CN104940952A - Macrolides compound and siRNA-containing composition and preparation method thereof - Google Patents
Macrolides compound and siRNA-containing composition and preparation method thereof Download PDFInfo
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- CN104940952A CN104940952A CN201510287200.4A CN201510287200A CN104940952A CN 104940952 A CN104940952 A CN 104940952A CN 201510287200 A CN201510287200 A CN 201510287200A CN 104940952 A CN104940952 A CN 104940952A
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- sirna
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Abstract
The invention belongs to the biological medicine field, and more specifically relates to a macrolides compound and siRNA-containing composition and a preparation method thereof. The composition comprises a siRNA sequence and the macrolides compound, and also comprises one or combination of more than two of cation lipid, phosphatide and cholesterol-polyethylene glycol. The macrolides compound and siRNA-containing composition can increase the pharmacokinetics characteristic for siRNA treatment, is capable of changing bio-distribution, intracellular intake, targeting property and transfection efficiency, and reducing adverse reaction.
Description
Technical field
The invention belongs to field of pharmaceutical biology, be specifically related to the composition and method of making the same comprising macrocyclic ester compound and siRNA.
Background technology
Before more than 10 years, RNA has disturbed since (RNA interference, RNAi) scientific discovery, and RNAi has become cytogenetic crucial actuator and illustrated the powerful of body growth, pathogenesis and aging.SiRNA becomes RNA to relevant protein bound.RISC by the guiding identification of siRNA and the messenger RNA of target gene of degrading (messenger RNA, mRNA), the expression of specific protein is suppressed, and then specificity causes gene silencing.The successful key of RNAi is whether siRNA intactly can be transfected into target cell, be combined with target gene mRNA, and wherein siRNA stability is in vivo significant.Research shows: be improve 2 important channels of transmitting stability in siRNA body by carrying out chemical modification to siRNA structure and carrying out carrier modification to siRNA skeleton.
There is when siRNA plays a role the sequence-specific of height and very strong targeting.RNAi has Cascaded amplification effect, double stranded RNA (the double-stranded RNA of minute quantity, dsRNA) mRNA that just degradable is more much bigger than self concentration,, but in body after injection siRNA, complex has to pass through systemic circulation system, avoid the filtration of renal glomerulus as far as possible, cytophagously to engulf, the degraded of serum albumin absorption and endogenous nucleic acid enzyme, and the delay of reticuloendothelial system (RES) could arrive target cell.The less stable of siRNA in serum, is easily degraded by Nuclease R Nase.After intravenous injection siRNA, siRNA is distributed to each large organ by blood circulation, experiences elimination simultaneously.SiRNA intravascular enters organizes interstitial (exosmosing), then arrives target cell across interstitial, is absorbed after arriving target cell by endocytosis.In the process, siRNA is embedded by the endocytosis vesicles that endosome starts, and must escape, be discharged in Cytoplasm from endosome, and is loaded on RISC and can plays a role.The outer physical arrangement blending penetration capacity and depend on target tissue of siRNA, and in its born of the same parents, intake is also relevant with the surface nature of siRNA and carrier.Biggest obstacle siRNA being delivered to target site is endosome to the degraded to siRNA and carrier of the embedding of siRNA and carrier and lysosome.
Naked siRNA its own band negative electricity, molecular weight is large, and polarity is strong, is difficult to through cell membrane, blood vessel endothelium, and be easily detained at spleen, in vivo easily by nuclease degradation, the half-life is short, and transfection efficiency is low, even can produce strong inflammatory reaction etc.Therefore, the performance of body internal stability to its curative effect of siRNA is most important.Utilize the assistance of various carrier, siRNA is delivered to by different means the emphasis that target cell is current research efficiently.
Summary of the invention
The present invention's technical scheme is to provide a kind of compositions comprising macrocyclic ester compound and siRNA, it is characterized in that, compositions comprises siRNA sequence and macrocyclic ester compound.
Preferably, described compositions also comprises one or more the combination in phospholipid and cholesterol-poly(oxyethylene glycol).
Preferably, calculate according to mass fraction in described compositions, described siRNA 1-5 part and macrocyclic ester Compound Compound 2-5 part.
Preferably, the siRNA sequence described in described compositions is HBV siRNA:
Positive-sense strand: [mC] CG [mU] GUG [mC] ACUUCGCUU [mC] A [dT] [dT];
Antisense strand: UGAAGCGAAGUG [mC] A [mC] ACGGUC.
Preferably, in described compositions, calculate according to mass fraction, described siRNA sequence 1-5 part, macrocyclic ester Compound Compound 1-5 part, phosphatidase 0-3 and lipid-Polyethylene Glycol 1-10.
Preferably, in described compositions, calculate according to mass fraction, described siRNA1 part, macrocyclic ester Compound Compound 2 parts, phosphatidase 0 .5 part and lipid-Polyethylene Glycol 3 parts.
Preferably, in described compositions, described phospholipid is selected from DPPE, DPPC, DPPG, PHOSPHATIDYL ETHANOLAMINE, lecithin, one or more in phosphatidylinositols and cardiolipin;
Described lipid-Polyethylene Glycol is selected from one or more in cholesterol-poly(oxyethylene glycol), two myristoyl glycerol polyethylene glycol, 1,2-bis-sub-oleoyl-cis-glycerol-3-phosphate ethanol ammonia, two palmitoylglycerol Polyethylene Glycol and two stearyl glycerol polyethylene glycol.
The structural formula of described T2C1 compound is as follows:
Described T2C1 compound is by Isosorbide-5-Nitrae, and 7,10-tetraazacyclododecanand and epoxide are obtained by reacting at 90 DEG C; Be placed in during reaction in glass container, Keep agitation, reaction mol ratio is 1:4, and reaction duration is 24-72 hour, is preferably 28-65 hour.Preferred epoxy alkyl glycidyl ether (the alkyl C of epoxide
12-C
14).
Preferably, described T2C1 compound weighs Isosorbide-5-Nitrae, and 7,10-tetraazacyclododecanand mixes with C12-14-alkyl glycidyl ether mg, hatches 43 hours synthesis T2C1 under then stirring at 92 DEG C.
Another technical scheme of the present invention is to provide the preparation method of the compositions comprising macrocyclic ester compound and siRNA, it is characterized in that, comprises the following steps:
(1) described siRNA sequence is dissolved in water or containing in the water of 9% sucrose, obtains solution one;
(2) take T2C1 compound, phospholipid, cholesterol or/and lipid-Polyethylene Glycol, and these components dissolved are obtained solution two in solvent;
(3) after mutually being mixed homogeneously with solution two by solution one, under room temperature, carry out vacuum filtration, solvent is wherein evaporated gradually;
(4) after most solvent evaporation, carry out homogenizing, obtain suspension;
(5) again high pressure homogenize, lyophilizing are carried out to suspension, form dry thing, described in obtaining, comprise the compositions of macrocyclic ester compound and siRNA.
Preferably, the preparation method of compositions, described solution one is 1:(0.25-0.8 with the volume ratio of solution two).
Preferably, the preparation method of compositions, described solvent is one or more the combination in methanol, ethanol, chloroform, acetone, ethyl acetate.
In described step (4), the granular size in the suspension after homogenizing is 40-240nm.
The compositions comprising 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 untoward reaction etc.
Detailed description of the invention
Embodiment 1 synthesizes compound T2C1 compound
Weigh 100mg1,4,7,10-tetraazacyclododecanand to mix with C12-14-alkyl glycidyl ether 726.8mg, under then stirring at 92 DEG C, hatch 43 hours synthesis T2C1.
Embodiment 2
Pharmaceutical formulation siRNA (siRNA+T2C1+ cholesterol-poly(oxyethylene glycol)+DPPE 1:1:1:1)
Prepare preparation: weigh a certain amount of 100mg T2C1, positive particle lipid DOTMA, phospholipid DPPE, cholesterol-poly(oxyethylene glycol) cholesterol Polyethylene Glycol, in glass container, mixing is dissolved in chloroform, removes chloroform under vacuo.Then add 300ml water, vortex concussion is soluble in water, and through homogenizing process, processing condition is 1000 bar, twice circulation (ATS homogenizer).
Embodiment 3
SiRNA:T2C1 cholesterol-poly(oxyethylene glycol): DPPE is 1:0.3:0.3:0.4.
Prepare preparation: weigh a certain amount of 100mg T2C1, positive particle lipid DOTMA, phospholipid DPPE, cholesterol-poly(oxyethylene glycol), in glass container, mixing is dissolved in ethanol, removes ethanol under vacuo.Then add 300ml water, vortex concussion is soluble in water, and through homogenizing process, processing condition is 1000 bar, twice circulation (ATS homogenizer).
Embodiment 4
SiRNA:T2C1: cholesterol-poly(oxyethylene glycol): DPPE is 1:0.5:0.1:0.4
Weigh a certain amount of 100mg T2C1, positive particle lipid DOTMA, phospholipid DPPE, cholesterol-poly(oxyethylene glycol) cholesterol Polyethylene Glycol, in glass container, mixing is dissolved in ethanol, removes ethanol under vacuo.Then add 300ml water, vortex concussion is soluble in water, and through homogenizing process, processing condition is 1000 bar, twice circulation (ATS homogenizer).
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, this embodiment is also not used to limit 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 scope of technical solution of the present invention.
Embodiment 5
HBV siRNA：
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 CO
2in (5%CO
2), overnight incubation in 37 DEG C of incubators.Morning cell covers the orifice plate area of 40%.0.5 μ L siRNA(1 μM of stock solution is diluted) with 10 μ L DMEM culture fluid.0.4 μ L transfecting formulations (, as table one, after part transfecting formulations transfection siRNA, the effect of inhibition of gene expression is as table two for its formula) is diluted with the DMEM culture fluid of 10 μ L.And at room temperature keep 5 minutes.The siRNA of mixed diluting and the transfecting formulations of dilution, vortex shakes 10 seconds, and keeps at room temperature 20 minutes.Add 20 μ L transfecting complexes in the hole containing 80 μ LDMEM culture fluid.At CO
2in (5%CO
2), continue in 37 DEG C of incubators to cultivate.
Cell 100 μ L PBS are washed once, then add 100 μ L (Turbocapture test kit, Qiagen Inc.) lysis buffer by the separation of mRNA: after transfection two days.The cellular products (80 μ L) of dissolving transfers to the seizure plate of the mRNA in 96 holes, at room temperature hatches 1 hour.Wash three times with 100 μ L lavation buffer solutions, then the elution buffer of 80 μ L joins in each hole, incubation 5 minutes at 65 DEG C.Elute soln (containing mRNA's) is transferred to the 96 new clear plates in hole.
Real-time RT-PCR: the mRNA that 3 μ L are separated is used for real-time RT-PCR.RT-PCR method adopts SYBR Green mono-step real-time RT-PCR test kit (SensiMix mono-step SYBR Green test kit, BIOLINE).Mix 11 μ L master mix (containing reverse transcriptase), the forward of 1 μ L and reverse primer (6 μMs), the SYBR Green of 0.3 μ L 50X and 2.7 μ L water.The temperature of reverse transcription reaction is at 42 DEG C, and after 30 minutes, 95 DEG C afterwards, 15 minutes for activating Tag polymerase; The temperature and time of PCR circulation is 95 DEG C, 15 seconds, 60 DEG C, 30 seconds, 72 DEG C, 20 seconds.With the change of Δ Δ CT methods analyst gene expression.
table 1: the effect of inhibition of gene expression after preparation transfection siRNA.Transfecting formulations transfection siRNA to HepG2.2.15 cell, drew materials after 48 hours, expressed change with real-time RT-PCR method analyzing gene.
table 2: the effect of inhibition of gene expression after preparation transfection siRNA.Transfecting formulations transfection siRNA to HepG2.2.15 cell, drew materials after 48 hours, expressed change with real-time RT-PCR method analyzing gene.
Claims (10)
1. comprise a compositions of macrocyclic ester compound and siRNA, it is characterized in that, compositions comprises siRNA sequence and macrocyclic ester compound.
2. comprise the compositions of macrocyclic ester compound and siRNA as claimed in claim 1, it is characterized in that, also comprise one or more the combination in phospholipid and cholesterol-poly(oxyethylene glycol).
3. comprise the compositions of macrocyclic ester compound and siRNA as claimed in claim 1, it is characterized in that, calculate according to mass fraction, described siRNA1-5 part and macrocyclic ester Compound Compound 2-5 part.
4. comprise the compositions of macrocyclic ester compound and siRNA as claimed in claim 1, it is characterized in that, described siRNA sequence is HBV siRNA:
Positive-sense strand: [mC] CG [mU] GUG [mC] ACUUCGCUU [mC] A [dT] [dT];
Antisense strand: UGAAGCGAAGUG [mC] A [mC] ACGGUC.
5. comprise the compositions of macrocyclic ester compound and siRNA as claimed in claim 1, it is characterized in that, calculate according to mass fraction, described siRNA sequence 1-5 part, macrocyclic ester Compound Compound 1-5 part, phosphatidase 0-3 and lipid-Polyethylene Glycol 1-10.
6. comprise the compositions of macrocyclic ester compound and siRNA as claimed in claim 1, it is characterized in that, calculate according to mass fraction, described siRNA1 part, macrocyclic ester Compound Compound 2 parts, phosphatidase 0 .5 part and lipid-Polyethylene Glycol 3 parts.
7. comprise the compositions of macrocyclic ester compound and siRNA as claimed in claim 1, it is characterized in that,
Described macrocyclic ester compound is T2C1 compound, and described phospholipid is selected from DPPE, DPPC, DPPG, PHOSPHATIDYL ETHANOLAMINE, lecithin, one or more in phosphatidylinositols and cardiolipin;
Described lipid-Polyethylene Glycol is selected from one or more in cholesterol-poly(oxyethylene glycol), two myristoyl glycerol polyethylene glycol, 1,2-bis-sub-oleoyl-cis-glycerol-3-phosphate ethanol ammonia, two palmitoylglycerol Polyethylene Glycol and two stearyl glycerol polyethylene glycol.
8. comprise the preparation method of the compositions of macrocyclic ester compound and siRNA as claimed in claim 1, it is characterized in that, comprise the following steps:
(1) described siRNA sequence is dissolved in water or containing in the water of 9% sucrose, obtains solution one;
(2) take macrocyclic ester compound, phospholipid, cholesterol or/and lipid-Polyethylene Glycol, and these components dissolved are obtained solution two in solvent;
(3) after mutually being mixed homogeneously with solution two by solution one, under room temperature, carry out vacuum filtration, solvent is wherein evaporated gradually;
(4) after most solvent evaporation, carry out homogenizing, obtain suspension;
(5) again high pressure homogenize, lyophilizing are carried out to suspension, form dry thing, described in obtaining, comprise the compositions of macrocyclic ester compound and siRNA.
9. be used for the treatment of the preparation method of the siRNA compositions of hepatitis B as claimed in claim 8, it is characterized in that, described solution one is 1:(0.25-0.8 with the volume ratio of solution two).
10. comprise the preparation method of the compositions of macrocyclic ester compound and siRNA as claimed in claim 8, it is characterized in that, described solvent is one or more the combination in methanol, ethanol, chloroform, acetone, ethyl acetate.
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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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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016188473A1 (en) * | 2015-05-28 | 2016-12-01 | 厦门成坤生物技术有限公司 | Sirna composition for treating viral hepatitis b |
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 |
-
2015
- 2015-05-29 CN CN201510287200.4A patent/CN104940952B/en active Active
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Patent 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 |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016188473A1 (en) * | 2015-05-28 | 2016-12-01 | 厦门成坤生物技术有限公司 | Sirna composition for treating viral hepatitis b |
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| HK1213497A1 (en) | 2016-07-08 |
| CN104940952B (en) | 2016-05-25 |
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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. |