of polymerization, number-average molecular weight and molecular weight distribution PDI see Table 2.The GPC spectrum is seen Fig. 3 (A) and Fig. 3 (B).
1H NMR spectrum is seen Fig. 4.
The sign of table 2 mPEG-PCL polymer
aGPC measures;
B1H NMR measures.
By table 2 as seen, according to
1H NMR result of calculation, mPEG
45-PCL
45Number-average molecular weight be 7130g/mol, corresponding block PCL
45Number-average molecular weight be 5130g/mol; MPEG
45-PCL
100Number-average molecular weight be 13400g/mol, corresponding block PCL
100Number-average molecular weight be 11400g/mol.
The corresponding mPEG of Fig. 3 (A)
45-PCL
45The corresponding mPEG of Fig. 3 (B)
45-PCL
100Two kinds of polymer present as we can see from the figure all is regular unimodal, and has the molecular weight distribution of relative narrower.
The corresponding mPEG of Fig. 4 (A)
45-PCL
45The corresponding mPEG of Fig. 4 (B)
45-PCL
100Can see mPEG through contrast
45-PCL
45And mPEG
45-PCL
100Have similar spectrum, be analyzed as follows: alphabetical a to the f labelling all proton signals of ownership mPEG-PCL, the triplet of the degree of polymerization of poly-epsilon-caprolactone by 4.02ppm (belong to polycaprolactone-C (O) OCH
2) with 3.63ppm unimodal (belong to poly glycol monomethyl ether-CH
2CH
2-) integral area ratio calculate.
(2) the synthetic and sign of poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer (mPEG-PCL-PPEEA)
With mPEG-PCL is initiator, stannous iso caprylate (Sn (Oct)
2) be the Preparation of Catalyst polymer, polyreaction is carried out in glove box.Before reaction, the mPEG-PCL macromole evocating agent is with exsiccant methylbenzene azeotropic twice, decompressing and extracting.Synthetic route such as Fig. 1 (D).The concrete steps of synthetic mPEG-b-PCL-b-PPEEABoc are as follows:
1) after the processing of the round-bottomed flask that will react and inflated with nitrogen dry, puts into glove box through repeatedly evacuation flame.
2) ratio in table 2 feeds intake: add mPEG-PCL, PEEABoc and THF in the 25mL flask, guarantee that the initial concentration of PEEABoc is 1mol/L.After stirring 30 minutes under 30 ℃, add Sn (Oct)
2
3) reaction after 3 hours concentrates reactant liquor, is deposited under 0 ℃ in ether/methanol mixed solvent of 10:1 (v/v), filters, and solid is drained, and promptly obtains mPEG-b-PCL-b-PPEEABoc.
4) get the above-mentioned polymer of 1g and be dissolved in the anhydrous THF of 10mL, the 6M HCl/THF solution that adds 20mL makes that the final concentration of HCl is 4M., after 2 hours reactant liquor is concentrated at 0 ℃ of following stirring reaction,, filter, drain solid, promptly obtain the mPEG-b-PCL-b-PPEEA polymer with cold ether sedimentation.
The different rate of charges of table 3 (mol ratio) synthesize the mPEG-PCL-PPEEABoc polymer
aThe digitized representation polymer basis in the polymer lower right corner
1The degree of polymerization that H NMR obtains
With gel permeation chromatography (GPC) method is the number-average molecular weight and the molecular weight distribution PDI (molecular weight distribution breadth index) of standard analysis mPEG-PCL-PPEEABoc copolymer with the polystyrene.It may be noted that because the mPEG-PCL-PPEEA behind the deprotection is difficult to characterize with GPC, so listed molecular weight and molecular weight distribution all are meant the mPEG-PCL-PPEEABoc that deprotection is preceding.To mPEG-PCL-PPEEABoc and mPEG-PCL-PPEEA carry out proton nmr spectra (
1H NMR) analyzes, measure its degree of polymerization and number-average molecular weight.The degree of polymerization of mPEG-PCL-PPEEABoc, number-average molecular weight and molecular weight distribution PDI see Table 4.The GPC spectrum of mPEG-PCL-PPEEABoc is seen Fig. 3 (C) and Fig. 3 (D).MPEG
45-b-PCL-b-PPEEABoc and mPEG
45-b-PCL-b-PPEEA's
1H NMR spectrum is seen Fig. 5.
The sign of table 4 polymer
aGPC measures;
B1H NMR measures.
By table 4 as seen, according to
1H NMR result of calculation, mPEG
45-PCL
45-PPEEABoc
7Number-average molecular weight be 9000g/mol, corresponding block PPEEABoc
7Number-average molecular weight be 1870g/mol; The mPEG of correspondence behind the deprotection
45-PCL
45-PPEEA
7Number-average molecular weight be 6550g/mol, corresponding block PPEEA
7Number-average molecular weight be 1,420g/mol; MPEG
45-PCL
100-PPEEABoc
12Number-average molecular weight be 16600g/mol, corresponding block PPEEABoc
12Number-average molecular weight be 3200g/mol; The mPEG of correspondence behind the deprotection
45-PCL
100-PPEEA
12Number-average molecular weight be 15840g/mol, corresponding block PPEEA
12Number-average molecular weight be 2430g/mol.
The corresponding mPEG of Fig. 3 (C)
45-b-PCL
45-b-PPEEABoc
7The corresponding mPEG of Fig. 3 (D)
45-b-PCL
100-b-PPEEABoc
12Polymer presents as we can see from the figure all is regular unimodal, and has the molecular weight distribution of relative narrower.Relatively can see that the molecular weight of mPEG-PCL-PPEEABoc polymer moves to the high molecular direction with mPEG-PCL macromole evocating agent (Fig. 3 (A) and Fig. 3 (B)).
MPEG before the corresponding deprotection of Fig. 5 (A)
45-b-PCL
45-b-PPEEABoc
7MPEG behind the corresponding deprotection of Fig. 5 (B)
45-b-PCL
45-b-PPEEA
7MPEG before the corresponding deprotection of Fig. 5 (C)
45-b-PCL
100-b-PPEEABoc
12MPEG behind the corresponding deprotection of Fig. 5 (D)
45-b-PCL
100-b-PPEEA
12Compare with Fig. 4 (A); several new signals have appearred in Fig. 5 (A) and Fig. 5 (C); as the proton signal of the methylene at the e place on the PCL segment of the multimodal correspondence at 1.40～1.48ppm place; obviously had more the proton signal of 9 protons (j) of methyl on the poly phosphate side group Boc blocking group, the signal that occurs at the 4.35ppm place corresponding to poly phosphate main chain section on-O-C
H 2-C
H 2The peak of 4 protons (g) of-O-, the proton peak that occurs at 4.20ppm and 3.41ppm then belong on the poly phosphate segment side group-O-C
H 2(h)-C
H 2(i)-signal of NH-.The segmental degree of polymerization of poly phosphate is according to the ratio calculating of proton (g) integral area on the poly phosphate main chain at 4.35ppm place among the HNMR figure and proton (b) integral area on the polyethylene glycol backbone at 3.63ppm place.The appearance at characteristic signal peak has proved the generation of block polymer on these poly phosphate segments.With mPEG
45-PCL
100-PPEEABoc
12Triblock polymer reacts the blocking group of sloughing Boc in anhydrous HCl/THF solution, after Fig. 5 (B) and Fig. 5 (D) have provided deprotection
1The variation of H-NMR spectrogram.Therefrom can find; the strong peak at the 1.48ppm place on the Boc group disappears; and a blunt peak has appearred at the 8.42ppm place; active hydrogen corresponding to the side chain amido; thereby the success that has proved deprotection is carried out; and the integral area of respective peaks calculating caprolactone and phosphate ester segment are kept perfectly from figure, degraded takes place wait side reaction, and corresponding segmental polymer is all less than changing.
1, the preparation of nano-particle
There is the hydrophobic interaction of hydrophobic block in amphipathic nature block polymer in aqueous solution, as long as copolymer concentration is higher than critical aggregate concentration and can forms nano-particle.
The method for preparing nano-particle has multiple, the simplest common be exactly solvent evaporation method, concrete grammar is: 10mg mPEG-PCL-PPEEA triblock copolymer is dissolved in the 1mL oxolane, stirred 1 hour under the room temperature, under agitation splash into the 10mL ultra-pure water with the speed of 60mL/h then, restir was taken out organic solvent after two hours under decompression, standardize solution obtains the nanoparticles solution of 1mg/mL to 10mL.
The mPEG for preparing 1mg/mL by said method respectively
45-PCL
45-PPEEA
7The mPEG of nanoparticles solution and 1mg/mL
45-PCL
100-PPEEA
12Nanoparticles solution.
2, the mensuration of critical aggregate concentration (CAC)
Be that the critical aggregate concentration that fluorescent probe characterizes the aggregation of amphipathic nature polyalcohol is the most common and effective method with the pyrene.
The nanoparticles solution of the 1mg/mL of above-mentioned preparation is diluted to a series of concentration, guarantees that simultaneously the concentration of pyrene under each concentration keeps 6 * 10
-7Mol/L is constant.Fixedly maximum emission wavelength scans the excitation spectrum of pyrene at 390nm.See Fig. 6 (A) and (B).
Fig. 6 is the excitation spectrum of pyrene in variable concentrations mPEG-PCL-PPEEA polymer solution.Fig. 6 (A) is mPEG
45-PCL
45-PPEEABoc
7Fig. 6 (B) is mPEG
45-PCL
100-PPEEABoc
12As seen from the figure, the variation tendency unanimity of two figure.Increase along with polymer concentration, significant variation has taken place in the fluorescence spectrum of pyrene probe, the intensity that at first is the peak strengthens gradually, next be its (0,0) absorption band from the 336nm red shift to 339nm, these variations are because pyrene is transferred to hydrophobic environment by water environment, mean the formation of the nano-particle of nucleocapsid structure.
In conjunction with above-mentioned excitation spectrum, the strength ratio at peak, i.e. I when calculating 339nm and 336nm under each polymer concentration
339/ I
336, with a series of I
339/ I
336To the logarithm mapping of corresponding concentration, see Fig. 6 (C), the Log among the figure refers in particular to Log
10
By Fig. 6 (C) as seen, two curves are all S-type, and under low concentration, the fluorescence intensity of polymer is basicly stable, and after reaching finite concentration, its ratio begins rapid increase, show that pyrene is optionally entered in the hydrophobic core by water environment, and this moment, nano-particle formed.When continue increasing polymer concentration, fluorescence intensity arrives a platform, no longer increase, this show copolymer in water fully self assembly to have formed with the polycaprolactone be the nano-particle of hydrophobic core.The CAC value can be determined by the intersection point of horizontal line and oblique line tangent line.
Utilize said method to try to achieve mPEG
45-PCL
45-PPEEA
7And mPEG
45-PCL
100-PPEEA
12The CAC value be respectively 3.57 * 10
-3With 2.68 * 10
-3Mg/mL.Can find that along with the increase of hydrophobic segment polycaprolactone, the CMC value of polymer reduces, and shows the enhancing with hydrophobic interaction, the easier formation of polymer micelle.The big or small direct reaction of CAC the stability of polymer beads in aqueous solution, its value shows more for a short time that the particulate stability of assembling is higher.The CAC value of the polymer among the present invention is compared with general micromolecule surfactant, and low nearly two orders of magnitude show this polymer stability preferably.
3, the form of nano-particle
Utilize the form of the formed nano-particle of transmission electron microscope observation mPEG-PCL-PPEEA copolymer.Shown in Fig. 7 (A) and Fig. 7 (B), the corresponding mPEG of Fig. 7 (A)
45-PCL
45-PPEEA
7The corresponding mPEG of Fig. 7 (B)
45-PCL
100-PPEEA
12
As seen from the figure, two kinds of formed nano-particle of mPEG-PCL-PPEEA copolymer all present regular spherical structure, and particle diameter is about 20nm and 60nm and size distribution homogeneous.
4, the particle diameter of nano-particle and zeta electromotive force
By model is that the dynamic light scattering detectable concentration of Malvern Zetasizer Nanao ZS90 is the particle diameter and the particle size distribution of the mPEG-PCL-PPEEA nano-particle of 0.1mg/mL.Fig. 7 (C) and Fig. 7 (D) are respectively polymer mPEG
45-PCL
45-PPEEA
7Particle diameter and the result of zeta electromotive force, Fig. 7 (E) and Fig. 7 (F) are respectively polymer mPEG
45-PCL
100-PPEEA
12Particle diameter and the result of zeta electromotive force.
MPEG
45-PCL
45-PPEEA
7The average diameter of nano-particle is 27.7nm, and particle size dispersion degree (PDI) is 0.20, and the zeta electromotive force is 32.3mV.MPEG
45-PCL
100-PPEEA
12Particulate average diameter is at 121nm, and particle size dispersion degree (PDI) is 0.09, and the zeta electromotive force is 45mV.Need indicatedly be, utilize the result of size ratio transmission electron microscope of dynamic light scattering measurement bigger than normal, this mainly be because dynamic light scattering measurement be the hydrodynamic radius of nano-particle in aqueous solution, be the particle diameter of nano-particle and transmission electron microscope is measured at dewatering state.But no matter which kind of measuring method shows that all the nano-particle of above-mentioned triblock polymer all has uniform particle size distribution.These positive charges are except can stable nanoparticles, and more significant feature is to play the effect of transmission siRNA in conjunction with siRNA.
5, the biocompatibility of mPEG-PCL-PPEEA nano-particle
The nanoparticles solution dilution of the 1mg/mL of above-mentioned preparation is 1.95-500 μ g/mL.Measure the particulate cytotoxicity of variable concentrations mPEG-PCL-PPEEA copolymer nano by the MTT method.Concrete test is: with the mPEG of variable concentrations
45-PCL
45-PEEP
7The mPEG of nanoparticles solution and variable concentrations
45-PCL
100-PEEP
12Nanoparticles solution respectively with HEK293 co-culture of cells after 24 hours, survey cell survival rate respectively, test in contrast with PEI.The survival rate of cell is seen Fig. 8 under the different disposal.
As seen from the figure, when polymer concentration increases to 500 μ g/mL from 1.95 μ g/mL, two kinds of nano-particle pair cell vigor all do not have significantly influence, and cell viability maintains about 100% always, shows that above-mentioned nano-particle has excellent biological compatibility.
SiRNA is a kind of double-chain small molecule RNA that is made up of more than 20 nucleotide, has negative charge, can form stabilized complex by the positive and negative charge effect with the mPEG-PCL-PPEEA nano-particle of positively charged.
N/P refers to the ratio of the negative charge of the amido positive charge of nano-particle and siRNA phosphate group.
1, the sign of nano-particle and siRNA complex
Method according to embodiment 2 prepares the mPEG that concentration is 5mg/mL
45-PCL
45-PEEP
7Nanoparticles solution.Green fluorescent protein GFP siRNA, sequence is GCAAGCTGACCCTGAAGTTCAT, available from Shanghai JiMa pharmacy Technology Co., Ltd, 10D GFP siRNA is dissolved in the water of 150 μ LDEPC processing, obtaining concentration is 20 μ MsiRNA solution.Above-mentioned nano-particle and above-mentioned siRNA solution respectively by different N/P than mixing, prepare different mPEG
45-PCL
45-PEEP
7Nano-particle and siRNA complex.
Particle diameter and particle size distribution by the above nano-particle of dynamic light scattering measurement or nano-particle and siRNA complex.Measure the electromotive force on the surface of above nano-particle or nano-particle and siRNA complex by Zeta electric potential.Fig. 9 (A) is when the N/P ratio is respectively 1:0,1:1,5:1, mPEG
45-PCL
45-PEEP
7The change of size of nano-particle and siRNA complex.MPEG when Fig. 9 (B) is respectively 1:0 and 1:1 for the N/P ratio
45-PCL
45-PEEP
7The surface potential of nano granule granule and siRNA complex changes.
From Fig. 9 (A) figure as seen, when N/P than being 1:0, when promptly having only nano-particle, the complex particle diameter is 121nm to the maximum, when N/P when being 1:1, complex particle diameter minimum is 98 ± 1.4nm.And along with the increase of N/P ratio, the complex particle diameter increases, and near the particle diameter of N/P than for 1:0 the time.From Fig. 9 (B) figure as seen, when N/P is 1:0, the surface potential maximum of complex, be 45mV, when the N/P ratio was 1:1, the composite surface current potential was reduced to-10.7 ± 0.76mV, showed that the mPEG-PCL-PPEEA nano-particle combines the formation complex by charge interaction with siRNA.
Different N/P is than the mPEG that forms
45-PCL
45-PEEP
7The particle diameter and the surface potential of nano-particle and siRNA complex see Table 5.
Table 5 different N/P is than the particle diameter and the surface potential of the complex that forms
2, the cytophagy of nano-particle and siRNA complex
1) mPEG
45-PCL
100-PPEEA
12The cytophagy of nano-particle and siRNA complex
0.5 μ L PKH26 dyestuff is joined the mPEG that 1mL concentration is 5mg/mL
45-PCL
100-PPEEA
12In the nanoparticles solution, room temperature is placed and is spent the night, and removes the PKH26 dyestuff that does not enter the nano-particle hydrophobic core with the ultra-pure water dialysis then.The green fluorescent protein GFP siRNA of fluorescent dye FAM labelling, sequence is GCAAGCTGACCCTGAAGTTCAT, available from Shanghai JiMa pharmacy Technology Co., Ltd, 10D GFP siRNA is dissolved in the 150 μ L DEPC treated waters, obtaining concentration is 20 μ M GFP siRNA solution.Above-mentioned nano-particle mixes by N/P=50 with above-mentioned siRNA solution, preparation mPEG
45-PCL
100-PPEEA
12Nano-particle and siRNA complex.
With the mPEG for preparing above
45-PCL
100-PPEEA
12Nano-particle and siRNA complex and HEK293 cell are 37 ℃ of co-cultivation after 2 hours, with Hoechst 33342 pair cell nuclear stainings, used under the 4% formalin room temperature fixed cell at last 30 minutes, and engulfed the result of nano-particle, see Figure 10 with the laser confocal microscope observation of cell.
Figure 10 has provided HEK293 cytophagy mPEG
45-PCL
100-PPEEA
12The result that the nano-particle laser confocal microscope is observed, part green in the cell is the siRNA of FAM labelling, and part red in the cell is the nano-particle of PKH26 labelling, and part blue in the cell is a nucleus.By three kinds of synergetic results of color as can be seen nano-particle separate in cell with siRNA, and nano-particle do not enter nucleus, siRNA mainly is distributed in the cell cytosol.
Among Figure 10, Figure 10 (A) is the laser confocal microscope photo under the 488nm exciting light, demonstration be the siRNA of FAM labelling; Figure 10 (B) is the laser confocal microscope photo under the 551nm exciting light, demonstration be the nano-particle of PKH26 labelling; Figure 10 (C) is the laser confocal microscope photo under the 346nm exciting light, demonstration be nucleus; Figure 10 (D) is the stack result of Figure 10 (A), Figure 10 (B) and 10 (C).Nano-particle separates in cell with siRNA as seen from the figure, and nano-particle do not enter nucleus, and siRNA mainly is distributed in the cell cytosol.Show mPEG
45-PCL
100-PPEEA
12Nano-particle can effectively be transported to siRNA in the cell, and can separate with siRNA, makes the effect of siRNA performance gene silencing.
2) mPEG
45-PCL
45-PPEEA
7The cytophagy of nano-particle and siRNA complex
Same step 1) the results are shown in Figure 11.
Among Figure 11, Figure 11 (A) is the laser confocal microscope photo under the 488nm exciting light, demonstration be the siRNA of FAM labelling; Figure 11 (B) is the laser confocal microscope photo under the 551nm exciting light, demonstration be the nano-particle of PKH26 labelling; Figure 11 (C) is the laser confocal microscope photo under the 346nm exciting light, demonstration be nucleus; Figure 11 (D) is the stack result of Figure 11 (A), Figure 11 (B) and 11 (C).
The mPEG that Figure 11 result displayed and Figure 10 show
45-PCL
100-PPEEA
12The cytophagy result of nano-particle is similar.
3, nano-particle and siRNA complex silent gene are expressed
1) nano-particle and siRNA complex are to the silence of green fluorescence protein gene
Green fluorescent protein GFP siRNA, sequence is GCAAGCTGACCCTGAAGTTCAT, available from Shanghai JiMa pharmacy Technology Co., Ltd.
300ng pEGFP-N2 plasmid (Clontech) is transfected into the HEK293 cell with Lipofectamine2000, is called HEK293-pEGFP-N2.Following several processing is established in experiment altogether:
100pmol green fluorescent protein GFP siRNA is dissolved among the 50 μ LOpti-MEM, then to the mPEG of the 5mg/mL that wherein adds 60 μ l
45-PCL
100-PPEEA
12, obtain the mPEG of N/P=50
45-PCL
100-PPEEA
12Nano-particle and siRNA complex solution.After solution at room temperature left standstill 20 minutes, in 6 hours the HEK293 cell of pEGFP-N2 plasmid (Clontech) that joined transfection.
100pmol green fluorescent protein GFP siRNA is dissolved among the 50 μ L Opti-MEM, then to the mPEG of the 5mg/mL that wherein adds 60 μ l
45-PCL
45-PPEEA
7, obtain the mPEG of N/P=50
45-PCL
45-PPEEA
7Nano-particle and siRNA complex solution.After solution at room temperature left standstill 20 minutes, in 6 hours the HEK293 cell of pEGFP-N2 plasmid (Clontech) that joined transfection.
With HEK293-pEGFP-N2 cell in contrast 1; With the complex transfection HEK293-pEGFP-N2 cell of Lipofectamine 2000 transfection LuciferasesiRNA, as positive control (final concentration of Luciferase siRNA is the 20pmol/ hole); With free Luciferase siRNA transfection HEK293-pEGFP-N2 cell, 2 (final concentration of Luciferase siRNA is the 20pmol/ hole) in contrast.With the fluorescence inverted microscope HEK293 cell of handling after 48 hours is taken pictures, photo is seen Figure 12.
Figure 12 (A) is contrast 1; The positive contrast of Figure 12 (B); Figure 12 (C) is contrast 2; The corresponding mPEG of Figure 12 (D)
45-PCL
100-PPEEA
12The corresponding mPEG of Figure 12 (E)
45-PCL
45-PPEEA
7
As seen from the figure, gene expression in the independent siRNA pair cell is obviously influence not, the siRNA of Lipofectamine2000 transfection has obviously reduced the expression of GFP in cell, and the mPEG-PCL-PPEEA nano-particle significantly reduces the expression of GFP in the cell under the concentration in 100pmol/ hole.In transfection in the HEK293 cell of pEGFP-N2 plasmid (Clontech) expression of GFP do 100%, transfection in the cell of pEGFP-N2 plasmid and independent siRNA the GFP expression be 99.3%, utilizing the interior GFP expression of cell of Lipofectamine 2000 GFP-transfected siRNA is 54.5%, utilizes mPEG
45-PCL
100-PPEEA
12The GFP expression is 51.5% in the cell of GFP-transfected siRNA, utilizes mPEG
45-PCL
45-PPEEA
7The GFP expression is 72.3% in the cell of GFP-transfected siRNA.
2) nano-particle and siRNA complex are to the silence of firefly luciferase gene
Luciferase is a LUC Photinus pyralis LUC Photinus pyralis FL, can send visible light by the catalytic reaction substrate, according to the reaction homogeneity of enzyme, and can quantitative mensuration Luciferase expression of gene level.
Luciferase siRNA, corresponding sequence is: CTTACGCTGAGTACTTCGA, available from Shanghai JiMa pharmacy Technology Co., Ltd.
300ng pGL3 plasmid (Promega) is transfected into the HEK293 cell with Lipofectamine2000, is called HEK293-pGL3.100pmol Luciferase siRNA is dissolved among the 50 μ l Opti-MEM,, obtains mPEG-PCL-PPEEA nano-particle and the siRNA complex solution of N/P=50 then to the mPEG-PCL-PPEEA of the 5mg/mL that wherein adds 60 μ l.After solution at room temperature left standstill 20 minutes, add different volumes to transfection in 6 hours the HEK293 cell of pGL3 plasmid, make the final concentration of Luciferase siRNA be respectively 20pmol/ hole, 50pmol/ hole, 100pmol/ hole, 150pmol/ hole, 200pmol/ hole.
With HEK293-pGL3 cell 1 (MOCK) in contrast; With the complex transfection HEK293-pGL3 cell of Lipofectamine 2000 transfection Luciferase siRNA, as positive control (final concentration of LuciferasesiRNA is the 20pmol/ hole); With free Luciferase siRNA transfection HEK293-pGL3 cell, 2 (final concentration of Luciferase siRNA is the 20pmol/ hole) in contrast.With the relative expression quantity of Veritas MicroplateLuminometer detection processing luciferase after 48 hours, see Figure 13.Figure 13 (A) is mPEG
45-PCL
100-PPEEA
12Figure 13 (B) is mPEG
45-PCL
45-PPEEA
7
By Figure 13 (A) as seen, the result shows that when N/P=50 along with the increase of siRNA dosage, the Luciferase expression reduces.MPEG
45-PCL
100-PPEEA
12Nano-particle transfection 150pmol siRNA compares with Lipofectamine2000 transfection 20pmol siRNA, and the expression of Luciferase is suitable in the cell.
By Figure 13 (B) as seen, the result shows when N/P=50, mPEG
45-PCL
45-PPEEA
7Nano-particle has and mPEG
45-PCL
100-PPEEA
12The ability of the transfection siRNA that nano-particle is similar, along with the increase of siRNA dosage, the Luciferasede expression reduces in the cell.
Under the situation in identical siRNA dosage (pmol/ hole), with different N/P than (N/P is 50,75,100) complex of preparation nano-particle and siRNA, with above-mentioned complex transfection HEK293-pGL3 cell, respectively with HEK293-pGL3 cell (MOCK), the HEK293-pGL3 cell of the free Luciferase siRNA of transfection and in contrast with the HEK293-pGL3 cell of Lipofectamine 2000 transfection Luciferase siRNA, detect the relative expression quantity that above-mentioned different composite thing is handled Luciferase after 48 hours respectively with Veritas Microplate Luminometer, see Figure 13 (C).
As seen from the figure, along with the increase of N/P ratio, the expression of Luciferase descends.
Claims (10)
1, the pharmaceutical carrier of a kind of siRNA, its active component are the positively charged nano-particle of polymer formation.
2, pharmaceutical carrier as claimed in claim 1 is characterized by: described nano-particle has carried out chemical modification or ligand modified.
3, pharmaceutical carrier as claimed in claim 1 is characterized by: described polymer is poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer.
4, pharmaceutical carrier as claimed in claim 3 is characterized by: described polycaprolactone is a poly-epsilon-caprolactone, and described poly phosphate is polymerized by the five-membered cyclic phosphate ester monomer of structural formula suc as formula (I):
5, pharmaceutical carrier as claimed in claim 4 is characterized by: in the described triblock copolymer, mid-block is described polycaprolactone, and an end-blocks is a poly glycol monomethyl ether, and another end-blocks is described poly phosphate; The degree of polymerization of described Polyethylene Glycol block is 45, and corresponding number-average molecular weight is 2000g/mol; The degree of polymerization of described polycaprolactone block is 45-100, and corresponding number-average molecular weight is 5,130-11,400g/mol; The degree of polymerization of described poly phosphate block is 7-12, and corresponding number-average molecular weight is 1,420-2,430g/mol.
6, pharmaceutical carrier as claimed in claim 5 is characterized by: described triblock copolymer is mPEG
45-PCL
45-PPEEA
7Or mPEG
45-PCL
100-PPEEA
12
7, a kind of poly glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer, described polycaprolactone are poly-epsilon-caprolactone, and described poly phosphate is polymerized by the five-membered cyclic phosphate ester monomer of structural formula suc as formula (I):
8, triblock copolymer as claimed in claim 7, it is characterized by: in the described triblock copolymer, mid-block is described polycaprolactone, an end-blocks is a poly glycol monomethyl ether, another end-blocks is described poly phosphate, the degree of polymerization of described poly glycol monomethyl ether block is 45, and corresponding number-average molecular weight is 2,000g/mol; The degree of polymerization of described polycaprolactone block is 45-100, and corresponding number-average molecular weight is 5,130-11,400g/mol; The degree of polymerization of described poly phosphate block is 7-12, and corresponding number-average molecular weight is 1,420-2,430g/mol.
9, as claim 7 or 8 described copolymers, it is characterized by: described triblock copolymer is mPEG
45-PCL
45-PPEEA
7Or mPEG
45-PCL
100-PPEEA
12
10, the method for a kind of synthesizing polyethylene glycol monomethyl ether-polycaprolactone-polyphosphate triblock copolymer, be to be macromole evocating agent with terminal hydroxy group poly glycol monomethyl ether-polycaprolactone, with the stannous iso caprylate is catalyst, obtains triblock copolymer by the monomeric ring-opening polymerization of annular phosphate;
Described terminal hydroxy group poly glycol monomethyl ether-polycaprolactone is to be initiator with the poly glycol monomethyl ether, is catalyst with the stannous iso caprylate, causes caprolactone monomer and be polymerized under bulk conditions;
Described annular phosphate monomer is by 2-chloro-2-oxygen-1,3, and 2-dioxaphospholane and N-tertbutyloxycarbonyl amido ethanol synthesis obtain;
Described 2-chloro-2-oxygen-1,3, the 2-dioxaphospholane obtains by ethylene glycol and Phosphorous chloride. reaction;
Described N-tertbutyloxycarbonyl amido ethanol (EABoc) obtains by ethanolamine and Bis(tert-butoxycarbonyl)oxide reaction.
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