CN104940952B - The composition and method of making the same that comprises macrocyclic ester compound and siRNA - Google Patents
The composition and method of making the same that comprises macrocyclic ester compound and siRNA Download PDFInfo
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- CN104940952B CN104940952B CN201510287200.4A CN201510287200A CN104940952B CN 104940952 B CN104940952 B CN 104940952B CN 201510287200 A CN201510287200 A CN 201510287200A CN 104940952 B CN104940952 B CN 104940952B
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Abstract
The invention belongs to biomedicine field, be specifically related to the composition and method of making the same that comprises macrocyclic ester compound and siRNA. Described composition comprises siRNA sequence and macrocyclic ester compound, also comprises one or more the combination in cation lipid, phosphatide and CPEG. The composition that comprises macrocyclic ester compound and siRNA of the present invention can improve the characteristics of pharmacokinetics of siRNA treatment, changes intake, targeting and transfection efficiency in bio distribution, born of the same parents, reduces bad reaction etc.
Description
Technical field
The invention belongs to medicine bioengineering field, the composition that is specifically related to comprise macrocyclic ester compound and siRNA andPreparation method.
Background technology
Since before more than 10 years, RNA disturbs (RNAinterference, RNAi) scientific discovery, RNAi has become cellThe crucial adjuster of heredity and the powerful of illustrating body growth, pathogenesis and aging. SiRNA ties to relevant proteinSynthetic RNA. RISC identifies by the guiding of siRNA and the mRNA (messengerRNA, mRNA) of the target gene of degrading, and makesThe expression of specific protein is suppressed, and then specificity causes gene silencing. Whether the successful key of RNAi is can be by siRNAIntactly be transfected into target cell, mRNA is combined with target gene, and wherein siRNA stability is in vivo significant. ResearchShow: be to improve in siRNA body to transmit by siRNA structure being carried out to chemical modification and siRNA skeleton being carried out to carrier modification2 important channels of stability.
When playing a role, siRNA there is sequence-specific and the very strong targeting of height. RNAi has cascade and amplifies effectShould, the double stranded RNA (double-strandedRNA, dsRNA) of minute quantity just degradable is more much bigger than self concentrationMRNA,, but inject after siRNA in body, compound must pass through systemic circulation system, avoids as far as possible the filter of renal glomerulusCross, cytophagous engulfing, the degraded of haemocyanin absorption and endogenous nuclease, and reticuloendothelial system (RES) is stagnantStay and could arrive target cell. The less stable of siRNA in serum, is easily degraded by Nuclease R Nase. Intravenous injectionAfter siRNA, siRNA is distributed to each large organ by blood circulation, and experience is eliminated simultaneously. SiRNA intravascular enters organizes interstitial(exosmosing), then arrives target cell across interstitial, after arrival target cell, absorbs by endocytosis. In this process, siRNA is by intensionThe endocytosis vesicles embedding that body starts, must escape from endosome, is discharged in cytoplasm, and is loaded on RISC and can brings into play workWith. The outer of siRNA blends the physical arrangement that penetration capacity depends on target tissue, and its born of the same parents in intake also with siRNA with carryThe surface nature of body is relevant. The biggest obstacle that siRNA is delivered to target site is the embedding of endosome to siRNA and carrier and moltenThe degraded of enzyme body to siRNA and carrier.
Naked siRNA self is electronegative, and molecular weight is large, and polarity is strong, is difficult to through cell membrane, blood vessel endothelium, easily at spleenBe detained, easily by nuclease degradation, the half-life is short in vivo, and transfection efficiency is low, even can produce strong inflammatory reactionDeng. Therefore, the body internal stability of siRNA is most important to the performance of its curative effect. Utilize the assistance of various carriers, siRNA is logicalCross different means and be delivered to efficiently the emphasis that target cell is current research.
Summary of the invention
Technical scheme of the present invention is to provide a kind of composition that comprises macrocyclic ester compound and siRNA, its featureBe, composition comprises siRNA sequence and macrocyclic ester compound.
Preferably, described composition also comprises one or more the combination in phosphatide and CPEG.
Preferably, in described composition, calculate described siRNA1-5 part and macrocyclic ester compound according to mass fractionCompound 2-5 part.
Preferably, the siRNA sequence described in described composition is HBVsiRNA:
Positive-sense strand: [mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT];
Antisense strand: UGAAGCGAAGUG[mC] A[mC] ACGGUC.
Preferably, in described composition, calculate described siRNA sequence 1-5 part, macrocyclic ester according to mass fractionCompound compound 1-5 part, phosphatidase 10-3 and lipid-polyethylene glycol 1-10.
Preferably, in described composition, calculate described siRNA1 part, the materialization of macrocyclic ester chemical combination according to mass fraction2 parts of compounds, 3 parts of phosphatidase 10 .5 part and lipid-polyethylene glycol.
Preferably, in described composition, described phosphatide is selected from DPPE, DPPC, DPPG, phosphatidyl-ethanolamine, lecithin, phosphorusOne or more in acyl inositol and cardiolipin;
Described lipid-polyethylene glycol is selected from CPEG, two myristoyl glycerol polyethylene glycol, 1,2-bis-In sub-oleoyl-cis-glycerol-3-phosphate ethanol ammonia, two palmityl glycerol polyethylene glycol and two stearyl glycerol polyethylene glycolOne or more.
The structural formula of described T2C1 compound is as follows:
Described T2C1 compound is by Isosorbide-5-Nitrae, and 7,10-tetraazacyclododecanand and epoxide obtain 90 DEG C of reactions;When reaction, be placed in glass container, continue to stir, reaction mol ratio is 1:4, and reaction duration is 24-72 hour, is preferably 28-65Hour. Preferred epoxy alkyl glycidol ether (the alkyl C of epoxide12-C14)。
Preferably, described T2C1 compound is to weigh Isosorbide-5-Nitrae, and 7,10-tetraazacyclododecanand and C12-14-alkyl shrink sweetOil ether mg mixes, then 92oC is hatched 43 hours synthetic T2C1 under stirring.
Another technical scheme of the present invention is to provide the preparation side of the composition that comprises macrocyclic ester compound and siRNAMethod, is characterized in that, comprises the following steps:
(1), in the water that described siRNA sequence is dissolved in to water or contains 9% sucrose, obtain solution one;
(2) take T2C1 compound, phosphatide, cholesterol or/and lipid-polyethylene glycol, and by these components dissolved in moltenIn agent, obtain solution two;
(3) after solution one is mixed mutually with solution two, under room temperature, carry out vacuum filtration, make solvent wherein byGradually evaporation;
(4) after most solvent evaporation, carry out homogeneous, obtain suspension;
(5) again suspension is carried out to high-pressure homogeneous, freeze-drying, form dry thing, described in obtaining, comprise macrocyclic ester compoundComposition with siRNA.
Preferably, the preparation method of composition, described solution one is 1:(0.25-0.8 with the volume ratio of solution two).
Preferably, the preparation method of composition, described solvent is one in methyl alcohol, ethanol, chloroform, acetone, ethyl acetatePlant or two or more combinations.
In described step (4), the granular size in the suspension after homogeneous is 40-240nm.
The composition that comprises macrocyclic ester compound and siRNA of the present invention can improve the pharmacokinetics spy of siRNA treatmentLevy, change intake, targeting and transfection efficiency in bio distribution, born of the same parents, reduce bad reaction etc.
Detailed description of the invention
Embodiment 1 synthetic compound T2C1 compound
Weigh 100mg1,4,7,10-tetraazacyclododecanand mixes with C12-14-alkyl glycidyl ether 726.8mg,Then 92oC is hatched 43 hours synthetic T2C1 under stirring.
Embodiment 2
Pharmaceutical formulation siRNA (siRNA+T2C1+ CPEG+DPPE1:1:1:1)
Prepare preparation: weigh a certain amount of 100mgT2C1, positive particle lipid DOTMA, phosphatide DPPE, cholesterol-poly-secondGlycol cholesterol polyethylene glycol mixes and is dissolved in chloroform in glass container, under vacuum, removes chloroform. Then add300ml water, vortex concussion is soluble in water, and through homogeneous processing, processing condition is 1000 bar, twice circulation (ATS homogenizer).
Embodiment 3
SiRNA:T2C1 CPEG: DPPE is 1:0.3:0.3:0.4.
Prepare preparation: weigh a certain amount of 100mgT2C1, positive particle lipid DOTMA, phosphatide DPPE, cholesterol-poly-Ethylene glycol mixes and is dissolved in ethanol in glass container, under vacuum, removes ethanol. Then add 300ml water, vortex concussion is moltenYu Shuizhong, through homogeneous processing, processing condition is 1000 bar, twice circulation (ATS homogenizer).
Embodiment 4
SiRNA:T2C1: CPEG: DPPE is 1:0.5:0.1:0.4
Weigh a certain amount of 100mgT2C1, positive particle lipid DOTMA, phosphatide DPPE, CPEG courage is solidAlcohol polyethylene glycol mixes and is dissolved in ethanol in glass container, under vacuum, removes ethanol. Then add 300ml water, vortex shakeSwing soluble in waterly, through homogeneous processing, processing condition is 1000 bar, twice circulation (ATS homogenizer).
Above-mentioned detailed description is for the illustrating of one of them possible embodiments of the present invention, this embodiment not in order toLimit the scope of the claims of the present invention, the equivalence that all the present invention of disengaging do is implemented or is changed, and all should be contained in the technology of the present inventionIn the scope of scheme.
Embodiment 5
HBVsiRNA：
Positive-sense strand: [mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT];
Antisense strand: UGAAGCGAAGUG[mC] A[mC] ACGGUC.
Formulation preparation method is with reference to embodiment 2.
Cell transfecting: HepG2.2.15 cell is inoculated in 96 orifice plates, at CO2In (5%CO2), in 37 DEG C of incubators, cultivateSpend the night. Morning cell covers 40% orifice plate area. Dilute 0.5 μ LsiRNA(1 with 10 μ LDMEM nutrient solutionsμ M stock solution). (it is filled a prescription as table one, and part transfecting formulations turns to dilute 0.4 μ L transfecting formulations with the DMEM nutrient solution of 10 μ LDye the effect of inhibition of gene expression after siRNA as table two). And at room temperature keep 5 minutes. The siRNA of mixed diluting and dilutionTransfecting formulations, vortex concussion 10 seconds, and keeping at room temperature 20 minutes. Add 20 μ L transfection complexs in cultivating containing 80 μ LDMEMIn the hole of liquid. At CO2In (5%CO2), in 37 DEG C of incubators, continue to cultivate.
The separation of mRNA: after transfection two days, cell is washed once with 100 μ L PBS, then add 100 μ L(Turbocapture kit, Qiagen company system) lysis buffer. The cellular products (80 μ L) of dissolving is transferred to one 96The seizure plate of the mRNA in hole, at room temperature hatches 1 hour. With 100 μ L lavation buffer solution washing three times, then the wash-out of 80 μ L is slowRush liquid and join in each hole, incubation 5 minutes at 65 DEG C. It is clear that elute soln (containing mRNA's) is transferred to 96 new holesClear plate.
Real-time RT-PCR: the mRNA that 3 μ L separate is used for real-time RT-PCR. RT-PCR method adopts SYBRGreen mono-step realTime RT-PCR kit (SensiMix mono-step SYBRGreen kit, BIOLINE). Mixing 11 μ L mastermix(containsReverse transcriptase), the forward of 1 μ L and reverse primer (6 μ M), the SYBRGreen of 0.3 μ L 50X and 2.7 μ L water. Reverse transcription is anti-The temperature of answering is at 42 ° of C, after 30 minutes, and 95 ° of C afterwards, 15 minutes for activating Tag polymerase; The temperature of PCR circulation and timeBetween be 95 ° of C, 15 seconds, 60 ° of C, 30 seconds, 72 ° of C, 20 seconds. With the variation of Δ Δ CT methods analyst gene expression.
Table 1: the effect of inhibition of gene expression after preparation transfection siRNA. SiRNA to HepG2.2.15 is thin in transfecting formulations transfectionBorn of the same parents, drew materials after 48 hours, by real-time RT-PCR methods analyst changes in gene expression.
Table 2: the effect of inhibition of gene expression after preparation transfection siRNA. SiRNA to HepG2.2.15 is thin in transfecting formulations transfectionBorn of the same parents, drew materials after 48 hours, by real-time RT-PCR methods analyst changes in gene expression.
Claims (7)
1. a composition that comprises macrocyclic ester compound and siRNA, is characterized in that, composition comprise siRNA sequence andMacrocyclic ester compound, also comprises one or more the combination in phosphatide and CPEG;
Described macrocyclic ester compound is T2C1 compound,
Described phosphatide is selected from one or more in DPPE, DPPC, DPPG, phosphatidyl-ethanolamine;
Described lipid-polyethylene glycol is selected from CPEG, 1, the sub-oleoyl of 2-bis--cis-glycerol-3-phosphate ethanol ammoniaIn one or more;
Wherein, calculate described siRNA sequence 1-5 part, T2C1 compound 1-5 part, phosphatidase 10-3 part and fat according to mass fractionMatter-polyethylene glycol 1-10 part.
2. the composition that comprises macrocyclic ester compound and siRNA as claimed in claim 1, is characterized in that, according to qualityUmber calculates, described siRNA1-5 part and T2C1 compound 2-5 part.
3. the composition that comprises macrocyclic ester compound and siRNA as claimed in claim 1, is characterized in that, describedSiRNA sequence is HBVsiRNA:
Positive-sense strand: [mC] CG[mU] GUG[mC] ACUUCGCUU[mC] A[dT] [dT];
Antisense strand: UGAAGCGAAGUG[mC] A[mC] ACGGUC.
4. the composition that comprises macrocyclic ester compound and siRNA as claimed in claim 1, is characterized in that, according to qualityUmber calculates, 2 parts of described siRNA1 part, macrocyclic ester compounds, 3 parts of phosphatidase 10 .5 part and lipid-polyethylene glycol.
5. the preparation method of the composition that comprises macrocyclic ester compound and siRNA as claimed in claim 1, its feature existsIn, comprise the following steps:
(1), in the water that described siRNA sequence is dissolved in to water or contains 9% sucrose, obtain solution one;
(2) take macrocyclic ester compound, phosphatide, cholesterol or/and lipid-polyethylene glycol, and by these components dissolved inIn solvent, obtain solution two;
(3) after solution one is mixed mutually with solution two, under room temperature, carry out vacuum filtration, make solvent wherein graduallyEvaporation;
(4) after most solvent evaporation, carry out homogeneous, obtain suspension;
(5) again suspension is carried out to high-pressure homogeneous, freeze-drying, forms dry thing, described in obtaining, comprise macrocyclic ester compound andThe composition of siRNA.
6. the preparation method of the siRNA composition that is used for the treatment of virus B hepatitis as claimed in claim 5, its feature existsIn, described solution one is 1:0.25-0.8 with the volume ratio of solution two.
7. the preparation method of the composition that comprises macrocyclic ester compound and siRNA as claimed in claim 5, its feature existsBe one or more the combination in methyl alcohol, ethanol, chloroform, acetone, ethyl acetate in, described solvent.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510287200.4A CN104940952B (en) | 2015-05-29 | 2015-05-29 | The composition and method of making the same that comprises macrocyclic ester compound and siRNA |
| HK16101646.8A HK1213497A1 (en) | 2015-05-29 | 2016-02-16 | Lipomacrocyclic compound and sirna-containing composition and preparation method thereof sirna |
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| CN201510287200.4A CN104940952B (en) | 2015-05-29 | 2015-05-29 | The composition and method of making the same that comprises macrocyclic ester compound and siRNA |
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| CN104922141B (en) * | 2015-05-28 | 2016-05-25 | 厦门成坤生物技术有限公司 | A kind of siRNA composition that is used for the treatment of virus B hepatitis |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103189057A (en) * | 2010-08-26 | 2013-07-03 | 崔坤元 | Lipomacrocycles and uses thereof |
| CN104107437A (en) * | 2013-06-09 | 2014-10-22 | 厦门成坤生物技术有限公司 | RNA interference composition for treating viral hepatitis type b and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103189057A (en) * | 2010-08-26 | 2013-07-03 | 崔坤元 | Lipomacrocycles and uses thereof |
| CN104107437A (en) * | 2013-06-09 | 2014-10-22 | 厦门成坤生物技术有限公司 | RNA interference composition for treating viral hepatitis type b and preparation method thereof |
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| CN104940952A (en) | 2015-09-30 |
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Denomination of invention: Macrolides compound and siRNA-containing composition and preparation method thereof Effective date of registration: 20180212 Granted publication date: 20160525 Pledgee: Xiamen Park Xisheng equity investment partnership (limited partnership) Pledgor: Xiamen Chengkun Biotechnology Co., Ltd. Registration number: 2018350000021 |
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Effective date of registration: 20181105 Address after: 361101 1-206F, unit 1, Zeng North Road, software park, torch high tech Zone, Xiamen, Fujian Patentee after: Xiamen Park Xisheng equity investment partnership (limited partnership) Address before: 361027 4, 2036 Weng Jiao Road, East Fu town, Haicang District, Xiamen, Fujian. Patentee before: Xiamen Chengkun Biotechnology Co., Ltd. |