药用高分子材料siRNA药物

姚老师作业：聚乳酸的本体聚合工艺，相应机理和反应方程,第六章 高分子纳米药物-siRNA药物,病变组织的基因活性,正常表达,过度表达,低表达,Emulate Drug?,抗体:Bind protein, but only extracellular epitopesLow throughput, high cost反义RNA/核酶:Stop gene expression (production), most proteinsChemical stabilization essentialBarriers: sequence selection, potency, toxicities, RNA干扰:Stop gene expression (production), most proteinsGood activity without chemical stabilizationRapidly replacing antisense: really better?,特异性基因下调手段,RNA干扰（RNA interference, RNAi）技术,功能：特异性地沉默内源或外源性靶基因影响：2001年Science杂志十大科学成就之二2002年Science杂志十大科学成就之首医学领域的应用：基因功能研究；基因敲除；基因治 疗；基因表达调控等; 抗病毒以及糖尿病、肥胖症和癌症、 黄斑变性的眼科疾病治疗等。,RNAi作用机制,Nature，2004，430：161,From H. Winkler, IBC Conference “RNA Interference”, San Diego, CA, Feb 2003,siRNA,dsRNA,siRNA,RNAse,shRNA,In Vitro,In Vivo,In Vivo,miRNA,Adapted From Dykxhoorn et al., Nature Reviews 4, 457, 2003,具有RNAi机制的药物,TargetID,siRNATarget ValidationGrowing,Clinical,LeadDiscovery &Optimization,Preclinical,siRNA药物: 革命性突破,Potential of siRNA-Based Therapy,Phase 1 Phase 2 Phase 3,RNA干扰疗法的潜力,siRNA药物迅速进入临床研究,难以被细胞摄取：大分子，阴离子,脱靶效应：非特异性沉默， 引起免疫刺激等,难以富集在肿瘤部位：体内不稳定， 半衰期短,1,2,3,Potential of siRNA-Based Therapy,RNA干扰疗法的潜力,siRNA药物临床引用的关键,Nature 2010, 468, p487, Nov. 25, 2010,Potential of siRNA-Based Therapy,RNA干扰疗法的潜力,siRNA给药技术成为关键挑战,表达siRNA的质粒DNA的传递病毒（Nature Biotechnology, 2002, 20: 1006-10） 非病毒类载体化学合成的siRNA的传递非病毒类载体：脂质体和高分子物理手段: electroporation, high pressure/high volume injection,Potential of siRNA-Based Therapy,RNA干扰疗法的潜力,siRNA的传递：体内RNAi技术应用的关键,脂质类,高分子纳米载体,抗体类,化学修饰siRNA,siRNA缀合物,Potential of siRNA-Based Therapy,RNA干扰疗法的潜力,siRNA的非病毒载体种类,DAN和siRNA传递的比较,化学修饰的siRNA,Nature，2004，432：173-178,化学修饰的siRNA,Challenge 1: 稳定Challenge 2: 长循环Challenge 3: 靶向Challenge 4: 渗透Challenge 5: 细胞摄取Challenge 6: 制剂化,基于纳米载体的 siRNA给药系统,Engineering Nanoparticles for siRNA Delivery,Extended CirculationTissue Targeting,Penetration,Cell internalization,脂质体类siRNA纳米载体,脂质体类siRNA纳米载体,Stable Nucleic Acid Lipid Particle (SNALP),Stable Nucleic Acid Lipid Particle (SNALP),Mean particle size: 14012 nmMean encapsulation efficiency: 93%3%Nucleic acid/lipid ratio: 424g/mol,siRNA-SNALP with a longer half-life in plasma and liver reduced serum HBV DNA titers in mouse model following intravenous injection,Nature Biotechnology 23, 1002 - 1007 (2005),ApoB-siRNA-SNALP resulted in silencing of ApoB mRNA expression in liver and significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels,Nature 441, 111-114 (4 May 2006),高分子类 siRNA给药系统,StericCoat(Inert),Active Core,ExposedTargetingLigands,CleavableLink(Shedding),Ligands,Steric polymer,+,siRNAPayload,水溶性阳离子高分子纳米载体:,NUCLEIC ACIDS RESEARCH 32 (19): Art. No. e149 2004,PNAS 2007, 104, 5715-5721,靶向修饰的水溶性阳离子高分子纳米载体:,Cancer Research 2005, 65, 8984-8992,PEG,JACS, 127, 1624-1625,靶向修饰的水溶性高分子-siRNA键合物:,JACS, 126 (42), 13612 -13613,Loading siRNA post nanoparticle formationPEG: prolonged blood circulationBiocompatibleDegradable, potentially intracellular siRNA releaseCo-delivery of chemotherapeutic drug for synergistic effect,亲水嵌段,疏水嵌段,带正电荷嵌段,阳离子胶束/siRNA复合纳米颗粒:,Sun TM, et al., ACS Nano, 2011, 5, 1483-1494,Days post-xenografting,12,16,20,24,28,32,36,40,44,Sun TM, et al., ACS Nano, 2011, 5, 1483-1494,-actin,Kim A.Woodrow, W. Mark Saltzman et al., Nature Materials, 2009, 8, 526 533Christopher J. Cheng,W. Mark Saltzman, Biomaterials, 2011, 32, 61946203,siRNA包载效率低下，仅为21%！1 g siRNA目前的价格为200万！,基于聚乙二醇-聚乳酸的siRNA纳米给药体系,Organic phase (polymer/lipid),aqueous phase (siRNA),Such nanoparticles, which is based FDA approved PEG-PLA, exhibit high encapsulation efficiency of siRNA (about 96%).,Removing the organic solvent,Second emulsion,More water,PEG-PLA/Cationic lipid/siRNAnanoparticles,First emulsion,Cationic Lipid Assisted Nanoparticles: CLAN,基于CLAN技术设计的siRNA给药系统,基于CLAN技术设计的靶向siRNA给药