基于核酸自组装纳米结构的siRNA药物递送研究进展

赵晓然¹
机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353
×
王晓亮¹
机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353
×
王颖¹
机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353
×
杨亲正¹
机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353
×
汤新景²
机构：北京大学药学院天然药物及仿生药物国家重点实验室，北京 100191
×

1. 齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353； 2. 北京大学药学院天然药物及仿生药物国家重点实验室，北京 100191

中图分类号: Q527,Q522,R34

DOI:10.16476/j.pibb.2019.0003

Research Progress of siRNA Drug Delivery Based on Self-Assembled Nanostructures of Nucleic Acids

ZHAO Xiao-Ran¹
Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
×
WANG Xiao-Liang¹
Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
×
WANG Ying¹
Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
×
YANG Qin-Zheng¹
Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China
×
TANG Xin-Jing²
Affiliation：State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
×

1. State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China； 2. State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

CLC: Q527,Q522,R34

DOI:10.16476/j.pibb.2019.0003

全文
图表
评论
参考文献
作者
出版信息

参考文献 1

WittrupA, LiebermanJ. Knocking down disease: a progress report on siRNA therapeutics. Nature Reviews Genetics, 2015, 16(9): 543-552

参考文献 2

KuS H, JoS D, LeeY K, et al. Chemical and structural modifications of RNAi therapeutics. Advanced Drug Delivery Reviews, 2016, 104(3): 16-28

参考文献 3

HannonG J. RNA interference. Nature, 2002, 418(6894): 244-251

参考文献 4

FireA, XuS, MontgomeryM K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391(6669): 806-811

参考文献 5

HoyS M. Patisiran: first global approval. Drugs, 2018, 78(15): 1625-1631

参考文献 6

WittrupA, LiebermanJ. Knocking down disease: a progress report on siRNA therapeutics. Nature Reviews Genetics, 2015, 16(9): 543-552

参考文献 7

WhiteheadK A, LangerR, AndersonD G. Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery, 2009, 8(2): 129-138

参考文献 8

LiS, HuangL. Nonviral gene therapy: promises and challenges. Gene Therapy, 2000, 7(1): 31-34

参考文献 9

LiuY P, BerkhoutB. miRNA cassettes in viral vectors: problems and solutions. BBA-Gene Regulatory Mechanisms, 2011, 1809(11): 732-745

参考文献 10

DuernerL J, SchwantesA, SchneiderI C, et al. Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue. Gene Therapy, 2008, 15(22): 1500-1510

参考文献 11

DescampsD, BenihoudK. Two key challenges for effective adenovirus-mediated liver gene therapy: innate immune responses and hepatocyte-specific transduction. Current Gene Therapy, 2009, 9(2): 115-127

参考文献 12

BurnettJ C, RossiJ J. RNA-based therapeutics: current progress and future prospects. Chemistry & Biology, 2012, 19(1): 60-71

参考文献 13

BertrandN, LerouxJ C. The journey of a drug-carrier in the body: an anatomo-physiological perspective. Journal of Controlled Release, 2012, 161(2): 152-163

参考文献 14

ZhangS, ZhaoB, JiangH, et al. Cationic lipids and polymers mediated vectors for delivery of siRNA. Journal of Controlled Release, 2007, 123(1): 1-10

参考文献 15

YuT, LiuX, Bolcato-BelleminA L, et al. An amphiphilic dendrimer for effective delivery of small interfering RNA and gene silencing in vitro and in vivo. Angewandte Chemie. International Ed. in English, 2012, 51(34): 8478-8484

参考文献 16

Ornelas-MegiattoC, WichP R, FrechetJ M. Polyphosphonium polymers for siRNA delivery: an efficient and nontoxic alternative to polyammonium carriers. Journal of the American Chemical Society, 2012, 134(4): 1902-1905

参考文献 17

OzpolatB, SoodA K, Lopez-BeresteinG. Liposomal siRNA nanocarriers for cancer therapy. Advanced Drug Delivery Reviews, 2014, 66: 110-116

参考文献 18

AndeyT, MarepallyS, PatelA, et al. Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model. Journal of Controlled Release, 2014, 184(7): 67-78

参考文献 19

LeeS H, ChungB H, ParkT G, et al. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing. Accounts of Chemical Research, 2012, 45(7): 1014-1025

参考文献 20

WightmanL, KircheisR, RösslerV, et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. Journal of Gene Medicine, 2001, 3(4): 362-372

参考文献 21

MuhonenP, TenniläT, AzhayevaE, et al. RNA interference tolerates 2′‐fluoro modifications at the argonaute2 cleavage site. Chemistry & Biodiversity, 2007, 4(5): 858-873

参考文献 22

LungwitzU, BreunigM T, GopferichA. Polyethylenimine-based non-viral gene delivery systems. European Journal of Pharmaceutics & Biopharmaceutics Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E V, 2005, 60(2): 247-266

参考文献 23

JudgeA D, SoodV, ShawJ R, et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature Biotechnology, 2005, 23(4): 457-462

参考文献 24

HwaK S, HoonJ J, Soo HyeonL, et al. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. Journal of Controlled Release Official Journal of the Controlled Release Society, 2008, 129(2): 107-116

参考文献 25

DanielaC, RossiJ J. The promises and pitfalls of RNA-interference-based therapeutics. Nature, 2009, 457(7228): 426-433

参考文献 26

Ballarin-GonzalezB, HowardK A. Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions. Advanced Drug Delivery Reviews, 2012, 64(15): 1717-1729

参考文献 27

JangM, HanH D, AhnH J. A RNA nanotechnology platform for a simultaneous two-in-one siRNA delivery and its application in synergistic RNAi therapy. Scientific Reports, 2016, 6: 32363

参考文献 28

JangM, KimJ H, NamH Y, et al. Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription. Nature Communications, 2015, 6: 7930

参考文献 29

MokH, LeeS H, ParkJ W, et al. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing. Nature Materials, 2010, 9(3): 272-278

参考文献 30

LeeS H, MokH, JoS, et al. Dual gene targeted multimeric siRNA for combinatorial gene silencing. Biomaterials, 2011, 32(9): 2359-2368

参考文献 31

HongC A, LeeS H, KimJ S, et al. Gene silencing by siRNA microhydrogels via polymeric nanoscale condensation. Journal of the American Chemical Society, 2011, 133(35): 13914-13917

参考文献 32

GoldL, JanjicN, JarvisT, et al. Aptamers and the RNA world, past and present. Cold Spring Harbor Perspectives in Biology, 2012, 4(3): 829-841

参考文献 33

ZhouJ, RossiJ. Aptamers as targeted therapeutics: current potential and challenges. Nature Reviews Drug Discovery, 2017, 16(3): 181-202

参考文献 34

JeongH, LeeS H, HwangY, et al. Multivalent aptamer-RNA conjugates for simple and efficient delivery of doxorubicin/siRNA into multidrug-resistant cells. Macromolecular Bioscience, 2017, 17(4), doi: 10.1002/mabi.201600343

参考文献 35

YooH, JungH, KimS A, et al. Multivalent comb-type aptamer-siRNA conjugates for efficient and selective intracellular delivery. Chemical Communications (Camb), 2014, 50(51): 6765-6767

参考文献 36

NakashimaY, AbeN, ItoY, et al. Nanostructured RNAs for RNA interference. Chemical Communications, 2011, 47(29): 8367-8369

参考文献 37

SonS, KimN, YouD G, et al. Antitumor therapeutic application of self-assembled RNAi-AuNP nanoconstructs: combination of VEGF-RNAi and photothermal ablation. Theranostics, 2017, 7(1): 9-22

参考文献 38

Yu-KyoungO, Tae GwanP. siRNA delivery systems for cancer treatment. Advanced Drug Delivery Reviews, 2009, 61(10): 850-862

参考文献 39

WhiteheadK A, RobertL, AndersonD G. Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery, 2009, 8(2): 129-138

参考文献 40

LvH, ZhangS, WangB, et al. Toxicity of cationic lipids and cationic polymers in gene delivery. Journal of Controlled Release Official Journal of the Controlled Release Society, 2006, 114(1): 100-109

参考文献 41

LeeH, Lytton-JeanA K, ChenY, et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nature Nanotechnology, 2012, 7(6): 389-393

参考文献 42

DingF, MouQ, MaY, et al. A crosslinked nucleic acid nanogel for effective siRNA delivery and antitumor therapy. Angewandte Chemie. International Ed. in English, 2018, 57(12): 3064-3068

参考文献 43

GrabowW W, ZakrevskyP, AfoninK A, et al. Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes. Nano Letters, 2011, 11(2): 878-887

参考文献 44

AfoninK A, KireevaM, GrabowW W, et al. Co-transcriptional assembly of chemically modified RNA nanoparticles functionalized with siRNAs. Nano Letters, 2012, 12(10): 5192-5195

参考文献 45

HongC A, JangB, JeongE H, et al. Self-assembled DNA nanostructures prepared by rolling circle amplification for the delivery of siRNA conjugates. Chemical Communications, 2014, 50(86): 13049-13051

参考文献 46

RenK, LiuY, WuJ, et al. A DNA dual lock-and-key strategy for cell-subtype-specific siRNA delivery. Nature Communications, 2016, 7: 13580

参考文献 47

RenK, ZhangY, ZhangX, et al. In situ SiRNA assembly in living cells for gene therapy with microRNA triggered cascade reactions templated by nucleic acids. ACS Nano, 2018, 12(11): 10797-10806

参考文献 48

SeyhanA A, VlassovA V, JohnstonB H. RNA interference from multimeric shRNAs generated by rolling circle transcription. Oligonucleotides, 2006, 16(4): 353-363

参考文献 49

DaubendiekS L, RyanK, KoolE T. Rolling-circle RNA synthesis: circular oligonucleotides as efficient substrates for T7 RNA polymerase. Journal of the American Chemical Society, 1995, 117(29): 7818-7819

参考文献 50

LeeJ B, HongJ, BonnerD K, et al. Self-assembled RNA interference microsponges for efficient siRNA delivery. Nature Materials, 2012, 11(4): 316-322

参考文献 51

RohY H, DengJ Z, DreadenE C, et al. A multi-RNAi microsponge platform for simultaneous controlled delivery of multiple small interfering RNAs. Angewandte Chemie. International Ed. in English, 2016, 55(10): 3347-3351

参考文献 52

LeeJ H, KuS H, KimM J, et al. Rolling circle transcription-based polymeric siRNA nanoparticles for tumor-targeted delivery. Journal of Controlled Release, 2017, 263: 29-38

参考文献 53

HanD, ParkY, NamH, et al. Enzymatic size control of RNA particles using complementary rolling circle transcription (cRCT) method for efficient siRNA production. Chemical Communications, 2014, 50(79): 11665-11667

参考文献 54

HanS, KimH, LeeJ B. Library siRNA-generating RNA nanosponges for gene silencing by complementary rolling circle transcription. Scientific Reports, 2017, 7(1): 10005

参考文献 55

KimH, JeongJ, KimD, et al. Bubbled RNA-based cargo for boosting RNA interference. Advanced Science, 2017, 4(8): 1600523

摘要

RNA干扰(RNA interference,RNAi)作为转录后调节机制，可靶向mRNA进行剪切降解从而发挥基因沉默效应. siRNA(small interference RNA)因其高效性和特异性而被广泛应用于药物研究中. 目前，研究者们已开发了多种阳离子载体用于siRNA递送. 但由于siRNA双链结构具有相对较强的刚性结构，且阴离子电荷密度较低，无法与阳离子载体形成稳定、致密的复合物，使得siRNA的应用仍面临诸多挑战，如细胞摄取率低、靶向特异性差、递送过程不稳定、潜在的细胞毒性以及易诱发免疫反应等. 近年来，核酸自组装纳米结构由于其结构灵活且负电荷密度较高而受到广泛关注，有望实现siRNA药物的高效递送和基因沉默. 本文综述了近年来基于核酸自组装纳米结构的siRNA递送的研究进展及其应用.

Abstract

RNAi (RNA interference), as a post-transcriptional regulatory mechanism, can achieve efficient gene silencing by the degradation of target mRNA. siRNA, a double strand RNA, is widely used in nucleic acid drug development due to its high efficiency and specificity based on RNAi mechanism. Currently, a variety of cationic carriers have been developed for siRNA delivery. However, due to the relatively strong rigid structure and relatively low anionic charge density, it is difficult for siRNA to form a stable and compact complex with cationic carriers. So the application of siRNA still faces many challenges, such as inefficient cellular uptake, a lack of specificity in cells and tissue, poor stability in delivery process, potential cytotoxicity and high initial immune response, etc. In recent years, nucleic acid self-assembled nanoparticles have attracted wide attention due to their flexible structures and high negative charge density, which will be very useful to achieve efficient delivery and gene silencing of siRNA drugs. This review focuses on recent progresses in the development of siRNA self-assembled nanostructures and their potential therapeutic applications.

关键词

RNA干扰；siRNA；基因沉默；核酸自组装；纳米核酸药物

Keywords

RNA interference; siRNA gene silencing; nucleic acid self-assembly; nucleic acid nanoparticle

杨亲正. E-mail: yqz@qlu.edu.cn

汤新景. E-mail: xinjingt@bjmu.edu.cn

siRNA(small interference RNA)是由长的RNA双链经Dicer酶剪切或经人工合成的长度为21~23碱基对的小干扰RNA(图1). 其可与ago2蛋白形成核酸/蛋白质基因沉默复合物(RNA-induced silencing complex，RISC)^[1]. 在启动基因沉默的过程中，siRNA的正义链首先被ago2蛋白剪切，并脱离RISC复合物；而反义链留在复合物中，进一步识别并剪切目标mRNA，实现目标靶基因的沉默^[2,3]. 自从1998年Fire等^[4]首次发现siRNA的基因沉默机制以来，siRNA已成为目前基因功能和核酸药物的研究和应用最为广泛的非编码RNA. 2018年，首款基于RNA干扰的siRNA药物onpattro (patisiran)获得美国FDA批准上市，并用于治疗由遗传性转甲状腺素蛋白淀粉样变性(hereditary transthyretin amyloidosis，hATTR)引起的周围神经疾病(多发性神经疾病，polyneuropathy)^[5]. 开发更有效的siRNA药物，来实现RNAi疗法用于治疗癌症、遗传性疾病和流行性病毒感染等疾病，已成为核酸药物的重要研究方向.

图1
siRNA基因沉默机制[6]

图1 siRNA基因沉默机制^[6]

Fig. 1 Gene silencing mechanism of siRNA^[6]

siRNA药物的研发中，高效递送系统的构建是其发挥沉默活性的关键^[7,8]. 目前常见的siRNA递送载体主要包括病毒载体^[9,10,11]和阳离子载体^{[12,13,14,15,16,17]}两大类. 病毒载体具有很高的转染效率，可以将shRNA表达质粒插入到细胞基因组中，实现长时间稳定表达shRNA. 但病毒转录系统的突变风险大、初始免疫应答高、组织分布无特异性且容易引发炎症. 阳离子载体包括带正电荷的肽、脂质体和聚合物(如PEG、壳聚糖、PEI、环糊精等)等. 通过静电电荷相互作用将siRNA包载制成致密纳米粒子，促进细胞对siRNA药物的摄取^[12]. 但siRNA链很短，导致其刚性结构较强且带负电荷较弱，与阳离子载体的结合不够稳定^[18,19]，从而需要更高浓度的siRNA药物或者更大的siRNA药物载体的量^[20,21,22]，因此存在较高的细胞毒性^{[23,24,25,26]}.

近年来，核酸自组装纳米载体改善了siRNA刚性结构强的问题，增大siRNA聚合物的负电荷，可与阳离子载体紧凑、稳定地结合. 同时，核酸自组装纳米载体极大地提高了siRNA的装载量. 在最新的研究中，核酸自组装纳米载体上进一步修饰核酸适配体、叶酸等靶向配体^[27,28]，可以不再借助阳离子载体，实现siRNA的高效递送和特异性基因沉默. 核酸自组装纳米递送载体包括三种组装策略：一是siRNA之间偶联自组装形成负电荷密度较高的siRNA多聚体. 二是由寡核苷酸自组装构建载体，并通过与siRNA药物杂交形成纳米核酸药物. 相比siRNA直接偶联自组装，此策略能够更加精准地控制siRNA纳米粒的形状、尺寸及表面性质，且大多不借助阳离子载体即可被细胞摄取. 三是设计包含有siRNA模板序列的环状DNA，通过滚环转录技术形成Poly-RNA. Poly-RNA再进一步自组装成RNA纳米微球. 相比前两者，该策略大大提高了siRNA的装载量，降低了由于阳离子载体和siRNA药物过量使用而引起的细胞毒性；同时紧凑致密的纳米结构增强了siRNA的核酸酶稳定性. 本文综述了上述三种基于核酸自组装纳米结构的siRNA递送平台、结构特征、递送机制及其应用.

1　基于核酸自组装的siRNA纳米递送平台
1.1　siRNA之间的偶联自组装
此策略是指siRNA之间通过简单的碱基互补配对、巯基化修饰或金纳米粒修饰自组装成高分子质量的聚合物. 该聚合物具有大的负电荷密度，仅需要少量的阳离子载体或弱阳离子载体便可形成更加致密、稳定的纳米粒复合物，有效克服血清降解，提高细胞摄取效率和基因沉默效率.
1.1.1　化学键交联的siRNA自组装
siRNA两条链的3′端或5′端的巯基化修饰策略已被广泛应用于siRNA偶联自组装研究当中. 2010年，Mok等^[29](图2a)首先在siRNA正义链和反义链的3′端分别进行巯基化修饰，再通过与交联剂作用首次合成具有可断裂二硫键和不可断裂键的化学交联siRNA自组装线性结构. 可断裂二硫键可在细胞质的还原性环境中发生断裂，解离出单个siRNA；而不可断裂交联剂修饰的siRNA需要通过Dicer酶的剪切得到单个siRNA. 与裸siRNA相比，这种通过硫醇基团-交联剂反应聚合构建的长链dsRNA可与阳离子载体缩合成更加致密稳定的聚合物. 2011年，Lee等^[30]又在上述研究成果的基础上构建了绿色荧光蛋白(green fluorescent protein，GFP)和血管内皮生长因子(vascular endothelial growth factor，VEGF)双基因靶向siRNA自组装结构. 研究发现，通过可断裂二硫键交联得到的双基因靶向siRNA多聚体比物理混合两种siRNA多聚体显示出更强的抑制作用，具有明显的协同沉默效应，可用于同时沉默多个上调基因.
图2 化学键交联的siRNA自组装
Fig. 2 Self assembly of siRNA based on chemical crosslinking
注：(a)具有可断裂、不可断裂交联臂的siRNA自组装纳米结构^[29]；(b)高度网络化的siRNA自组装微孔纳米结构^[31].
Hong等^[31](图2b)首次利用巯基化修饰构建了一种高度网络化的siRNA自组装纳米微孔水凝胶. 首先在siRNA正义链和反义链的3′端分别进行巯基化修饰，再与交联剂作用，形成单链线型和Y型结构的siRNA聚合物. 随后通过碱基互补配对构建出多种高负电荷密度的siRNA微孔纳米结构，仅利用弱阳离子聚合物LPEI(MW. 2 500)即可将siRNA纳米凝胶缩合成结构致密的siRNA纳米药物. 该siRNA纳米药物细胞毒性相对较小、免疫应答较低、且具有良好的细胞摄取效率和基因沉默活性.
siRNA自组装多聚体虽然提高了血清稳定性和细胞摄取能力，但缺乏靶向特异性和选择性. 为解决这一问题，研究人员将具有特定三级结构并且可与靶标分子特异性结合的核酸适配体与siRNA缀合^[32,33,34]. 2014年Yoo等^[35](图3a)构建了一种MUC1核酸适配体-siRNA梳状结构. 即在siRNA反义链的5′和3′修饰硫醇基团，通过硫醇-马来酰亚胺反应，将多条反义链的5′端和3′端偶联. 同时，siRNA的正义链缀合MUC1适配体序列，通过正义链与反义链互补杂交，构建由多个MUC1适配体修饰的多聚siRNA梳状结构. 在细胞质中的还原性环境作用下，二硫键发生断裂并释放siRNA，进而发挥siRNA的靶基因沉默活性. MUC1核酸适配体靶向恶性腺癌细胞中高表达的MUC1蛋白，与siRNA缀合可实现细胞的靶向性给药治疗. 而梳状结构增加了适配体与靶蛋白的接触机会，促进siRNA药物的摄取，同时减少阳离子载体的使用，大大降低了细胞毒性和免疫应答.
图3 siRNA之间偶联自组装
Fig. 3 Self assembly of siRNA
注：(a)核酸适配体-siRNA梳状结构^[35]；(b)通过碱基互补配对制备siRNA自组装多聚体^[36]；(c)金纳米粒介导的siRNA自组装纳米结构^[37].
1.1.2　碱基互补配对的自组装
Nakashima等^[36](图3b)利用RNA寡聚核苷酸单链，通过碱基间的互补配对组装成Y型三聚体RNA和十字形的四聚体RNA. 通过Dicer酶的剪切作用，缓慢释放出siRNA. 由于这种多聚RNA分子具有多个分支，可以同时靶向不同基因的siRNA，并且可以精准控制几种siRNA的比例. 分支状多聚RNA结构的致密性可有效保护siRNA不被核酸酶降解，提高了siRNA的稳定性. 并且相比于线性自组装siRNA，分枝状多聚RNA结构被Dicer酶降解的速率更慢，HeLa细胞中的基因沉默活性评价表明，十字形四聚体RNA在5天内仍具有很强的沉默效果.
1.1.3　基于金纳米粒的siRNA自组装
金纳米粒具有独特的光学性质、良好的生物相容性、易于表面功能化等优势，已被用于核酸递送以及光热治疗. 2017年，Son等^[37](图3c)提出了基于金纳米粒的siRNA自组装技术. 首先将一定数量的抗血管内皮生长因子(VEGF)的siRNA正义链/反义链与金纳米粒结合，然后通过碱基互补配对将RNAi-AuNP组装成多种几何纳米结构. 借助聚乙烯亚胺(PEI)对RNAi-AuNPs复合物进一步包载，形成更加致密的纳米簇形式，将siRNA的抑制血管生成作用和AuNP的光热效应相结合. PC-3荷瘤小鼠体内实验表明，抗VEGF的siRNA在肿瘤初期抑制了血管的生成，AUNP光热活性进一步使残存肿瘤完全消融.
1.2　siRNA的DNA/RNA载体的自组装
传统的阳离子载体与siRNA之间非特异性的电荷相互作用存在诸多缺点，粒子形状、大小、表面化学性质不均一，因此在体内存在多种生物分布和药代动力学，血液半衰期短以及缺乏体内和体外研究之间的相关性^[38,39,40]. 为了克服目前阳离子载体存在的问题，研究者们利用寡核苷酸通过化学合成法和酶促合成法制备的纳米粒作为载体，再通过与siRNA杂化构建siRNA纳米药物. 此策略利用碱基序列的可编程性精准控制纳米粒子大小和结构，从而改善siRNA纳米药物的组织特异性和细胞毒性.
1.2.1　化学合成法
2012年，Lee等^[41](图4a)报道了利用四面体DNA纳米结构(oligonucleotide nanoparticles, ONPs)靶向递送siRNA的活性研究. 该纳米结构由6条DNA链和siRNA自组装而成. 由于ONPs表面带有强烈的负电荷而无法入膜，因此利用叶酸(FA)修饰来促进纳米粒的细胞摄取，且不需要阳离子转染试剂，便可实现KB异种移植瘤内靶基因的沉默，而未组装成纳米结构的裸siRNA因无法进入KB细胞而无任何基因沉默活性. 同时，研究者可通过精准控制该核酸纳米粒子的尺寸及靶向配体(如多肽和叶酸)的密度和空间取向来提高siRNA的细胞摄取率和基因沉默活性.
图4 基于化学合成法制备siRNA纳米递送载体
Fig. 4 Preparation of siRNA nanometer delivery carrier based on chemical synthesis
注：(a)通过化学合成法制备的寡核苷酸与siRNA自组装成四面体DNA/RNA纳米结构^[41]；(b)siRNA与梳状DNA-聚己内酯自组装形成球状微水凝胶^[42].
然而，以往的研究中，siRNA通常暴露在纳米结构之外，容易被酶降解. 受阳离子载体能够压缩和保护载体内siRNA这一功能的启发，Ding等^[42](图4b)构建了一种梳状DNA-聚己内脂包裹siRNA的核酸自组装纳米结构. 首先通过二苯并环辛烷基修饰的DNA链(DBCO-DNA)与叠氮修饰的聚己内酯，在二甲基亚砜中的无铜点击反应构建了一种DNA-聚己内酯梳状结构(DNA-grafted polycaprolactone，DNA-g-PCL). 同时，在siRNA序列两侧设计2条可与DNA-g-PCL上的DNA互补的悬垂单链. 通过碱基配对，siRNA与DNA-g-PCL自组装成尺寸大小可控的纳米凝胶结构. 这种纳米凝胶具有良好的稳定性和高抗核酸酶降解能力，增强细胞摄取效率，能够更有效地将siRNA递送至肿瘤部位，并且在体内和体外均表现良好的靶基因沉默作用.
1.2.2　酶促合成法
寡核苷酸纳米结构的组装需要多条寡核苷酸链，除了用化学合成法之外，还可以用酶促的方法合成. 酶促法相对化学合成法，制备成本低、制备过程稳定. 2011年，Grabow等^[43](图5a)利用酶促反应通过体外转录产生长链RNA后折叠吻合制备六边形siRNA纳米结构. 此类结构的优势在于结构单元和折叠方式简单，在较低RNA组装浓度下即可具有良好的热力学稳定性，且结构灵活，可控制多种siRNA的结合. 2012年，Afonin等^[44]在前期研究的基础上，利用2΄-F-dUMPs对RNA链进行修饰，进一步提高了血清稳定性和核酸酶稳定性. 通过在转录体系中额外加入Mn²⁺，优化了转录和自组装条件. 所获得的siRNA纳米粒在MDA-MB-231细胞中具有良好的GFP基因沉默效果.
图5 基于酶促合成法制备siRNA纳米递送载体
Fig. 5 Preparation of siRNA nanometer delivery carrier based on enzymic synthesis
注：(a)体外转录产生的poly-RNA与6条siRNA自组装成稳定的纳米环^[43]；(b)基于滚环扩增技术的DNA/siRNA自组装Y型纳米结构^[45].
滚环扩增(rolling circle amplification, RCA)是酶法制备DNA纳米递送载体的另一实例. 2014年，Hong等^[45](图5b)报道了基于RCA法制备DNA纳米载体用于siRNA递送的研究. 首先设计了一种包含3个发卡结构的Y型DNA模板，在Phi 29 DNA聚合酶催化下，扩增出包含200多个Y型模板单元的DNA长链. 随后利用PstⅠ酶对扩增序列的特定位点进行剪切生产DNA片段，然后这些片段DNA经过退火自组装成Y形DNA纳米结构. Y形纳米结构的每个臂有一段悬垂序列，可与叶酸修饰的siRNAs杂交. KB细胞中的活性评价显示，Y-FA-siRNA纳米结构增强了siRNA的递送效率，并具有剂量依赖性的基因沉默效应，而FA-siRNA没有任何沉默效果.
siRNA的靶向选择性和特异性是成功实现siRNA核酸药物治疗的关键，也是目前RNAi治疗面临的重要问题. 为解决这一问题，Ren等^[46](图6a)基于滚环扩增技术，设计出了一种“智能锁钥”机制来递送siRNA，且对人白血病细胞(CSM细胞)具有靶向特异性. 首先“三角梯”DNA结构单元(triangular rung unit，TRU)与siRNA杂交形成siRNA-TRU结构，然后再与滚环扩增产生的含有MNAzyme特异性酶切位点的长链DNA自组装形成寡核苷酸纳米载体(oligonucleotide nano vehicle,ONV). 具有MNAzyme核酸酶活性的sgc4f和可靶向酪氨酸激酶7受体(RTK7)的sgc8c两种核酸适配体作为siRNA递送系统的“锁”，ONV作为递送系统的“钥匙”，平均长度0.6 μm，能装载约84条siRNA. 当sgc8c和sgc4f同时存在时，siRNA-ONV可与CEM细胞特异性结合. siRNA-ONV“锁钥”机制为RNAi疗法的开发提供了新的思路.
图6 靶向特异性siRNA纳米递送载体
Fig. 6 Target specific siRNA assembled nanometer carrier
注：(a)基于“锁钥”机制的siRNA纳米载体；(b)基于microRNA引发的原位链置换级联反应的siRNA纳米载体^[46,47].
2018年，Ren等^[47](图6b)又设计了一种DNA/RNA自组装(DNA nanomachine，DNM)siRNA纳米递送平台. 首先ssDNA (D/D′)与特异性RNA (R/R′)杂交生成具有凸起结构的DR、D′R′，在滚环扩增形成的DNA支架上相邻排列形成DNM平台. 随后DNM与叶酸修饰的PEI形成稳定的纳米复合物，促进靶细胞的特异性摄取，并协助DNM从溶酶体逃逸至细胞质中. 细胞质内的miR-21与特异性R链杂交，打开凸起环，触发DR和D′R′之间的链置换级联反应，释放出siRNA(RR′)和miR-21. 释放出的miR-21触发原位的链置换级联反应连续释放活性siRNA. 该平台在时间和空间上都具有精确的可控性，siRNA生成效率高，递送过程稳定且具有良好的原位特异性. 研究人员以血管内皮生长因子(VEGF)为沉默靶点研究DNM平台的基因治疗效果. 结果表明，DNM平台对VEGF的mRNA水平和蛋白质表达水平均有良好的基因沉默作用，可有效抑制体内肿瘤的增殖.
1.3　Poly-RNA的自组装
近年来，基于poly-RNA自组装的siRNA递送系统吸引着越来越多研究者的注意. Poly-RNA是指环状DNA在RNA聚合酶的催化下，通过滚环转录技术(rolling circle transcription,RCT)^[48,49]合成重复shRNA序列的串联长RNA单链. RNA长链折叠并组装到焦磷酸镁表面，形成紧密的海绵微球结构. 此微球结构包含多达几十万拷贝的siRNA单元. 相比前两种自组装策略，该策略的siRNA装载量进一步提高，且致密的海绵微球结构提高了siRNA的核酸酶稳定性和血清稳定性，有效的降低了脱靶效应和细胞毒性.
1.3.1　滚环转录
2012年，Lee等^[50](图7a)首次利用滚环转录技术制备了RNA的海绵微球结构，并在表面包载一层带正电的聚乙烯亚胺(PEI)，球体从2 μm被进一步压缩至200 nm，从而可被细胞更有效地摄取. 被PEI包载后的纳米微球结构致密，可防止内部siRNA的核酸酶降解. 当纳米球进入细胞后，细胞质中的Dicer酶将shRNA剪切成siRNA，从而发挥siRNA对靶基因的沉默功能. 2016年，该课题组^[51]基于RCT技术，构建出多靶向RNAi海绵微球结构. 研究人员通过控制环状DNA模板的类型和比例，利用RCT技术，将靶向不同基因poly-RNA以精确的比例自组装成海绵微球结构，随后利用PEI将RNAi微球压缩成更加致密的纳米粒子，进而被细胞摄取. 多序列的siRNA微海绵球能够同时递送多种siRNA，因此可实现多个基因同时沉默.
图7 滚环转录法制备siRNA海绵微球纳米平台
Fig. 7 Preparation of siRNA microsponge platform based on RCT method
注：(a)基于滚环转录的自组装RNAi微球纳米结构^[50]；(b)胆固醇和叶酸修饰siRNA海绵微球纳米结构^[28].
2015年，Jang等^[28](图7b)在RCT技术的基础上，先后引入分别由胆固醇修饰和叶酸修饰的DNA与poly-RNA杂交配对. 胆固醇修饰的DNA通过疏水性缩合作用，将RNA微球压缩成更加致密、紧凑的纳米粒. 叶酸修饰的DNA使纳米粒与叶酸受体阳性细胞具有较高的结合效率，提高其细胞靶向性和选择特异性. 该siRNA纳米平台的递送过程不再借助阳离子载体，降低了免疫反应与细胞毒性. 研究表明，该siRNA纳米平台在SKOV3-RFP细胞和SKOV3-RFP肿瘤异种移植小鼠体内均有明显的靶向RFP基因沉默效应. 2016年，该课题组^[27]基于上述研究结果，进一步构建了与叶酸-DNA-胆固醇杂交的双靶向RNA纳米微球. 纳米微球结构中包含2种siRNA，通过叶酸-DNA-胆固醇对纳米粒进一步压缩，无需借助阳离子载体就可实现2种mRNA的有效沉默，且具有良好的协同作用.
2017年，Lee等^[52]利用RCT技术合成了重复串联siRNA反义链的极长RNA，然后与DNA正义链通过退火进行杂交，形成RAPSI (RCT and annealing-generated polymeric siRNA)纳米花结构，再利用巯基化乙二醇壳聚糖(thiol-modified glycol chitosan，TGC)进行包载压缩. RAPSI/tGC进入细胞后，被细胞质中的RNase H酶识别剪切，活性siRNA被释放，靶向沉默PC-3细胞中VEGF基因的表达. RAPSI/Tgc具有较好的核酸酶稳定性和抗阴离子竞争的能力. 同时，巯基化乙二醇壳聚糖的包载增强了纳米粒的肿瘤靶向能力.
1.3.2　互补滚环转录
互补滚环转录(cRCT)是研究者们对RNA滚环转录技术的延伸. 2014年，Han等^[53](图8a)首次提出互补滚环转录法. 此方法是利用两个互补环状DNA模板，通过体外滚环转录得到poly-RNA. poly-RNA再通过进一步碱基互补配对自组装成海绵微球结构. 海绵微球在阳离子载体辅助下被递送进细胞，并被Dicer酶进一步剪切成活性siRNA，进而发挥基因沉默效果. 与前面提到的海绵微球结构有所不同的是，通过cRCT技术得到的海绵微球结构能够通过控制T7 RNA聚合酶的浓度来调节微球的大小. 当RNA聚合酶的浓度从1 U/μl增加到40 U/μl时，微球的粒径可从5 μm缩小到400 nm. 2017年，Han等^[54](图8b)进一步优化转录体系中DNA模板与RNA聚合酶的比例，实现了cRCT技术产生的siRNA纳米粒对CCK-8中GFP基因的沉默效应. 研究发现，当DNA模板与RNA聚合酶的比例为0.5 μmol·L^-1/RP80时，形成的RNA纳米粒最小. 纳米粒通过阳离子脂质体递送到细胞中，能够对GFP基因产生一定的沉默效果，且无细胞毒性. Kim等^[55](图8c)在前面工作的基础上又制备了鼓泡RNA纳米微球(BRC)，即2个poly-RNA存在不完全配对序列，引入多个双切割位点，在Dicer酶的作用下释放功能性siRNA，同时避免非功能性短dsRNA的产生. 同样通过调整模板DNA与T7RNA聚合酶的比例，合理控制纳米粒的大小. BRCs被证实在体内和体外均有良好的GFP基因沉默活性.
图8 互补滚环转录法制备siRNA海绵微球纳米结构
Fig. 8 Preparation of siRNA nanosponges using cRCT method
注：(a)通过控制T7 RNA聚合酶的浓度来调节微球的大小^[53]；(b)siRNA纳米微球文库^[54]；(c)鼓泡型RNA纳米微球^[55].
2　总结与展望
RNA干扰技术的发现被认为是分子医学领域最令人兴奋的重大突破之一. 这种由siRNA形成的RISC复合物触发的靶基因转录后调节机制，可以靶向特定mRNA进行剪切降解，从而发挥基因沉默效果. 理论上，几乎所有的基因都可被siRNA所调控. siRNA在哺乳动物癌症和遗传病等基因相关疾病的功能研究及治疗方面具有广阔的前景. 开发一种安全、高效的siRNA递送系统仍然是临床应用所面临的关键问题. 过去几年，研究者在siRNA递送和基因沉默研究方面取得了重大进展，相继报道了多种不同类型的载体用于siRNA的有效递送，但同时存在着许多挑战，包括细胞摄取效率低、靶向性差、易引发脱靶效应、细胞毒性和免疫反应. 因此，改善siRNA的递送问题是实现其高效基因沉默的关键.
核酸自组装纳米载体具有极强的结构灵活性，通过核苷酸之间多种组合和排列顺序，依据化学交联和碱基互补配对方式可人为设计多种不同的RNA纳米结构，并赋予其多种特殊的功能. 同时，相比天然siRNA，核酸自组装纳米结构负电荷密度更强，仅与弱阳性离子载体即可形成更致密、稳定的纳米复合物，甚至可以避免阳离子载体的使用. 因此，核酸自组装纳米结构递送系统可有效克服siRNA刚性结构较强和负电荷密度较低等的物理缺陷，提高siRNA递送效率和基因沉默效率，减弱细胞毒性和免疫反应.
尽管核酸自组装纳米递送系统取得了较好的进展，但其临床应用仍有待解决的问题. 例如siRNA的溶酶体逃逸问题，需要研究不同核酸纳米结构的溶酶体逃逸机制，使siRNA能够有效的从溶酶体释放到细胞质中，进而发挥其基因沉默效果. 同时，自组装siRNA纳米药物的体内循环、肝拦截和代谢、以及靶向富集等方面有待深入研究. 核酸自组装纳米系统在生物医学领域的应用才刚刚起步，作为新一代siRNA递送载体，核酸自组装纳米系统在RNA干扰疗法领域将具有广阔的应用前景.
YANG Qin-Zheng. E-mail: yqz@qlu.edu.cn
TANG Xin-Jing. E-mail: xinjingt@bjmu.edu.cn
参考文献
- 1
  Wittrup A, Lieberman J. Knocking down disease: a progress report on siRNA therapeutics. Nature Reviews Genetics, 2015, 16(9): 543-552
- 2
  Ku S H, Jo S D, Lee Y K, et al. Chemical and structural modifications of RNAi therapeutics. Advanced Drug Delivery Reviews, 2016, 104(3): 16-28
- 3
  Hannon G J. RNA interference. Nature, 2002, 418(6894): 244-251
- 4
  Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391(6669): 806-811
- 5
  Hoy S M. Patisiran: first global approval. Drugs, 2018, 78(15): 1625-1631
- 6
  Wittrup A, Lieberman J. Knocking down disease: a progress report on siRNA therapeutics. Nature Reviews Genetics, 2015, 16(9): 543-552
- 7
  Whitehead K A, Langer R, Anderson D G. Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery, 2009, 8(2): 129-138
- 8
  Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Therapy, 2000, 7(1): 31-34
- 9
  Liu Y P, Berkhout B. miRNA cassettes in viral vectors: problems and solutions. BBA-Gene Regulatory Mechanisms, 2011, 1809(11): 732-745
- 10
  Duerner L J, Schwantes A, Schneider I C, et al. Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue. Gene Therapy, 2008, 15(22): 1500-1510
- 11
  Descamps D, Benihoud K. Two key challenges for effective adenovirus-mediated liver gene therapy: innate immune responses and hepatocyte-specific transduction. Current Gene Therapy, 2009, 9(2): 115-127
- 12
  Burnett J C, Rossi J J. RNA-based therapeutics: current progress and future prospects. Chemistry & Biology, 2012, 19(1): 60-71
- 13
  Bertrand N, Leroux J C. The journey of a drug-carrier in the body: an anatomo-physiological perspective. Journal of Controlled Release, 2012, 161(2): 152-163
- 14
  Zhang S, Zhao B, Jiang H, et al. Cationic lipids and polymers mediated vectors for delivery of siRNA. Journal of Controlled Release, 2007, 123(1): 1-10
- 15
  Yu T, Liu X, Bolcato-Bellemin A L, et al. An amphiphilic dendrimer for effective delivery of small interfering RNA and gene silencing in vitro and in vivo. Angewandte Chemie. International Ed. in English, 2012, 51(34): 8478-8484
- 16
  Ornelas-Megiatto C, Wich P R, Frechet J M. Polyphosphonium polymers for siRNA delivery: an efficient and nontoxic alternative to polyammonium carriers. Journal of the American Chemical Society, 2012, 134(4): 1902-1905
- 17
  Ozpolat B, Sood A K, Lopez-Berestein G. Liposomal siRNA nanocarriers for cancer therapy. Advanced Drug Delivery Reviews, 2014, 66: 110-116
- 18
  Andey T, Marepally S, Patel A, et al. Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model. Journal of Controlled Release, 2014, 184(7): 67-78
- 19
  Lee S H, Chung B H, Park T G, et al. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing. Accounts of Chemical Research, 2012, 45(7): 1014-1025
- 20
  Wightman L, Kircheis R, Rössler V, et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. Journal of Gene Medicine, 2001, 3(4): 362-372
- 21
  Muhonen P, Tennilä T, Azhayeva E, et al. RNA interference tolerates 2′‐fluoro modifications at the argonaute2 cleavage site. Chemistry & Biodiversity, 2007, 4(5): 858-873
- 22
  Lungwitz U, Breunig M T, Gopferich A. Polyethylenimine-based non-viral gene delivery systems. European Journal of Pharmaceutics & Biopharmaceutics Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E V, 2005, 60(2): 247-266
- 23
  Judge A D, Sood V, Shaw J R, et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature Biotechnology, 2005, 23(4): 457-462
- 24
  Hwa K S, Hoon J J, Soo Hyeon L, et al. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. Journal of Controlled Release Official Journal of the Controlled Release Society, 2008, 129(2): 107-116
- 25
  Daniela C, Rossi J J. The promises and pitfalls of RNA-interference-based therapeutics. Nature, 2009, 457(7228): 426-433
- 26
  Ballarin-Gonzalez B, Howard K A. Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions. Advanced Drug Delivery Reviews, 2012, 64(15): 1717-1729
- 27
  Jang M, Han H D, Ahn H J. A RNA nanotechnology platform for a simultaneous two-in-one siRNA delivery and its application in synergistic RNAi therapy. Scientific Reports, 2016, 6: 32363
- 28
  Jang M, Kim J H, Nam H Y, et al. Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription. Nature Communications, 2015, 6: 7930
- 29
  Mok H, Lee S H, Park J W, et al. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing. Nature Materials, 2010, 9(3): 272-278
- 30
  Lee S H, Mok H, Jo S, et al. Dual gene targeted multimeric siRNA for combinatorial gene silencing. Biomaterials, 2011, 32(9): 2359-2368
- 31
  Hong C A, Lee S H, Kim J S, et al. Gene silencing by siRNA microhydrogels via polymeric nanoscale condensation. Journal of the American Chemical Society, 2011, 133(35): 13914-13917
- 32
  Gold L, Janjic N, Jarvis T, et al. Aptamers and the RNA world, past and present. Cold Spring Harbor Perspectives in Biology, 2012, 4(3): 829-841
- 33
  Zhou J, Rossi J. Aptamers as targeted therapeutics: current potential and challenges. Nature Reviews Drug Discovery, 2017, 16(3): 181-202
- 34
  Jeong H, Lee S H, Hwang Y, et al. Multivalent aptamer-RNA conjugates for simple and efficient delivery of doxorubicin/siRNA into multidrug-resistant cells. Macromolecular Bioscience, 2017, 17(4), doi: 10.1002/mabi.201600343
- 35
  Yoo H, Jung H, Kim S A, et al. Multivalent comb-type aptamer-siRNA conjugates for efficient and selective intracellular delivery. Chemical Communications (Camb), 2014, 50(51): 6765-6767
- 36
  Nakashima Y, Abe N, Ito Y, et al. Nanostructured RNAs for RNA interference. Chemical Communications, 2011, 47(29): 8367-8369
- 37
  Son S, Kim N, You D G, et al. Antitumor therapeutic application of self-assembled RNAi-AuNP nanoconstructs: combination of VEGF-RNAi and photothermal ablation. Theranostics, 2017, 7(1): 9-22
- 38
  Yu-Kyoung O, Tae Gwan P. siRNA delivery systems for cancer treatment. Advanced Drug Delivery Reviews, 2009, 61(10): 850-862
- 39
  Whitehead K A, Robert L, Anderson D G. Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery, 2009, 8(2): 129-138
- 40
  Lv H, Zhang S, Wang B, et al. Toxicity of cationic lipids and cationic polymers in gene delivery. Journal of Controlled Release Official Journal of the Controlled Release Society, 2006, 114(1): 100-109
- 41
  Lee H, Lytton-Jean A K, Chen Y, et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nature Nanotechnology, 2012, 7(6): 389-393
- 42
  Ding F, Mou Q, Ma Y, et al. A crosslinked nucleic acid nanogel for effective siRNA delivery and antitumor therapy. Angewandte Chemie. International Ed. in English, 2018, 57(12): 3064-3068
- 43
  Grabow W W, Zakrevsky P, Afonin K A, et al. Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes. Nano Letters, 2011, 11(2): 878-887
- 44
  Afonin K A, Kireeva M, Grabow W W, et al. Co-transcriptional assembly of chemically modified RNA nanoparticles functionalized with siRNAs. Nano Letters, 2012, 12(10): 5192-5195
- 45
  Hong C A, Jang B, Jeong E H, et al. Self-assembled DNA nanostructures prepared by rolling circle amplification for the delivery of siRNA conjugates. Chemical Communications, 2014, 50(86): 13049-13051
- 46
  Ren K, Liu Y, Wu J, et al. A DNA dual lock-and-key strategy for cell-subtype-specific siRNA delivery. Nature Communications, 2016, 7: 13580
- 47
  Ren K, Zhang Y, Zhang X, et al. In situ SiRNA assembly in living cells for gene therapy with microRNA triggered cascade reactions templated by nucleic acids. ACS Nano, 2018, 12(11): 10797-10806
- 48
  Seyhan A A, Vlassov A V, Johnston B H. RNA interference from multimeric shRNAs generated by rolling circle transcription. Oligonucleotides, 2006, 16(4): 353-363
- 49
  Daubendiek S L, Ryan K, Kool E T. Rolling-circle RNA synthesis: circular oligonucleotides as efficient substrates for T7 RNA polymerase. Journal of the American Chemical Society, 1995, 117(29): 7818-7819
- 50
  Lee J B, Hong J, Bonner D K, et al. Self-assembled RNA interference microsponges for efficient siRNA delivery. Nature Materials, 2012, 11(4): 316-322
- 51
  Roh Y H, Deng J Z, Dreaden E C, et al. A multi-RNAi microsponge platform for simultaneous controlled delivery of multiple small interfering RNAs. Angewandte Chemie. International Ed. in English, 2016, 55(10): 3347-3351
- 52
  Lee J H, Ku S H, Kim M J, et al. Rolling circle transcription-based polymeric siRNA nanoparticles for tumor-targeted delivery. Journal of Controlled Release, 2017, 263: 29-38
- 53
  Han D, Park Y, Nam H, et al. Enzymatic size control of RNA particles using complementary rolling circle transcription (cRCT) method for efficient siRNA production. Chemical Communications, 2014, 50(79): 11665-11667
- 54
  Han S, Kim H, Lee J B. Library siRNA-generating RNA nanosponges for gene silencing by complementary rolling circle transcription. Scientific Reports, 2017, 7(1): 10005
- 55
  Kim H, Jeong J, Kim D, et al. Bubbled RNA-based cargo for boosting RNA interference. Advanced Science, 2017, 4(8): 1600523

基本信息

DOI:10.16476/j.pibb.2019.0003

中图分类号: Q527,Q522,R34

引用信息

赵晓然,王晓亮,王颖等.基于核酸自组装纳米结构的siRNA药物递送研究进展[J].生物化学与生物物理进展,2019,46(06):533-544.

ZHAO Xiao-Ran,WANG Xiao-Liang,WANG Ying,et al.Research Progress of siRNA Drug Delivery Based on Self-Assembled Nanostructures of Nucleic Acids[J].Progress in Biochemistry and Biophysics,2019,46(06):533-544.

基金信息

国家自然科学基金（21877001、81821004）和科技部国家重大新药创制项目（2017ZX09303013）资助.

This work was supported by grants from The National Natural Science Foundation of China(21877001, 81821004), National Major Scientific and Technological Special Project for “Significant New Drug Development”(2017ZX09303013).

稿件历史

纸刊出版 : 2019-06-20
收稿日期 : 2019-01-03
录用日期 : 2019-04-04

赵晓然

机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353

Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China

王晓亮

机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353

Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China

王颖

机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353

Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China

杨亲正

机构：齐鲁工业大学(山东省科学院)生物工程学院生物基材料与绿色造纸国家重点实验室，济南 250353

Affiliation：State Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China

汤新景

机构：北京大学药学院天然药物及仿生药物国家重点实验室，北京 100191

Affiliation：State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F001.png

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F002.png

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F003.png

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F004.png

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F005.png

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F006.png

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F007.png

html/pibbcn/20190003/alternativeImage/5cdf017f-8352-41b7-afab-a2ab56103cf1-F008.png

图1 siRNA基因沉默机制^[6]

Fig. 1 Gene silencing mechanism of siRNA^[6]

图2 化学键交联的siRNA自组装

Fig. 2 Self assembly of siRNA based on chemical crosslinking

图3 siRNA之间偶联自组装

Fig. 3 Self assembly of siRNA

图4 基于化学合成法制备siRNA纳米递送载体

Fig. 4 Preparation of siRNA nanometer delivery carrier based on chemical synthesis

图5 基于酶促合成法制备siRNA纳米递送载体

Fig. 5 Preparation of siRNA nanometer delivery carrier based on enzymic synthesis

图6 靶向特异性siRNA纳米递送载体

Fig. 6 Target specific siRNA assembled nanometer carrier

图7 滚环转录法制备siRNA海绵微球纳米平台

Fig. 7 Preparation of siRNA microsponge platform based on RCT method

图8 互补滚环转录法制备siRNA海绵微球纳米结构

Fig. 8 Preparation of siRNA nanosponges using cRCT method



image /

无注解

(a)具有可断裂、不可断裂交联臂的siRNA自组装纳米结构^[29]；(b)高度网络化的siRNA自组装微孔纳米结构^[31].

(a)核酸适配体-siRNA梳状结构^[35]；(b)通过碱基互补配对制备siRNA自组装多聚体^[36]；(c)金纳米粒介导的siRNA自组装纳米结构^[37].

(a)通过化学合成法制备的寡核苷酸与siRNA自组装成四面体DNA/RNA纳米结构^[41]；(b)siRNA与梳状DNA-聚己内酯自组装形成球状微水凝胶^[42].

(a)体外转录产生的poly-RNA与6条siRNA自组装成稳定的纳米环^[43]；(b)基于滚环扩增技术的DNA/siRNA自组装Y型纳米结构^[45].

(a)基于“锁钥”机制的siRNA纳米载体；(b)基于microRNA引发的原位链置换级联反应的siRNA纳米载体^[46,47].

(a)基于滚环转录的自组装RNAi微球纳米结构^[50]；(b)胆固醇和叶酸修饰siRNA海绵微球纳米结构^[28].

(a)通过控制T7 RNA聚合酶的浓度来调节微球的大小^[53]；(b)siRNA纳米微球文库^[54]；(c)鼓泡型RNA纳米微球^[55].

参考文献

1
Wittrup A, Lieberman J. Knocking down disease: a progress report on siRNA therapeutics. Nature Reviews Genetics, 2015, 16(9): 543-552
2
Ku S H, Jo S D, Lee Y K, et al. Chemical and structural modifications of RNAi therapeutics. Advanced Drug Delivery Reviews, 2016, 104(3): 16-28
3
Hannon G J. RNA interference. Nature, 2002, 418(6894): 244-251
4
Fire A, Xu S, Montgomery M K, et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391(6669): 806-811
5
Hoy S M. Patisiran: first global approval. Drugs, 2018, 78(15): 1625-1631
6
Wittrup A, Lieberman J. Knocking down disease: a progress report on siRNA therapeutics. Nature Reviews Genetics, 2015, 16(9): 543-552
7
Whitehead K A, Langer R, Anderson D G. Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery, 2009, 8(2): 129-138
8
Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Therapy, 2000, 7(1): 31-34
9
Liu Y P, Berkhout B. miRNA cassettes in viral vectors: problems and solutions. BBA-Gene Regulatory Mechanisms, 2011, 1809(11): 732-745
10
Duerner L J, Schwantes A, Schneider I C, et al. Cell entry targeting restricts biodistribution of replication-competent retroviruses to tumour tissue. Gene Therapy, 2008, 15(22): 1500-1510
11
Descamps D, Benihoud K. Two key challenges for effective adenovirus-mediated liver gene therapy: innate immune responses and hepatocyte-specific transduction. Current Gene Therapy, 2009, 9(2): 115-127
12
Burnett J C, Rossi J J. RNA-based therapeutics: current progress and future prospects. Chemistry & Biology, 2012, 19(1): 60-71
13
Bertrand N, Leroux J C. The journey of a drug-carrier in the body: an anatomo-physiological perspective. Journal of Controlled Release, 2012, 161(2): 152-163
14
Zhang S, Zhao B, Jiang H, et al. Cationic lipids and polymers mediated vectors for delivery of siRNA. Journal of Controlled Release, 2007, 123(1): 1-10
15
Yu T, Liu X, Bolcato-Bellemin A L, et al. An amphiphilic dendrimer for effective delivery of small interfering RNA and gene silencing in vitro and in vivo. Angewandte Chemie. International Ed. in English, 2012, 51(34): 8478-8484
16
Ornelas-Megiatto C, Wich P R, Frechet J M. Polyphosphonium polymers for siRNA delivery: an efficient and nontoxic alternative to polyammonium carriers. Journal of the American Chemical Society, 2012, 134(4): 1902-1905
17
Ozpolat B, Sood A K, Lopez-Berestein G. Liposomal siRNA nanocarriers for cancer therapy. Advanced Drug Delivery Reviews, 2014, 66: 110-116
18
Andey T, Marepally S, Patel A, et al. Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model. Journal of Controlled Release, 2014, 184(7): 67-78
19
Lee S H, Chung B H, Park T G, et al. Small-interfering RNA (siRNA)-based functional micro- and nanostructures for efficient and selective gene silencing. Accounts of Chemical Research, 2012, 45(7): 1014-1025
20
Wightman L, Kircheis R, Rössler V, et al. Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. Journal of Gene Medicine, 2001, 3(4): 362-372
21
Muhonen P, Tennilä T, Azhayeva E, et al. RNA interference tolerates 2′‐fluoro modifications at the argonaute2 cleavage site. Chemistry & Biodiversity, 2007, 4(5): 858-873
22
Lungwitz U, Breunig M T, Gopferich A. Polyethylenimine-based non-viral gene delivery systems. European Journal of Pharmaceutics & Biopharmaceutics Official Journal of Arbeitsgemeinschaft Für Pharmazeutische Verfahrenstechnik E V, 2005, 60(2): 247-266
23
Judge A D, Sood V, Shaw J R, et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nature Biotechnology, 2005, 23(4): 457-462
24
Hwa K S, Hoon J J, Soo Hyeon L, et al. Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. Journal of Controlled Release Official Journal of the Controlled Release Society, 2008, 129(2): 107-116
25
Daniela C, Rossi J J. The promises and pitfalls of RNA-interference-based therapeutics. Nature, 2009, 457(7228): 426-433
26
Ballarin-Gonzalez B, Howard K A. Polycation-based nanoparticle delivery of RNAi therapeutics: adverse effects and solutions. Advanced Drug Delivery Reviews, 2012, 64(15): 1717-1729
27
Jang M, Han H D, Ahn H J. A RNA nanotechnology platform for a simultaneous two-in-one siRNA delivery and its application in synergistic RNAi therapy. Scientific Reports, 2016, 6: 32363
28
Jang M, Kim J H, Nam H Y, et al. Design of a platform technology for systemic delivery of siRNA to tumours using rolling circle transcription. Nature Communications, 2015, 6: 7930
29
Mok H, Lee S H, Park J W, et al. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing. Nature Materials, 2010, 9(3): 272-278
30
Lee S H, Mok H, Jo S, et al. Dual gene targeted multimeric siRNA for combinatorial gene silencing. Biomaterials, 2011, 32(9): 2359-2368
31
Hong C A, Lee S H, Kim J S, et al. Gene silencing by siRNA microhydrogels via polymeric nanoscale condensation. Journal of the American Chemical Society, 2011, 133(35): 13914-13917
32
Gold L, Janjic N, Jarvis T, et al. Aptamers and the RNA world, past and present. Cold Spring Harbor Perspectives in Biology, 2012, 4(3): 829-841
33
Zhou J, Rossi J. Aptamers as targeted therapeutics: current potential and challenges. Nature Reviews Drug Discovery, 2017, 16(3): 181-202
34
Jeong H, Lee S H, Hwang Y, et al. Multivalent aptamer-RNA conjugates for simple and efficient delivery of doxorubicin/siRNA into multidrug-resistant cells. Macromolecular Bioscience, 2017, 17(4), doi: 10.1002/mabi.201600343
35
Yoo H, Jung H, Kim S A, et al. Multivalent comb-type aptamer-siRNA conjugates for efficient and selective intracellular delivery. Chemical Communications (Camb), 2014, 50(51): 6765-6767
36
Nakashima Y, Abe N, Ito Y, et al. Nanostructured RNAs for RNA interference. Chemical Communications, 2011, 47(29): 8367-8369
37
Son S, Kim N, You D G, et al. Antitumor therapeutic application of self-assembled RNAi-AuNP nanoconstructs: combination of VEGF-RNAi and photothermal ablation. Theranostics, 2017, 7(1): 9-22
38
Yu-Kyoung O, Tae Gwan P. siRNA delivery systems for cancer treatment. Advanced Drug Delivery Reviews, 2009, 61(10): 850-862
39
Whitehead K A, Robert L, Anderson D G. Knocking down barriers: advances in siRNA delivery. Nature Reviews Drug Discovery, 2009, 8(2): 129-138
40
Lv H, Zhang S, Wang B, et al. Toxicity of cationic lipids and cationic polymers in gene delivery. Journal of Controlled Release Official Journal of the Controlled Release Society, 2006, 114(1): 100-109
41
Lee H, Lytton-Jean A K, Chen Y, et al. Molecularly self-assembled nucleic acid nanoparticles for targeted in vivo siRNA delivery. Nature Nanotechnology, 2012, 7(6): 389-393
42
Ding F, Mou Q, Ma Y, et al. A crosslinked nucleic acid nanogel for effective siRNA delivery and antitumor therapy. Angewandte Chemie. International Ed. in English, 2018, 57(12): 3064-3068
43
Grabow W W, Zakrevsky P, Afonin K A, et al. Self-assembling RNA nanorings based on RNAI/II inverse kissing complexes. Nano Letters, 2011, 11(2): 878-887
44
Afonin K A, Kireeva M, Grabow W W, et al. Co-transcriptional assembly of chemically modified RNA nanoparticles functionalized with siRNAs. Nano Letters, 2012, 12(10): 5192-5195
45
Hong C A, Jang B, Jeong E H, et al. Self-assembled DNA nanostructures prepared by rolling circle amplification for the delivery of siRNA conjugates. Chemical Communications, 2014, 50(86): 13049-13051
46
Ren K, Liu Y, Wu J, et al. A DNA dual lock-and-key strategy for cell-subtype-specific siRNA delivery. Nature Communications, 2016, 7: 13580
47
Ren K, Zhang Y, Zhang X, et al. In situ SiRNA assembly in living cells for gene therapy with microRNA triggered cascade reactions templated by nucleic acids. ACS Nano, 2018, 12(11): 10797-10806
48
Seyhan A A, Vlassov A V, Johnston B H. RNA interference from multimeric shRNAs generated by rolling circle transcription. Oligonucleotides, 2006, 16(4): 353-363
49
Daubendiek S L, Ryan K, Kool E T. Rolling-circle RNA synthesis: circular oligonucleotides as efficient substrates for T7 RNA polymerase. Journal of the American Chemical Society, 1995, 117(29): 7818-7819
50
Lee J B, Hong J, Bonner D K, et al. Self-assembled RNA interference microsponges for efficient siRNA delivery. Nature Materials, 2012, 11(4): 316-322
51
Roh Y H, Deng J Z, Dreaden E C, et al. A multi-RNAi microsponge platform for simultaneous controlled delivery of multiple small interfering RNAs. Angewandte Chemie. International Ed. in English, 2016, 55(10): 3347-3351
52
Lee J H, Ku S H, Kim M J, et al. Rolling circle transcription-based polymeric siRNA nanoparticles for tumor-targeted delivery. Journal of Controlled Release, 2017, 263: 29-38
53
Han D, Park Y, Nam H, et al. Enzymatic size control of RNA particles using complementary rolling circle transcription (cRCT) method for efficient siRNA production. Chemical Communications, 2014, 50(79): 11665-11667
54
Han S, Kim H, Lee J B. Library siRNA-generating RNA nanosponges for gene silencing by complementary rolling circle transcription. Scientific Reports, 2017, 7(1): 10005
55
Kim H, Jeong J, Kim D, et al. Bubbled RNA-based cargo for boosting RNA interference. Advanced Science, 2017, 4(8): 1600523