CN105078889A - Liposome delivery system for treating cartilage diseases and preparation method of liposome delivery system - Google Patents

Abstract

The invention belongs to the technical field of medicine. In order to solve the lack of an effective and safe cartilage siRNA delivery system and limit the application of RNAi in the treatment of cartilage diseases, the invention provides a cartilage siRNA liposome delivery system and a preparation method thereof. Including liposome, liposome modification and small nucleic acid drug, liposome is phosphatidylcholine, cholesterol, Dlin-KC2-DMA; liposome modification is polyethylene glycol lipid conjugate, small nucleic acid Drugs for Ihh? siRNA. The invention modifies the liposome nano-based cartilage RNA interference delivery system, and the formed complex has positive charges, is safe, small in size and can be distributed into the full-thickness cartilage tissue. Carrier experiments have confirmed that siRNA can be delivered into chondrocytes, and the corresponding genes in chondrocytes can be successfully knocked out, breaking through the bottleneck of gene knockout in cartilage tissue; it can be used as a targeted gene to treat PTOA.

Description

A liposome delivery system for treating cartilage diseases and its preparation method

technical field

The invention belongs to the technical field of medicine, and in particular relates to a liposome delivery system for treating cartilage diseases and a preparation method thereof.

Background technique

Indian Hedgehog (Ihh) is associated with Post-traumatic osteoarthritis (PTOA), and the expression of Ihh is increased in the early stage of knee anterior cruciate ligament (ACL) injury, and ACL injury will eventually develop into PTOA. In animal experiments, the use of conditional gene knockout mice to knock out the Ihh gene in the articular cartilage of adult mice can slow down ACL injury and reduce the occurrence of PTOA. Therefore, knocking out the Ihh gene can be used as a targeted gene to treat PTOA. However, the current Ihh gene knockout technology cannot be used to treat PTOA in animals other than transgenic mice and humans, and chemical Ihh inhibitors can cause serious side effects, including: holoprosencephaly, cleft lip and palate, limb development Obstacles etc. Therefore, it is very necessary to find a safe and effective method to knock out the Ihh gene in articular cartilage to treat PTOA.

The surface layer of articular cartilage is very dense, composed of chondrocytes and extracellular matrix, and the latter is mainly composed of II collagen and proteoglycan surrounded by it. Some clinical phase I-III experimental studies have confirmed that silencing specific genes by means of RNA interference can effectively treat human diseases (PignatelloR, SarpietroMG, CastelliF. ,GuoB,WuH,TangT,ZhangBT,ZhengL,HeY,YangZ,PanX,ChowH,ToK,LiY,LiD,WangX,WangY,LeeK,HouZ,DongN,LiG,LeungK,HungL,HeF,ZhangL,QinL. . NatMed 2012;18(2):307-14). However, in view of the structural characteristics of articular cartilage and its surface has a large amount of negative charges, siRNA alone cannot enter chondrocytes. Currently, there is still a lack of effective and safe cartilage siRNA delivery system, which limits the application of RNAi in the treatment of cartilage diseases. .

Liposome has good biocompatibility and has been widely used as a drug carrier, which can make the encapsulated drug have better stability and reduce the toxic and side effects of the drug to a certain extent. A liposome nano-delivery system (LipidNanoparticle, LNP) is relatively mature in the field of bone tissue, which is a bone-targeted 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. AdeliverysystemtargetingboneformationsurfacetofacilitateRNAi-basedanabolictherapy.NatMed.2012;18(2):307-14), but due to Bone tissue and cartilage tissue are different tissues. The liposomes of this type of liposome nano-delivery system carry bone-targeting molecules, which can bind liposomes to bone tissue in a directional manner, but cannot bind to tissues other than bone. Therefore, this type of liposome nano-delivery system cannot be applied to cartilage tissue.

The application number is 201110156949.7, and the title is the invention patent of bone-targeted delivery system and preparation method based on small nucleic acid drug osteogenesis therapy. It provides a bone-targeted delivery system and preparation method based on small nucleic acid drug osteogenesis therapy. The bone-targeted delivery system includes liposomes, bone-targeted molecules and small nucleic acid drugs, and the bone-targeted molecules are selected from bisphosphonates, 8 aspartic acid polypeptide repeat sequences, 6 aspartic acid-serine -one or more of the serine polypeptide repeat sequence and the aptamer screened for osteoblast-like cells, the small nucleic acid drug is selected from the mimetic of small interfering ribonucleic acid and microribonucleic acid with the function of promoting bone formation and One or more of the microRNA blocking agents. Compared with the traditional bone-targeted delivery system, the present invention has stronger specificity and specificity, high transfection efficiency of small nucleic acid drugs, and can achieve higher silencing efficiency. The bone-targeted delivery system prepared by this patent is intramuscular or intravenous injection of liposomes, and then liposomes bring liposomes into bone tissue through targeting molecules, thereby acting on bone tissue, but the bone-targeted delivery system Unable to target liposomes into cartilage tissue.

Contents of the invention

In order to solve the current lack of effective and safe cartilage siRNA delivery system, which limits the application of RNAi in the treatment of cartilage diseases, the present invention provides a liposome delivery system for treating cartilage diseases and a preparation method thereof.

The present invention is achieved by the following technical scheme: a liposome delivery system for treating cartilage diseases, including liposomes, liposome modifications and small nucleic acid drugs, the liposomes are phosphatidylcholine DPPC, Cholesterol, Dlin-KC2-DMA; The liposome modification is polyethylene glycol lipid conjugate C-PEG, and the small nucleic acid drug is IhhsiRNA; The concentration of the phosphatidylcholine DPPC is 35mg/ml, The mass percentage of liposomes and liposome modifications is 11.72%; the cholesterol concentration is 9.5 mg/ml, and the mass percentage of liposomes and liposome modifications is 23.85%; polyethylene glycol lipid conjugated The concentration of C-PEG is 50mg/ml, accounting for 16.74% of the mass percentage of liposomes and liposome modifications; the concentration of Dlin-KC2-DMA is 28.5mg/ml, accounting for liposomes and liposome modifications The mass percentage is 47.69%. The target sequence of the small nucleic acid drug is SEQ ID NO: 2, which is the 1708-1734 base sequence of IhhsiRNA.

所述IhhsiRNA在Genebank中SequenceName：ACCNM_053384.1，碱基序列为SEQIDNO：1，其正义链为：5'-rGrArArGrGrArGrCrCrUrGrGrArUrGrUrCrCrUrUrGrCrCAC-3'；反义链为：5'-rGrUrGrGrCrArArGrGrArCrArUrCrCrArGrGrCrUrCrCrUrUrCrCrC-3'；本发明选用IhhsiRNA The 1708th-1734th nucleotide sequence in the nucleotide sequence was used as the target sequence.

sense strand start site 2085 start of antisense strand 2109 length of justice strand 25 Antisense strand length 27 Sense strand molecular weight 7980.9 Molecular weight of antisense strand 8583.2 total molecular weight 16564.0 GC ratio 57.9

The preparation method of the liposome delivery system for treating cartilage diseases comprises the following steps:

(1) Mixed liposomes: Mix 2ul phosphatidylcholine DPPC, 15ul cholesterol, 2ul polyethylene glycol lipid conjugates, 10ul Dlin-KC2-DMA, then add ethanol to dissolve the liposomes to form A liquid;

(2) Add 20uM, 10ul IhhsiRNA to 55ul citrate buffer to form solution B;

(3) Add liquid A into liquid B and mix evenly to form AB mixed liquid;

(4) Add 400ul of PBS buffer solution to the AB mixture for washing, and then centrifuge at 12000rr for 5min in an ultrafiltration centrifuge tube;

(5) Repeat washing and centrifugation with PBS for 3 times, and the 80-100ul liquid obtained after centrifugation is the cartilage siRNA liposome delivery system.

The concentration of the citrate buffer is 50mM, and the pH is 4.

The invention modifies the liposome nano-based cartilage RNA interference delivery system (LipidNanoparticle (LNP)-basedcartilageRNAiDeliverySystem). The packaging forms a LNP-siRNA complex system that is positively charged, safe, and small enough to distribute into full-thickness cartilage tissue. The present invention injects the liposome containing siRNA into the joint cavity, utilizes the mutual attraction of negative and positive charges to deliver the liposome in a disguised form to the articular chondrocytes, and successfully knocks out the Ihh gene in the chondrocytes. The invention safely and effectively knocks out the Ihh gene in articular cartilage, and can be used as a targeted gene to treat PTOA. Carrier experiments confirmed that the modified LNP could deliver Ihh-siRNA into chondrocytes, and successfully knocked out the Ihh gene in chondrocytes. It breaks through the bottleneck of gene knockout in cartilage tissue and solves this problem. The invention safely and effectively knocks out the Ihh gene in articular cartilage, and can be used as a targeted gene to treat PTOA.

What the liposome of the present invention adopts is universal liposome, can deliver any siRNA, and siRNA is a kind of small RNA molecule, and different protein can knock out the gene of this protein by its corresponding siRNA, make this protein not express. For example, siRNA of Ihh protein can knock out the gene of Ihh protein, so that the expression of Ihh protein decreases or even does not express. After the present invention delivers IhhsiRNA into articular cartilage through liposomes, the PCR method is used to confirm that siRNA has played an effect, and it is confirmed in a disguised form that the liposome-encapsulated siRNA can enter articular cartilage and play a role. The use of conditional transgenic mice has confirmed that knocking out the Ihh gene can inhibit the occurrence of osteoarthritis; Ihh-siRNA is currently mainly used for the treatment of joint diseases, and because the surface of articular cartilage carries negative charges, it can make it into the joints through the effect of charges. Luminal liposomes move toward the surface of the articular cartilage instead of spreading around.

In order to illustrate the liposome delivery system for the treatment of cartilage diseases of the present invention, it is further described as follows in conjunction with the accompanying drawings:

Figure 1 is a schematic diagram of the structure of a liposome delivery system for the treatment of cartilage diseases. In the figure: the sphere is composed of DLin-KC2-DMA, DPPC, cholesterol and C16PEG2000Ceramide; C16PEG2000Ceramide is amphiphilic, and one end is lipophilic, so it can Inserted into the membrane structure, PEG2000 is partially hydrophilic, so it faces the water phase; DPPC and cholesterol form the membrane structure; the chemical structure of DLin-KC2-DMA is similar to DPPC, and participates in the formation of the membrane structure; the inside of the sphere is wrapped with IhhsiRNA. The nanoparticle with this chemical composition has high efficiency, that is, the dosage is lower than that of similar compounds to have a good effect.

Fig. 2 is a diagram of the experimental results of liposome delivery of siRNA into chicken hypertrophic chondrocytes, the results show that the liposome delivery system of the present invention can deliver siRNA into cells, while simple siRNA cannot enter cells.

Figure 3 is a confocal microscope showing that liposomes can package small molecules such as siRNA or beacon and send them into chondrocytes, where the white circle area shows red fluorescence. In Figure 3, GAPDH-beacon is used, which When entering the cell, it does not fluoresce by itself, but if there is GAPDH RNA, then GAPDH-Beacon can bind to GAPDH and emit fluorescence. Intra-articular injection of liposome and GAPDH-beacon in rats, 48 hours later, the rats were sacrificed, the knee articular cartilage was removed to make frozen sections, observed under a confocal microscope, the liposome and GAPDH-beacon group could be seen There is red fluorescence in the chondrocytes, which means that the liposome can deliver GAPDH-beacon into the cartilage tissue cells. However, GAPDH-beacon alone could not enter chondrocytes, and no fluorescence was found in the control group under confocal microscope observation.

Figure 4 is the in vivo fluorescence molecular tomography showing that liposomes can deliver Beacon into chondrocytes: inject liposomes and GAPDH-beacon complexes in the right knee joint cavity of rats, and inject GAPDH-beacon alone in the left knee joint. The fluorescence molecular tomography system scanned the knee joints of rats at different time points at a wavelength of 680nm, and found that there was fluorescence in the right knee joint for up to a week, but there was no fluorescence in the left knee joint, indicating that liposomes could deliver GAPDH-beacon entered rat articular chondrocytes, but GAPDH-beacon alone could not enter. The test result shows that the liposome delivery system prepared by the present invention can deliver siRNA or beacon into chondrocytes.

Figure 5 is the knockout rate of Ihh after liposomes were used to deliver IhhsiRNA into rat articular cartilage for 1 week: the complex of liposome and IhhsiRNA was injected into the right knee joint cavity of the rat, and the left knee joint was injected with independent IhhsiRNA . One week later, the rats were sacrificed, the left and right knee articular cartilages were scraped, and total RNA was extracted. qPCR showed that the mRNA level of Ihh in the right knee articular chondrocytes was less than 60% of that in the left knee joint. That is, the liposome can successfully deliver IhhsiRNA into the articular chondrocytes of living animals, and the IhhsiRNA can function normally, knocking out the Ihh gene in the knee joint.

Description of drawings

Figure 1 is a schematic diagram of the structure of liposomes; Figure 2 is the experimental results of liposome delivery of siRNA into chicken hypertrophic chondrocytes, in which DAPI is blue fluorescence, showing the position of the nucleus; Figure 3 is liposome delivery of Beacon into Confocal microscope display results of chondrocytes in cartilage tissue; Figure 4 is the in vivo fluorescent molecular tomography display results of liposome delivery of Beacon into chondrocytes; Figure 5 is liposome delivery of IhhsiRNA into rat articular cartilage 1 One week later, the schematic diagram of the knockout results of Ihh. Figure 6 is the molecular formula of phosphatidylcholine (DPPC); Figure 7 is the molecular formula of cholesterol; Figure 8 is the molecular formula of polyethylene glycol lipid conjugate (C16PEG2000Ceramide); Figure 9 is the prepared LNP-siRNA complex system Real-time PCR detection results of IhhmRNA in rats after treatment; FIG. 10 is HE staining of articular cartilage treated by the prepared LNP-siRNA complex system in rats with articular cartilage damage.

Detailed ways

Embodiment 1: A liposome delivery system for the treatment of cartilage diseases, including liposomes, liposome modifications and small nucleic acid drugs, the liposomes are phosphatidylcholine DPPC, cholesterol, Dlin-KC2 -DMA; the liposome modification is polyethylene glycol lipid conjugate (C16PEG2000Ceramide) C-PEG, and the small nucleic acid drug is IhhsiRNA; its target sequence is SEQ ID NO: 2, which is the 1708-1734th part of IhhsiRNA Base sequence; the phosphatidylcholine DPPC concentration is 35mg/ml, accounting for 11.72% of the mass percent of liposomes and liposome modifications; the cholesterol concentration is 9.5mg/ml, accounting for liposomes and lipids The mass percentage of body modification is 23.85%; The concentration of polyethylene glycol lipid conjugate C-PEG is 50mg/ml, accounting for 16.74% of the mass percentage of liposome and liposome modification; Dlin-KC2-DMA The concentration is 28.5mg/ml, accounting for 47.69% of the mass percentage of liposomes and liposome modifications.

A method for preparing a liposome delivery system for treating cartilage diseases, that is, a LNP-siRNA complex system, comprising the following steps:

(1) Mixed liposomes: mix 2ul phosphatidylcholine DPPC, 15ul cholesterol, 2ul polyethylene glycol lipid conjugates, 10ul Dlin-KC2-DMA, then add 6ul ethanol to fully dissolve the liposomes , to form A solution, a total of 35ul;

(2) Add 20uM and 10ul of IhhsiRNA to 55ul of citrate buffer to form solution B, totaling 65ul;

(3) Slowly add liquid A to liquid B, vibrate with an oscillator, and mix them evenly to form a mixed liquid of AB;

(4) Add 400ul of PBS to the AB mixture for washing, a total of 500ul, and then centrifuge at 12000r for 5min in an ultrafiltration centrifuge tube;

(5) Repeat washing and centrifugation with PBS for 3 times, and the 80-100ul liquid obtained after centrifugation is the cartilage siRNA liposome delivery system.

The concentration of the citrate buffer is 50mM, and the pH is 4.

Experimental example 1: Verification of liposome delivery system into chondrocytes

1. Liposome delivery of siRNA into chicken hypertrophic chondrocytes

Experimental materials: 1/3 chondrocytes of 17-day chicken embryo sternum, lipid nanoparticle (LNP), negative control siRNA (NegativeControlsiRNA). 0.3% EDTA-free trypsin, 0.9% collagenase, 0.3% hyaluronidase [Type I, Sigma, Cat. No. H3506-1G].

Experimental method: The LNP-siRNA complex was prepared as described in Example 1. Obtain chondrocytes from 1/3 of the sternum of 15 17-day-old chicken embryos, cut them into pieces, put them in a 50ml centrifuge tube, and then add 1ml of each of the three digestive solutions, 3ml in total (digestive solution preparation: 0.3% EDTA-free pancreatic Protease, 0.9% collagenase, 0.3% hyaluronidase (prepared by volume 1:1:1), placed in a 37°C water bath for 30 minutes, carefully sucked up the upper liquid, and then added 1ml each of the three digestive solutions , a total of 3ml, incubate in a 37°C water bath for 1-1.5 hours, shake once every 10 minutes; then add the same volume of culture medium to stop the enzyme, add a total of 6ml of culture medium to react. It is then filtered to remove undigested clumps and impurities. The filtrate was centrifuged at 1100rpm for 5 minutes, and the supernatant was removed; the precipitate obtained by centrifugation was added to 10ml of cell culture medium, and the cells were mixed by pipetting and then seeded on a plate. The medium was changed every 2 days, and when the cells adhered to the wall and reached 80% of the plate, they were passaged and seeded on a 6-well plate. When the cells grew to 60-80% again, 40ul of LNP-siRNA complex was added to the experimental group, and only 40ul of 5uM siRNA was added to the control group. After 24 hours, the medium was changed and observed under a microscope.

Preparation of cell culture medium: F-12 (500ml) + 10% fetal bovine serum FetalBovineSerumfromGIBCO (50ml) + [P+S] (penicillin + streptomycin) (5ml).

The results show that: Fluorescence microscope detection is shown in Figure 2. Since NegativeControl siRNA itself carries green fluorescence, green fluorescence is observed in the cytoplasm of the experimental group (LNP-siRNA group) under the fluorescence microscope, indicating that siRNA has entered the cell, that is, LNP can effectively Can deliver siRNA into cells. No fluorescence was seen in the control group (Free-siRNA group), and the simple siRNA without carrier could not enter the cell cytoplasm.

2. Liposome delivery of Beacon into chondrocytes in mouse cartilage tissue

Experimental materials: 6 mice, lipid nanoparticles (LNP), GAPDH.beacon.

Experimental method: The method described in Example 1 was used to prepare the LNP-GAPDH.beacon complex. The mice were injected with 40ul of LNP-GAPDH.beacon complex into the right knee joint, and the same dose of GAPDH.beacon was injected into the left knee joint. After 48 hours, the mice were sacrificed, the articular cartilage of the mouse knees were scraped, frozen sections were prepared, and observed under a confocal microscope. Observations showed that red fluorescence could be seen in the articular cartilage of the right knee of the mouse, but no fluorescence could be observed in the articular cartilage of the left knee.

The structure of Beacon, the molecular beacon, is a stem-loop double-labeled oligonucleotide probe that forms a hairpin structure of about 8 bases at the 5' and 3' ends. The nucleic acid sequences at both ends are complementary and paired, and the label is at one end. The fluorophore is in close proximity to the quencher labeled at the other end and therefore does not fluoresce. The photons generated after the fluorophore is excited are quenched by the quencher, and the energy generated by the fluorophore is released in the form of infrared instead of visible light, so when the GAPDH-Beacon is transported into the cell through LNP, it binds to the mRNA of GAPDH , you can see fluorescence under infrared light, which shows the reliability of LNP. When using GAPDH-Beacon, use a confocal microscope to see the fluorescence at 680nm, or scan the rat at 680nm by FMT to see the fluorescence directly.

The LNP-GAPDH.beacon complex was injected into the right knee joint, and red fluorescence was observed under a confocal microscope, indicating that LNP can effectively deliver GAPDH.beacon into the articular cartilage. There was pure GAPDH.beacon in the left knee joint, and there was no fluorescence under the confocal microscope, indicating that pure GAPDH.beacon could not enter the articular cartilage. The experimental results are shown in Figure 3.

In Figure 3, GAPDH-beacon is used, which enters the cell and does not fluoresce by itself, but if there is GAPDH mRNA, GAPDH-Beacon can bind to GAPDH mRNA and emit fluorescence. Inject liposomes and GAPDH-beacon into the joint cavity of the mice. After 48 hours, the mice were sacrificed, and the knee articular cartilage was taken to make frozen sections and observed under a confocal microscope. The liposomes and GAPDH-beacon group could be seen There is red fluorescence in the chondrocytes, which means that the liposome can deliver GAPDH-beacon into the cartilage tissue cells. However, GAPDH-beacon alone could not enter the chondrocytes, so the control group injected with simple GAPDH.beacon did not find fluorescence under confocal microscope observation.

Experimental Example 2: Verification of the expression of IhhmRNA in rats after the prepared LNP-siRNA complex was systematically treated:

Experimental materials: 10 Wistar rats. Lipid nanoparticle (LNP), Ihh.siRNA solution: take 10ul of Ihh.siRNA with a concentration of 20uM, dilute it to 40ul for use.

Experimental method: The LNP-Ihh.siRNA complex was prepared as described in Example 1. 40ul of the LNP-Ihh.siRNA complex was injected into the right knee joint of the rat, and 40ul of the Ihh.siRNA solution was injected into the left knee joint. After 48 hours, the rats were sacrificed, the whole knee articular cartilage of the rats was scraped, and the total RNA was extracted, reverse-transcribed into cDNA, and detected by Real-time PCR.

IhhmRNA primers: sense strand: 5'- CAGGAAGGACCCATTCCGTC- 3' ; antisense strand: 5'- AAGTCACAAACCCAGGTCCC- 3' .

The results showed that the expression of IhhmRNA decreased by 80% in the rat right knee joint compared with the left knee joint. It shows that LNP can effectively deliver Ihh.siRNA into the articular cartilage, and play an effect so that the expression of IhhmRNA decreases. The result is shown in Figure 9.

Experimental Example 3: Experimental verification of the therapeutic effect of the prepared LNP-siRNA complex system on articular cartilage injury in rats:

Experimental materials: 30 adult wistar rats, divided into three groups, 10 in each group. Lipid nanoparticle (LNP), Ihh.siRNA solution: take 10ul of Ihh.siRNA with a concentration of 20uM, dilute it to 40ul for use.

Experimental method: LNP and Ihh.siRNA were prepared according to the method described in Example 1 to form a LNP-Ihh.siRNA complex. The 30 rats were divided into 3 groups, 10 in each group. The first group was: the sham operation group. After opening the right knee joint capsule of the rats, the anterior cruciate ligament (ACL) was not cut off, and re-sutured. From the second day, 40ul of Ihh.siRNA solution was injected into the right knee joint every week, that is, the Sham+siRNA group; the second group was: the experimental group, after opening the right knee joint capsule of the rats, cutting the anterior cruciate ligament (ACL) layer by layer Suture, and inject LNP-Ihh.siRNA complex 40ul every week from the second day after operation, that is, ACLT+LNP.siRNA group; the third group is: the control group, after the right knee joint capsule of the rat is opened, the anterior cruciate ligament is cut off (ACL) was sutured layer by layer, and 40ul of Ihh.siRNA solution was injected weekly into the right knee joint from the second day after operation, that is, the ACLT+siRNA group. All the rats were sacrificed 8 weeks after the operation, and the right knee joint was fixed in formalin and decalcified, and sliced.

The results of HE staining are shown in Figure 10. The results showed that in the Sham+siRNA group, the articular surface was intact without damage, in the ACLT+siRNA group, the articular surface was severely damaged, and the cells were arranged in disorder, while in the ACLT+LNP.siRNA group, the articular cartilage The damage is mild, and the cells are clustered. It shows that after the ACL is cut, compared with the control group, the injection of LNP-Ihh.siRNA complex into the joint cavity can effectively reduce the cartilage damage.

sequence listing

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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.