CN102441177A - 用于siRNA传递的纳米泡载体 - Google Patents
用于siRNA传递的纳米泡载体 Download PDFInfo
- Publication number
- CN102441177A CN102441177A CN2011103964787A CN201110396478A CN102441177A CN 102441177 A CN102441177 A CN 102441177A CN 2011103964787 A CN2011103964787 A CN 2011103964787A CN 201110396478 A CN201110396478 A CN 201110396478A CN 102441177 A CN102441177 A CN 102441177A
- Authority
- CN
- China
- Prior art keywords
- sirna
- nanometer bubble
- block copolymer
- gas
- nanometer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108020004459 Small interfering RNA Proteins 0.000 title claims abstract description 54
- 239000002101 nanobubble Substances 0.000 title abstract 3
- 239000000969 carrier Substances 0.000 title 1
- 229920001400 block copolymer Polymers 0.000 claims description 13
- -1 cation lipid Chemical class 0.000 claims description 13
- 239000002202 Polyethylene glycol Substances 0.000 claims description 12
- 239000007789 gas Substances 0.000 claims description 12
- 229920001223 polyethylene glycol Polymers 0.000 claims description 12
- 230000036760 body temperature Effects 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 4
- 229960004692 perflenapent Drugs 0.000 claims description 4
- NJCBUSHGCBERSK-UHFFFAOYSA-N perfluoropentane Chemical compound FC(F)(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)F NJCBUSHGCBERSK-UHFFFAOYSA-N 0.000 claims description 4
- 229920000151 polyglycol Polymers 0.000 claims description 3
- 239000010695 polyglycol Substances 0.000 claims description 3
- 229910018503 SF6 Inorganic materials 0.000 claims description 2
- UBAZGMLMVVQSCD-UHFFFAOYSA-N carbon dioxide;molecular oxygen Chemical compound O=O.O=C=O UBAZGMLMVVQSCD-UHFFFAOYSA-N 0.000 claims description 2
- IJDNQMDRQITEOD-UHFFFAOYSA-N n-butane Chemical class CCCC IJDNQMDRQITEOD-UHFFFAOYSA-N 0.000 claims description 2
- 229910052757 nitrogen Inorganic materials 0.000 claims description 2
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 claims description 2
- 229960004065 perflutren Drugs 0.000 claims description 2
- SFZCNBIFKDRMGX-UHFFFAOYSA-N sulfur hexafluoride Chemical compound FS(F)(F)(F)(F)F SFZCNBIFKDRMGX-UHFFFAOYSA-N 0.000 claims description 2
- 229960000909 sulfur hexafluoride Drugs 0.000 claims description 2
- 230000005540 biological transmission Effects 0.000 claims 2
- 239000003570 air Substances 0.000 claims 1
- 238000000338 in vitro Methods 0.000 abstract description 4
- 238000001727 in vivo Methods 0.000 abstract description 3
- 230000000144 pharmacologic effect Effects 0.000 abstract description 2
- 241000700721 Hepatitis B virus Species 0.000 description 20
- 238000001890 transfection Methods 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- ATUOYWHBWRKTHZ-UHFFFAOYSA-N dimethylmethane Natural products CCC ATUOYWHBWRKTHZ-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001294 propane Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- JBDDMGRYUVEJCV-UHFFFAOYSA-N 2-amino-n,n-bis(2-hydroxyethyl)acetamide Chemical compound NCC(=O)N(CCO)CCO JBDDMGRYUVEJCV-UHFFFAOYSA-N 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-M Bromide Chemical compound [Br-] CPELXLSAUQHCOX-UHFFFAOYSA-M 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- REYJJPSVUYRZGE-UHFFFAOYSA-N Octadecylamine Chemical compound CCCCCCCCCCCCCCCCCCN REYJJPSVUYRZGE-UHFFFAOYSA-N 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 1
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Natural products O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 210000000026 apposition eye Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 208000006359 hepatoblastoma Diseases 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- WGYKZJWCGVVSQN-UHFFFAOYSA-N mono-n-propyl amine Natural products CCCN WGYKZJWCGVVSQN-UHFFFAOYSA-N 0.000 description 1
- QMDUPVPMPVZZGK-UHFFFAOYSA-N n,n-dimethyloctadecan-1-amine;hydrobromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[NH+](C)C QMDUPVPMPVZZGK-UHFFFAOYSA-N 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 229940041022 streptomycins Drugs 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种纳米泡载体，可以在体外和体内传递siRNA，并且在携带siRNA的纳米泡经过部位或作用部位施加超声手段，可以促进siRNA的释放，并起到相应的药理作用。
Description
技术领域
本发明涉及医药领域，具体涉及用于siRNA传递的纳米泡载体。
背景技术
siRNA中文名称是小干扰脱氧核糖核酸，是基于基因沉默(RNAi)技术而设计的一种核酸活性成分。siRNA是人工合成的双链RNA，是一种小RNA分子(21-25核苷酸)，由Dicer(RNAaseIII家族中对双链RNA具有特异性的酶)加工而成。siRNA是siRISC的主要成员，激发与之互补的目标mRNA的沉默。siRNA无论得到的方法如何，如化学合成、体外转录、用RNAaseIII消化长片断双链RNA制备siRNA、siRNA表达载体、病毒载体、siRNA表达框架，因为它们的分子结构相同，理化性质也一样，只是产品形式可能有所不同，即保存在什么溶液中。
siRNA自发现以来已获得大量关注，但一直没有作为新药上市，最重要的问题是siRNA作为核酸分子，很容易被体内核酸酶降解，并且siRNA从注射部位到达作用部位要经历很多过程途径，进入细胞后，还有从内涵体或溶酶体中逃逸，进入胞浆，才能发挥作用。siRNA的特殊性使其制剂研究非常困难。
发明内容
本发明人出乎意料地发现我们设计的一种纳米泡载体，可以在体外和体内传递siRNA，并且在携带siRNA的纳米泡经过部位或作用部位施加超声手段，可以促进siRNA的释放，并起到相应的药理作用。
本发明公开的用于siRNA传递的纳米泡载体，其特征是纳米泡由聚酯-聚乙二醇嵌段共聚物和阳离子脂质构成外壳，内部充填气体或在正常体温下转化为气体的液体。
本发明中的siRNA根据所表达的功能特点而设计核苷酸序列，并通过生物技术公司合成得到。siRNA的功能包括各种疾病的治疗用途，例如各种病毒性肝炎、肿瘤、心血管疾病、免疫性疾病。
本发明中的聚酯-聚乙二醇嵌段共聚物选自聚乳酸-聚乙二醇嵌段共聚物、聚乳酸羟基乙酸-聚乙二醇嵌段共聚物，优选的是聚乳酸羟基乙酸-聚乙二醇嵌段共聚物。聚乳酸羟基乙酸-聚乙二醇嵌段共聚物中聚乳酸羟基乙酸嵌段的分子量范围是1000～20000，优选的是2000～10000，更优选的是3000～10000；聚乙二醇嵌段的分子量范围是500～10000，优选的是1000～5000，更优选的是1500～3000。聚乳酸-聚乙二醇嵌段共聚物和聚乳酸羟基乙酸-聚乙二醇嵌段共聚物可以从市场上购买得到；或根据文献合成方法，也可以自行合成得到。
本发明中的阳离子脂质选自1，2-二油酰-3-三甲基胺基丙烷、双十八烷基二甲基溴化铵、双十烷基二甲基溴化铵、双十二烷基二甲基溴化铵、尿嘧啶脂质衍生物、1，4-二氢吡啶脂质衍生物、二甲基羟乙基胺丙胺碳酰胆固醇酯、3β-(N-(N’，N’-二甲基氨基乙烷)碳酰)胆固醇、O，O’-双十四酰-N-三甲基氨基乙酰二乙醇胺盐酸盐、十六烷基三甲基溴化铵(CTAB)、苯扎氯胺、鱼精蛋白、硬脂胺、二油酰磷脂酰乙醇胺。这些阳离子脂质都可以从市场上得到。
本发明中的气体选自空气、、氮气、氧气、二氧化碳、全氟丙烷、全氟丁烷、六氟化硫。
本发明中的在正常体温下转化为气体的液体是全氟戊烷。全氟戊烷的沸点是29.5℃。
本发明公开的用于siRNA传递的纳米泡载体，其特征是纳米泡粒径范围是20～700纳米，优选的是50～500纳米，更优选的是50～300纳米，进一步优选的是50～200纳米。
由于siRNA的水溶性，siRNA无法包裹在纳米泡内部，而只能通过静电作用，与纳米泡外壳中的阳离子脂质发生相互作用而进行包裹。本发明基于siRNA和纳米泡的特点还专门设计了用于siRNA传递的纳米泡载体的制备方法如下：
(1)将聚酯-聚乙二醇嵌段共聚物和阳离子脂质溶解在有机溶剂中；
(2)将含siRNA的水溶液在搅拌下加入上述溶液中；
(3)挥发或透析除去有机溶剂得到含siRNA的胶束溶液；
(4)在胶束溶液中加入气体或在正常体温下转化为气体的液体，并持续超声，形成纳米泡。
上述制备方法中，有机溶剂优选的是和水互溶的有机溶剂，选自乙醇、甲醇、丙酮、异丙醇、二甲基甲酰胺、四氢呋喃、二氧六环、二甲基亚砜。加入的siRNA水溶液占分散介质的有机溶液体积的5～80％，优选的是10～50％。
附图说明
图1.ELISA检测HBsAg OD值
具体实施方式
实施例1.载siRNA的纳米泡
将80mg聚乳酸羟基乙酸-聚乙二醇(PLGA8000-PEG15000)、10mg1，2-二油酰-3-三甲基胺基丙烷共同溶解于40mL二甲基甲酰胺中，在磁力搅拌条件下加入5mg siRNA，转移至截留分子量为7000的透析袋，于4L水中透析48小时，每隔12小时更换新鲜水，透析完成后，取出透析袋内的溶液，在水浴超声条件下，加入气体或在正常体温下转化为气体的液体，并持续超声0.5小时，形成纳米泡。
siRNA可选自各种治疗疾病用siRNA，如针对乙型肝炎病毒(HBV)的序列：
序列1：ACCCUUAUAAAGAAUUUGGdAdG
dGdCUGGGAAUAUUUCUUAAACC
序列2：GCUGUGCCUUGGGUGGCUUdTdT
dTdTCGACACGGAACCCACCGAA
序列3：UACCGCAGAGUCUAGACUCdTdT
dTdTAUGGCGUCUCAGAUCUGAG
还有抗肿瘤siRNA，如针对血管内皮生长因子(VEGF)的siRNA。
实验例1.载siRNA的纳米泡的体外转染实验
1材料和方法
1.1材料和仪器
HepG2.2.15细胞株(HBV DNAayw亚型转染肝母细胞瘤HepG2细胞的细胞系，可分泌HBV各种病毒标志物及完整的Dane颗粒)，DMEM混合培养液(含10％胎牛血清，380μg/mlG418，3％谷氨酰胺，100U/ml青霉素，100μg/ml链霉素)，乙肝病毒表面抗原、核心抗原检测试剂盒。酶标仪，实时荧光定量PCR仪，高速台式离心机，超低温冰箱。
载siRNA的纳米泡由实施例1方法制备，siRNA序列为
序列1：ACCCUUAUAAAGAAUUUGGdAdG
dGdCUGGGAAUAUUUCUUAAACC。
采用的是全氟戊烷充填。
1.3HBV的siRNA干扰试验
HepG2.2.15细胞接种到96孔板，转染前一天换无抗生素培养基培养24小时。设置三组，包括载siRNA的纳米泡、Lipofectamine 2000(阳性对照)、空白未给药组，分别设3复孔，继续培养至72小时。
1.4ELISA检测表抗与核抗及RT-PCR检测HBV DNA的表达
HBV表面抗原和核心抗原的检测，参照试剂盒标准操作程序执行；并用RNA抽提试剂盒分别提取各组转染后的总RNA，进行RT-PCR的检测。
2结果与讨论
2.1乙肝表面抗原检测
HepG2.2.15细胞与载siRNA的纳米泡或其他对照一起孵育72小时，并在加入5分钟后，37℃水浴超声10分钟，双波长450/690nm读取OD值。根据诊断试剂盒的判断标准，阴性对照OD值≤0.1且阳性对照OD值≥0.8时实验正常，否则实验无效，由此判断本实验有效。本发明中载siRNA的纳米泡能明显干扰HBsAg的复制，明显优于阳性对照(图1)。
2.2RT-PCR定量检测HBV DNA的表达
参照2.1的结果对抗HBsAg有效的实验组进行了RT-PCR检测(表1)。载siRNA的纳米泡组可见明显的DNA复制被抑制，证明其具有干扰HBV效应，明显优于阳性对照。
表1.HepG2.2.15细胞中HBV DNA拷贝数
实验例2.载siRNA的纳米泡的体内治疗实验
1材料与方法
1.1材料与仪器
采用实验例1中的载siRNA纳米泡，HBV转染小鼠模型(C57)，实时荧光定量PCR仪。高速台式离心机，超低温冰箱。
1.2HBV转染小鼠模型的建立
取4-8周18-19g的C57小鼠5只，采用水动力转染法静脉快速(4～8s)注射含10μgHBV基因组质粒的生理盐水(体积为小鼠体重的10％)，饲养两天待用。
1.3给药方案及血样的制备
将小鼠分为三组，每组三只：阴性对照，给药组，给药超声组。阴性组不做任何处理，给药组小鼠将载siRNA纳米泡经尾静脉注射给药0.1mL。给药超声组在静脉注射载siRNA纳米泡完毕30min后，将给药组小鼠肝部位浸入100W超声波清洗器37℃水浴中连续超声30秒，分别于第五天及第十天眼眶取血0.15mL，静置3小时后于离心机上经5000rpm离心20分钟，用移液器取血清置于0.5mL离心管放置超低温冰箱-70℃，待测。
1.4血清样品的测定
将血清样品用实时荧光定量PCR仪测定含量。
2结果与讨论
2.1HBV转染小鼠血清中HBV DNA含量变化
HBV小鼠血清样品经PCR扩增仪进行扩增，扩增后HBV DNA含量反映药物抑制HBVDNA复制效果。数值越小表明LAPE自组装体抑制HBV DNA复制效果越好。
表2.HBV转染小鼠血液HBV DNA含量
由于PCR扩增仪测定HBV DNA含量会有一定误差，因此未给药的阴性对照组在5天后或10天HBV DNA含量会有所下降和上升，而且HBV DNA有一个自然下降的趋势。由表2可见HBV转染小鼠经给药后血液中HBV DNA含量下降非常明显，超声后由于促进siRNA从纳米泡中释放，转染效果更强。
Claims (4)
1.一种用于siRNA传递的纳米泡载体，其特征是纳米泡由聚酯-聚乙二醇嵌段共聚物和阳离子脂质构成外壳，内部充填气体或在正常体温下转化为气体的液体。
2.如权利要求1所述的用于siRNA传递的纳米泡载体，其特征是聚酯-聚乙二醇嵌段共聚物选自聚乳酸-聚乙二醇嵌段共聚物、聚乳酸羟基乙酸-聚乙二醇嵌段共聚物。
3.如权利要求1所述的用于siRNA传递的纳米泡载体，其特征是气体选自空气、氮气、氧气、二氧化碳、全氟丙烷、全氟丁烷、六氟化硫。
4.如权利要求1所述的用于siRNA传递的纳米泡载体，其特征是在正常体温下转化为气体的液体是全氟戊烷。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103964787A CN102441177A (zh) | 2011-12-05 | 2011-12-05 | 用于siRNA传递的纳米泡载体 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103964787A CN102441177A (zh) | 2011-12-05 | 2011-12-05 | 用于siRNA传递的纳米泡载体 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102441177A true CN102441177A (zh) | 2012-05-09 |
Family
ID=46004432
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103964787A Pending CN102441177A (zh) | 2011-12-05 | 2011-12-05 | 用于siRNA传递的纳米泡载体 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102441177A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI476005B (zh) * | 2013-01-31 | 2015-03-11 | Univ Nat Yang Ming | 具有超音波觸發釋藥功能以及造影功能的奈米級與微米級氣泡 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008148223A1 (en) * | 2007-06-07 | 2008-12-11 | Agriculture And Agri-Food Canada | Nanocarrier based plant transfection and transduction |
| CN101822838A (zh) * | 2009-03-05 | 2010-09-08 | 无锡纳奥生物医药有限公司 | 靶向识别肿瘤细胞的纳米药物载体材料及其制备和应用 |
-
2011
- 2011-12-05 CN CN2011103964787A patent/CN102441177A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008148223A1 (en) * | 2007-06-07 | 2008-12-11 | Agriculture And Agri-Food Canada | Nanocarrier based plant transfection and transduction |
| CN101822838A (zh) * | 2009-03-05 | 2010-09-08 | 无锡纳奥生物医药有限公司 | 靶向识别肿瘤细胞的纳米药物载体材料及其制备和应用 |
Non-Patent Citations (1)
| Title |
|---|
| 王双苗: "新型基因传递系统", 《中国优秀硕士学位论文全文数据库.医药卫生科技辑》, no. 10, 15 October 2011 (2011-10-15), pages 060 - 69 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI476005B (zh) * | 2013-01-31 | 2015-03-11 | Univ Nat Yang Ming | 具有超音波觸發釋藥功能以及造影功能的奈米級與微米級氣泡 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zeng et al. | Current strategies for exosome cargo loading and targeting delivery | |
| Xu et al. | Rational designs of in vivo CRISPR-Cas delivery systems | |
| Han et al. | Overview and update on methods for cargo loading into extracellular vesicles | |
| Pan et al. | Development of a microRNA delivery system based on bacteriophage MS2 virus‐like particles | |
| van Den Berg et al. | Advances with RNAi-based therapy for hepatitis B virus infection | |
| Żak et al. | Lipid nanoparticles for organ-specific mRNA therapeutic delivery | |
| Carmona et al. | Controlling HBV replication in vivo by intravenous administration of triggered PEGylated siRNA-nanoparticles | |
| CN102438978B (zh) | 聚胺衍生物 | |
| Scomparin et al. | Achieving successful delivery of oligonucleotides—From physico-chemical characterization to in vivo evaluation | |
| Tarn et al. | Antisense oligonucleotide-based therapy of viral infections | |
| JP2024507482A (ja) | イオン化可能な脂質分子、その製造方法及び脂質ナノ粒子の製造における応用 | |
| Schlenk et al. | Recent developments and perspectives on gene therapy using synthetic vectors | |
| CN103974719A (zh) | 用于递送免疫原编码rna的聚乙二醇化脂质体 | |
| Baptista et al. | mRNA, a revolution in biomedicine | |
| Neagoe et al. | Efficient siRNA delivery system using carboxilated single-wall carbon nanotubes in cancer treatment | |
| Ivacik et al. | Countering hepatitis B virus infection using RNAi: how far are we from the clinic? | |
| Qin et al. | RGD peptide-based lipids for targeted mRNA delivery and gene editing applications | |
| CN103329852A (zh) | 一种hbv持续性感染及纤维化小鼠模型建立 | |
| Ely et al. | In vitro transcribed mRNA for expression of designer nucleases: Advantages as a novel therapeutic for the management of chronic HBV infection | |
| Peng et al. | Exosome and virus infection | |
| Weinberg et al. | Progress in the use of RNA interference as a therapy for chronic hepatitis B virus infection | |
| Mozaffari et al. | Amphiphilic peptides for efficient siRNA delivery | |
| Fan et al. | Triton X-100-modified adenosine triphosphate-responsive siRNA delivery agent for antitumor therapy | |
| Matsuzaka et al. | Extracellular vesicles as novel drug-delivery systems through intracellular communications | |
| Golubovic et al. | Bioinspired lipid nanocarriers for RNA delivery |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120509 |