CN105078889A - Liposome delivery system for treating cartilage diseases and preparation method of liposome delivery system - Google Patents
Liposome delivery system for treating cartilage diseases and preparation method of liposome delivery system Download PDFInfo
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Abstract
The invention belongs to the technical field of medicine; in order to solve the problem that no efficient and safe cartilage siRNA delivery system is provided for limiting application of RNAi in treating cartilage diseases, the invention provides a cartilage siRNA liposome delivery system and a preparation method of the delivery system. The delivery system consists of lipidosome, a lipidosome modifier and a small nucleic acid drug, wherein the lipidosome is phosphatidylcholine, cholesterol or Dlin-KC2-DMA; the lipidosome modifier is a polyethylene glycol lipid conjugate; and the small nucleic acid drug is Ihh siRNA. According to the invention, a liposome nano based cartilage RNA interference delivery system is modified, and the formed composite has positive charges, is safe and small in volume, and can be distributed in a full-lamellar cartilage tissue. Carrier experiments verify that siRNA can be delivered into cartilage cells to successful knock out corresponding genes inside the cartilage cells, therefore a bottleneck of gene knockout in the cartilage tissue is broken through; and the siRNA, as a target gene, can be used for treating PTOA (post-traumatic osteoarthritis).
Description
Technical field
The invention belongs to medical art, be specifically related to a kind of liposome delivery system being used for the treatment of cartilage disease and preparation method thereof.
Background technology
Indian hedgehog protein (IndianHedgehog, Ihh) with osteoarthritis after traumatic (Post-traumaticosteoarthritis, PTOA) relevant, at anterior cruciate ligament of knee joint (AnteriorCruciateLigament, ACL) the commitment Ihh damaged expresses and increases, and develops into PTOA the most at last after ACL damage.In zoopery, use the conditional gene knockout mice Ihh gene knocked out in adult mice articular cartilage can slow down ACL damage and cause reducing of PTOA.Therefore, knocking out Ihh gene can as a target gene to treat PTOA.But, current Ihh gene Knockout cannot use the treatment of other animals beyond transgenic mice and human body PTOA, and the Ihh inhibitor of chemistry can produce serious toxic and side effects, comprising: holoprosencephaly, harelip, limb development obstacle etc.Therefore, be necessary very much at present to find one safely and effectively method to knock out in articular cartilage Ihh gene to treat PTOA.
Articular cartilage top layer is very fine and close, be made up of, and the latter forms primarily of II collagen with by the proteoglycan that it holds chondrocyte and extracellular matrix.The experimentation of some clinical I-III phases confirms that the reticent specific gene of mode disturbed by RNA can play effective therapeutical effect (PignatelloR to human body diseases; SarpietroMG, CastelliF.Synthesisandbiologicalevaluationofanewpolymeri cconjugateandnanocarrierwithosteotropicproperties.JFunct BiomaterMarch2012; 3 (1): 79-99.ZhangG, GuoB, WuH, TangT; ZhangBT, ZhengL, HeY, YangZ; PanX, ChowH, ToK, LiY; LiD, WangX, WangY, LeeK; HouZ, DongN, LiG, LeungK; HungL, HeF, ZhangL, QinL.Adeliverysystemtargetingboneformationsurfacetofacil itateRNAi-basedanabolictherapy.NatMed2012; 18 (2): 307-14).But, in view of the organizational structure feature of articular cartilage, its surface is again with a large amount of negative charge, and independent siRNA can not enter in chondrocyte, still lack effectively at present and the cartilage siRNA delivery system of safety, limit the application of RNAi in treatment cartilage disease.
Liposome has good biocompatibility, is widely used as pharmaceutical carrier, the medicine of encapsulating can be made to have good stability, and can reduce the toxic and side effects of medicine to a certain extent.What use comparative maturity in osseous tissue field is a kind of liposome nano-delivery system (LipidNanoparticle, LNP), and it is Bone targeting liposome (ZhangG; GuoB, WuH, TangT; ZhangBT, ZhengL, HeY; YangZ, PanX, ChowH; ToK, LiY, LiD; WangX, WangY, LeeK; HouZ; DongN, LiG, LeungK; HungL; HeF, ZhangL, QinL.Adeliverysystemtargetingboneformationsurfacetofacil itateRNAi-basedanabolictherapy.NatMed.2012; 18 (2): 307-14), but due to the tissue that osseous tissue and cartilaginous tissue are different, Bone targeting molecule is carried above the liposome of this lipoid plastid nano-delivery system, directionally liposome can be combined with osseous tissue, and cannot with bone beyond tissue bond, therefore this lipoid plastid nano-delivery system can not apply to cartilaginous tissue.
Application number is 201110156949.7, and name is called the patent of invention of the Bone targeting delivery system and preparation method thereof based on small nucleic acids medicine osteogenic treatment, provides a kind of Bone targeting delivery system based on small nucleic acids medicine osteogenic treatment and preparation method thereof.This Bone targeting delivery system comprises liposome, Bone targeting molecule and small nucleic acids medicine, described Bone targeting molecule is selected from one or more in Diphosphonate, 8 aspartic acid polypeptide repetitive sequences, 6 aspartic acid-Ser-Ser polypeptide repetitive sequences and the aptamer that filters out for osteoblast, and described small nucleic acids medicine is selected from one or more in the blocker with the small interference ribonucleic acid of promoting bone growing function, the analogies of miRNA and miRNA.The present invention compares traditional Bone targeting delivery system and has stronger specificity and specificity, and small nucleic acids medicine transfection efficiency is high, can reach higher silence efficiency.And the Bone targeting delivery system prepared by this patent is liposome intramuscular injection or intravenous injection, then liposome brings liposome into osseous tissue by targeted molecular, thus act on osseous tissue, but liposome targeting cannot enter in cartilaginous tissue by this Bone targeting delivery system.
Summary of the invention
The present invention lacks effectively and the cartilage siRNA delivery system of safety at present in order to solve, and limits the application of RNAi in treatment cartilage disease, provides a kind of liposome delivery system being used for the treatment of cartilage disease and preparation method thereof.
The present invention is realized by following technical scheme: a kind of liposome delivery system being used for the treatment of cartilage disease, comprises liposome, liposome trim and small nucleic acids medicine, and described liposome is phosphatidylcholine DPPC, cholesterol, Dlin-KC2-DMA; Described liposome trim is polyethyleneglycol lipid conjugate C-PEG, and described small nucleic acids medicine is IhhsiRNA; Described phosphatidylcholine DPPC concentration is 35mg/ml, and the mass percent accounting for liposome and liposome trim is 11.72%; Cholesterol concentration is 9.5mg/ml, and the mass percent accounting for liposome and liposome trim is 23.85%; Polyethyleneglycol lipid conjugate C-PEG concentration is 50mg/ml, and the mass percent accounting for liposome and liposome trim is 16.74%; The concentration of Dlin-KC2-DMA is 28.5mg/ml, and the mass percent accounting for liposome and liposome trim is 47.69%.The target sequence of described small nucleic acids medicine is SEQIDNO:2, i.e. the 1708-1734 bit base sequence of IhhsiRNA.
Described IhhsiRNA SequenceName:ACCNM_053384.1 in Genebank, base sequence is SEQIDNO:1, and its positive-sense strand is: 5'-rGrArArGrGrArGrCrCrUrGrGrArUrGrUrCrCrUrUrGrCrCAC-3'; Antisense strand is: 5'-rGrUrGrGrCrArArGrGrArCrArUrCrCrArGrGrCrUrCrCrUrUrCrCr C-3'; The present invention to select in IhhsiRNA base sequence 1708-1734 bit base sequence as target sequence.
| The beginning site of positive-sense strand | 2085 | The beginning starting point of antisense strand | 2109 |
| The length of positive-sense strand | 25 | The length of antisense strand | 27 |
| The molecular weight of positive-sense strand | 7980.9 | The molecular weight of antisense strand | 8583.2 |
| Total molecular weight | 16564.0 | GC ratio | 57.9 |
The preparation method being used for the treatment of the liposome delivery system of cartilage disease comprises the following steps:
(1) Coliposomes: mixed by the Dlin-KC2-DMA of 2ul phosphatidylcholine DPPC, 15ul cholesterol, 2ul polyethyleneglycol lipid conjugate, 10ul, then add ethanol by liposome dissolving, forms A liquid;
(2) IhhsiRNA of 20uM, 10ul is added the citrate buffer of 55ul, form B liquid;
(3) A liquid is added mix homogeneously in B liquid, form AB mixed liquor;
(4) in AB mixed liquor, the PBS buffer solution of 400ul is added, then the centrifugal 5min of 12000r in ultra-filtration centrifuge tube;
(5) use centrifugal 3 times of PBS repeated washing, centrifugal rear obtained 80-100ul liquid is cartilage siRNA liposome delivery system.
The concentration of described citrate buffer is 50mM, pH is 4.
The cartilage RNA that the present invention has modified based on liposome nanometer disturbs delivery system (LipidNanoparticle (LNP)-basedcartilageRNAiDeliverySystem).Packaging forms LNP-siRNA composite system, and this complex is with positive charge, and safety, volume are little can be distributed in full-thickness cartilage tissue.The present invention enters joint intracavity the liposome injection including siRNA, utilize negative and positive electric charge to attract each other and by liposome in a disguised form targeted delivery enter articular chondrocytes, the Ihh gene in successful knockout chondrocyte.The present invention knocks out Ihh gene in articular cartilage safely and effectively, can treat PTOA as a target gene.Confirmed by vehicle experiments, Ihh-siRNA can send into chondrocyte by the LNP after modification, the Ihh gene in successful knockout chondrocyte.Breach the bottleneck of gene knockout in cartilaginous tissue, solve this difficult problem.The present invention knocks out Ihh gene in articular cartilage safely and effectively, can treat PTOA as a target gene.
What liposome of the present invention adopted is general liposome, and can send any siRNA, siRNA is a kind of small RNA molecular, different albumen all by its corresponding siRNA by the gene knockout of this albumen, this albumen is not expressed.Such as, the siRNA of Ihh albumen by the gene knockout of Ihh albumen, can make Ihh protein expression decline and does not even express.After IhhsiRNA is sent articular cartilage by liposome by the present invention, confirm that siRNA has played effect by PCR method, in a disguised form can enter articular cartilage after proved liposome siRNA and can play a role.Confirm to knock out the generation that Ihh gene can suppress osteoarthritis through service condition transgenic mice; Ihh-siRNA is mainly used in the treatment of joint disease at present, and carries negative charge due to articular cartilage surface, by the effect of electric charge, the liposome entering articular cavity can be made to move to articular cartilage surface, instead of spread everywhere.
For the liposome delivery system being used for the treatment of cartilage disease of the present invention is described, further illustrate as follows by reference to the accompanying drawings:
Fig. 1 is the structural representation of the liposome delivery system being used for the treatment of cartilage disease, in figure: spheroid is for be made up of DLin-KC2-DMA, DPPC, cholesterol and C16PEG2000Ceramide; C16PEG2000Ceramide is amphipathic, one end lipophilic, so can insert membrane structure, the part of PEG2000 is hydrophilic, so towards aqueous phase; DPPC and cholesterol component film structure; The chemical constitution of DLin-KC2-DMA is similar with DPPC, participates in component film structure; Ball interior parcel IhhsiRNA.The nano-particle of this chemical composition has high efficiency, and namely dosage just has good effect lower than similar compound.
Fig. 2 is the experimental result picture that liposome delivery siRNA enters the hypertrophic chondrocyte of chicken, and result shows liposome delivery system of the present invention and siRNA can be sent in cell, and simple siRNA cannot enter in cell.
Fig. 3 is that this micromolecular of siRNA or beacon can be packed by Laser Scanning Confocal Microscope display liposome, and send in chondrocyte, wherein the display of white circle region is red fluorescence, in Fig. 3, use GAPDH-beacon, it enters in cell, self does not fluoresce, if but there is the RNA of GAPDH, so GAPDH-Beacon just could combine with GAPDH, and sends fluorescence.At intraarticular injection liposome and the GAPDH-beacon of rat, after 48 hours, put to death rat, its knee cartilage is gone to cook frozen section, observe under Laser Scanning Confocal Microscope, there is red fluorescence in the chondrocyte can seeing liposome and GAPDH-beacon group, namely represent that liposome can be sent GAPDH-beacon and enter in cartilaginous tissue cell.And independent GAPDH-beacon cannot enter chondrocyte, matched group is observed and is not found fluorescence under Laser Scanning Confocal Microscope.
Fig. 4 is that living body fluorescent molecule tomoscan display liposome can be sent Beacon and enters chondrocyte: at right Injection in knuckle articular cavity liposome and the GAPDH-beacon complex of rat, left knee joint injects independent GAPDH-beacon, at the double knee joint of fluorescence molecule tomographic system 680nm wavelength place scanning different time points rat, find have fluorescence to exist in right knee joint, and fluorescence existence is one week, and do not have in left knee joint, show that liposome can be sent GAPDH-beacon and enter rat articular chondrocytes, and independent GAPDH-beacon cannot enter.Illustrated by this testing result: siRNA or beacon can send in chondrocyte by the liposome delivery system prepared by the present invention.
IhhsiRNA is sent cartilage of rats after 1 week for applying liposome by Fig. 5, and Ihh knocks out rate: at right Injection in knuckle articular cavity liposome and the IhhsiRNA complex of rat, and left knee joint injects independent IhhsiRNA.After one week, put to death rat, respectively scraping left and right knee cartilage, extract total serum IgE, qPCR shows the mRNA level in-site not enough left kneed 60% of Ihh in right knee cartilage cell.Namely IhhsiRNA can successfully send in living animal articular chondrocytes by liposome, and IhhsiRNA can normally play a role, and knocks out the Ihh gene in knee joint.
Accompanying drawing explanation
Fig. 1 is the structural representation of liposome; Fig. 2 is the experimental result picture that liposome delivery siRNA enters the hypertrophic chondrocyte of chicken, and in figure, DAPI is blue-fluorescence, showed cell nuclear location; Fig. 3 is the Laser Scanning Confocal Microscope display result figure that liposome delivery Beacon enters the chondrocyte in cartilaginous tissue; Fig. 4 is the living body fluorescent molecule tomoscan display result figure that liposome delivery Beacon enters chondrocyte; Fig. 5 is that IhhsiRNA was sent cartilage of rats after 1 week by liposome, and Ihh knocks out result schematic diagram.Fig. 6 is the molecular formula of phosphatidylcholine (DPPC); Fig. 7 is the molecular formula of cholesterol; Fig. 8 is the molecular formula of polyethyleneglycol lipid conjugate (C16PEG2000Ceramide); Fig. 9 is the Real-timePCR testing result figure of IhhmRNA in rat body after prepared LNP-siRNA composite system process rat; Figure 10 is the articular cartilage HE colored graph of the rat body intra-articular cartilage injury in treating of prepared LNP-siRNA composite system.
Detailed description of the invention
Embodiment 1: a kind of liposome delivery system being used for the treatment of cartilage disease, comprises liposome, liposome trim and small nucleic acids medicine, and described liposome is phosphatidylcholine DPPC, cholesterol, Dlin-KC2-DMA; Described liposome trim is polyethyleneglycol lipid conjugate (C16PEG2000Ceramide) C-PEG, and described small nucleic acids medicine is IhhsiRNA; Its target sequence is SEQIDNO:2, i.e. the 1708-1734 bit base sequence of IhhsiRNA; Described phosphatidylcholine DPPC concentration is 35mg/ml, and the mass percent accounting for liposome and liposome trim is 11.72%; Cholesterol concentration is 9.5mg/ml, and the mass percent accounting for liposome and liposome trim is 23.85%; Polyethyleneglycol lipid conjugate C-PEG concentration is 50mg/ml, and the mass percent accounting for liposome and liposome trim is 16.74%; The concentration of Dlin-KC2-DMA is 28.5mg/ml, and the mass percent accounting for liposome and liposome trim is 47.69%.
Be used for the treatment of the liposome delivery system of cartilage disease and the preparation method of LNP-siRNA composite system, comprise the following steps:
(1) Coliposomes: the Dlin-KC2-DMA of 2ul phosphatidylcholine DPPC, 15ul cholesterol, 2ul polyethyleneglycol lipid conjugate, 10ul is mixed, then adds 6ul ethanol, liposome is fully dissolved, forming A liquid, is 35ul altogether;
(2) IhhsiRNA of 20uM, 10ul is added the citrate buffer of 55ul, forming B liquid, is 65ul altogether;
(3) A liquid is slowly added in B liquid, oscillator vibrates, by its mix homogeneously, form AB mixed liquor;
(4) in AB mixed liquor, the PBS washing of 400ul is added, 500ul, the then centrifugal 5min of 12000r in ultra-filtration centrifuge tube altogether;
(5) use centrifugal 3 times of PBS repeated washing, centrifugal rear obtained 80-100ul liquid is cartilage siRNA liposome delivery system.
The concentration of described citrate buffer is 50mM, pH is 4.
Experimental example 1: checking liposome delivery system enters chondrocyte
1. liposome delivery siRNA enters the hypertrophic chondrocyte of chicken
Experiment material: on 17 days Embryo Gallus domesticus breastbones 1/3 chondrocyte, lipid nanometer microgranule (LNP), negative control siRNA(NegativeControlsiRNA).0.3% containing the trypsin of EDTA, the collagenase of 0.9%, the hyaluronidase [I type, Sigma company, article No. H3506-1G] of 0.3%.
Experimental technique: method preparation LNP-siRNA complex as described in Example 1.Obtain the chondrocyte of on 15 17 days Embryo Gallus domesticus breastbones 1/3, cut into pieces, be put in 50ml centrifuge tube, then three kinds of each 1ml of Digestive system are added, 3ml(Digestive system preparation altogether: 0.3% containing the trypsin of EDTA, the collagenase of 0.9%, the hyaluronidase by volume 1:1:1 preparation of 0.3%), be placed in 37 DEG C of water baths after 30 minutes, careful absorption supernatant liquid, and then add three kinds of each 1ml of Digestive system, 3ml altogether, 1-1.5 hour is hatched, every vibration in 10 minutes once in 37 DEG C of water baths; Then the culture fluid adding same volume stops enzyme, adds culture fluid 6ml altogether and reacts.Then cross and filter indigested agglomerate and impurity.Filtrate under 1100rpm centrifugal 5 minutes, removing supernatant; Centrifugal gained precipitation adds 10ml cell culture fluid, and then piping and druming mixing cell is inoculated on flat board.Liquid is changed once in 2 days in interval, grows to when accounting for dull and stereotyped 80%, go down to posterity, be inoculated on 6 orifice plates until cell attachment.When cell grows to 60-80% again, experimental group adds LNP-siRNA complex 40ul, and matched group only adds the siRNA40ul of 5uM.Liquid is changed, basis of microscopic observation after 24 hours.
The preparation of cell culture fluid: F-12 (500ml)+10% hyclone FetalBovineSerumfromGIBCO (50ml)+[P+S] (penicillin+streptomycin) (5ml).
Result shows: fluorescence microscope detects sees Fig. 2, due to the self-contained green fluorescence of NegativeControlsiRNA, therefore observe under fluorescence microscope in experimental group (LNP-siRNA group) cell bag slurry and there is green fluorescence, show that siRNA enters in cell, namely siRNA can available energy send in cell by LNP.Matched group (Free-siRNA group) has no fluorescence, and the simple siRNA not adding carrier cannot enter cell cytosol.
2. liposome delivery Beacon enters the in-house chondrocyte of Mouse cartilage
Experiment material: mice 6, lipid nanometer microgranule (LNP), GAPDH.beacon.
Experimental technique: adopt method preparation LNP-GAPDH.beacon complex described in embodiment 1.Injection LNP-GAPDH.beacon complex 40ul, the GAPDH.beacon of injection same dose in left knee joint in the right knee joint of mice.After 48 hours, put to death mice, scraping mice knee cartilage, prepares frozen section, observes under Laser Scanning Confocal Microscope.Observe display: the right knee cartilage of mice can see red fluorescence, and left knee cartilage cannot observe fluorescence.
The structure of Beacon and molecular beacon is a kind of stem ring double labelling oligonucleotide probe of the hairpin structure about 5 ' and 3 ' end self formation 8 bases, the nucleic acid array complementation pairing at two ends, the fluorophor being marked at one end is tightly close with the quenching group being marked at the other end, therefore can not produce fluorescence.The photon produced after fluorophor is excited is quenched agent cancellation, the energy produced by fluorophor discharges with infrared instead of visible ray form, so enter after in cell when transporting GAPDH-Beacon by LNP, mRNA with GAPDH combines, just can see fluorescence under infrared light, namely describe the reliability of LNP.When using GAPDH-Beacon, use Laser Scanning Confocal Microscope to see fluorescence at 680nm place, or see fluorescence intuitively by FMT 680nm place scanning rat.
In right knee joint, injection LNP-GAPDH.beacon complex, observes red fluorescence under Laser Scanning Confocal Microscope, shows that GAPDH.beacon can send in articular cartilage by LNP effectively.Be simple GAPDH.beacon in left knee joint, unstressed configuration under Laser Scanning Confocal Microscope, show that simple GAPDH.beacon can not enter in articular cartilage, experimental result is as Fig. 3.
In figure 3, use GAPDH-beacon, it enters in cell, and self does not fluoresce, if but there is the mRNA of GAPDH, so GAPDH-Beacon just can combine with GAPDHmRNA, and sends fluorescence.At intraarticular injection liposome and the GAPDH-beacon of mice, after 48 hours, put to death mice, get its knee cartilage and cook frozen section, observe under Laser Scanning Confocal Microscope, there is red fluorescence in the chondrocyte can seeing liposome and GAPDH-beacon group, namely represent that liposome can be sent GAPDH-beacon and enter in cartilaginous tissue cell.And independent GAPDH-beacon cannot enter chondrocyte, therefore matched group is injected simple GAPDH.beacon and is observed under Laser Scanning Confocal Microscope and do not find fluorescence.
Experimental example 2: the checking to IhhmRNA expression in rat body after prepared LNP-siRNA composite system process rat:
Experiment material: Wistar rat 10.Lipid nanometer microgranule (LNP), Ihh.siRNA solution: get the Ihh.siRNA10ul that concentration is 20uM, for subsequent use after being diluted to 40ul.
Experimental technique: method preparation LNP-Ihh.siRNA complex as described in Example 1.Injection LNP-Ihh.siRNA complex 40ul in Rat Right knee joint, in left knee joint, injection Ihh.siRNA solution 40ul injects left knee joint.After 48 hours, put to death rat, scraping rat complete knee joint cartilage, after extracting total serum IgE, reverse transcription becomes cDNA, carries out Real-timePCR detection.
IhhmRNA primer: positive-sense strand:
5'-cAGGAAGGACCCATTCCGTC-
3'; Antisense strand:
5'-aAGTCACAAACCCAGGTCCC-
3'.
Result shows: Rat Right knee joint is compared with left knee joint, and the expression of IhhmRNA have dropped 80%.Illustrate LNP can effectively just Ihh.siRNA send in articular cartilage, and play effect, IhhmRNA expressed and declines.Result is as Fig. 9.
Experimental example 3: the experimental verification to the rat body intra-articular cartilage injury in treating effect of prepared LNP-siRNA composite system:
Experiment material: adult wistar rat 30, is divided into three groups, often organizes 10.Lipid nanometer microgranule (LNP), Ihh.siRNA solution: get the Ihh.siRNA10ul that concentration is 20uM, for subsequent use after being diluted to 40ul.
Experimental technique: LNP and Ihh.siRNA is prepared into LNP-Ihh.siRNA complex according to method described in embodiment 1.30 rats are divided into 3 groups, often organize each 10, first group is: sham operated rats, after Rat Right capsula articularis genus is opened, do not cut off anterior cruciate ligament (ACL), again sew up, second day after operation plays injection Ihh.siRNA solution 40ul weekly and injects right knee joint, i.e. Sham+siRNA group; Second group is: experimental group, after being opened by Rat Right capsula articularis genus, cuts off anterior cruciate ligament (ACL) layer-by-layer suture afterwards, and second day after operation rises injects weekly LNP-Ihh.siRNA complex 40ul, i.e. ACLT+LNP.siRNA group; 3rd group is: matched group, after being opened by Rat Right capsula articularis genus, cuts off anterior cruciate ligament (ACL) layer-by-layer suture afterwards, and second day after operation plays injection Ihh.siRNA solution 40ul weekly and injects right knee joint, i.e. ACLT+siRNA group.Within postoperative 8 weeks, put to death whole rat, get after right knee joint formalin fixes decalcification, slice row.
HE coloration result is shown in Figure 10, and result shows, and in Sham+siRNA group, articular surface is complete without destroying, and in ACLT+siRNA group, articular surface damage is serious, and cell arrangement is mixed and disorderly, and in ACLT+LNP.siRNA group, articular cartilage damage is comparatively light, cell cluster.After illustrating that ACL cuts off, compared with matched group, injecting LNP-Ihh.siRNA complex in articular cavity can alleviate cartilage injury effectively.
sequence table
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<120> liposome delivery system being used for the treatment of cartilage disease and preparation method thereof
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The sequence of <223>IHHsiRNA
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1261tagcctcctgcttcgcagctgtgtctgaccaccatctggctcagttggccttctggcccc
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1681ggtaggctctctttccctagagaccttaagacccagctagctctgactgcgattcacaca
1741ccccacctacctgcacaggaaggacccattccgtctgccttcggactgcttactctagtg
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1861ctgcccgcaggtgggctcacacctcagttgatgctgctagattccctaggagccagcagg
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<210>2
<211>27
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<213> artificial sequence
<223> target sequence
<400>2
1aagacccagctagctctgactgcgatt
Claims (4)
1. a cartilage siRNA liposome delivery system, comprises liposome, liposome trim and small nucleic acids medicine, it is characterized in that: described liposome is phosphatidylcholine DPPC, cholesterol, Dlin-KC2-DMA; Described liposome trim is polyethyleneglycol lipid conjugate C-PEG, and described small nucleic acids medicine is IhhsiRNA; Described phosphatidylcholine DPPC concentration is 35mg/ml, and the mass percent accounting for liposome and liposome trim is 11.72%; Cholesterol concentration is 9.5mg/ml, and the mass percent accounting for liposome and liposome trim is 23.85%; Polyethyleneglycol lipid conjugate C-PEG concentration is 50mg/ml, and the mass percent accounting for liposome and liposome trim is 16.74%; The concentration of Dlin-KC2-DMA is 28.5mg/ml, and the mass percent accounting for liposome and liposome trim is 47.69%.
2. a kind of cartilage siRNA liposome delivery system according to claim 1, is characterized in that: the target sequence of described small nucleic acids medicine is SEQIDNO:2, i.e. the 1708-1734 bit base sequence of IhhsiRNA.
3. a kind of cartilage siRNA liposome delivery system according to claim 1, is characterized in that: preparation method comprises the following steps:
(1) Coliposomes: mixed by the Dlin-KC2-DMA of 2ul phosphatidylcholine DPPC, 15ul cholesterol, 2ul polyethyleneglycol lipid conjugate, 10ul, then add ethanol by liposome dissolving, forms A liquid;
(2) IhhsiRNA of 20uM, 10ul is added the citrate buffer of 55ul, form B liquid;
(3) A liquid is added mix homogeneously in B liquid, form AB mixed liquor;
(4) in AB mixed liquor, the PBS buffer solution of 400ul is added, then the centrifugal 5min of 12000r in ultra-filtration centrifuge tube;
(5) use centrifugal 3 times of PBS repeated washing, centrifugal rear obtained 80-100ul liquid is cartilage siRNA liposome delivery system.
4. according to a kind of cartilage siRNA liposome delivery system that claim 2 is stated, it is characterized in that: the concentration of described citrate buffer is 50mM, pH is 4.
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