CN104027816B - Co-loaded adriamycin and siRNA (small interfering ribose nucleic acid) carrier capable of removing pegylation and synthesis method thereof - Google Patents

Co-loaded adriamycin and siRNA (small interfering ribose nucleic acid) carrier capable of removing pegylation and synthesis method thereof Download PDF

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CN104027816B
CN104027816B CN201410268060.1A CN201410268060A CN104027816B CN 104027816 B CN104027816 B CN 104027816B CN 201410268060 A CN201410268060 A CN 201410268060A CN 104027816 B CN104027816 B CN 104027816B
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CN104027816A (en
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姚煜
钱军民
徐明辉
胥伟军
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Xian Jiaotong University
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Abstract

The invention provides a co-loaded adriamycin and siRNA (small interfering ribose nucleic acid) carrier capable of removing pegylation and a synthesis method thereof. Firstly, cystamine modification is carried out on terminated methoxyl polyethylene glycol, so that cystamine modified mPEG is obtained; then a carbodiimide method is utilized for bonding an RAFT (reversible additive fragment transfer) polymerization chain transfer agent onto the end of a cystamine modified mPEG chain, so that a macromolecule RAFT chain transfer agent; then RAFT polymerization is carried out, so that a terminated methoxyl polyethylene glycol-poly(N-(3-dimethylamine propyl) methacrylamide)-poly(N-t-butyloxycarboryl propylene hydrazide) triblock copolymer is obtained; finally a protective group (namely t-butyloxycarboryl) is removed, so that a terminated methoxyl polyethylene glycol-poly(N-(3-dimethylamine propyl) methacrylamide)-polypropylene hydrazide triblock copolymer is obtained. The synthesis method has the advantages that raw materials are available, reaction conditions are mild, molecular weight of a polymer and length of a chain of each block in the obtained carrier can be easily controlled, after adriamycin is loaded, nanoparticles can be self assembled, pharmaceutical effect is high, and co-loading of a chemotherapeutic drug and a gene-based drug is realized.

Description

A kind of remove Pegylation common loading amycin and siRNA carrier and synthetic method thereof
Technical field
The invention belongs to biomedical materials field, be specifically related to a kind of remove Pegylation common loading amycin and siRNA carrier and synthetic method thereof.
Background technology
Chemotherapy is one of main policies of clinical cancer therapy, and it mainly utilizes blood circulation that Cytotoxic chemicals is delivered to diseased region, brings toxic and side effects inevitably to normal cell in health simultaneously.The curative effect of chemotherapeutics and toxic and side effects are all closely related with application dosage.Therefore, how to improve chemotherapeutical medicine curative effect and avoid its toxic and side effects to be the problem that chemotherapy of tumors is concerned about most all the time.At present, exploitation molecular targeted agents and drug delivery system just become study hotspot.Wherein, molecular targeted agents because of develop difficulty and clinical efficacy be far inferior to the problems such as expection, be in progress relatively slow; And based on the drug delivery system of biomaterial because drug loading is large, be suitable for that medicament categories is many, good biocompatibility, the advantage such as biodegradable, become research emphasis, develop the carrier formats such as nanoparticle, nanogel, polymer-drug conjugate, nano-micelle.In drug delivery system exploitation, generally by polyethyleneglycol modified carrier, with avoid carrier in blood circulation time disposed fast by immune system, be beneficial to the enrichment of carrier in tumor area.But, polyethyleneglycol modifiedly also reduce carrier and cell interaction, make carrier enter to escape in tumor cell or in endosome through endocytosis and enter Cytoplasm and become difficulty.Visible, the Polyethylene Glycol that carrier is modified is removed in good time, raising curative effect of medication will be beneficial to.
Amycin is the antitumor drug of extensive use clinically, all has good curative effect to pulmonary carcinoma, breast carcinoma, bladder cancer etc.The same with most drug, amycin water solublity and selectivity inequality, many clinically exist the shortcomings such as bioavailability is low, the half-life is short, toxic and side effects is obvious in the form of its hydrochloride salt, serious its clinical efficacy of restriction.Therefore, be loaded in various forms of pharmaceutical carrier, can be effectively alleviated/overcome these shortcomings.But this still cannot solve the drug resistance problems of amycin.The generation of drug resistance is normally because certain albumen of tumor cells expression is as pumped extracellular to avoid its toxic action by medicine during P-gp albumen.In recent years, gene therapy is regarded as the promising method improving chemotherapeutic efficacy and fundamentally eliminate drug resistance, as siRNA method.The resistance protein that it can be produced by reticent tumor cell, realizes the gathering of chemotherapeutics in tumor cell, plays therapeutical effect.But, genomic medicine send a challenge difficult problem especially because its usual bear electricity, very easily removed by immune system and cannot therapeutical effect be played.
Summary of the invention
The object of the present invention is to provide a kind of remove Pegylation common loading amycin and siRNA carrier and synthetic method thereof, this synthetic method raw material is easy to get, and reaction condition is gentle, in the carrier obtained each section of polymer molecular weight and chain length easy to control, can nanoparticle be self-assembled into after carrying amycin, drug effect is high, achieves the common loading of chemotherapeutics and genomic medicine.
For achieving the above object, the present invention can go the common loading amycin of Pegylation and the synthetic method of siRNA carrier, comprises the steps:
1) synthesis of mPEG macromole RAFT agent:
Cystamine end methoxy poly (ethylene glycol) is dissolved in anhydrous organic solvent, then under nitrogen atmosphere protection, add the RAFT agent of a band carboxyl, DMAP and dicyclohexylcarbodiimide wherein, again room temperature reaction 24 hours ~ 72 hours, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain mPEG macromole RAFT agent; Wherein, the mol ratio of cystamine end methoxy poly (ethylene glycol), the RAFT agent being with a carboxyl, DMAP and dicyclohexylcarbodiimide is 1:(2 ~ 20): 1:(4 ~ 40);
2) synthesis of mPEG-S-S-PDMAPMA-CTA:
MPEG macromole RAFT agent, N-(3-dimethylamine propyl) Methacrylamide and initiator are dissolved in anhydrous and oxygen-free organic solvent, then in 60 DEG C ~ 65 DEG C reactions 24 hours ~ 48 hours, obtain reactant liquor; Reactant liquor by cold diethyl ether precipitation, then by precipitate and separate and vacuum drying, obtains mPEG-S-S-PDMAPMA-CTA after concentrated; Wherein, the mol ratio of mPEG macromole RAFT agent and N-(3-dimethylamine propyl) Methacrylamide is 10:(100 ~ 600);
3) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the water of 0 ~ 4 DEG C, then under agitation acryloyl chloride organic solution is dripped, adjust ph to 8 ~ 9 simultaneously, and room temperature reaction 4 ~ 10 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride organic solution is 1:1.1;
4) synthesis of mPEG-S-S-PDMAPMA-PBocAAH-CTA:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides and initiator are dissolved in anhydrous and oxygen-free organic solvent, then in 60 DEG C ~ 65 DEG C reactions 24 hours ~ 48 hours, obtain reactant liquor; Reactant liquor by cold diethyl ether precipitation, then by precipitate and separate and vacuum drying, obtains mPEG-S-S-PDMAPMA-PBocAAH-CTA after concentrated; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA and N-tertbutyloxycarbonyl propylene hydrazides is 5:(100 ~ 500);
5) the tertbutyloxycarbonyl protecting group on mPEG-S-S-PDMAPMA-PBocAAH-CTA is removed, namely obtain common loading amycin and the siRNA carrier that can remove Pegylation.
Described cystamine end methoxy poly (ethylene glycol) adopts following steps synthesis:
A) by molecular weight be end methoxy poly (ethylene glycol), the dibutyl tin laurate and 2 of 800 ~ 4000Da, 2'-dithio diethyl isocyanates is dissolved in dry toluene, under nitrogen atmosphere in 85 DEG C of reactions 48 hours, then solid is settled out with anhydrous n-hexane, collect solid vacuum drying, obtain the mPEG that 2,2'-dithio diethyl is isocyanate-modified; Wherein, the mol ratio of holding methoxy poly (ethylene glycol), dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:(0.02 ~ 0.08): (3 ~ 8);
B) by 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain cystamine end methoxy poly (ethylene glycol).
Described step 1) in the RAFT agent of a band carboxyl be S-1-dodecyl-S'-(α, α '-dimethyl-α "-acetic acid) trithiocarbonate, 4-cyano group-4-[(dodecyl sulfanyl thiocarbonyl group) sulfanyl] valeric acid, 4-cyano group-4-(thiobenzoyl) valeric acid or 4-cyanopentanoic acid dithiobenzoic acid.
Described step 1) in dissolve 5g ~ 40g cystamine end methoxy poly (ethylene glycol) in often liter of anhydrous organic solvent, step 2) in dissolve the mPEG macromole RAFT agent of 50g ~ 150g, step 4 in often liter of anhydrous and oxygen-free organic solvent) in the mPEG-S-S-PDMAPMA-CTA of often liter of anhydrous and oxygen-free organic solvent dissolution 50g ~ 200g.
Described step 2) in the mol ratio of N-(3-dimethylamine propyl) Methacrylamide and initiator be (100 ~ 600): 1, step 4) mol ratio of middle N-tertbutyloxycarbonyl propylene hydrazides and initiator is (100 ~ 500): 1.
Described step 2) and step 4) in anhydrous and oxygen-free organic solvent be anhydrous and oxygen-free dioxane, anhydrous and oxygen-free oxolane or anhydrous and oxygen-free dimethyl formamide.
Described step 2) and step 4) in initiator be azodiisobutyronitrile or 4,4'-azo two (cyanopentanoic acid).
Described step 5) by the method that the tertbutyloxycarbonyl protecting group on mPEG-S-S-PDMAPMA-PBocAAH-CTA removes be:
MPEG-S-S-PDMAPMA-CTA is dissolved in anhydrous organic solvent, is added drop-wise in the trifluoroacetic acid and dichloromethane mixed solvent that volume ratio is 1:1 in 0 DEG C, then rises to room temperature reaction 5 hours; Solvent is removed in distilling under reduced pressure, in the solid obtained, add sodium bicarbonate aqueous solution again and carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, namely obtains common loading amycin and the siRNA carrier that can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA and trifluoroacetic acid is 1:(15 ~ 40).
Described step 1) and step 5) in anhydrous organic solvent be anhydrous methylene chloride, anhydrous chloroform, anhydrous dioxane or anhydrous tetrahydro furan.
A kind of common loading amycin of gone to Pegylation of described method synthesis and siRNA carrier, chemical name is end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-polyacrylic hydrazide triblock copolymer, and structural formula is:
Described step 3) in drip acryloyl chloride organic solution in solvent be chloroform, dichloromethane or dioxane.
Described step a) in dissolve the end methoxy poly (ethylene glycol) of 5g ~ 30g, step b in every 100 milliliters of dry toluenes) in dissolve the isocyanate-modified mPEG of 2, the 2'-dithio diethyl of 5g ~ 20g in every 100 ml distilled waters.
Described step 5) in dissolve the mPEG-S-S-PDMAPMA-CTA of 50g ~ 200g in often liter of anhydrous organic solvent;
Compared with prior art, beneficial effect of the present invention is:
The Polyethylene Glycol section that the present invention can realize carrier stealth is linked together by disulfide bond and other two sections (side hydrazides section and cation sections), the reproducibility environment sensitive that this disulfide bond brings homoglutathion concentration in tumor cell, can glutathion be responded and rupture, remove Polyethylene Glycol section, be beneficial to carrier to escape in Cytoplasm from endosome, avoid lysosomal zymolysis, therefore, the present invention can improve effect of drugs.And the carrier of the present invention's synthesis can be water-soluble, therefore, can carry out in water when the side hydrazides section of carrier of the present invention loads amycin and cation section loading siRNA, avoid organic solvent residual or organic solvent to the potential side effect of siRNA.In addition, carrier of the present invention forms hydrazone key by carbonyl on its side hydrazides group and amycin, is bonded on carrier, because of amycin hydrophobicity, gives carrier-amycin conjugate and presents amphipathic character, therefore, it is possible to be self-assembled into nanoparticle; Meanwhile, the hydrazone key of keyed jointing amycin, to tumor inner acidic environment sensitive, once medicine-carried system enters tumor cell, just can discharge medicine very soon, improves medicine effective concentration in the short time, thus improves curative effect of medication.Carrier of the present invention loading amycin after due to can self assembly formed nanoparticle, and the cation section being positioned at triblock copolymer interlude can form complex with siRNA by electrostatic interaction, be covered on kernel nanoparticle, obtain the constitutionally stable nuclear shell structure nano grain that outermost layer is the stealthy layer of Polyethylene Glycol, realize the common loading of chemotherapeutics and genomic medicine.
Finally, raw material of the present invention is easy to get, and reaction temperature is below 100 DEG C, and therefore, reaction condition is gentle, and each section of polymer molecular weight and chain length easy to control.
Accompanying drawing explanation
Fig. 1 is the common loading amycin of gone to Pegylation in embodiments of the invention 1 and the synthetic route schematic diagram of siRNA carrier.
Fig. 2 is common loading amycin and the siRNA carrier of gone to Pegylation prepared by embodiment 1 1h-NMR spectrogram.
Fig. 3 is the grain size distribution that the common loading amycin of gone to Pegylation that synthesizes of embodiment 1 and siRNA carrier load self-assembled nanometer grain after amycin.
Fig. 4 is the laser co-focusing fluorescence photo that nanoparticle that the common loading amycin of gone to Pegylation that synthesizes of embodiment 1 and siRNA carrier carry amycin and FAM labelling siRNA altogether enters in breast cancer cell MCF-7; Wherein, a is the nuclear fluorescence photo of DAPI dyeing, and b is the doxorubicin fluorescence photo at nucleus place, and c is the fluorescence photo after the siRNA of FAM labelling enters cell, and d figure is the constitutional diagram of a-c.
Detailed description of the invention
Below in conjunction with accompanying drawing, the invention will be further described.
See Fig. 1; the present invention carries out cystamineization modified keyed jointing micromolecule RAFT agent to mono methoxy polyethylene glycol; obtain Polyethylene Glycol macromole RAFT agent; with cationic monomer N-(3-dimethylamine propyl) Methacrylamide and the monomer N-tertbutyloxycarbonyl propylene hydrazides of protecting group tertbutyloxycarbonyl can be removed for raw material; through processes such as two step RAFT polymerizations and removal protecting groups, obtained end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-polyacrylic hydrazide triblock copolymer can removing Polyethylene Glycol.
Make row further further combined with drawings and Examples of the present invention to this law to describe in detail, but be not limited to this.
Embodiment 1:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 800Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, react 48 hours at 85 DEG C under nitrogen atmosphere, then precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, obtain the mPEG that 2,2'-dithio diethyl is isocyanate-modified; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.02:4, dissolves the mPEG of 10g in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 5g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous methylene chloride, then under nitrogen atmosphere protection, add S-1-dodecyl-S'-(α wherein, α '-dimethyl-α "-acetic acid) trithiocarbonate (DDACT), DMAP (DMAP) and dicyclohexylcarbodiimide (DCC); again room temperature reaction 48 hours, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG, DDACT, DMAP and DCC is 1:10:1:20, dissolves 20g cystamine mPEG in often liter of anhydrous methylene chloride;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
MPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and azodiisobutyronitrile are dissolved in anhydrous and oxygen-free dioxane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and azodiisobutyronitrile is 10:200:1, dissolves the mPEG-S-S-CTA of 50g in often liter of anhydrous and oxygen-free dioxane;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 0 DEG C, then the acryloyl chloride chloroformic solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 10 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride chloroformic solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and azodiisobutyronitrile are dissolved in anhydrous and oxygen-free dioxane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and azodiisobutyronitrile is 5:100:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 150g in often liter of anhydrous and oxygen-free dioxane;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-PBocAAH-CTA is dissolved in anhydrous methylene chloride, drip in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then room temperature reaction is risen to 5 hours, reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, namely common loading amycin and siRNA carrier that product can remove Pegylation is obtained, wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:15, dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 50g in often liter of mixed solvent.
The common loading amycin of gone to Pegylation of the present embodiment synthesis and the chemical name of siRNA carrier are end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-polyacrylic hydrazide triblock copolymer, its 1h-NMR spectrogram is shown in Fig. 2.As can be seen from Figure 2, the chemical shift of each copolymer component proton all obtains good qualification, shows that the synthesis of this copolymer is successful.
After the common loading amycin of gone to the Pegylation of the present embodiment synthesis and siRNA carrier load amycin, present amphipathic character, can be self-assembled into as nanoparticle in water, the grain size distribution that dynamic light scattering method records is shown in Fig. 3.As seen from Figure 3, particle size distribution range is 100-300 nanometer, and concentrate on 200 nanometers, particle size distribution range is narrow, more satisfactory.
The nanoparticle formed after the common loading amycin of gone to the Pegylation of the present embodiment synthesis and siRNA carrier load the Pgp siRNA of amycin and green fluorescence agent FAM labelling altogether effectively can enter in cell through endocytosis.Its stowage is by genomic medicine and common loading amycin and the siRNA carrier mixing of loading gone to the Pegylation after amycin, is mixed to form complex, realizes the loading of genomic medicine through turbula shaker.
B in Fig. 4 a is the fluorescence that the nucleus of DAPI dyeing sends, R in Fig. 4 b is the fluorescence that the amycin at nucleus place sends, G in Fig. 4 c is the fluorescence sent after the Pgp siRNA of FAM labelling enters cell, Fig. 4 d is obtained after being combined by Fig. 4 a-4c, as can be seen from Fig. 4 d, after the common loading amycin of Pegylation and siRNA carrier can be gone to carry the Pgp siRNA of amycin and FAM labelling altogether, amycin and Pgp siRNA all can effectively enter in breast cancer cell MCF-7, around the nucleus being distributed in breast cancer cell MCF-7, the carrier of visible synthesis can be used for the common loading of amycin and siRNA and sends.
Embodiment 2:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 2000Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, react 48 hours under 85 DEG C of blanket of nitrogen under nitrogen atmosphere, then precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, obtain the mPEG that 2,2'-dithio diethyl is isocyanate-modified; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.04:4; The mPEG of 20g is dissolved in the dry toluene of every 100 milliliters;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 15g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous chloroform, then under nitrogen atmosphere protection, add S-1-dodecyl-S'-(α wherein, α '-dimethyl-α "-acetic acid) trithiocarbonate (DDACT), DMAP (DMAP) and dicyclohexylcarbodiimide (DCC); again room temperature reaction 48 hours, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG, DDACT, DMAP and DCC is 1:5:1:10, dissolves the cystamine mPEG of 20g in often liter of anhydrous chloroform;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
MPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and 4,4'-azo two (cyanopentanoic acid) are dissolved in anhydrous and oxygen-free dioxane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and 4,4'-azo two (cyanopentanoic acid) is 10:300:1, dissolves the mPEG-S-S-CTA of 150g in often liter of anhydrous and oxygen-free dioxane;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 4 DEG C, then the acryloyl chloride dichloromethane solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 4 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride dichloromethane solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and 4,4'-azo two (cyanopentanoic acid) are dissolved in anhydrous and oxygen-free dioxane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and 4,4'-azo two (cyanopentanoic acid) is 5:150:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 200g in often liter of anhydrous and oxygen-free dioxane;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-PBocAAH-CTA is dissolved in anhydrous dioxane, during to drip volume ratio be the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then rises to room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, that is: 4 to common loading amycin and the siRNA carrier that can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:40, dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 50g in often liter of mixed solvent.
Embodiment 3:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 2000Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, react 48 hours under 85 DEG C of blanket of nitrogen under nitrogen atmosphere, then precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, namely the mPEG that 2,2'-dithio diethyl is isocyanate-modified is obtained; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.05:5; The mPEG of 10g is dissolved in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 10g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous tetrahydro furan, then under nitrogen atmosphere protection, add S-1-dodecyl-S'-(α wherein, α '-dimethyl-α "-acetic acid) trithiocarbonate (DDACT), DMAP (DMAP) and dicyclohexylcarbodiimide (DCC); again room temperature reaction 48 hours, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, lyophilizing after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of mPEG, DDACT, DMAP and DCC is 1:15:1:30, dissolves the cystamine mPEG of 25g in often liter of anhydrous tetrahydro furan;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
MPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and azodiisobutyronitrile are dissolved in anhydrous and oxygen-free oxolane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and azodiisobutyronitrile is 10:400:1, dissolves the mPEG-S-S-CTA of 125g in often liter of anhydrous and oxygen-free oxolane;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 2 DEG C, then the acryloyl chloride dioxane solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 8 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride dioxane solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and azodiisobutyronitrile are dissolved in anhydrous and oxygen-free oxolane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and azodiisobutyronitrile is 5:150:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 120g in often liter of anhydrous and oxygen-free oxolane;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-CTA is dissolved in anhydrous tetrahydro furan, drips in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then rise to room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, namely obtains common loading amycin and the siRNA carrier that can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:15, dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 50g in often liter of mixed solvent.
Embodiment 4:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 4000Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, under nitrogen atmosphere in 85 DEG C of reactions 48 hours, then precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, namely the mPEG that 2,2'-dithio diethyl is isocyanate-modified is obtained; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.02:4; The mPEG of 5g is dissolved in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 10g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous methylene chloride, then under nitrogen atmosphere protection, add S-1-dodecyl-S'-(α wherein, α '-dimethyl-α "-acetic acid) trithiocarbonate (DDACT), DMAP (DMAP) and dicyclohexylcarbodiimide (DCC); again room temperature reaction 48 hours, obtain in reactant liquor, add cold diethyl ether and make to separate out solid in reactant liquor; Collect after solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG, DDACT, DMAP and DCC is 1:8:1:16, dissolves 25g cystamine mPEG in often liter of anhydrous methylene chloride;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
MPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and azodiisobutyronitrile are dissolved in anhydrous and oxygen-free dioxane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and azodiisobutyronitrile is 10:200:1, dissolves the mPEG-S-S-CTA of 130g in often liter of anhydrous and oxygen-free dioxane;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 0 DEG C, then the acryloyl chloride chloroformic solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 6 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride chloroformic solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and azodiisobutyronitrile are dissolved in anhydrous and oxygen-free dioxane, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and azodiisobutyronitrile is 5:500:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 60g in often liter of anhydrous and oxygen-free dioxane;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-CTA is dissolved in anhydrous methylene chloride, drips in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then to rise after room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, namely obtains common loading amycin and siRNA carrier that product can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:20, dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 70g in often liter of mixed solvent.
Embodiment 5:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 4000Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, react 48 hours under 85 DEG C of blanket of nitrogen under nitrogen atmosphere, then precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, namely the mPEG that 2,2'-dithio diethyl is isocyanate-modified is obtained; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.03:4; The mPEG of 5g is dissolved in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 10g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous chloroform, then under nitrogen atmosphere protection, add S-1-dodecyl-S'-(α wherein, α '-dimethyl-α "-acetic acid) trithiocarbonate (DDACT), DMAP (DMAP) and dicyclohexylcarbodiimide (DCC); again room temperature reaction 48 hours, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid precipitation in reactant liquor, lyophilizing after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG, DDACT, DMAP and DCC is 1:12:1:24, dissolves the cystamine mPEG of 35g in often liter of anhydrous chloroform;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
By mPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dimethyl formamide, then in 60 DEG C of reactions 48 hours, reactant liquor is obtained; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and 4,4'-azo two (cyanopentanoic acid) is 10:600:1, dissolves the mPEG-S-S-CTA of 50g in often liter of anhydrous and oxygen-free dimethyl formamide;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 2 DEG C, then the acryloyl chloride dioxane solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 8 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride dioxane solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and 4,4'-azo two (cyanopentanoic acid) are dissolved in anhydrous and oxygen-free dimethyl formamide, then in 60 DEG C of reactions 48 hours, obtain reactant liquor; Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and 4,4'-azo two (cyanopentanoic acid) is 5:400:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 50g in often liter of anhydrous and oxygen-free dimethyl formamide.
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-CTA is dissolved in anhydrous methylene chloride, drips in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then rise to room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, namely obtains common loading amycin and siRNA carrier that product can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:30, dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 50g in often liter of mixed solvent.
Embodiment 6:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 2000Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, react 48 hours under 85 DEG C of blanket of nitrogen under nitrogen atmosphere, then precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, namely the mPEG that 2,2'-dithio diethyl is isocyanate-modified is obtained; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.06:4; The mPEG of 8g is dissolved in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 12g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous dioxane, then under nitrogen atmosphere protection, add S-1-dodecyl-S'-(α wherein, α '-dimethyl-α "-acetic acid) trithiocarbonate (DDACT), DMAP (DMAP) and dicyclohexylcarbodiimide (DCC); again room temperature reaction 48 hours, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG:DDACT:DMAP:DCC is 1:15:1:30, dissolves the cystamine mPEG of 32g in often liter of anhydrous dioxane;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
By mPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dimethyl formamide, then in 60 DEG C of reactions 48 hours, obtain reactant liquor, reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA:DMAPMA:4,4'-azo two (cyanopentanoic acid) is 10:420:1, dissolves the mPEG-S-S-CTA of 130g in often liter of anhydrous and oxygen-free dimethyl formamide;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 0 DEG C, then the acryloyl chloride chloroformic solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 10 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride chloroformic solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and 4,4'-azo two (cyanopentanoic acid) are dissolved in anhydrous and oxygen-free dimethyl formamide, then in 60 DEG C of reactions 48 hours, obtain reactant liquor.Reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and 4,4'-azo two (cyanopentanoic acid) is 5:180:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 145g in often liter of anhydrous and oxygen-free dimethyl formamide;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-PBocAAH-CTA is dissolved in anhydrous tetrahydro furan, drips in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then to rise to after room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, namely obtains common loading amycin and siRNA carrier that product can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:18, dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 60g in often liter of mixed solvent.
Embodiment 7:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 1200Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, under nitrogen atmosphere in 85 DEG C of reactions 48 hours, precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, namely the mPEG that 2,2'-dithio diethyl is isocyanate-modified is obtained; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.07:6; The mPEG of 25g is dissolved in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally the reactant liquor obtained is obtained after dialysis and lyophilizing the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 20g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous methylene chloride, then under nitrogen atmosphere protection, add 4-cyano group-4-[(dodecyl sulfanyl thiocarbonyl group) sulfanyl] valeric acid, DMAP (DMAP) and dicyclohexylcarbodiimide (DCC) wherein, little at room temperature reaction 48 again, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG, 4-cyano group-4-[(dodecyl sulfanyl thiocarbonyl group) sulfanyl] valeric acid, DMAP and DCC is 1:8:1:16, dissolves the cystamine mPEG of 28g in often liter of anhydrous methylene chloride;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
By mPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dioxane, then in 63 DEG C of reactions 48 hours, obtain reactant liquor, reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and 4,4'-azo two (cyanopentanoic acid) is 10:450:1, dissolves the mPEG-S-S-CTA of 130g in often liter of anhydrous and oxygen-free dioxane;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 0 DEG C, then the acryloyl chloride chloroformic solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 10 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride chloroformic solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
By mPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dioxane, then in 63 DEG C of reactions 48 hours, obtain reactant liquor, reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and 4,4'-azo two (cyanopentanoic acid) is 5:320:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 110g in often liter of anhydrous and oxygen-free dioxane;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-PBocAAH-CTA is dissolved in anhydrous chloroform, drips in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then to rise to after room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, and namely last lyophilizing obtains common loading amycin and the siRNA carrier that product can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:21, and often liter of mixed solvent transverse and longitudinal dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 90g.
Embodiment 8:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 1200Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, under nitrogen atmosphere in 85 DEG C of reactions 48 hours, precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, namely the mPEG that 2,2'-dithio diethyl is isocyanate-modified is obtained; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.08:6; The mPEG of 30g is dissolved in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally the reactant liquor obtained is obtained after dialysis and lyophilizing the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 20g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous methylene chloride, then under nitrogen atmosphere protection, add 4-cyano group-4-(thiobenzoyl) valeric acid, DMAP (DMAP) and dicyclohexylcarbodiimide (DCC) wherein, again room temperature reaction 24 hours, obtain reactant liquor liquid; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG, 4-cyano group-4-(thiobenzoyl) valeric acid, DMAP and DCC is 1:2:1:4, dissolves the cystamine mPEG of 5g in often liter of anhydrous methylene chloride;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
By mPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dioxane, then in 65 DEG C of reactions 24 hours, obtain reactant liquor, reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and 4,4'-azo two (cyanopentanoic acid) is 10:100:1, dissolves the mPEG-S-S-CTA of 130g in often liter of anhydrous and oxygen-free dioxane;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 2 DEG C, then the acryloyl chloride chloroformic solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 10 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride chloroformic solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
By mPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dioxane, then in 65 DEG C of reactions 24 hours, obtain reactant liquor, reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and 4,4'-azo two (cyanopentanoic acid) is 5:320:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 110g in often liter of anhydrous and oxygen-free dioxane;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-PBocAAH-CTA is dissolved in anhydrous chloroform, drips in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then to rise to after room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, and namely last lyophilizing obtains common loading amycin and the siRNA carrier that product can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:21, and often liter of mixed solvent transverse and longitudinal dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 160g.
Embodiment 9:
1) synthesis of cystamine end methoxy poly (ethylene glycol):
Be end methoxy poly (ethylene glycol) (being abbreviated as mPEG), the dibutyl tin laurate and 2 of 1200Da by the molecular weight of drying, 2'-dithio diethyl isocyanates is dissolved in dry toluene, under nitrogen atmosphere in 85 DEG C of reactions 48 hours, precipitate 3 times in anhydrous n-hexane, the solid vacuum drying obtained, namely the mPEG that 2,2'-dithio diethyl is isocyanate-modified is obtained; Wherein, the mol ratio of the mPEG added, dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:0.08:6; The mPEG of 30g is dissolved in every 100 milliliters of dry toluenes;
By 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally the reactant liquor obtained is obtained after dialysis and lyophilizing the cystamine end methoxy poly (ethylene glycol) (being called for short cystamine mPEG) of white powder; Wherein, the mPEG that 2, the 2'-dithio diethyl of 20g are isocyanate-modified is dissolved in every 100 ml distilled waters;
2) synthesis of mPEG macromole RAFT agent:
Cystamine mPEG is dissolved in anhydrous methylene chloride, then under nitrogen atmosphere protection, add 4-cyanopentanoic acid dithiobenzoic acid, DMAP (DMAP) and dicyclohexylcarbodiimide (DCC) wherein, little at room temperature reaction 72 again, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain faint yellow solid product mPEG macromole RAFT agent (being called for short mPEG-S-S-CTA); Wherein, the mol ratio of cystamine mPEG, 4-cyanopentanoic acid dithiobenzoic acid, DMAP and DCC is 1:20:1:40, dissolves the cystamine mPEG of 40g in often liter of anhydrous methylene chloride;
3) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent is held:
By mPEG-S-S-CTA, N-(3-dimethylamine propyl) Methacrylamide (DMAPMA) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dioxane, then in 63 DEG C of reactions 35 hours, obtain reactant liquor, reactant liquor precipitates with cold diethyl ether after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide) di-block copolymer RAFT agent (being called for short mPEG-S-S-PDMAPMA-CTA); Wherein, the mol ratio of mPEG-S-S-CTA, DMAPMA and 4,4'-azo two (cyanopentanoic acid) is 10:150:1, dissolves the mPEG-S-S-CTA of 130g in often liter of anhydrous and oxygen-free dioxane;
4) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the solution forming 1mol/L in the water of 2 DEG C, then the acryloyl chloride chloroformic solution of 5mol/L is under agitation dripped, use sodium hydrate aqueous solution adjust ph to 8 ~ 9 of 2mol/L simultaneously, and room temperature reaction 10 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride chloroformic solution is 1:1.1; Recrystallization adopts dichloromethane solvent;
5) synthesis of methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer is held:
By mPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides (BocAAH) and 4,4'-azo two (cyanopentanoic acid) is dissolved in anhydrous and oxygen-free dioxane, then in 63 DEG C of reactions 35 hours, obtain reactant liquor, reactant liquor precipitates by cold diethyl ether precipitation after concentrated, again by precipitate and separate and vacuum drying, obtain end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-poly-(N-tertbutyloxycarbonyl propylene hydrazides) triblock copolymer (being called for short mPEG-S-S-PDMAPMA-PBocAAH-CTA); Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA, BocAAH and 4,4'-azo two (cyanopentanoic acid) is 5:320:1, dissolves the mPEG-S-S-PDMAPMA-CTA of 110g in often liter of anhydrous and oxygen-free dioxane;
6) protecting group---the tertbutyloxycarbonyl on triblock copolymer is removed:
MPEG-S-S-PDMAPMA-PBocAAH-CTA is dissolved in anhydrous chloroform, drips in volume ratio is the anhydrous trifluoroacetic acid of 1:1 and the mixed solvent of dichloromethane in 0 DEG C, then to rise to after room temperature reaction 5 hours; Reaction terminates rear distilling under reduced pressure and removes solvent, add in the solid obtained again mass concentration be 1% sodium bicarbonate aqueous solution carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, and namely last lyophilizing obtains common loading amycin and the siRNA carrier that product can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:21, and often liter of mixed solvent transverse and longitudinal dissolves the mPEG-S-S-PDMAPMA-PBocAAH-CTA of 200g.
In above-described embodiment 1 ~ 9, cold diethyl ether temperature is-20 DEG C, and freeze temperature is-40 DEG C, and vacuum drying temperature is at 30 DEG C; When reaction system concentrates, be concentrated to 1/1 to two/3rd of original volume.
In the present invention, cystamine end methoxy poly (ethylene glycol) is the end product 2-amino-ethyl two sulfur second ammonia ester Polyethylene Glycol that application number is the Chinese patent embodiment 4 of 201310229284.7.
The present invention can load amycin and siRNA simultaneously, makes chemotherapeutics and genomic medicine play curative effect simultaneously, improves oncotherapy effect and reduces toxic and side effects.In addition, each section of polymer molecular weight and chain length is easy to control, reaction condition is gentle, raw material is easy to get in carrier of the present invention, is water solublity before medicine carrying, has amphipathic, can be self-assembled into nanoparticle after medicine carrying.

Claims (8)

1. can go the common loading amycin of Pegylation and a synthetic method for siRNA carrier, it is characterized in that, comprise the steps:
1) synthesis of mPEG macromole RAFT agent:
Cystamine end methoxy poly (ethylene glycol) is dissolved in anhydrous organic solvent, then under nitrogen atmosphere protection, add the RAFT agent of a band carboxyl, DMAP and dicyclohexylcarbodiimide wherein, again room temperature reaction 24 hours ~ 72 hours, obtain reactant liquor; In reactant liquor, add cold diethyl ether make to separate out solid in reactant liquor, collect after the solid in reactant liquor is separated, obtain mPEG macromole RAFT agent; Wherein, the mol ratio of cystamine end methoxy poly (ethylene glycol), the RAFT agent being with a carboxyl, DMAP and dicyclohexylcarbodiimide is 1:(2 ~ 20): 1:(4 ~ 40);
Described cystamine end methoxy poly (ethylene glycol) adopts following steps synthesis:
A) by molecular weight be end methoxy poly (ethylene glycol), the dibutyl tin laurate and 2 of 800 ~ 4000Da, 2'-dithio diethyl isocyanates is dissolved in dry toluene, under nitrogen atmosphere in 85 DEG C of reactions 48 hours, then solid is settled out with anhydrous n-hexane, collect solid vacuum drying, obtain the mPEG that 2,2'-dithio diethyl is isocyanate-modified; Wherein, the mol ratio of holding methoxy poly (ethylene glycol), dibutyl tin laurate and 2,2'-dithio diethyl isocyanates is 1:(0.02 ~ 0.08): (3 ~ 8);
B) by 2, the isocyanate-modified mPEG of 2'-dithio diethyl is dissolved in distilled water, then 60 DEG C of reactions 6 hours, finally by the reactant liquor that obtains through dialysis and lyophilizing, obtain cystamine end methoxy poly (ethylene glycol);
The RAFT agent of a described band carboxyl is S-1-dodecyl-S'-(α, α '-dimethyl-α "-acetic acid) trithiocarbonate, 4-cyano group-4-[(dodecyl sulfanyl thiocarbonyl group) sulfanyl] valeric acid, 4-cyano group-4-(thiobenzoyl) valeric acid or 4-cyanopentanoic acid dithiobenzoic acid;
2) synthesis of mPEG-S-S-PDMAPMA-CTA:
MPEG macromole RAFT agent, N-(3-dimethylamine propyl) Methacrylamide and initiator are dissolved in anhydrous and oxygen-free organic solvent, then in 60 DEG C ~ 65 DEG C reactions 24 hours ~ 48 hours, obtain reactant liquor; Reactant liquor by cold diethyl ether precipitation, then by precipitate and separate and vacuum drying, obtains mPEG-S-S-PDMAPMA-CTA after concentrated; Wherein, the mol ratio of mPEG macromole RAFT agent and N-(3-dimethylamine propyl) Methacrylamide is 10:(100 ~ 600);
3) synthesis of N-tertbutyloxycarbonyl propylene hydrazides:
Tertbutyloxycarbonyl hydrazine is dissolved in the water of 0 DEG C ~ 4 DEG C, then under agitation acryloyl chloride organic solution is dripped, adjust ph to 8 ~ 9 simultaneously, and room temperature reaction 4 hours ~ 10 hours, finally by obtain product dichloromethane extraction, recrystallization vacuum drying, obtain N-tertbutyloxycarbonyl propylene hydrazides; Wherein, the mol ratio of the acryloyl chloride in tertbutyloxycarbonyl hydrazine and acryloyl chloride organic solution is 1:1.1;
4) synthesis of mPEG-S-S-PDMAPMA-PBocAAH-CTA:
MPEG-S-S-PDMAPMA-CTA, N-tertbutyloxycarbonyl propylene hydrazides and initiator are dissolved in anhydrous and oxygen-free organic solvent, then in 60 DEG C ~ 65 DEG C reactions 24 hours ~ 48 hours, obtain reactant liquor; Reactant liquor by cold diethyl ether precipitation, then by precipitate and separate and vacuum drying, obtains mPEG-S-S-PDMAPMA-PBocAAH-CTA after concentrated; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-CTA and N-tertbutyloxycarbonyl propylene hydrazides is 5:(100 ~ 500);
5) the tertbutyloxycarbonyl protecting group on mPEG-S-S-PDMAPMA-PBocAAH-CTA removed, namely obtain common loading amycin and the siRNA carrier that can remove Pegylation, its structural formula is:
2. according to claim 1ly go the common loading amycin of Pegylation and the synthetic method of siRNA carrier, it is characterized in that: described step 1) in dissolve 5g ~ 40g cystamine end methoxy poly (ethylene glycol) in often liter of anhydrous organic solvent, step 2) in dissolve the mPEG macromole RAFT agent of 50g ~ 150g, step 4 in often liter of anhydrous and oxygen-free organic solvent) in the mPEG-S-S-PDMAPMA-CTA of often liter of anhydrous and oxygen-free organic solvent dissolution 50g ~ 200g.
3. according to claim 1ly go the common loading amycin of Pegylation and the synthetic method of siRNA carrier, it is characterized in that: described step 2) in the mol ratio of N-(3-dimethylamine propyl) Methacrylamide and initiator be (100 ~ 600): 1, step 4) mol ratio of middle N-tertbutyloxycarbonyl propylene hydrazides and initiator is (100 ~ 500): 1.
4. according to claim 1ly go the common loading amycin of Pegylation and the synthetic method of siRNA carrier, it is characterized in that: described step 2) and step 4) in anhydrous and oxygen-free organic solvent be anhydrous and oxygen-free dioxane, anhydrous and oxygen-free oxolane or anhydrous and oxygen-free dimethyl formamide.
5. according to claim 1ly go the common loading amycin of Pegylation and the synthetic method of siRNA carrier, it is characterized in that: described step 2) and step 4) in initiator be azodiisobutyronitrile or 4,4'-azo two (cyanopentanoic acid).
6. according to claim 1ly go the common loading amycin of Pegylation and the synthetic method of siRNA carrier; it is characterized in that, described step 5) by the method that the tertbutyloxycarbonyl protecting group on mPEG-S-S-PDMAPMA-PBocAAH-CTA removes be:
MPEG-S-S-PDMAPMA-PBocAAH-CTA is dissolved in anhydrous organic solvent, is added drop-wise in the trifluoroacetic acid and dichloromethane mixed solvent that volume ratio is 1:1 in 0 DEG C, then rises to room temperature reaction 5 hours; Solvent is removed in distilling under reduced pressure, in the solid obtained, add sodium bicarbonate aqueous solution again and carry out neutralisation treatment, then loading molecular cut off is that the ammonia being 0.25% to mass concentration in the bag filter of 3500Da is dialysed 48 hours, last lyophilizing, namely obtains common loading amycin and the siRNA carrier that can remove Pegylation; Wherein, the mol ratio of mPEG-S-S-PDMAPMA-PBocAAH-CTA and trifluoroacetic acid is 1:(15 ~ 40).
7. according to claim 6ly go the common loading amycin of Pegylation and the synthetic method of siRNA carrier, it is characterized in that: described step 1) and step 5) in anhydrous organic solvent be anhydrous methylene chloride, anhydrous chloroform, anhydrous dioxane or anhydrous tetrahydro furan.
8. the common loading amycin as gone to the Pegylation of the method synthesis in claim 1 ~ 7 as described in any one claim and siRNA carrier, it is characterized in that, chemical name is end methoxy poly (ethylene glycol)-poly-(N-(3-dimethylamine propyl) Methacrylamide)-polyacrylic hydrazide triblock copolymer, and structural formula is:
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