CN107298729A - A kind of N of hydroxyl modification, cationic polymer of N dimethylaminos and its preparation method and application - Google Patents

A kind of N of hydroxyl modification, cationic polymer of N dimethylaminos and its preparation method and application Download PDF

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CN107298729A
CN107298729A CN201710367726.2A CN201710367726A CN107298729A CN 107298729 A CN107298729 A CN 107298729A CN 201710367726 A CN201710367726 A CN 201710367726A CN 107298729 A CN107298729 A CN 107298729A
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cationic polymer
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CN107298729B (en
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董岸杰
周俊辉
邓联东
张建华
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Tianjin University
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    • C08F8/00—Chemical modification by after-treatment
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    • C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
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    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/32—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals
    • C08F220/325—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals containing glycidyl radical, e.g. glycidyl (meth)acrylate

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Abstract

The present invention relates to a kind of N of hydroxyl modification, the cationic polymer of N dimethylaminos and its preparation method and application, the N of its hydroxyl modified, N dimethylaminos are N, N, and the hydroxypropyl thioether of N dimethyl ethyls 2, cationic polymer is homopolymer or copolymer；The relative molecular mass of described cationic polymer is 5,000 40000.Described cationic polymer is polymer when being amphipathic copolymer, it is self-assembly of polycation nano grain of the average grain diameter less than 200nm in aqueous, the presence of surface hydroxyl can significantly improve the biocompatibility of the polycation, and cell compatibility is preferable and without haemolysis.Nanometer particle loading DNA, siRNA or polypeptide, the protide bio-pharmaceutical formed, kernel can load hydrophobic chemicals, for medicine delivery.As siRNA carrier, with preferable application prospect.

Description

A kind of N of hydroxyl modification, the cationic polymer of N- dimethylaminos and its preparation side Method and application
Technical field
The present invention relates to a kind of N of hydroxyl modification, the cationic polymer of N- dimethylaminos and preparation method thereof and should With belonging to functional polymer technology.
Technical background
SiRNA is a kind of short-chain nucleic acids for being capable of specific downregulation gene expression.In theory, by rationally designing, siRNA Can be with any gene in silence body, people can fundamentally solve the difficult disease of many puzzlement mankind using this biotechnology Disease, such as hepatitis B, cancer, skin disease.Although siRNA has extremely wide clinical treatment potentiality, and siRNA is from clinic Using an also very big segment distance.As exogenous molecule, there are many barriers for being difficult to overcome in siRNA in delivery problems Hinder.First, siRNA is the exogenous large biological molecule with negative electrical charge, and it is difficult and the vivo medicine-feeding directly by cellular uptake SiRNA can be recognized and removed by internal immune system quickly afterwards.Therefore, siRNA needs to be delivered into by assistant carrier In the cell of particular organization, so as to realize the therapeutic purposes of its silence target gene.The many of mode is directly administered for siRNA Deficiency, the research of siRNA delivery vectors turns into focus.
Cationic polymer is the important siRNA delivery vectors of a class, and it can be made by electrostatic in aqueous with siRNA With being bound to each other to form stable nano-particle, while having the advantages that to prepare simple, transfection efficiency high., in the world in 2008 One siRNA medicine (CALAA-01) based on cationic polymer enters clinical experimental stage.Although, cationic polymer energy Enough effective delivering siRNA, but its electropositive can cause serious toxic side effect.At present, the common practice of people is poly- Bonded PEG or the biocompatibility of cationic polymer is improved on cation by the way of wrapping up layer by layer.Although but such as This, cationic polymer is still difficult to meet the requirement of clinical practice, it is necessary to develop reduction cation for the biocompatibility of carrier The effective ways of polymer toxicity.
The content of the invention
In order to effectively reduce the toxicity of cationic polymer, promote clinical application, we exist with reference to alcohols material High-compatibility feature in living organism, prepares a kind of N of hydroxyl modification, the cationic polymer of N- dimethylaminos, with Phase realizes the high-efficiency delivery of the biomolecule such as DNA, siRNA and effectively improves the biocompatibility of cationic polymer.
The present invention is to be realized by the following technical programs：
A kind of N of hydroxyl modification, the cationic polymer of N- dimethylaminos, the N of its hydroxyl modified, N- dimethylaminos are N, N, N- dimethyl ethyl -2- hydroxyl-propyls thioether (hereinafter referred to as HDMA), cationic polymer are homopolymer or copolymer； The relative molecular mass of described cationic polymer is 5000-40000.
Described cationic polymer, its cationic polymer is homopolymer, i.e., wherein bonded HDMA construction unit Gross mass accounts for the 100% of cationic polymerization amount of substance.
Described cationic polymer, its cationic polymer is copolymer, wherein bonded HDMA construction unit is total Quality accounts for the 10%~90% of cationic polymerization amount of substance.
Described cationic polymer, the copolymer described in it, for the amphipathic copolymer containing hydrophobic structure unit, The gross mass of hydrophobic structure unit accounts for the 10%~60% of cationic polymerization amount of substance.
Described cationic polymer, the copolymer described in it, for the homopolymer (PHDMA) of the construction unit with HDMA The amphipathic three block copolymer constituted with nonionic, hydrophilic polymers (B) and hydrophobic section (R)；Wherein, the phase of PHDMA blocks It is 2000~10000 to molecular mass, nonionic, hydrophilic polymers B relative molecular mass is 2000-10000, hydrophobic section R Relative molecular mass be 2000~20000；Weight/mass percentage composition of the PHDMA blocks in triblock copolymer be 10%~ 70%.
Described cationic polymer, B sections of the nonionic, hydrophilic polymers of the amphipathic three block copolymer described in it Selected from polyethylene glycol (PEG), poly- carboxybetaine methacrylate or phosphocholine.
Described cationic polymer, the copolymer described in it is PEG-R-PHDMA type block copolymers, and structural formula is such as Under：
Wherein, PHDMA is polymethylacrylic acid (N, N, N- dimethyl ethyl -2- hydroxyl-propyls thioether) ester, naIt is its knot Structure unit number, is 8-41 integer；N is the construction unit number of polyethylene glycol, is 45-227 integer；Hydrophobic section R is esters list The homopolymer or copolymer of body or vinyl monomer, esters monomer be selected from caprolactone, lactide, lactide, 4- (ethylene ketal)- Caprolactone, vinyl monomer is selected from methacrylic acid -2- (3,5- dioxanes -4- (2,4,6- trimethoxy-benzene)) propyl ester, methyl-prop Olefin(e) acid (diisopropylaminoethyl) ethyl ester.
The N of the hydroxyl modification of the present invention, the preparation method of the cationic polymer of N- dimethylaminos, with vinyl polymer For hydrophobic section：Monohydroxy PEG is dissolved in dichloromethane, micromolecule chain transfer agent S-1- dodecyls-S '-(α, α "-two is added Methyl-α "-acetic acid) trithiocarbonate (CTAm), it is subsequently added dicyclohexylcarbodiimide and DMAP, room temperature Under the conditions of react after concentrate, in ice ether precipitate, suction filtration, vacuum drying, obtain macromole evocating agent PEG-CTAm；In reaction Macromole evocating agent is added in device, after dmso solution, adds at hydrophobic vinyl monomer, 30-80 DEG C and reacts 24 After hour, add at methyl propenoic acid glycidyl ether, 30-80 DEG C and react 24 hours, concentration is precipitated, suction filtration in ice ether, Vacuum drying, obtains the block copolymer of the side base containing oxirane；Gained copolymer is dissolved in tetrahydrofuran, N, N- bis- is added Methylamino ethyl mercaptan, room temperature reaction is stayed overnight；Concentration, is precipitated, suction filtration in ice ether, and vacuum drying is obtained with vinyl polymer For the triblock copolymer of hydrophobic section.
The N of the hydroxyl modification of the present invention, the preparation method of the cationic polymer of N- dimethylaminos, it is characterized in that can drop Solution polyester is hydrophobic section：Monohydroxy PEG is added in reactor, esters monomer is then added in the reactor, catalyst is added Stannous octoate, is reacted at 100-130 DEG C and stays overnight, product dichloromethane is dissolved, and is precipitated in ice ether, suction filtration, and vacuum is done It is dry to obtain PEG-R polymer；PEG-R is dissolved in dichloromethane, micromolecule chain transfer agent CTAm is added, is subsequently added two hexamethylenes Reaction is stayed overnight under base carbodiimide and DMAP, room temperature condition, is concentrated, and is precipitated in ice ether, suction filtration, and vacuum is done It is dry, obtain macromole evocating agent PEG-R-CTAm；PEG-R-CTAm and methyl propenoic acid glycidyl ether are added in reactor, Then add after being reacted 24 hours at DMSO, 30 DEG C and concentrate, precipitated in ice ether, suction filtration, vacuum drying obtains second containing epoxy The block copolymer of alkane side base；Gained block copolymer is dissolved in tetrahydrofuran, N, N- dimethylamino ethyl mercaptans, room temperature is added Concentrate, precipitated in ice ether, suction filtration after reaction overnight, vacuum drying obtains the three block using degradable polyester as hydrophobic section Copolymer.
The N of the hydroxyl modification of the present invention, the cationic polymer of N- dimethylaminos is applied to load DNA, siRNA or many Peptide, protide bio-pharmaceutical, for medicine delivery.
Specific detailed description is as follows：
A kind of N of hydroxyl modification, the cationic polymer of N- dimethylaminos, the N of described hydroxyl modified, N- diformazan ammonia Base is N, and N, N- dimethyl ethyl -2- hydroxyl-propyls thioether (is abbreviated as HDMA) in the present invention, and described cationic polymer can To be homopolymer or copolymer, wherein the gross mass of bonded HDMA construction unit account for cationic polymerization amount of substance 10%~ 100%, the relative molecular weight of described cationic polymer is 5000-40000.
HDMA structure is as shown in Equation 1：
X is the group of bonded HDMA and macromolecular main chain in formula 1, can be the groups such as ester group, ehter bond, acid amides, preferably ester Base.
HDMA can by N, opening between N- dimethylaminos ethyl mercaptan and epoxide group and formed.
HDMA side bases can load DNA, siRNA, polypeptide and egg as a kind of cationic structural unit by Electrostatic Absorption Electronegative material such as white, plays a part of absorption carriage or separation.The presence of hydroxyl is further characterized in that, cation can be reduced Toxicity.
Described cationic polymer, is characterized in the homopolymer that a kind of construction unit by bonded HDMA is constituted, i.e., bonded The gross mass of HDMA construction unit accounts for the 100% of cationic polymerization amount of substance.
Described cationic polymer, is characterized in a kind of copolymer, is bonded HDMA construction unit and other structures list The copolymer that member is formed, can be random copolymer, block copolymer, graft copolymer, radial copolymer etc.；It is wherein bonded The gross mass of HDMA construction unit accounts for the 10%~90% of cationic polymerization amount of substance,
Described copolymer, is characterized in the amphipathic copolymer containing hydrophobic structure unit, hydrophobic structure unit Gross mass accounts for the 10%~60% of cationic polymerization amount of substance.
The further preferred amphipathic three block copolymer of described copolymer, is the cationic polymer with HDMA side bases (PHDMA) amphipathic three block copolymer constituted with nonionic, hydrophilic polymers (B) and hydrophobic section (R)；Wherein, PHDMA The relative molecular weight of block is 2000-10000, and the relative molecular weight of B block is 2000-10000, hydrophobic section R average molecular Measure as 2000-20000；Weight/mass percentage composition of the PHDMA blocks in triblock copolymer is 10%-70%.
Above-mentioned nonionic, hydrophilic polymers (B) are selected from polyethylene glycol (PEG), poly- carboxybetaine methacrylic acid Ester, phosphocholine.
Described amphipathic three block copolymer, further preferred PEG-R-PHDMA types block copolymer.Specifically, PEG-R-PHDMA can be the structure shown in formula 2, wherein, PHDMA is polymethylacrylic acid (N, N, N- dimethyl ethyl -2- hydroxyls Base-propyl group thioether) ester.
N is the construction unit number of polyethylene glycol in formula 2, is 45-227 integer；naIt is polymethylacrylic acid (N, N, N- bis- Methylethyl -2- hydroxyl-propyls thioether) ester construction unit number, be 8-41 integer.
The polymer of hydrophobic section R in above-mentioned copolymer, selection degradable poly esters hydrophobic polymer or vinyl monomer； It is preferred that caprolactone (CL), lactide (GA) lactide (LA), 4- (ethylene ketal)-caprolactone (BMCL), methacrylic acid -2- (3,5- dioxanes -4- (2,4,6- trimethoxy-benzene)) propyl ester (TTMA), methacrylic acid (diisopropylaminoethyl) ethyl ester (DPA) Homopolymer or copolymer.
Described PEG-R-PHDMA type block copolymers, the preferred TTMA of hydrophobic section R homopolymer (PTTMA), molecular weight is 2000-10000。
Preparation method using vinyl polymer as the triblock copolymer of hydrophobic section is：Polyethylene glycol is dissolved in dichloromethane Alkane, adds micromolecule chain transfer agent S-1- dodecyls-S '-(α, α "-dimethyl-α "-acetic acid) trithiocarbonate (CTAm), It is subsequently added after being reacted under dicyclohexylcarbodiimide and DMAP, room temperature condition and concentrates, is precipitated in ice ether, Suction filtration, vacuum drying, obtains macromole evocating agent PEG-CTAm；Macromole evocating agent is added in the reactor, it is sub- with dimethyl After sulfone dissolving, add after being reacted 24 hours at hydrophobic vinyl monomer, such as TTMA or DPA, 30-80 DEG C, add metering system Acid glycidyl ether, is reacted 24 hours at 30-80 DEG C, and concentration is precipitated, suction filtration in ice ether, vacuum drying, obtains containing epoxy The block copolymer of ethane side base；Gained copolymer is dissolved in tetrahydrofuran, N is added, N- dimethylamino ethyl mercaptans, room temperature is anti- It should stay overnight.Concentration, is precipitated, suction filtration in ice ether, and vacuum drying obtains the triblock copolymer using vinyl polymer as hydrophobic section Thing.
Preparation method using degradable polyester as the triblock copolymer of hydrophobic section is：PEG is added in reactor, then Esters monomer, such as CL or LA are added in the reactor, are added to react at stannous octoate, 100-130 DEG C and are stayed overnight, by product dichloro Methane dissolves, and is precipitated in ice ether, suction filtration, and vacuum drying obtains PEG-R polymer；PEG-R is dissolved in dichloromethane, plus Enter micromolecule chain transfer agent CTAm, be subsequently added under dicyclohexylcarbodiimide and DMAP, room temperature condition and react Overnight, concentrate, precipitated in ice ether, suction filtration, vacuum drying obtains macromole evocating agent PEG-R-CTAm；By PEG-R- CTAm and methyl propenoic acid glycidyl ether are added in reactor, are then added after being reacted 24 hours at DMSO, 30 DEG C and are concentrated, Precipitated in ice ether, suction filtration, vacuum drying obtains the block copolymer of the side base containing oxirane；Gained block copolymer is molten In tetrahydrofuran, concentrate, precipitated in ice ether, suction filtration, very after adding N, N- dimethylamino ethyl mercaptans, room temperature reaction overnight Sky is dried, and obtains the triblock copolymer using degradable polyester as hydrophobic section.
Heretofore described copolymer carries stronger positive charge, available for load DNA, siRNA or polypeptide, protide Bio-pharmaceutical, for medicine delivery.Wherein described amphipathic copolymer, it is small that it is self-assembly of average grain diameter in aqueous In 200nm polycation nano grain, the presence of surface hydroxyl can significantly improve the biocompatibility of the polycation, cell Compatibility is preferable and without haemolysis.Nanometer particle loading DNA, siRNA or polypeptide, the protide bio-pharmaceutical formed, it is interior Core can load hydrophobic chemicals, for medicine delivery.
Research shows that the amphipathic copolymer nano particle formed loads siRNA, subcutaneously noted in cellular level and mouse Higher gene silencing efficiency can be produced by penetrating, as siRNA carrier, with preferable application prospect.
Brief description of the drawings
The hydrogen nuclear magnetic resonance spectrogram of the block copolymer of the side base containing oxirane prepared by Fig. 1 embodiments 1, it was demonstrated that closed Into the precursor polymer structure containing pendant epoxy be consistent with designed.
The cationic amphiphilic block copolymer ETA-1 of hydroxyl modified prepared by Fig. 2 embodiments 1 proton nmr spectra Figure, it was demonstrated that each blocking group and molecular weight in its block structure, is consistent, specific data are shown in Table 1 with design structure.
Fig. 3 ETA-1 nanoparticles siRNA loads gel electrophoresis figure, it was demonstrated that nanoparticle can preferably load siRNA.
Fig. 4 ETA-1 nanoparticle electromicroscopic photographs.Prove that ETA-1 can be self-assembly of the nanometers of 100nm once in water Grain.
Fig. 5 loads siRNA ETA-1 nanoparticle electromicroscopic photographs.The ETA-1 nanoparticles loaded after siRNA are still spherical in shape, grain Footpath is declined slightly.
The cytotoxicity of Fig. 6 loads siRNA ETA-1 nanoparticles, it is seen then that cytoactive is all more than 80%, with very Good biocompatibility.
Fig. 7 ETA-1 hemolytic experiment results.As a result illustrate that the copolymer haemolysis amount is close with PBS, do not produce substantially Raw haemolysis.
The transfection efficiency figure of Fig. 8 loads siRNA ETA-1 nanoparticles, at N/P ratio more than 15, Gene silencing efficacy is equal Higher than commercially available transfection reagent Lipo2000, with good siRNA delivery functions.
The Gene silencing efficacy figure of Fig. 9 loads siRNA ETA-1 nanoparticles liver in vivo.As a result prove in 2mg/Kg Under dosage, the target gene that the carrier can substantially in silence liver cell shows preferable vivo gene delivery effect.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area Personnel further understand the present invention, but the invention is not limited in any way.It is pointed out that to the ordinary skill people in field For member, the various modifications and improvements can be made under the premise of present inventive concept is not departed from.These belong to the guarantor of the present invention Protect scope.Below only by taking the preparation of triblock copolymer as an example.
Embodiment 1：Amphipathic three block cation copolymer ETA-1 synthesis
By the poly glycol monomethyl ether (mPEG of 2g molecular weight 20002k) dichloromethane is dissolved in, add 0.5g small molecule chains Transfer agent CTAm (Macromolecules 2006,39,1724-1730), be subsequently added dicyclohexylcarbodiimide (DCC) and Concentrate, precipitated in ice ether, suction filtration after being reacted under DMAP (DMAP), room temperature condition, vacuum drying obtains big Molecule RAFT reagents PEG2k-CTAm；1g macromole evocating agents are added in the reactor, after being dissolved with dimethyl sulfoxide (DMSO) (DMSO), Add after being reacted 24 hours at 3.85g TTMA, 30-80 DEG C, add anti-at 2g methyl propenoic acid glycidyl ether, 30-80 DEG C Answer 24 hours, concentrate, precipitated in ice ether, suction filtration, vacuum drying obtains the block copolymer of the side base containing oxirane, its Nuclear magnetic spectrogram such as Fig. 1.Take 3g resulting polymers to be dissolved in tetrahydrofuran, add 0.8g N, N- dimethylamino ethyl mercaptans, room temperature is anti- It should stay overnight.Concentration, is precipitated, suction filtration in ice ether, and vacuum drying obtains PEG-R-PHDMA type block copolymer ETA-1, its Nuclear magnetic spectrogram such as Fig. 2.
Embodiment 2-6
Embodiment 2-6 synthetic method is identical with the device of embodiment 1 and reaction condition, only changes monomer kind on this basis The molecular weight of class, inventory and rate of charge, i.e. three sections of three block cation copolymer.The specific molecular weight information of resulting polymers Such as table 1.
The structural information of copolymer prepared by the embodiment 1-11 of table 1
Embodiment 7-8
By the method for embodiment 1, except that without TTMA monomers, using PEG2k- CTAm directly triggers methacrylic acid to contract Water glycerin ether polymerize, and PEG and polymethylacrylic acid glycidol ether block copolymer is obtained, then with-dimethylamino second sulphur Alcohol reacts, the bonded upper HDMA of side base.By changing mPEG molecular weight and methyl propenoic acid glycidyl ether monomer consumption, obtain not With block ratio and the PEG-PHDMA block copolymers of different molecular weight, EA-1 and EA-2 are shown in Table 1.
Embodiment 9
By the method for embodiment 1, replace mPEG with the methanol of identical molal quantity, prepare RAFT reagent Cs H3- CTAm, uses CH3- CTAm triggers TTMA polymerizations, triggers methyl propenoic acid glycidyl ether to polymerize again, obtains diblock copolymer, then with N, N- diformazans Epoxy ring-opening addition reaction on aminoothyl mercaptan and gained random copolymer, you can obtain TTMA and methacrylic acid (N, N, N- dimethyl ethyl -2- hydroxyl-propyls thioether) ester amphipathic nature block polymer PT-PA.
Embodiment 10
By the method for embodiment 9, CH is used3- CTAm triggers TTMA and methyl propenoic acid glycidyl ether polymerization simultaneously, obtains nothing Copolymer is advised, then with N, N- dimethylaminos ethyl mercaptan and the epoxy ring-opening addition reaction on gained random copolymer, you can To TTMA and methacrylic acid (N, N, N- dimethyl ethyl -2- hydroxyl-propyls thioether) amphipathic random copolymer P (T/A) of ester - 1, the relative molecular mass that wherein content of TTMA construction units is 10%, P (T/A) -1 is 35000.
The consumption of methyl propenoic acid glycidyl ether monomer is adjusted, or uses different comonomers, you can difference is obtained The amphipathic random copolymer of component.
Embodiment 11
By the method for embodiment 10, other monomers are added without, CH is used3- CTAm directly triggers methyl propenoic acid glycidyl ether Polymerization, obtains the homopolymer PMHDMA-25 of methacrylic acid (N, N, N- dimethyl ethyl -2- hydroxyl-propyls thioether) ester, molecule Amount 25000；Regulate and control the ratio of monomer and initiator, can obtain different molecular weight PMHDMA.
The amphipathic three block copolymer ECA-1 for the polycation that embodiment 12 is modified containing polyhydroxy synthesis
By 1g mPEG2kAdd in reactor, 2g CL monomers are then added in the reactor, be eventually adding two drops octanoic acid sub- Tin, is reacted at 100-130 DEG C and stays overnight, product dichloromethane is dissolved, and is precipitated in ice ether, and suction filtration, vacuum drying is obtained PEG-PCL polymer.By 2g PEG2k- PCL is dissolved in dichloromethane, adds 0.5g micromolecule chain transfer agent CTAm, is subsequently added React and stay overnight under dicyclohexylcarbodiimide (DCC) and DMAP (DMAP), room temperature condition, concentrate, in ice ether Middle precipitation, suction filtration, vacuum drying obtains Macro RAFT agent PEG2k-PCL-CTAm.Take 1g PEG2k- PCL-CTAm and 2g Methyl propenoic acid glycidyl ether add in reactor, then add dense after reaction a period of time at 10ml DMSO, 30 DEG C Contracting, is precipitated, suction filtration in ice ether, and vacuum drying obtains the block copolymer of the side base containing oxirane.Take 3g resulting polymers It is dissolved in tetrahydrofuran, concentrates, precipitated in ice ether after adding 0.8g N, N- dimethylamino ethyl mercaptans, room temperature reaction overnight, Suction filtration, vacuum drying, obtains the three block cation copolymer ECA-1 of hydroxyl modified.
Embodiment 13-18
Synthetic method is identical with the device of embodiment 7 and reaction condition, on this basis only change monomeric species, inventory with And the molecular weight of rate of charge, i.e. three sections of three block cation copolymer.Resulting polymers concrete outcome information such as table 2.
The structural information of copolymer prepared by the embodiment 12-18 of table 2
Embodiment 19：The preparation of blank nanoparticle
The accurate block copolymer ETA-1 for weighing 10mg, is dissolved in 1mL tetrahydrofuran, and block copolymer solution is slow In the pure water (can also be PBS or physiological saline) for being added drop-wise to 8mL stirrings, it is stirred at room temperature after 3h, centrifuges, clear liquid is certainly Assemble nanometer grain dispersion liquid, particle diameter and the distribution of different time points nanoparticle solution are tested using DLS.With in nanoparticle dispersion liquid Nitrogen content be unit, nanoparticle dispersion liquid is diluted and obtains the nanoparticle dispersion liquids of 1mM nitrogen concentrations, it is standby.
Embodiment 20：SiRNA load
A 1.5ml EP pipe is taken, the 6ul 269ng/ul siRNA aqueous solution is added into pipe, 38.4ul 1mM nitrogen is taken The ETA-1 nanoparticle dispersion liquids of concentration, then add 355.60 aqueous solution of the μ l containing DMEM, and 20ml is stood after mixing and is loaded SiRNA nanoparticle dispersion liquid.
Embodiment 21：Transfection experiment
HepG-2 cells are inoculated with 24 orifice plates, cell density is 1 × 104Cells/well, cell is stable expression fluorescein The HepG-2 cells of zymoprotein (luciferase), culture volume is 0.5mL.Cultivate after 18h, cell confluency reaches 50%.Before transfection, culture medium is changed into DMEM culture mediums.ETA-1 containing 1.0 μ g siRNA prepared by 100 μ L embodiments 14 Nanoparticle solution is added into 24 orifice plates, and each sample sets 3 parallel holes.37 DEG C and 5%CO2Under conditions of, cell is trained 4h is supported, liquid in hole is then taken out, is changed to the DMEM low sugar culture mediums containing 2%FBS, 0.5mL/ holes.Cultivate after 48h, discard hole In liquid, with PBS 2 times, then add 200 μ L gene lysis buffers to every hole, freeze thawing 1 time has cracked cell Entirely.After piping and druming completely, suction out cell pyrolysis liquid and managed to new EP, centrifuge (12000rpm, 30s), supernatant is suctioned out.To 10 μ L Added in supernatant after 50 μ L substrates, relative light units are determined using fluorescence photometer (Synergy HT, BioTek, USA).With The concentration of BCA (Pierce, USA) kit measurement total protein.The luciferase expressed by the cell of any reagent will not be added Luciferase protein amount divided by do not add the luciferase that the cell of any reagent is expressed that albumen is expressed as benchmark, vehicle group Protein content is the relative suppression efficiency that can obtain protein expression.
Embodiment 22- embodiments 33
The different copolymer of selection, nanoparticle is prepared according to the method for embodiment 19, is loaded according to the method for embodiment 20 SiRNA, then transfected by the operating method of embodiment 21 in different cell line, as a result such as table 3：
Gene load, cell compatibility and the gene silencing efficiency of cationic polymer prepared by table 3
In gene silencing efficiency experiment in table, siRNA dosage is 50nM.
Embodiment 30：Toxicity test
1 × 10 is inoculated with 96 orifice plates4Individual HepG-2 cells/wells, are 100 μ L, 5%CO2,37 DEG C per hole culture volume Under the conditions of cultivate 24h.The load nanoparticle solution that 50 μ L contain 0.2 μ g siRNA is added, is added respectively while setting in zeroing hole, hole Enter the complex solution that same volume is separately added into DMEM culture mediums, MTT and dimethyl sulfoxide (DMSO), control wells, DMEM cultures Base, MTT and dimethyl sulfoxide (DMSO), 5%CO2, 48h is cultivated under the conditions of 37 DEG C.20 μ L MTT solution (5mg/mL) are added per hole, Continue 5%CO2, 37 DEG C of culture 4h.Supernatant in sample well is discarded, 150 μ L DMSO are added into every hole, low speed concussion is placed in 10min is vibrated on instrument, crystal is fully dissolved.Each hole light absorption value is measured at enzyme-linked immunosorbent assay instrument 490nm.Calculate each Sample well cell relative viability, gained cell viability is more than 80%.Example is listed in such as table 3.
Embodiment 35：Hemolytic experiment
Prepare 2% red blood cell, from rabbit hearts extract blood 5ml, add 0.2ml anti-coagulants EDTA, washed with PBS, from The heart, removes the leucocyte on surface, does not show red to supernatant, abandons after supernatant the PBS for taking 1ml to add 50ml and produce.By material Material PBS solution adjustment concentration is 50,30,20,15,10 and 5.Every time using before red blood cell, washed with PBS, until in supernatant Without substantially red (slightly yellow), the μ l of substrate 200 are drawn, 10ml is diluted to and uses.By the 2% of 0.5ml material and 0.5ml it is red thin Cell lysis liquid is mixed, static 3h under room temperature condition, centrifuges 3min with 10050r/min afterwards.100 μ l are taken out on 96 orifice plates, are used 570nm wavelength detecting.Positive control and negative control are used as using water and PBS respectively.Each 2, sample is parallel.
Hemolysis rate (%)=(sample absorption-negative control absorbs)/(positive control absorption-negative control absorbs) × 100%.Hemolysis rate is considered as haemolysis more than 5%.
Embodiment 36：Experiment in vivo
Male C57BL/6 mouse (18-20g) are taken, the siRNA containing anti-ApoB prepared ETA-1 nanoparticles are disperseed Liquid is injected into Mice Body by way of intravenous injection.Dosage is 1mg/Kg.Mouse is put to death after 48h, liver is taken out, The ApoB of mouse content is detected by RT-PCR.As a result accompanying drawing 9 is seen.

Claims (10)

1. a kind of N of hydroxyl modification, the cationic polymer of N- dimethylaminos, it is characterized in that the N of hydroxyl modified, N- diformazan ammonia Base is N, N, and N- dimethyl ethyl -2- hydroxyl-propyls thioethers (hereinafter referred to as HDMA), cationic polymer is homopolymer or copolymerization Thing；The relative molecular mass of described cationic polymer is 5000-40000.
2. cationic polymer as claimed in claim 1, it is characterized in that cationic polymer is homopolymer, i.e., it is wherein bonded The gross mass of HDMA construction unit accounts for the 100% of cationic polymerization amount of substance.
3. cationic polymer as claimed in claim 1, it is characterized in that cationic polymer is copolymer, wherein bonded HDMA The gross mass of construction unit account for the 10%~90% of cationic polymerization amount of substance.
4. cationic polymer as claimed in claim 3, it is characterized in that described copolymer, to contain hydrophobic structure unit Amphipathic copolymer, the gross mass of hydrophobic structure unit accounts for the 10%~60% of cationic polymerization amount of substance.
5. cationic polymer as claimed in claim 4, it is characterized in that described copolymer, for the construction unit with HDMA Homopolymer (PHDMA) and nonionic, hydrophilic polymers (B) and hydrophobic section (R) composition amphipathic three block copolymer；Its In, the relative molecular mass of PHDMA blocks is 2000~10000, and nonionic, hydrophilic polymers B relative molecular mass is 2000-10000, hydrophobic section R relative molecular mass are 2000~20000；Quality of the PHDMA blocks in triblock copolymer Percentage composition is 10%~70%.
6. cationic polymer as claimed in claim 5, it is characterized in that the nonionic of described amphipathic three block copolymer B sections of hydrophilic polymer is selected from polyethylene glycol (PEG), poly- carboxybetaine methacrylate or phosphocholine.
7. cationic polymer as claimed in claim 6, is total to it is characterized in that described copolymer is PEG-R-PHDMA types block Polymers, structural formula is as follows：
Wherein, PHDMA is polymethylacrylic acid (N, N, N- dimethyl ethyl -2- hydroxyl-propyls thioether) ester, naIt is its structure list First number, is 8-41 integer；N is the construction unit number of polyethylene glycol, is 45-227 integer；Hydrophobic section R be esters monomer or The homopolymer or copolymer of vinyl monomer, esters monomer be selected from caprolactone, lactide, lactide, 4- (ethylene ketal)-oneself in Ester, vinyl monomer is selected from methacrylic acid -2- (3,5- dioxanes -4- (2,4,6- trimethoxy-benzene)) propyl ester, methacrylic acid (diisopropylaminoethyl) ethyl ester.
8. the N of the hydroxyl modification of claim 7, the preparation method of the cationic polymer of N- dimethylaminos, it is characterized in that, with Vinyl polymer is hydrophobic section：Monohydroxy PEG is dissolved in dichloromethane, add micromolecule chain transfer agent S-1- dodecyls- S '-(α, α "-dimethyl-α "-acetic acid) trithiocarbonate (CTAm), is subsequently added dicyclohexylcarbodiimide and 4- diformazan ammonia Concentrate, precipitated in ice ether, suction filtration after being reacted under yl pyridines, room temperature condition, vacuum drying obtains macromole evocating agent PEG- CTAm；Macromole evocating agent is added in the reactor, after dmso solution, adds hydrophobic vinyl monomer, 30-80 After being reacted 24 hours at DEG C, add at methyl propenoic acid glycidyl ether, 30-80 DEG C and react 24 hours, concentration, in ice ether Precipitation, suction filtration, vacuum drying obtains the block copolymer of the side base containing oxirane；Gained copolymer is dissolved in tetrahydrofuran, N is added, N- dimethylamino ethyl mercaptans, room temperature reaction is stayed overnight；Concentration, in ice ether precipitate, suction filtration, vacuum drying, obtain with Vinyl polymer is the triblock copolymer of hydrophobic section.
9. the N of the hydroxyl modification of claim 7, the preparation method of the cationic polymer of N- dimethylaminos, it is characterized in that can Degraded polyester is hydrophobic section：Monohydroxy PEG is added in reactor, esters monomer is then added in the reactor, catalysis is added Agent stannous octoate, reacts at 100-130 DEG C and stays overnight, product dichloromethane is dissolved, and is precipitated in ice ether, suction filtration, vacuum It is dried to obtain PEG-R polymer；PEG-R is dissolved in dichloromethane, micromolecule chain transfer agent CTAm is added, is subsequently added two rings Reaction is stayed overnight under hexyl carbodiimide and DMAP, room temperature condition, is concentrated, and is precipitated in ice ether, suction filtration, vacuum Dry, obtain macromole evocating agent PEG-R-CTAm；PEG-R-CTAm and methyl propenoic acid glycidyl ether are added into reactor In, then add after being reacted 24 hours at DMSO, 30 DEG C and concentrate, precipitated in ice ether, suction filtration, vacuum drying obtains containing ring The block copolymer of oxidative ethane side base；Gained block copolymer is dissolved in tetrahydrofuran, addition N, N- dimethylamino ethyl mercaptans, Concentrate, precipitated in ice ether, suction filtration after room temperature reaction overnight, vacuum drying obtains three using degradable polyester as hydrophobic section Block copolymer.
10. the N of claim 1 hydroxyl modification, the cationic polymer of N- dimethylaminos is applied to load DNA, siRNA or many Peptide, protide bio-pharmaceutical, for medicine delivery.
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