藥用高分子材料sirna藥物

1、姚老師作業(yè)：聚乳酸的本體聚合工藝，相應(yīng)機(jī)理和反應(yīng)方程,第六章 高分子納米藥物-siRNA藥物,,病變組織的基因活性,正常表達(dá),過度表達(dá),低表達(dá),Emulate Drug?,抗體:Bind protein, but only extracellular epitopesLow throughput, high cost反義RNA/核酶:Stop gene expression (production), most protein

2、sChemical stabilization essentialBarriers: sequence selection, potency, toxicities, … RNA干擾:Stop gene expression (production), most proteinsGood activity without chemical stabilizationRapidly replacing antisense:

3、 really better?,特異性基因下調(diào)手段,,RNA干擾（RNA interference, RNAi）技術(shù),功能：特異性地沉默內(nèi)源或外源性靶基因影響：2001年《Science》雜志十大科學(xué)成就之二2002年《Science》雜志十大科學(xué)成就之首醫(yī)學(xué)領(lǐng)域的應(yīng)用：基因功能研究；基因敲除；基因治 療；基因表達(dá)調(diào)控等; 抗病毒以及糖尿病、肥胖癥和癌癥、

4、 黃斑變性的眼科疾病治療等。,,RNAi作用機(jī)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.,

5、Nature Reviews 4, 457, 2003,具有RNAi機(jī)制的藥物,,TargetID,,siRNATarget ValidationGrowing,Clinical,LeadDiscovery &Optimization,Preclinical,,,,siRNA藥物: 革命性突破,,Potential of siRNA-Based Therapy,Phase 1 Phase 2

6、 Phase 3,,,,,,,,,,,,,,,,,,,,,RNA干擾療法的潛力,siRNA藥物迅速進(jìn)入臨床研究,,難以被細(xì)胞攝取：大分子，陰離子,脫靶效應(yīng)：非特異性沉默， 引起免疫刺激等,難以富集在腫瘤部位：體內(nèi)不穩(wěn)定， 半衰期短,,,,1,2,3,Potential of siRNA-B

7、ased Therapy,RNA干擾療法的潛力,siRNA藥物臨床引用的關(guān)鍵,,Nature 2010, 468, p487, Nov. 25, 2010,Potential of siRNA-Based Therapy,RNA干擾療法的潛力,siRNA給藥技術(shù)成為關(guān)鍵挑戰(zhàn),,表達(dá)siRNA的質(zhì)粒DNA的傳遞病毒（Nature Biotechnology, 2002, 20: 1006-10） 非病毒類載體化學(xué)合成的siRNA的傳

8、遞非病毒類載體：脂質(zhì)體和高分子物理手段: electroporation, high pressure/high volume injection,Potential of siRNA-Based Therapy,RNA干擾療法的潛力,siRNA的傳遞：體內(nèi)RNAi技術(shù)應(yīng)用的關(guān)鍵,,脂質(zhì)類,,,,高分子納米載體,抗體類,化學(xué)修飾siRNA,siRNA綴合物,Potential of siRNA-Based Therapy,RNA

9、干擾療法的潛力,siRNA的非病毒載體種類,,,DAN和siRNA傳遞的比較,,,,化學(xué)修飾的siRNA,Nature，2004，432：173-178,,化學(xué)修飾的siRNA,Challenge 1: 穩(wěn)定Challenge 2: 長循環(huán)Challenge 3: 靶向Challenge 4: 滲透Challenge 5: 細(xì)胞攝取Challenge 6: 制劑化,基于納米載體的 siRNA給藥系統(tǒng),,,,,,,,,,,,,,

10、,,,,,,,,,,,Engineering Nanoparticles for siRNA Delivery,Extended CirculationTissue Targeting,Penetration,Cell internalization,,,脂質(zhì)體類siRNA納米載體,,脂質(zhì)體類siRNA納米載體,,Stable Nucleic Acid Lipid Particle (SNALP),,Stable Nucleic Ac

11、id Lipid Particle (SNALP),Mean particle size: 140±12 nmMean encapsulation efficiency: 93%±3%Nucleic acid/lipid ratio: 42±4?g/?mol,,siRNA-SNALP with a longer half-life in plasma and liver reduced serum HB

12、V 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 pr

13、otein, serum cholesterol and low-density lipoprotein levels,Nature 441, 111-114 (4 May 2006),,高分子類 siRNA給藥系統(tǒng),,,StericCoat(Inert),Active Core,ExposedTargetingLigands,CleavableLink(Shedding),Ligands,Steric polymer,,+

14、,siRNAPayload,水溶性陽離子高分子納米載體:,,NUCLEIC ACIDS RESEARCH 32 (19): Art. No. e149 2004,PNAS 2007, 104, 5715-5721,,靶向修飾的水溶性陽離子高分子納米載體:,Cancer Research 2005, 65, 8984-8992,,PEG,JACS, 127, 1624-1625,靶向修飾的水溶性高分子-siRNA鍵合物:,,,JACS,

15、 126 (42), 13612 -13613,Loading siRNA post nanoparticle formationPEG: prolonged blood circulationBiocompatibleDegradable, potentially intracellular siRNA releaseCo-delivery of chemotherapeutic drug for synergistic ef

16、fect,,親水嵌段,疏水嵌段,帶正電荷嵌段,,,,,陽離子膠束/siRNA復(fù)合納米顆粒:,,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

17、-1494,β-actin,Kim A.Woodrow, W. Mark Saltzman et al., Nature Materials, 2009, 8, 526 – 533Christopher J. Cheng, W. Mark Saltzman, Biomaterials, 2011, 32, 6194–6203,siRNA包載效率低下，僅為21%！1 g siRNA目前的價格為200萬！,基于聚乙二醇-聚乳酸

18、的siRNA納米給藥體系,,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,,

19、,,,Second emulsion,More water,PEG-PLA/Cationic lipid/siRNAnanoparticles,First emulsion,Cationic Lipid Assisted Nanoparticles: CLAN,基于CLAN技術(shù)設(shè)計的siRNA給藥系統(tǒng),,基于CLAN技術(shù)設(shè)計的靶向siRNA給藥系統(tǒng),,Nature Biotechnol. 23, 709–717 (2005),,抗體類