CN102949729B - Active targeting type small interfering ribonucleic acid (siRNA) delivery carrier and preparation method thereof - Google Patents

Active targeting type small interfering ribonucleic acid (siRNA) delivery carrier and preparation method thereof Download PDF

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CN102949729B
CN102949729B CN201210475752.4A CN201210475752A CN102949729B CN 102949729 B CN102949729 B CN 102949729B CN 201210475752 A CN201210475752 A CN 201210475752A CN 102949729 B CN102949729 B CN 102949729B
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sirna
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沈折玉
吴爱国
任文智
马雪华
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Ningbo Institute of Material Technology and Engineering of CAS
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Abstract

The invention discloses an active targeting type small interfering ribonucleic acid (siRNA) delivery carrier, which is obtained by respectively covalently coupling a biodegradable high-molecular compound used as a carrier skeleton with a target molecule and an amino compound which are used as carrier side chains. The carrier can be self-assembled with siRNA to form a compound nanoparticle, and the siRNA is tightly wrapped to prevent nuclease from degrading the siRNA. In addition, the target molecule in the carrier can be interacted in specificity with an antigen or acceptor on the surface of a tumour cell, so that the carrier-siRNA compound nanoparticle can be precisely conveyed to a tumour part in a targeting mode, and enters a cancer cell through the antigen or receptor mediated endocytosis, the generation of a gene silencing compound is induced in the cell through the siRNA, mRNA is degraded through sequence specificity, and the expression quantity of interest proteins is reduced, so that cancer cells are killed and the purpose of cancer tumour treatment is realized. The invention also provides a preparation method of the active targeting type small interfering ribonucleic acid delivery carrier.

Description

Initiatively targeting type small interference ribonucleic acid delivery vehicles and preparation method thereof
Technical field
The present invention relates to a kind of small interference ribonucleic acid delivery vehicles, be specifically related to a kind of initiatively targeting type small interference ribonucleic acid delivery vehicles and preparation method thereof.
Background technology
It is the sequence-specific PTGS mechanism [Nature1998,391,806-811] being caused by double-stranded RNA that RNA disturbs.The small interference ribonucleic acid (siRNA) that length is about 20 base pairs is the effector molecule that RNA disturbs, in born of the same parents, induction generates gene silencing complex, sequence-specific degraded mRNA, thereby activity [the Nat Rev Genet2002 of blocking-up target gene, 3,737-747].Utilize siRNA to make gene silence and regulate destination protein function, in the gene therapy of a lot of diseases, having huge application potential, as neurodegenerative diseases, cancer, infectious disease etc.SiRNA has demonstrated huge application prospect as a kind of novel drugs, some biopharmaceutical companys have been devoted to siRNA medicament research and development at present, but also do not have in the world at present a routine siRNA medicine successfully to go on the market, this is mainly because the plasma half-life of siRNA is short, under physiological condition, be easily degraded, be difficult to be delivered to Cytoplasm, and lack targeting ability, therefore, it is the critical bottleneck of its success listing that the targeting of siRNA transports, design and synthetic siRNA safely and effectively transport carrier has become the important directions of current siRNA medicament research and development [Mol Pharmaceut2009, 6, 686-695, Nat Med2007,13,372-377].
In order to overcome an above-mentioned difficult problem, the siRNA delivery vehicles of having reported at present mainly contains following four classes: 1) cationic-liposome class; 2) cationic polymer class; 3) polypeptide class conjugate; 4) antibody fusion protein system.These delivery systems can protect siRNA not by nuclease degradation, be conducive to siRNA and absorbed by target cell and there is vivo gene reticent effect [Nature2009,457,426-433; J Control Release2010,144,251-258].Wherein, the nanoparticle being formed by amphipathic cationic polymer self assembly has been proved to be able to delivery of plasmid DNA, antisense oligonucleotide and siRNA, is a most promising class in nucleic acid delivery vehicles.Such carrier can load nucleic acid by ionic interaction, protect it not by nuclease degradation, improve cellular uptake [Nat Mater2006,5,79179-6 simultaneously; Biomacromolecules2007,8,1028-1037; Biomaterials2007,28,5358-5368].
Singapore's biological engineering and the tight teach problem group of the Yang Yi of nanotechnology research institute have been reported a kind of cation nanometer particle and PEGization derivant thereof, energy delivery of plasmid DNA, in a series of cancerous cell, obtain high gene expression rate [Biomaterials2007,28,5358-5368].The Wang Jun of China Science & Technology University teach problem group has been reported a kind of biodegradable three amphipathic block cation copolymers, this copolymer as siRNA delivery vehicles energy targeting acid ceramidase gene with treatment cancer, this triblock copolymer is methyl PEG-PCL-poly--2-amine ethyl phosphonic acid ethylene glycol [Biomaterials2011,32,3124-3133].The Tamara Minko of state university of New Jersey teach problem group has been reported a kind of multifunctional novel triblock copolymer nano-carrier, can be used for effective siRNA carries and gene silencing, this copolymer is polyamide-polyethylene glycol-lysine [ACS Nano2011,5,1877-1887].But these siRNA delivery vehicles are under physiological condition, still good not to the targeting of target site.
On siRNA delivery vehicles, connect target molecule, can improve the targeting of siRNA delivery vehicles to target site.The Luo Ying of Peking University teach problem group reported being cross-linked of a kind of neutrality dendroid siRNA delivery system, this dendroid siRNA delivery system main body is polyamide dendroid polymer, hydrazides and acetylgalactosamine on its terminal amino group, are modified, dendritic after modification can form complex by ionic interaction with siRNA, finally with glutaraldehyde, complex is cross-linked, with protection, siRNA is not degraded, in system, galactosamine can play receptor-mediated active targeting, improve targeted and Gene silencing efficacy [the Bioconjugate Chem2012 of siRNA, 23, 174-183].But the binding ability of this carrier and siRNA is strong not, finally need to use glutaraldehyde to be cross-linked complex, to protect siRNA not to be degraded.
According to the domestic and international present Research analysis of above-mentioned siRNA delivery vehicles, design and synthetic siRNA safely and effectively transport carrier, improve siRNA is the major trend of research to the targeting of tumor tissues.
Summary of the invention
The invention provides a kind of initiatively targeting type small interference ribonucleic acid delivery vehicles and preparation method thereof, this active targeting type small interference ribonucleic acid delivery vehicles can effectively avoid siRNA by nuclease degradation, by siRNA high specific targeted to tumor locus.
A kind of initiatively targeting type small interference ribonucleic acid delivery vehicles, by the biodegradable macromolecular compound as carrier framework with obtain as target molecule and the amino-compound covalent coupling of carrier side chain;
Described macromolecular compound, target molecule and amino-compound contain the reactive functionality that covalent coupling can occur;
Described macromolecular compound be have carboxyl or amino reactive functionality, biodegradable, the natural macromolecular compound of no cytotoxicity or the macromolecular compound of synthetic;
Described target molecule is for to have the interactional molecule of specificity with tumor cell;
Described amino-compound is secondary amine, tertiary amine or quarternary ammonium salt compound.
In the present invention; described amino-compound is secondary amine, tertiary amine or quarternary ammonium salt compound molecule; this quasi-molecule is positively charged under pH neutrallty condition; there is strong binding ability with siRNA; thereby make siRNA delivery vehicles and be wrapped up formation carrier-siRNA complex nanometer granule in conjunction with siRNA, protection siRNA is not degraded in course of conveying in vivo.Described target molecule can interact with the antigen of tumor cell surface or receptor generation specificity, realize by carrier-siRNA complex nanometer granule accurately targeted to tumor locus, enter cancerous cell by antigen or receptor-mediated endocytosis, in born of the same parents, pass through the generation of the reticent complex of siRNA induced gene, sequence-specific degraded mRNA, destination protein expression is lowered, thereby reached the object of killing cancerous cell treatment tumor.
Described macromolecular compound be have carboxyl or the reactive functionality such as amino, biodegradable, the natural macromolecular of no cytotoxicity or the macromolecule of synthetic.Described macromolecular compound contains the reactive functionality such as carboxyl or amino, can further modify with target molecule and amino-compound; The biodegradable of carrier framework and no cytotoxicity are the essential conditions as drug conveying carrier.Described macromolecular compound is preferably sodium alginate, chitosan or polylysine, and more preferably natural polymer sodium alginate or chitosan, most preferably be sodium alginate.
Well known in the artly can be applied to the present invention with the antigen of tumor cell surface or the interactional target molecule of receptor generation specificity, for described target molecule can successfully be connected on described carrier framework, target molecule is preferably monoclonal antibody, folic acid part, galactosamine part or RGD small peptide, the small peptide of described RGD small peptide for containing arginine, glycine and aspartic acid (Arg-Gly-Asp) sequence.
For described amino-compound can be connected on described carrier framework, contain-NH of described amino-compound 2, the reactive functionality such as aldehyde radical or carboxyl.In addition, described amino-compound can also play the effect of proton sponge, enter after cancerous cell by endocytosis at described carrier-siRNA complex nanometer granule, the proton pump on endosome surface can pump into the proton in Cytoplasm in endosome, due to the proton sponge effect of complex nanometer granule, the proton pumping into can be absorbed by carrier-siRNA complex nanometer granule, due to the raising of osmotic pressure, water in Cytoplasm can continue to enter endosome, thereby endosome burst and discharge complex nanometer granule, fleeing from lysosomal pathway.Described amino-compound is preferably at least one in histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide, 2-(glyoxal ethyline base) ethamine, 4-imidazole formaldehyde, histidine and imidazole lactic acid.Taking skeleton macromole as sodium alginate, amino-compound is example as histamine, target molecule as galactosamine, galactosamine-sodium alginate-histamine initiatively targeted siRNA mechanism as shown in Figure 1.
In the present invention, the amino-compound connecting on described carrier framework is 0.05-5.0 with the number of target molecule ratio: 1, be preferably 0.1-1.0: 1.Now, described amino-compound can be efficiently by ionic interaction in conjunction with siRNA, form carrier-siRNA complex nanometer granule, and described target molecule also can be with higher efficiency selectively targeted tumor cell, improve the targeted effect of siRNA.
The preparation method of the active targeting type small interference ribonucleic acid delivery vehicles described in the present invention also provides, comprising:
(1) make in described macromolecular compound and described amino-compound and described target molecule any one generation covalent coupling react and obtain intermediate;
(2) make in intermediate that step (1) obtains and described amino-compound and described target molecule another that covalent coupling occurs and react, after reacting completely, the active targeting type small interference ribonucleic acid delivery vehicles described in purified obtaining.
In the time that the reactive functionality of described macromolecular compound is carboxyl (as sodium alginate), the preparation method of described active targeting type siRNA delivery vehicles comprises the following steps:
(1) activate the carboxyl in described macromolecular compound with EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide), react with the covalent coupling of carboxyl by amino that covalent coupling is a kind of has an amino target molecule on macromolecular skeleton, as the part such as monoclonal antibody or galactosamine, obtain intermediate;
(2) one or more amino-compounds of covalent coupling on the intermediate obtaining in step (1), as histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide or 2-(glyoxal ethyline base) ethamine, obtain described active targeting type small interference ribonucleic acid delivery vehicles.Taking skeleton macromole as sodium alginate, amino-compound is example as histamine, target molecule as galactosamine, synthetic reaction schematic diagram is as shown in Figure 2.
Covalent coupling reaction described in step (1) is that the functional groups amino in target molecule forms reacting of covalent chemical bond with functional group's carboxyl of macromolecular skeleton, and, under the catalysis of EDC, carboxyl and amino form the chemical reaction of amide group.The consumption of the EDC using is excessive, for activating the carboxyl of macromolecular skeleton.
As preferably, described macromolecular compound is sodium alginate;
Described amino-compound is at least one in histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide and 2-(glyoxal ethyline base) ethamine;
Covalent coupling reaction described in step (1) is carried out under the catalysis of EDC, the consumption mol ratio of described sodium alginate and EDC is 1: 100～1200, wherein, in single sodium alginate molecule, the number of contained alduronic acid construction unit is 100～1200.Now, in adding EDC, can also add NHS (N-hydroxy-succinamide), make the intermediate reaction after itself and EDC activated carboxyl form a kind of metastable NHS Acibenzolar, the consumption of described NHS and the consumption of EDC are roughly equal.
In the time that the macromolecular reactive functionality of siRNA delivery vehicles skeleton is amino (as chitosan, polylysine), the preparation method of described active targeting type siRNA delivery vehicles mainly comprises the following steps:
(1) by aldehyde radical and amino one or more amino-compounds of coupling on macromole that react, as 4-imidazole formaldehyde, or with the carboxyl in EDC and NHS activating ammonia based compound, by a kind of amino-compound of coupling on macromole that reacts amino and carboxyl, as histidine or imidazole lactic acid;
(2) with the carboxyl in EDC and NHS activation target molecule, by a kind of target molecule of coupling on macromole that reacts amino and carboxyl, as the part such as monoclonal antibody or folic acid.
Covalent coupling reaction described in step (1) is that functional group's aldehyde radical or the carboxyl in amino-compound forms reacting of covalent chemical bond with the functional groups amino of macromolecular skeleton.Aldehyde radical can form schiff bases at normal temperatures with amino reacting without catalysis; Carboxyl and the amino chemical reaction that forms amide group need the catalysis of EDC and NHS.In the time that amino-compound contains aldehyde radical, in macromolecular skeleton, the coupling amount of amino-compound can be controlled by the addition of amino-compound; In the time that amino-compound contains carboxyl, with the carboxyl in EDC and NHS activating ammonia based compound, the addition of the amino-compound that in macromolecular skeleton, the coupling amount of amino-compound can be activated by carboxyl is controlled.With the carboxyl in EDC and NHS activation target molecule, in the coupling reaction of macromolecular skeleton, the addition of the target molecule that carboxyl has been activated is excessive, makes the amino all coupling target molecules of residue in macromolecular skeleton in step (2).Therefore, on macromolecular skeleton the amino-compound of modifying and the number ratio of target molecule be to control by the coupling amount of amino-compound in step (1).
As preferably, the macromolecular compound described in step (1) is chitosan or polylysine;
Described amino-compound is at least one in histidine and imidazole lactic acid;
The consumption mol ratio of described macromolecular compound and amino-compound is 1: 100～1200.
Compared with the existing technology, beneficial effect of the present invention is embodied in:
(1), by the effect of target molecule, improved the target function of siRNA delivery vehicles to tumor cell;
(2) by having the effect of the amino-compound of strong binding ability with siRNA, improve the charging ratio of carrier to siRNA, and the effect that has improved the proton sponge that complex nanometer granule that carrier and siRNA form plays in endosome, to flee from lysosome.
Brief description of the drawings
Fig. 1 is the initiatively mechanism figure of targeted siRNA of galactosamine-sodium alginate-histamine;
Fig. 2 is galactosamine-sodium alginate-histamine synthetic reaction schematic diagram;
Fig. 3 is the infrared spectrogram of galactosamine-sodium alginate-histamine of making of embodiment 1;
The size comparison diagram of carrier/hTERT-siRNA complex nanometer granule when Fig. 4 is different N/P than (N/P is than representing in carrier the mol ratio of phosphine in amino and siRNA phosphoric acid skeleton), wherein, GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate-histamine that embodiment 1 makes, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan-histidine that embodiment 9 makes;
Fig. 5 be different N/P than time carrier/hTERT-siRNA complex nanometer granule Zeta potential comparison diagram, wherein, GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate-histamine that embodiment 1 makes, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan-histidine that embodiment 9 makes;
Fig. 6 is that carrier/FAM-siRNA complex nanometer granule (N/P=4) is by the quantitative analysis figure of HepG2 or HeLa cellular uptake, GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate-histamine that embodiment 1 makes, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan-histidine that embodiment 9 makes;
Fig. 7 is the survival rate of HepG2 or HeLa cell after transfection GAL-ALG-HA/hTERT-siRNA or FA-C S-HIS/hTERT-siRNA, blank group is the cell (getting its cell survival rate is 100%) that there is no transfection siRNA, GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate-histamine that embodiment 1 makes, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan-histidine that embodiment 9 makes.*P＜0.001，**P＜0.001。
Detailed description of the invention
Embodiment 1
Get 80mL4.95mg/mL sodium alginate (2.0mmol alduronic acid construction unit altogether, construction unit molecular weight is 198.11) aqueous solution, ice bath; Add 0.383g (2.0mmol) EDC hydrochlorate and 2.5mL0.08mmol/mL galactosamine solution (0.2mmol), room temperature reaction 4h; Then, add 2.5mL0.6mmol/mL histamine solution (1.5mmol), continue reaction 12h; Galactosamine-sodium alginate-histamine the macromole of the bag filter of holding back with 3500 molecular weight to synthesized carries out purification, gets 1mL and dries the concentration of calculating gained macromole solution.By concentrated galactosamine-the sodium alginate after the purification-histamine macromole solution solution that makes 10mg/mL, thereby make a kind of initiatively siRNA delivery vehicles galactosamine-sodium alginate-histamine of liver cancer targeting.
The preparation method of the GAL-ALG-HA/hTERT-siRNA complex nanometer granule of different N/P ratio is as follows: with HEPES buffer (20mM, pH7.4) the GAL-ALG-HA solution of preparation variable concentrations, prepare the hTERT-siRNA solution of 20 μ g/mL with DEPC water, the above-mentioned siRNA solution of 50 μ L is added respectively to the GAL-ALG-HA solution of the above-mentioned variable concentrations of 100 μ L, mix rear room temperature and cultivate 30min, can obtain GAL-ALG-HA/hTERT-siRNA complex nanometer granule.The size of complex nanometer granule and Zeta potential all use Malvern ParticleSizer (Nano ZS) to detect.
The quantitative analysis method of HepG2 cellular uptake GAL-ALG-HA/FAM-siRNA complex nanometer granule is as follows: by 7mL cell (5.0 × 10 5cells/mL) be seeded in the culture dish of Φ 90mm × 20mm, after incubated overnight, culture medium replaced with to the culture medium of the GAL-ALG-HA/FAM-siRNA complex nanometer granule that contains 0.1mg/mL, continue to cultivate 1-6 hour.Then, cell is washed to twice with PBS, use trypsin treatment 3min, centrifugal 5.0min (removing complex nanometer granule) under 2000 × g, gained cytolysis, in the DMSO of 1.0mL, is used to the fluorescence intensity of fluorescence spectrophotometer measurement gained sample.
After transfection GAL-ALG-HA/hTERT-siRNA, the analytical method of the survival rate of HepG2 cell is as follows: 5000 cells are inoculated in every hole in 96 orifice plates, after incubated overnight, culture medium is removed, use PBS washed twice, every hole adds the GAL-ALG-HA/hTERT-siRNA complex nanometer granule (0.1mg/mL) of 0.1mL, continue to cultivate 1 hour, then wash once with PBS, add complete medium to cultivate 47 hours, every hole adds the MTT solution (5mg/mL) of 20 μ L again, cultivate 4 hours, finally culture medium is removed, add the DMSO of 150 μ L.Measure the light absorption value of every hole solution under 570nm by microplate reader, the cell survival rate of blank group is defined as 100%.
As shown in Figure 3, wave number is 1260 and 1700cm to the synthetic macromolecular infrared spectrogram of gained galactosamine-sodium alginate-histamine -1the absworption peak at place is respectively in amide-C-N-and-the infrared signature peak of C=O, carboxyl of this explanation EDC activation successfully reacts generation amide with amino.Wave number is 1470 and 1660cm -1the absworption peak at place is respectively in histamine-CH 2with the infrared signature peak of-C=C-, this explanation histamine is successfully coupled in sodium alginate skeleton.Wave number is 2850cm -1the absworption peak at place is the infrared signature peak of C-H adjacent with amino in galactosamine, and this explanation galactosamine is successfully coupled in sodium alginate skeleton.
Different N/P than time GAL-ALG-HA/hTERT-siRNA complex nanometer granule size contrast as shown in Figure 4, wherein GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate-histamine.The size of complex nanometer granule reduces along with the increase of N/P ratio, and in the time that N/P ratio reaches 4, the size of complex nanometer granule is down to minimum, is about 130nm.
Different N/P than time GAL-ALG-HA/hTERT-siRNA complex nanometer granule Zeta potential contrast as shown in Figure 5, wherein GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate-histamine.The Zeta potential of complex nanometer granule raises along with the increase of N/P ratio, and in the time that N/P ratio reaches 8, it is the highest that the Zeta potential of complex nanometer granule rises to, and is about 6.5mV.
GAL-ALG-HA/FAM-siRNA complex nanometer granule (N/P=4) by the quantitative analysis of HepG2 cellular uptake as shown in Figure 6, along with the increase of complex nanometer granule and HepG2 culture time, the amount of cellular uptake nanoparticle increases, in the time that the cultivation time reaches 4 hours, the amount of cellular uptake nanoparticle reaches maximum substantially.This result proves that carrier galactosamine-sodium alginate-histamine GAL-ALG-HA can be used as effective delivery vehicles of siRNA.
As shown in Figure 7, after transfection GAL-ALG-HA/hTERT-siRNA, the survival rate of HepG2 cell is blank 41 ± 6.3%.This result shows that carrier galactosamine-sodium alginate-histamine GAL-ALG-HA can effectively be delivered in cell by siRNA and suppress the expression of mRNA, thereby suppresses the growth of HepG2 cell.
Embodiment 2
Change 0.6mmol/mL histamine solution in embodiment 1 into 0.6mmol/mL histamine alcohol dihydrochloride solution, other preparation conditions are constant, can make another kind of initiatively siRNA delivery vehicles galactosamine-sodium alginate-histamine alcohol of liver cancer targeting.
Embodiment 3
Change 0.6mmol/mL histamine solution in embodiment 1 into 0.6mmol/mL5-aminooimidazole-4-carboxamide hydrochloride solution, other preparation conditions are constant, can make the another kind of initiatively siRNA delivery vehicles galactosamine-sodium alginate-aminoimidazole carboxyamine of liver cancer targeting.
Embodiment 4
Change 0.6mmol/mL histamine solution in embodiment 1 into 0.6mmol/mL2-(glyoxal ethyline base) ethamine dihydrochloride solution, other preparation conditions are constant, can make another kind of initiatively siRNA delivery vehicles galactosamine-sodium alginate-methylimidazolyl ethamine of liver cancer targeting.
Embodiment 5
0.08mmol/mL galactosamine solution in embodiment 1 is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 0.08mmol/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-sodium alginate-histamine of the gliomatous siRNA delivery vehicles of targeting.
Embodiment 6
0.08mmol/mL galactosamine solution in embodiment 2 is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 0.08mmol/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-sodium alginate-histamine of the gliomatous siRNA delivery vehicles of targeting alcohol.
Embodiment 7
0.08mmol/mL galactosamine solution in embodiment 3 is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 0.08mmol/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-sodium alginate-aminoimidazole carboxyamine of the gliomatous siRNA delivery vehicles of targeting.
Embodiment 8
0.08mmol/mL galactosamine solution in embodiment 4 is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 0.08mmol/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-sodium alginate-methylimidazolyl of the gliomatous siRNA delivery vehicles of targeting ethamine.
Embodiment 9
(1) coupling amino-compound histidine on chitosan skeleton
Weigh 0.645g (4mmol glucosamine construction unit) chitosan (molecular weight ranges is 11-15 ten thousand), make it to be scattered in 30mL ultra-pure water, adjust pH makes it whole dissolvings.Ice bath after dissolving, then add 0.383g (2mmol) EDC hydrochlorate and 0.230g (2mmol) NHS, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve completely, add 10mL31mg/mL (2mmol) histidine solution, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to chitosan-histidine macromole of synthesized, by concentrated chitosan-histidine macromole the solution after the purification solution that makes 30mg/mL.
(2) coupling target molecule folic acid on chitosan skeleton
Get the above-mentioned chitosan-histidine of 20mL macromole solution (30mg/mL), after ice bath, add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve completely, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.Folic acid-chitosan-histidine the macromole of the bag filter of holding back with 3000 molecular weight to synthesized carries out purification, by concentrated folic acid-the chitosan after the purification-histidine macromole solution solution that makes 30mg/mL, thereby make a kind of initiatively siRNA delivery vehicles folic acid-chitosan-histidine of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.
The preparation method of the FA-CS-HIS/hTERT-siRNA complex nanometer granule of different N/P ratio is as follows: with HEPES buffer (20mM, pH7.4) the FA-CS-HIS solution of preparation variable concentrations, prepare the hTERT-siRNA solution of 20 μ g/mL with DEPC water, the above-mentioned siRNA solution of 50 μ L is added respectively to the FA-CS-HIS solution of the above-mentioned variable concentrations of 100 μ L, mix rear room temperature and cultivate 30min, can obtain FA-CS-HIS/hTERT-siRNA complex nanometer granule.The size of complex nanometer granule and Zeta potential all use Malvern ParticleSizer (Nano ZS) to detect.
The quantitative analysis method of HeLa cellular uptake FA-CS-HIS/FAM-siRNA complex nanometer granule is as follows: by 7mL cell (5.0 × 10 5cells/mL) be seeded in the culture dish of Φ 90mm × 20mm, after incubated overnight, culture medium replaced with to the culture medium of the FA-CS-HIS/FAM-siRNA complex nanometer granule that contains 0.1mg/mL, continue to cultivate 1-6 hour.Then, cell is washed to twice with PBS, use trypsin treatment 3min, centrifugal 5.0min (removing complex nanometer granule) under 2000 × g, gained cytolysis, in the DMSO of 1.0mL, is used to the fluorescence intensity of fluorescence spectrophotometer measurement gained sample.
After transfection GAL-ALG-HA/hTERT-siRNA, the analytical method of the survival rate of HepG2 cell is as follows: 5000 cells are inoculated in every hole in 96 orifice plates, after incubated overnight, culture medium is removed, use PBS washed twice, every hole adds the GAL-ALG-HA/hTERT-siRNA complex nanometer granule (0.1mg/mL) of 0.1mL, continue to cultivate 1 hour, then wash once with PBS, add complete medium to cultivate 47 hours, every hole adds the MTT solution (5mg/mL) of 20 μ L again, cultivate 4 hours, finally culture medium is removed, add the DMSO of 150 μ L.Measure the light absorption value of every hole solution under 570nm by microplate reader, the cell survival rate of blank group is defined as 100%.
Different N/P than time FA-CS-HIS/hTERT-siRNA complex nanometer granule size contrast as shown in Figure 4, wherein FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan-histidine.The size of complex nanometer granule reduces along with the increase of N/P ratio, and in the time that N/P ratio reaches 4, the size of complex nanometer granule is down to minimum, is about 210nm.
Different N/P than time FA-CS-HIS/hTERT-siRNA complex nanometer granule Zeta potential contrast as shown in Figure 5, wherein FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan-histidine.The Zeta potential of complex nanometer granule raises along with the increase of N/P ratio, and in the time that N/P ratio reaches 8, it is the highest that the Zeta potential of complex nanometer granule rises to, and is about 4.6mV.
FA-CS-HIS/FAM-siRNA complex nanometer granule (N/P=4) by the quantitative analysis of HeLa cellular uptake as shown in Figure 6, along with the increase of complex nanometer granule and HeLa culture time, the amount of cellular uptake nanoparticle increases, in the time that the cultivation time reaches 4 hours, the amount of cellular uptake nanoparticle reaches maximum substantially.This result proves that carrier folic acid-chitosan-histidine FA-CS-HIS can be used as effective delivery vehicles of siRNA.
As shown in Figure 7, after transfection FA-CS-HIS/hTERT-siRNA, the survival rate of HeLa cell is blank 36 ± 4.1%.This result shows that carrier folic acid-chitosan-histidine FA-CS-HIS can effectively be delivered in cell by siRNA and suppress the expression of mRNA, thereby suppresses the growth of HeLa cell.
Embodiment 10
Change 31mg/mL histidine solution in embodiment 9 steps (1) into 31.2mg/mLL-β-imidazole lactic acid solution, other preparation conditions are constant, can make the another kind of initiatively siRNA delivery vehicles folic acid-chitosan-imidazole lactic acid of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.
Embodiment 11
(1) coupling amino-compound 4-imidazole formaldehyde on chitosan skeleton
Weigh 0.645g (4mmol glucosamine construction unit) chitosan (molecular weight ranges is 11-15 ten thousand), make it to be scattered in 30mL ultra-pure water, adjust pH makes it whole dissolvings.Then add 10mL19mg/mL (2mmol) 4-imidazole formaldehyde solution, continue reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to chitosan-imidazole formaldehyde macromole of synthesized, by concentrated chitosan-imidazole formaldehyde macromole the solution after the purification solution that makes 30mg/mL.
(2) coupling target molecule folic acid on chitosan skeleton
Get the above-mentioned chitosan-imidazole formaldehyde of 20mL macromole solution (30mg/mL), after ice bath, add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve completely, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.Folic acid-chitosan-imidazole formaldehyde the macromole of the bag filter of holding back with 3000 molecular weight to synthesized carries out purification, by concentrated folic acid-the chitosan after the purification-imidazole formaldehyde macromole solution solution that makes 30mg/mL, thereby make a kind of initiatively siRNA delivery vehicles folic acid-chitosan-imidazole formaldehyde of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.
Embodiment 12
20mL66mg/mL (3mmol) folic acid solution in embodiment 9 steps (2) is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 2.0mL5.0mg/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-chitosan-histidine of the gliomatous siRNA delivery vehicles of targeting.
Embodiment 13
20mL66mg/mL (3mmol) folic acid solution in embodiment 10 is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 2.0mL5.0mg/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-chitosan-imidazole lactic acid of the gliomatous siRNA delivery vehicles of targeting.
Embodiment 14
20mL66mg/mL (3mmol) folic acid solution in embodiment 11 steps (2) is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 2.0mL5.0mg/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-chitosan-imidazole formaldehyde of the gliomatous siRNA delivery vehicles of targeting.
Embodiment 15
(1) coupling amino-compound histidine on polylysine skeleton
Weigh 0.731g (4mmol lysine construction unit) polylysine (molecular weight ranges is 1.5-3 ten thousand), make it to be dissolved in 30mL ultra-pure water, ice bath adds 0.383g (2mmol) EDC hydrochlorate and 0.230g (2mmol) NHS, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve completely, add 10mL31mg/mL (2mmol) histidine solution, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to polylysine-histidine macromole of synthesized, by concentrated polylysine-histidine macromole the solution after the purification solution that makes 30mg/mL.
(2) coupling target molecule folic acid on polylysine skeleton
Get the above-mentioned polylysine-histidine of 20mL macromole solution (30mg/mL), after ice bath, add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve completely, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.Folic acid-polylysine-histidine the macromole of the bag filter of holding back with 3000 molecular weight to synthesized carries out purification, by concentrated folic acid-the polylysine after the purification-histidine macromole solution solution that makes 30mg/mL, thereby make a kind of initiatively siRNA delivery vehicles folic acid-polylysine-histidine of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.
Embodiment 16
Change 31mg/mL histidine solution in embodiment 15 steps (1) into 31.2mg/mLL-β-imidazole lactic acid solution, other preparation conditions are constant, can make the another kind of initiatively siRNA delivery vehicles folic acid-polylysine-imidazole lactic acid of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.
Embodiment 17
(1) coupling amino-compound 4-imidazole formaldehyde on polylysine skeleton
Weigh 0.731g (4mmol lysine construction unit) polylysine (molecular weight ranges is 1.5-3 ten thousand), make it to be dissolved in 30mL ultra-pure water, then add 10mL19mg/mL (2mmol) 4-imidazole formaldehyde solution, continue reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to polylysine-imidazole formaldehyde macromole of synthesized, by concentrated polylysine-imidazole formaldehyde macromole the solution after the purification solution that makes 30mg/mL.
(2) coupling target molecule folic acid on polylysine skeleton
Get the above-mentioned polylysine-imidazole formaldehyde of 20mL macromole solution (30mg/mL), after ice bath, add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve completely, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.Folic acid-polylysine-imidazole formaldehyde the macromole of the bag filter of holding back with 3000 molecular weight to synthesized carries out purification, by concentrated folic acid-the polylysine after the purification-imidazole formaldehyde macromole solution solution that makes 30mg/mL, thereby make a kind of initiatively siRNA delivery vehicles folic acid-polylysine-imidazole formaldehyde of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.
Embodiment 18
20mL66mg/mL (3mmol) folic acid solution in embodiment 15 steps (2) is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 2.0mL5.0mg/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-polylysine-histidine of the gliomatous siRNA delivery vehicles of targeting.
Embodiment 19
20mL66mg/mL (3mmol) folic acid solution in embodiment 16 is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 2.0mL5.0mg/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-polylysine-imidazole lactic acid of the gliomatous siRNA delivery vehicles of targeting.
Embodiment 20
20mL66mg/mL (3mmol) folic acid solution in embodiment 17 steps (2) is changed into anti-glial fibrillary acidic protein (GFAP) antibody-solutions of 2.0mL5.0mg/mL, other preparation conditions are constant, can make the another kind of initiatively anti-GFAP antibody-polylysine-imidazole formaldehyde of the gliomatous siRNA delivery vehicles of targeting.

Claims (9)

1. a targeting type small interference ribonucleic acid delivery vehicles initiatively, is characterized in that, by the biodegradable macromolecular compound as carrier framework with obtain as target molecule and the amino-compound covalent coupling of carrier side chain;
Described macromolecular compound, target molecule and amino-compound contain the reactive functionality that covalent coupling can occur;
Described macromolecular compound be have carboxyl or amino reactive functionality, biodegradable, the natural macromolecular compound of no cytotoxicity or the macromolecular compound of synthetic;
Described target molecule is for to have the interactional molecule of specificity with tumor cell;
Described amino-compound is selected from histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide, 2-(2-methylimidazolyl) at least one in ethamine, 4-imidazole formaldehyde, histidine and imidazole lactic acid.
2. active targeting type small interference ribonucleic acid delivery vehicles according to claim 1, is characterized in that, described macromolecular compound is selected from sodium alginate, chitosan or polylysine.
3. active targeting type small interference ribonucleic acid delivery vehicles according to claim 1, is characterized in that, described target molecule is selected from monoclonal antibody, folic acid part, galactosamine part or RGD small peptide.
4. active targeting type small interference ribonucleic acid delivery vehicles according to claim 1, is characterized in that, the consumption mol ratio of described macromolecular compound and amino-compound is 1:100～1200.
5. active targeting type small interference ribonucleic acid delivery vehicles according to claim 1, is characterized in that, described amino-compound is 0.05～5.0:1 with the number of target molecule ratio.
6. active targeting type small interference ribonucleic acid delivery vehicles according to claim 5, is characterized in that, described amino-compound is 0.1～1.0:1 with the number of target molecule ratio.
7. a preparation method for the active targeting type small interference ribonucleic acid delivery vehicles as described in claim 1～6 any one, is characterized in that, comprising:
(1) by described macromolecular compound and described amino-compound and described target molecule, any one covalent coupling occurs reacts and obtain intermediate;
(2) make in intermediate that step (1) obtains and described amino-compound and described target molecule another that covalent coupling occurs and react, after reacting completely, the active targeting type small interference ribonucleic acid delivery vehicles described in purified obtaining.
8. the preparation method of active targeting type small interference ribonucleic acid delivery vehicles according to claim 7, is characterized in that, described macromolecular compound is sodium alginate;
Described amino-compound is histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide and 2-(2-methylimidazolyl) at least one in ethamine;
Covalent coupling reaction described in step (1) is carried out under the catalysis of EDC, and the consumption mol ratio of described sodium alginate and EDC is 1:100～1200.
9. the preparation method of active targeting type small interference ribonucleic acid delivery vehicles according to claim 7, is characterized in that, the macromolecular compound described in step (1) is chitosan or polylysine;
Described amino-compound is at least one in histidine, imidazole lactic acid and 4-imidazole formaldehyde;
Described macromolecular compound and the amount ratio of amino-compound are 1:100～1200.
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CN102316858A (en) * 2008-02-26 2012-01-11 阿帕玛生物科技公司 Engineered tunable nanoparticles for delivery of therapeutics, diagnostics, and experimental compounds and related compositions for therapeutic use
CN102665758A (en) * 2009-12-09 2012-09-12 Ibc药品公司 Dock-and-lock (DNL) Complexes For Delivery of Interference RNA

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CN101020725B (en) * 2007-03-23 2010-09-15 天津大学 Amphiphilic chitosan quatermary ammonium salt with long alkane radical and its preparation
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102316858A (en) * 2008-02-26 2012-01-11 阿帕玛生物科技公司 Engineered tunable nanoparticles for delivery of therapeutics, diagnostics, and experimental compounds and related compositions for therapeutic use
CN102665758A (en) * 2009-12-09 2012-09-12 Ibc药品公司 Dock-and-lock (DNL) Complexes For Delivery of Interference RNA

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