CN102949729A - 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 PDFInfo
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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
Technical field
The present invention relates to a kind of small interference ribonucleic acid delivery vehicles, be specifically related to a kind of active 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] that is 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, induce to generate the 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 adjusting destination protein function, in the gene therapy of a lot of diseases, huge application potential is arranged, such 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 the siRNA medicament research and development at present, but also there is not in the world at present a routine siRNA medicine successfully to go on the market, this mainly is because the plasma half-life of siRNA is short, easily be degraded under the physiological condition, be difficult to be delivered to Cytoplasm, and lack the targeting ability, therefore, it is the critical bottleneck of its success listing that the targeting of siRNA transports, design and synthetic safely and effectively siRNA transport carrier has become the important directions of present 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 by target cell picked-up and have vivo gene reticent effect [Nature2009,457,426-433; J Control Release2010,144,251-258].Wherein, the nanoparticle that is 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 the 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 nanotechnology research institute tight teach problem group of Yang Yi have been reported a kind of cation nanometer particle and PEGization derivant thereof, the 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 the siRNA delivery vehicles can targeting the acid ceramidase gene with the 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 the state university in 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, and are still good not to the targeting of target site.
Connect target molecule at the siRNA delivery vehicles, can improve the siRNA delivery vehicles to the targeting of target site.The Luo Ying of Peking University teach problem group has been reported a kind of crosslinked dendroid siRNA delivery system of neutrality; this dendroid siRNA delivery system main body is polyamide dendroid polymer; hydrazides and acetylgalactosamine have been modified on its terminal amino group; dendritic after the modification can form complex by ionic interaction with siRNA; carry out crosslinked with glutaraldehyde to complex at last; siRNA is not degraded with protection; galactosamine can play receptor-mediated active targeting in the system; 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, needs at last to use glutaraldehyde that complex is carried out crosslinked, and siRNA is not degraded with protection.
According to the domestic and international present Research analysis of above-mentioned siRNA delivery vehicles, design and synthetic safely and effectively siRNA transport carrier, raising siRNA is the major trend of research to the targeting of tumor tissues.
Summary of the invention
The invention provides a kind of active 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, with siRNA high specific targeted to tumor locus.
A kind of active targeting type small interference ribonucleic acid delivery vehicles is by obtaining as the biodegradable macromolecular compound of carrier framework and target molecule and amino-compound covalent coupling as the 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.
Among the present invention; described amino-compound is secondary amine, tertiary amine or quarternary ammonium salt compound molecule; this quasi-molecule is positively charged under the pH neutrallty condition; with siRNA strong binding ability is arranged; thereby make the siRNA delivery vehicles wrap up formation carrier-siRNA complex nanometer granule in conjunction with siRNA and with it, protection siRNA is not degraded in the course of conveying in vivo.Described target molecule can interact with antigen or the receptor generation specificity of tumor cell surface, realization with 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, make the downward modulation of destination protein expression, thereby reach the purpose of kill cancer 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 are sodium alginate.
Well known in the artly can both be applied to the present invention with antigen or the interactional target molecule of receptor generation specificity of tumor cell surface, for described target molecule can successfully be connected on the described carrier framework, target molecule is preferably monoclonal antibody, folic acid part, galactosamine part or RGD small peptide, and described RGD small peptide is for containing the small peptide of arginine, glycine and aspartic acid (Arg-Gly-Asp) sequence.
For so that described amino-compound can be connected on the described carrier framework, described amino-compound contains-NH
2, the reactive functionality such as aldehyde radical or carboxyl.In addition, described amino-compound can also play the effect of proton sponge, after described carrier-siRNA complex nanometer granule enters cancerous cell by endocytosis, the proton pump on endosome surface can pump into the proton in the Cytoplasm in the endosome, because the proton sponge effect of complex nanometer granule, the proton that pumps into can absorb by suppressed by vector-siRNA complex nanometer granule, because the raising of osmotic pressure, water in the Cytoplasm can continue to enter endosome, thereby endosome is burst and discharge complex nanometer granule, flee from lysosomal pathway.Described amino-compound is preferably at least a in histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide, 2-(glyoxal ethyline base) ethamine, 4-imidazole formaldehyde, histidine and the imidazole lactic acid.Take the 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.
Among the present invention, the amino-compound that connects on the described carrier framework is 0.05-5.0 with the number of target molecule ratio: 1, be preferably 0.1-1.0: 1.At this moment, 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 with the selectively targeted tumor cell of higher efficient, improve the targeted effect of siRNA.
The present invention also provides the preparation method of described active targeting type small interference ribonucleic acid delivery vehicles, comprising:
(1) makes in described macromolecular compound and described amino-compound and the described target molecule any one that covalent coupling reaction occurs and obtain intermediate;
(2) make that covalent coupling reaction occurs for another in intermediate that step (1) obtains and described amino-compound and the described target molecule, after reacting completely, purifiedly obtain described active targeting type small interference ribonucleic acid delivery vehicles.
When the reactive functionality of described macromolecular compound is carboxyl (such as sodium alginate), the preparation method of described active targeting type siRNA delivery vehicles may further comprise the steps:
(1) with the carboxyl in the described macromolecular compound of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) activation, by a kind of target molecule with amino of covalent coupling reaction covalent coupling on macromolecular skeleton amino and carboxyl, such as parts such as monoclonal antibody or galactosamines, obtain intermediate;
(2) one or more amino-compounds of covalent coupling on the intermediate that step (1) obtains, such 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.Take the skeleton macromole as sodium alginate, amino-compound is example as histamine, target molecule as galactosamine, the synthetic reaction schematic diagram is as shown in Figure 2.
Covalent coupling reaction described in the step (1) forms the reaction of covalent chemical bond for functional group's carboxyl of the functional groups amino in the target molecule and macromolecular skeleton, namely under the catalysis of EDC, carboxyl and amino form the chemical reaction of amide group.The consumption of employed EDC is excessive, is used for the carboxyl of activation macromolecular skeleton.
As preferably, described macromolecular compound is sodium alginate;
Described amino-compound is at least a in histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide and 2-(glyoxal ethyline base) ethamine;
Covalent coupling reaction described in the 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, and wherein, the number of contained alduronic acid construction unit is 100～1200 in the single sodium alginate molecule.At this moment, when adding EDC, can also add NHS (N-hydroxy-succinamide), make the intermediate reaction behind itself and the EDC activated carboxyl form a kind of metastable NHS Acibenzolar, the consumption of described NHS and the consumption of EDC get final product about equally.
When the macromolecular reactive functionality of siRNA delivery vehicles skeleton is amino (such as chitosan, polylysine), the preparation method of described active targeting type siRNA delivery vehicles mainly may further comprise the steps:
(1) by aldehyde radical reaction one or more amino-compound of coupling on macromole with amino, such as the 4-imidazole formaldehyde, perhaps with the carboxyl in EDC and the NHS activating ammonia based compound, by a kind of amino-compound of reaction coupling on macromole amino and carboxyl, such as histidine or imidazole lactic acid;
(2) with the carboxyl in EDC and the NHS activation target molecule, by a kind of target molecule of reaction coupling on macromole amino and carboxyl, such as parts such as monoclonal antibody or folic acid.
Covalent coupling reaction described in the step (1) forms the reaction of covalent chemical bond for the functional groups amino of the functional group's aldehyde radical in the amino-compound or carboxyl and macromolecular skeleton.The reaction of aldehyde radical and amino need not catalysis and can form at normal temperatures schiff bases; Carboxyl and the amino chemical reaction that forms amide group need the catalysis of EDC and NHS.When amino-compound contained aldehyde radical, the coupling amount of amino-compound can be controlled by the addition of amino-compound in the macromolecular skeleton; When amino-compound contained carboxyl, with the carboxyl in EDC and the NHS activating ammonia based compound, the addition of the amino-compound that the coupling amount of amino-compound can be activated by carboxyl in the macromolecular skeleton was controlled.With the carboxyl in EDC and the 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 the macromolecular skeleton in the step (2).Therefore, on the macromolecular skeleton the number ratio of the amino-compound of modifying and target molecule be to control by the coupling amount of amino-compound in the step (1).
As preferably, the macromolecular compound described in the step (1) is chitosan or polylysine;
Described amino-compound is at least a in histidine and the imidazole lactic acid;
The consumption mol ratio of described macromolecular compound and amino-compound is 1: 100～1200.
Compare with prior art, 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 with siRNA the effect of the amino-compound of strong binding ability being arranged, improved the charging ratio of carrier to siRNA, and the effect that has improved the proton sponge that carrier and the formed complex nanometer granule of siRNA play in endosome, to flee from lysosome.
Description of 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 of making of embodiment 1-histamine;
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 the carrier mol ratio of phosphine in amino and the siRNA phosphoric acid skeleton), wherein, GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate of making of embodiment 1-histamine, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan of making of embodiment 9-histidine;
Fig. 5 be different N/P than the time carrier/hTERT-siRNA complex nanometer granule the Zeta potential comparison diagram, wherein, GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate of making of embodiment 1-histamine, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan of making of embodiment 9-histidine;
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 of making of embodiment 1-histamine, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan of making of embodiment 9-histidine;
Fig. 7 is the survival rate of HepG2 or HeLa cell behind transfection GAL-ALG-HA/hTERT-siRNA or the FA-C S-HIS/hTERT-siRNA, the blank group is the cell (getting its cell survival rate is 100%) that does not have transfection siRNA, GAL-ALG-HA is siRNA delivery vehicles galactosamine-sodium alginate of making of embodiment 1-histamine, and FA-CS-HIS is siRNA delivery vehicles folic acid-chitosan of making of embodiment 9-histidine.*P＜0.001，**P＜0.001。
The specific embodiment
Get 80mL4.95mg/mL sodium alginate (be total to 2.0mmol alduronic acid construction unit, the 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; The bag filter of holding back with 3500 molecular weight carries out purification, gets 1mL and dry the concentration of calculating gained macromole solution galactosamine-sodium alginate of being synthesized-histamine macromole.With the concentrated solution that makes 10mg/mL of the galactosamine-sodium alginate behind the purification-histamine macromole solution, thereby make a kind of initiatively siRNA delivery vehicles galactosamine-sodium alginate of liver cancer targeting-histamine.
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 GAL-ALG-HA solution that the above-mentioned siRNA solution of 50 μ L is added respectively the above-mentioned variable concentrations of 100 μ L, room temperature is cultivated 30min behind the mixing, can obtain the 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: with 7mL cell (5.0 * 10
5Cells/mL) be seeded in the culture dish of Φ 90mm * 20mm, after the incubated overnight, culture medium replaced with 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, with PBS cell is washed twice, use trypsin treatment 3min, centrifugal 5.0min (removing complex nanometer granule) under 2000 * g in the DMSO of 1.0mL, uses the fluorescence intensity of fluorescence spectrophotometer measurement gained sample with the gained cytolysis.
The analytical method of the survival rate of HepG2 cell is as follows behind the transfection GAL-ALG-HA/hTERT-siRNA: 5000 cells are inoculated in every hole in 96 orifice plates, after the incubated overnight, culture medium is removed, use the 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, adding complete medium cultivated 47 hours, every hole adds the MTT solution (5mg/mL) of 20 μ L again, cultivated 4 hours, and at last culture medium was removed, add the DMSO of 150 μ L.Measure the light absorption value of every hole solution under 570nm with microplate reader, the cell survival rate of blank group is defined as 100%.
Synthetic gained galactosamine-sodium alginate-macromolecular infrared spectrogram of histamine as shown in Figure 3, wave number is 1260 and 1700cm
-1The absworption peak at place is respectively in the amide-C-N-and-the infrared signature peak of C=O, carboxyl and amino of this explanation EDC activation successfully react the generation amide.Wave number is 1470 and 1660cm
-1The absworption peak at place is respectively in the histamine-CH
2With the infrared signature peak of-C=C-, this explanation histamine successfully is coupled in the sodium alginate skeleton.Wave number is 2850cm
-1The absworption peak at place is the infrared signature peak of C-H adjacent with amino in the galactosamine, and this explanation galactosamine successfully is coupled in the sodium alginate skeleton.
Different N/P than the time GAL-ALG-HA/hTERT-siRNA complex nanometer granule the 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 when the N/P ratio reached 4, the size of complex nanometer granule was down to minimum, was about 130nm.
Different N/P than the time GAL-ALG-HA/hTERT-siRNA complex nanometer granule the 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 when the N/P ratio reached 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, increase along with complex nanometer granule and HepG2 culture time, the amount of cellular uptake nanoparticle increases, when the cultivation time reached 4 hours, the amount of cellular uptake nanoparticle reached 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, the survival rate of HepG2 cell is 41 ± 6.3% of blank behind the transfection GAL-ALG-HA/hTERT-siRNA.This result shows that carrier galactosamine-sodium alginate-histamine GAL-ALG-HA can effectively be delivered to siRNA in the cell and suppresses the expression of mRNA, thereby suppresses the growth of HepG2 cell.
Change 0.6mmol/mL histamine solution among the embodiment 1 into 0.6mmol/mL histamine alcohol dihydrochloride solution, other preparation conditions are constant, can make the another kind of initiatively siRNA delivery vehicles galactosamine-sodium alginate of liver cancer targeting-histamine alcohol.
Embodiment 3
Change 0.6mmol/mL histamine solution among the 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 of liver cancer targeting-aminoimidazole carboxyamine.
Change 0.6mmol/mL histamine solution among the embodiment 1 into 0.6mmol/mL2-(glyoxal ethyline base) ethamine dihydrochloride solution, other preparation conditions are constant, can make the another kind of initiatively siRNA delivery vehicles galactosamine-sodium alginate of liver cancer targeting-methylimidazolyl ethamine.
Embodiment 5
0.08mmol/mL galactosamine solution among the embodiment 1 is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-histamine.
0.08mmol/mL galactosamine solution among the embodiment 2 is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-histamine alcohol.
Embodiment 7
0.08mmol/mL galactosamine solution among the embodiment 3 is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-aminoimidazole carboxyamine.
0.08mmol/mL galactosamine solution among the embodiment 4 is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-methylimidazolyl ethamine.
Embodiment 9
(1) coupling amino-compound histidine on the chitosan skeleton
Weighing 0.645g (4mmol glucosamine construction unit) chitosan (molecular weight ranges is 11-15 ten thousand) makes it to be scattered in the 30mL ultra-pure water, and adjust pH makes it whole dissolvings.Ice bath after the dissolving, then add 0.383g (2mmol) EDC hydrochlorate and 0.230g (2mmol) NHS, continue magnetic agitation, dissolve rear adding 10mL31mg/mL (2mmol) histidine solution fully until EDC hydrochlorate and NHS, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to the chitosan that synthesized-histidine macromole, with the concentrated solution that makes 30mg/mL of the chitosan behind the purification-histidine macromole solution.
(2) coupling target molecule folic acid on the chitosan skeleton
Get the above-mentioned chitosan of 20mL-histidine macromole solution (30mg/mL), add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS behind the ice bath, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve fully, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to folic acid-chitosan of being synthesized-histidine macromole, with the concentrated solution that makes 30mg/mL of the folic acid-chitosan behind the purification-histidine macromole solution, thereby make a kind of initiatively siRNA delivery vehicles folic acid-chitosan of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.-histidine.
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 FA-CS-HIS solution that the above-mentioned siRNA solution of 50 μ L is added respectively the above-mentioned variable concentrations of 100 μ L, room temperature is cultivated 30min behind the mixing, can obtain the 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: with 7mL cell (5.0 * 10
5Cells/mL) be seeded in the culture dish of Φ 90mm * 20mm, after the incubated overnight, culture medium replaced with 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, with PBS cell is washed twice, use trypsin treatment 3min, centrifugal 5.0min (removing complex nanometer granule) under 2000 * g in the DMSO of 1.0mL, uses the fluorescence intensity of fluorescence spectrophotometer measurement gained sample with the gained cytolysis.
The analytical method of the survival rate of HepG2 cell is as follows behind the transfection GAL-ALG-HA/hTERT-siRNA: 5000 cells are inoculated in every hole in 96 orifice plates, after the incubated overnight, culture medium is removed, use the 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, adding complete medium cultivated 47 hours, every hole adds the MTT solution (5mg/mL) of 20 μ L again, cultivated 4 hours, and at last culture medium was removed, add the DMSO of 150 μ L.Measure the light absorption value of every hole solution under 570nm with microplate reader, the cell survival rate of blank group is defined as 100%.
Different N/P than the time FA-CS-HIS/hTERT-siRNA complex nanometer granule the 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 when the N/P ratio reached 4, the size of complex nanometer granule was down to minimum, was about 210nm.
Different N/P than the time FA-CS-HIS/hTERT-siRNA complex nanometer granule the 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 when the N/P ratio reached 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, increase along with complex nanometer granule and HeLa culture time, the amount of cellular uptake nanoparticle increases, when the cultivation time reached 4 hours, the amount of cellular uptake nanoparticle reached 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, the survival rate of HeLa cell is 36 ± 4.1% of blank behind the transfection FA-CS-HIS/hTERT-siRNA.This result shows that carrier folic acid-chitosan-histidine FA-CS-HIS can effectively be delivered to siRNA in the cell and suppresses the expression of mRNA, thereby suppresses the growth of HeLa cell.
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 of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.-imidazole lactic acid.
Embodiment 11
(1) coupling amino-compound 4-imidazole formaldehyde on the chitosan skeleton
Weighing 0.645g (4mmol glucosamine construction unit) chitosan (molecular weight ranges is 11-15 ten thousand) makes it to be scattered in the 30mL ultra-pure water, and 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 the chitosan that synthesized-imidazole formaldehyde macromole, with the concentrated solution that makes 30mg/mL of the chitosan behind the purification-imidazole formaldehyde macromole solution.
(2) coupling target molecule folic acid on the chitosan skeleton
Get the above-mentioned chitosan of 20mL-imidazole formaldehyde macromole solution (30mg/mL), add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS behind the ice bath, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve fully, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to folic acid-chitosan of being synthesized-imidazole formaldehyde macromole, with the concentrated solution that makes 30mg/mL of the folic acid-chitosan behind the purification-imidazole formaldehyde macromole solution, thereby make a kind of initiatively siRNA delivery vehicles folic acid-chitosan of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.-imidazole formaldehyde.
Embodiment 12
20mL66mg/mL (3mmol) folic acid solution in embodiment 9 steps (2) is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-histidine.
Embodiment 13
20mL66mg/mL (3mmol) folic acid solution among the embodiment 10 is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-imidazole lactic acid.
Embodiment 14
20mL66mg/mL (3mmol) folic acid solution in embodiment 11 steps (2) is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-imidazole formaldehyde.
Embodiment 15
(1) coupling amino-compound histidine on the polylysine skeleton
Weighing 0.731g (4mmol lysine construction unit) polylysine (molecular weight ranges is 1.5-3 ten thousand), make it to be dissolved in the 30mL ultra-pure water, ice bath adds 0.383g (2mmol) EDC hydrochlorate and 0.230g (2mmol) NHS, continue magnetic agitation, dissolve rear adding 10mL31mg/mL (2mmol) histidine solution fully until EDC hydrochlorate and NHS, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to the polylysine that synthesized-histidine macromole, with the concentrated solution that makes 30mg/mL of the polylysine behind the purification-histidine macromole solution.
(2) coupling target molecule folic acid on the polylysine skeleton
Get the above-mentioned polylysine of 20mL-histidine macromole solution (30mg/mL), add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS behind the ice bath, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve fully, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to folic acid-polylysine of being synthesized-histidine macromole, with the concentrated solution that makes 30mg/mL of the folic acid-polylysine behind the purification-histidine macromole solution, thereby make a kind of initiatively siRNA delivery vehicles folic acid-polylysine of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.-histidine.
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 of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.-imidazole lactic acid.
Embodiment 17
(1) coupling amino-compound 4-imidazole formaldehyde on the polylysine skeleton
Weighing 0.731g (4mmol lysine construction unit) polylysine (molecular weight ranges is 1.5-3 ten thousand) makes it to be dissolved in the 30mL ultra-pure water, then adds 10mL19mg/mL (2mmol) 4-imidazole formaldehyde solution, continues reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to the polylysine that synthesized-imidazole formaldehyde macromole, with the concentrated solution that makes 30mg/mL of the polylysine behind the purification-imidazole formaldehyde macromole solution.
(2) coupling target molecule folic acid on the polylysine skeleton
Get the above-mentioned polylysine of 20mL-imidazole formaldehyde macromole solution (30mg/mL), add 0.575g (3mmol) EDC hydrochlorate and 0.345g (3mmol) NHS behind the ice bath, continue magnetic agitation, after EDC hydrochlorate and NHS dissolve fully, add again 20mL66mg/mL (3mmol) folic acid solution, remove ice bath reaction 12h.The bag filter of holding back with 3000 molecular weight carries out purification to folic acid-polylysine of being synthesized-imidazole formaldehyde macromole, with the concentrated solution that makes 30mg/mL of the folic acid-polylysine behind the purification-imidazole formaldehyde macromole solution, thereby make a kind of initiatively siRNA delivery vehicles folic acid-polylysine of the targeting brain cancer, renal carcinoma, breast carcinoma, pulmonary carcinoma, ovarian cancer, uterus carcinoma, nasopharyngeal carcinoma etc.-imidazole formaldehyde.
Embodiment 18
20mL66mg/mL (3mmol) folic acid solution in embodiment 15 steps (2) is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-histidine.
Embodiment 19
20mL66mg/mL (3mmol) folic acid solution among the embodiment 16 is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-imidazole lactic acid.
20mL66mg/mL (3mmol) folic acid solution in embodiment 17 steps (2) is changed into anticol matter original fiber acid 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 of the gliomatous siRNA delivery vehicles of targeting-imidazole formaldehyde.
Claims (9)
1. a targeting type small interference ribonucleic acid delivery vehicles initiatively is characterized in that, by obtaining as the biodegradable macromolecular compound of carrier framework and target molecule and amino-compound covalent coupling as the 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.
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, it is characterized in that, described amino-compound is selected from least a in histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide, 2-(glyoxal ethyline base) ethamine, 4-imidazole formaldehyde, histidine and the imidazole lactic acid.
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 ratio of target molecule.
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 ratio of target molecule.
7. the preparation method such as each described active targeting type small interference ribonucleic acid delivery vehicles of claim 1～6 is characterized in that, comprising:
(1) any one generation covalent coupling reaction in described macromolecular compound and described amino-compound and the described target molecule is obtained intermediate;
(2) make that covalent coupling reaction occurs for another in intermediate that step (1) obtains and described amino-compound and the described target molecule, after reacting completely, purifiedly obtain described active targeting type small interference ribonucleic acid delivery vehicles.
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 at least a in histamine, histamine alcohol, 5-aminoimidazole-4-carbozamide and 2-(glyoxal ethyline base) ethamine;
Covalent coupling reaction described in the 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 the step (1) is chitosan or polylysine;
Described amino-compound is at least a in histidine, imidazole lactic acid and the 4-imidazole formaldehyde;
The consumption mol ratio of described macromolecular compound and amino-compound is 1: 100～1200.
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| CN108850779A (en) * | 2018-07-06 | 2018-11-23 | 浙江工商大学 | PEG-ACS/M-siRNA nano-complex and its application and the method for reducing histamine content in fish meal storage |
| CN114480377A (en) * | 2022-01-27 | 2022-05-13 | 浙江浥眸生物科技有限公司 | Brush-shaped nucleic acid assembly, composite nanoparticles and application of brush-shaped nucleic acid assembly and composite nanoparticles |
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| 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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| CN108850779A (en) * | 2018-07-06 | 2018-11-23 | 浙江工商大学 | PEG-ACS/M-siRNA nano-complex and its application and the method for reducing histamine content in fish meal storage |
| CN108850779B (en) * | 2018-07-06 | 2022-02-08 | 浙江工商大学 | PEG-ACS/M-siRNA nano-composite and application thereof, and method for reducing histamine content in fish meal storage |
| CN114480377A (en) * | 2022-01-27 | 2022-05-13 | 浙江浥眸生物科技有限公司 | Brush-shaped nucleic acid assembly, composite nanoparticles and application of brush-shaped nucleic acid assembly and composite nanoparticles |
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