免疫纳米微粒靶向胰腺癌细胞输送siRNA的方法研究

 目的：构建一种向胰腺癌细胞安全、高效输送siRNA的免疫纳米载体。方法：检测纳米载体IONP-PEI（非靶向组）及其与siRNA复合物的表征;通过琼脂糖凝胶电泳检测siRNA结合力、MTS法检测细胞活力和流式细胞术检测转染率以确定其复合siRNA的最佳N/P比值;细胞免疫荧光和普鲁士蓝染色观察scFvCD44v6偶联IONP-PEI的靶向纳米载体（靶向组）在细胞内分布;流式细胞术、荧光显微镜观察、real-time PCR和Western blotting检测靶向组和非靶向组的转染率和转染siKRAS后的干扰效果。结果：IONP和PEI的最适质量比为0.75;纳米载体复合siRNA的最佳N/P比值为20;IONP-PEI/siRNA复合物的电位为（21.73±8.07）mV,粒径为（51.3±2.2）nm。荧光显微镜显示,非靶向组和靶向组转染后均在细胞内,靶向组的转染率为（89.75±1.81）%,高于非靶向组的（59.87±4.52）%,且靶向组的荧光强度高于非靶向组。靶向组的KRAS mRNA的相对表达量为（34.02±615）%,低于非靶向组的（51.09±6.70）%;Western blotting显示靶向组的KRAS蛋白表达量低于非靶向组。结论：非靶向组和靶向组均能够将siRNA转染进细胞内,且靶向组具有更高的转染效率和更好的干扰基因表达效果。本课题构建的scFvCD44v6-IONP-PEI是一种高效、安全和靶向识别胰腺癌细胞的免疫纳米载体。     

聚乙烯亚胺-聚乙二醇-siRNA纳米复合物的制备和理化性质的研究

目的 探索聚乙烯亚胺-聚乙二醇(PEI-PEG)-siRNA纳米复合物的制备方法及其理化性质,以提高siRNA的细胞转染率.方法 设计合成了PEI-PEG共聚物基因载体,使其与针对细胞表面受体CD44v6的siRNA形成纳米复合物.通过粒径与电位测定、凝胶阻滞电泳、扫描电镜、流式细胞仪测定等方法,观察不同N/P比的纳米复合物的复合效果、表面形态和大小、基因转染率等.结果 电镜下纳米复合物呈近球形、大小较一致、分散良好的纳米颗粒.N/P=5、10时复合物粒径分别为(174.6±1.2)nm,(267.7±1.8)nm.当N/P超过10时纳米复合物粒径减小,zeta电位为正值且增大.此时,siRNA被PEI-PEG完全复合,产生一种荧光淬灭作用.流式细胞仪结果表明纳米复合物的基因转染率随着N/P的增加而增大.当N/P比为30时,其转染率为(75.6±9.2)%.结论 PEI-PEG是一种有潜力的阳离子基因载体,它的制备为下一步体外实验及动物实验提供了条件.     

CD147单抗介导的基因治疗纳米颗粒的肺癌细胞靶向性研究

目的:本研究采用靶向CD147的单克隆抗体对纳米基因载体颗粒进行靶向修饰后,进行针对肺癌细胞的蛋白激酶Cε(protein kinase Cε,PKCε)小干扰RNA基因治疗,观察其对肺癌细胞增殖和迁移能力的抑制效果。方法:制作可靶向CD147蛋白的磁性纳米基因载体。激光扫描共聚焦显微镜观察肺癌细胞CD147表达量。分别设立CP组、CN组和LP组复合物,按每6孔板孔质粒总量250 ng进行细胞转染。另设CD147靶向载体对照CA组和未转染细胞的对照(control)组。激光扫描共聚焦显微镜观察纳米造影剂的细胞内吞效果。实时荧光定量PCR检测PKCε的mRNA表达。Western blot法检测PKCε、Ki67、MMP3、Wnt1和GAPDH的蛋白表达。平板克隆形成实验检测细胞的增殖能力。Transwell法检测细胞的迁移能力。结果:免疫荧光法染色观察证实,人肺癌A549细胞的胞膜高表达CD147蛋白。CP组细胞中siRNA高效进入A549细胞,质粒内吞效率大于CN组和LP组。CP组、CN组、LP组和CA组的A549细胞中PKCε的mRNA相对表达量分别为control组的(9.76±0.18)%、(98.51±0.32)%、(99.17±0.16)%和(99.68±0.11)%,CP组与control组间的差异有统计学显著性(P0.05),CN组、LP组与control组间的差异无统计学显著性。CP组PKCε、Ki-67、MMP3及Wnt1蛋白的表达量明显降低,CN组和LP组与对照组之间的蛋白表达量的差异无统计学显著性。CP组的克隆形成数量明显少于control组,差异具有统计学显著性(P0.05)。CN组、LP组和CA组的有效克隆数量与control组相比差异没有统计学显著性。CP组的过膜细胞数量明显少于control组,差异具有统计学显著性(P0.05)。CN组、LP组和CA组的数量与control组相比差异没有统计学显著性。结论:靶向CD147修饰的纳米基因载体,可以对肺癌细胞进行高效的PKCε-siRNA基因治疗,实现对肺癌细胞增殖和迁移能力的高效抑制。     

敲低lncRNA LINC01296调节PD-L1抑制鼻咽癌细胞免疫逃逸的机制研究

目的 研究敲低长链非编码RNA(lncRNA)LINC01296调节程序性死亡配体-1(PD-L1)抑制鼻咽癌细胞免疫逃逸的机制。方法 分离培养人外周血淋巴细胞后与人鼻咽癌细胞CNE-2Z共培养,同时取C57BL/6小鼠皮下注射CNE-2Z细胞构建鼻咽癌移植瘤模型24只,均随机分为对照组、lncRNA LINC01296敲低组[转染lncRNA LINC01296小干扰RNA(siRNA)]、阴性对照组(转染lncRNA LINC01296 siRNA阴性对照和空载质粒)、lncRNA LINC01296敲低+PD-L1过表达组(转染lncRNA LINC01296 siRNA和PD-L1过表达质粒),分组转染后,流式细胞术检测人外周血淋巴细胞中活化CD8⁺T细胞比例;细胞计数试剂盒-8法检测人外周血淋巴细胞对CNE-2Z细胞杀伤率;测量移植瘤小鼠肿瘤体积;免疫荧光染色检测移植瘤小鼠肿瘤组织CD8和PD-L1阳性表达;实时荧光PCR实验检测CNE-2Z细胞和肿瘤组织lncRNA LINC01296、PD-L1信使RNA(mRNA)表达;蛋白印迹实验检测CNE-2Z...     

靶向干扰GPx1基因对人胶质母细胞瘤细胞生长和迁移的影响

目的:探讨靶向上调和下调谷胱甘肽过氧化物酶1(GPx1)对人胶质母细胞瘤细胞株(U87MG、U118MG)增殖、迁移和侵袭能力的影响。方法:合成针对GPx1的siRNA和构建、鉴定pc DNA3.1-GPx1重组质粒,分别用LipofectamineTM2000脂质体瞬时转染人胶质母细胞瘤细胞株U87MG和U118MG。Real-time PCR检测U87MG和U118MG中GPx1 mRNA表达。Western blotting法检测转染后GPx1蛋白表达,用MTS检测法和Transwell实验分别检测转染后细胞的活力、迁移及侵袭能力的变化。结果:siRNA干扰U87MG细胞后在24 h、48 h和72 h对细胞活力的抑制率分别为25.9%、35.7%和34.8%,较对照组明显降低(P0.05);质粒转染U118MG细胞后在24 h、48 h和72 h对细胞活力的促进率分别为22.7%、45.8%和39.8%,较对照组明显增加(P0.05);Transwell结果显示经siRNA干扰,U87MG细胞迁移抑制率为41.6%±8.2%,细胞侵袭抑制率为40.4%±10.1%,经siRNA干扰的细胞迁移和侵袭能力明显下降(P0.05);质粒转染后细胞迁移促进率为55.8%±9.8%,细胞侵袭促进率为60.8%±9.2%,经质粒转染的细胞的迁移和侵袭能力显著增强(P0.05)。结论:siRNA下调GPx1表达可抑制人胶质母细胞瘤细胞株U87MG的生长、迁移和侵袭;相反,质粒上调GPx1表达可促进人胶质母细胞瘤细胞株U118MG的生长、迁移和侵袭。     

SiRNA沉默JAB1表达抑制肝癌细胞增殖

 目的 观察沉默Jab1基因表达对人肝癌细胞HepG-2增殖的影响。方法 利用RNA干扰技术构建PAVU6-JAB1 siRNA干扰质粒,将其转染HepG-2细胞,采用Real-time PCR、Western blot等方法对Jab1表达进行检测,WST-8、流式细胞分析对细胞增殖的影响。结果 成功构建PAVU6- JAB1 siRNA干扰质粒,转染HepG-2细胞后Jab1蛋白表达量较对照组明显降低,P27kip1蛋白表达量明显升高 (P<0.01)。转染96 h后能显著抑制细胞增殖活性,由空载体转染组的99.6%?1.4%降至70.8%?1.6%。结论 构建靶向Jab1基因的干扰质粒能下调Jab1基因的表达,上调P27kip1蛋白水平,并抑制HepG-2细胞在体外的增殖活性。     

纳米微粒介导Bcl-xl小干扰RNA诱导人肺腺癌细胞凋亡

目的 探讨纳米微粒介导的Bcl-xl小干扰RNA(siRNA)对人肺腺癌A549细胞凋亡的影响.方法 制备包载Bcl-xl siRNh的纳米微粒,体外培养人肺腺癌A549细胞.以纳米微粒介导将人工合成的Bcl-xl siRNA转染细胞后,将细胞分为4组:生理盐水组、纳米微粒组、siRNA-c-纳米微粒组及siRNA-纳米微粒组,其中siRNA-c-纳米微粒组及siRNA-纳米微粒组又分为0.1、0.2、0.4、0.8 μmol/L4个浓度组.应用TUNEL法检测肺癌细胞凋亡;RT-PCR半定量检测Bcl-xl mRNA的表达;免疫细胞化学染色法检测Bcl-xl蛋白的表达.结果 转染Bcl-xl siRNA后,各处理浓度组的肺腺癌细胞凋亡指数随浓度的增加而逐渐升高[0.1、0.2、0.4、0.8 μmol/L组分别为(19.7±2.6)%、(32.4±5.5)%、(46.0±5.3)%和(55.4±5.9)%],与生理盐水组、纳米微粒组及同浓度的siRNA-c-纳米微粒组相比,差异均有统计学意义(P均<0.05).转染浓度为0.8 μmol/L时,肺腺癌细胞内Bcl-xl mRNA及其蛋白的表达明显降低(P<0.05),Bcl-xl蛋白表达阳性单位下降至0.0787±0.0233;Bcl-xl mRNA表达下降至0.856±0.242,与其它各组差异有统计学意义(P<0.05).结论 纳米微粒能有效介导Bcl-xl siRNA进入肺腺癌细胞.Bcl-xl siRNA能够特异性减少肺腺癌细胞的Bcl-xl的mRNA和蛋白的表达,抑制肿瘤细胞增殖及诱导细胞凋亡.     

人鳞状细胞癌细胞株(A431)中NET-1基因对癌细胞增殖和浸润的影响

目的 探讨小分子干扰核酸(siRNA)对皮肤鳞状细胞癌(简称皮肤鳞癌)细胞株(A431)中NET-1基因的抑制作用,及其基因对癌细胞增殖、浸润的影响.方法 构建针对人NET-1基因的siRNA NET-1真核表达载体(pU6H1-GFP-siRNA NET-1),转染A431细胞后通过半定量RT-PCR检测细胞中NET-1 mRNA水平以筛选较有效的siRNA NET-1.同时设置针对NET-1的正、反义真核表达载体和随机序列对照组siRNA表达载体,体外瞬时转染A431细胞,RT-PCR、Western blot分别检测癌细胞内NET-1 mRNA和蛋白的表达,经免疫荧光染色在激光共聚焦显微镜下观察NET-1蛋白在细胞内的表达;四甲基偶氮唑盐(MTT)法和流式细胞仪分别榆测A431细胞增殖与占不同细胞周期中细胞增殖指数(PI);划痕试验和Transwell迁移试验分别检测A431细胞的迁移和侵袭能力.结果 测序证实编码NET-1序列的siRNA已经插入载体pU6H1-GFP中U6和H1两个启动子之间,其他载体测序结果 符合设计要求.pU6H1-GFP载体转染A431细胞后其转染率达到80%.转染siRNA NET-1和反义NET-1后分别与未转染组比较,A431细胞中NET-1 mRNA分别减少72%和62%(t值分别为-36.01,-17.65;均P<0.05)和蛋白表达水平分别减少61%和69%(t值分别为-21.13,-33.14;均P<0.05);在转染48 h后能显著抑制A431细胞的增殖、迁移和浸润(均P<0.05).在转染对照组siRNA后并未见到明显的抑制效果.转染正义NET-1后,能分别增加细胞内52%NET-1 mRNA和49%蛋白表达水平(t值分别为12.49,13.98,均P<0.01).结论 靶向NET-1的siRNA真核表达载体能特异、有效的下调NET-1基因蛋白的表达,并抑制A431细胞增殖、迁移和浸润.进而证明内源性NET-1基因的功能与A431细胞增殖、迁移、浸润有关.靶向NET-1的siRNA显示基因抑制效率比反义核酸技术更好.     

RNA干扰LOXL2基因对头颈部鳞状细胞癌凋亡及PD-L1表达的影响

目的:探讨抑制赖氨酰氧化酶样蛋白2(LOXL2)基因表达对头颈部鳞状细胞癌(HNSCC)增殖凋亡及PD-L1表达的影响研究。方法:通过Western blot检测人HNSCC细胞YCU-H891、YCU-N861和KB中LOXL2的蛋白表达。以Lipo-fectamineTM2000为载体,参照其说明将合成的LOXL2 siRNA转染KB细胞,转染48 h后,Western blot检测LOXL2、PD-L1、STAT3、p-STAT3、PCNA和Bax的蛋白表达; CCK8及流式细胞仪分别检测细胞活力及凋亡率。结果:LOXL2在YCU-H891、YCU-N861和KB中表达均升高。转染LOXL2 siRNA的KB细胞LOXL2的蛋白表达明显降低,与空白组和NC组比较差异均具有统计学意义(P0. 05)。转染LOXL2 siRNA的KB细胞活力明显降低,凋亡率明显升高,PD-L1、p-STAT3和PCNA的蛋白表达均明显降低,Bax的蛋白表达明显升高,与NC组比较,差异均具有统计学意义(P0. 05)。结论:RNA干扰抑制LOXL2基因可降低HNSCC细胞活力和诱导凋亡,下调PD-L1表达,机制可能与抑制STAT3信号通路有关。     

川芎嗪和siRNA沉默转化生长因子β1干预大鼠肝星状细胞 结缔组织生长因子的表达

背景：作者前期研究发现川芎嗪可以通过抑制肝星状细胞的增殖、阻抑p38MARK信号通路、下调结缔组织生长因子的表达等多途径发挥抗肝纤维化的作用。一般认为转化生长因子β1是促进纤维化发生最重要的细胞因子,结缔组织生长因子是转化生长因子β1的下游效应介质,siRNA靶向抑制转化生长因子β1可能成为治疗肝纤维化的新方法。 目的：观察川芎嗪及化学合成的siRNA转化生长因子β1对大鼠肝星状细胞结缔组织生长因子表达的影响。 方法：体外培养大鼠肝星状细胞,从3条siRNA转化生长因子β1中筛选一条有效的基因沉默片段,然后利用脂质体转染试剂共同转染培养24 h的细胞作为转染组,并设计空白对照组、转染试剂组、川芎嗪组及联合治疗组。 结果与结论：与空白对照组相比,转染组、川芎嗪组、联合治疗组Ⅰ、Ⅲ型胶原及结缔组织生长因子mRNA表达均下调(P < 0.05)。在转染组、川芎嗪组及联合治疗组,结缔组织生长因子蛋白表达均下调(P < 0.05);培养上清液中Ⅰ型胶原和Ⅲ型胶原含量均降低(P < 0.05)。结果表明,siRNA 沉默转化生长因子β1基因能下调结缔组织生长因子的表达,减少Ⅰ、Ⅲ型胶原的表达。川芎嗪也能下调结缔组织生长因子的表达,但对Ⅰ、Ⅲ型胶原表达的抑制作用更加显著,提示川芎嗪抗肝纤维化可能是多种作用靶点。     

IFN-γ上调人胃癌细胞株PD-L1表达

 目的：研究不同人胃癌细胞株表面程序性死亡配体1（PD-L1）表达及干扰素γ（IFN-γ）对其表达的诱导情况。方法：分别培养不同人胃癌细胞株AGS、BGC823、MGC803和SGC7901,经不同浓度IFN-γ、作用不同时间后,利用流式细胞术及real-time PCR检测胃癌细胞株PD-L1在蛋白和mRNA水平的表达情况。结果：流式细胞术显示AGS、BGC823、MGC803和SGC7901细胞PD-L1蛋白表达率分别为（1.567±0.109）%、（2.640±0.577）%、（1.760±0.236）%和（16.030±1.289）%;不同胃癌细胞株对IFN-γ反应不同,经不同浓度IFN-γ刺激后均可不同程度上调PD-L1的表达（P<0.05）;real-time PCR显示10 μg/L IFN-γ刺激各胃癌细胞株12 h后PD-L1 mRNA水平上调（P<0.05）。结论：胃癌细胞株组成性表达PD-L1;IFN-γ可上调胃癌细胞株PD-L1的表达,呈现浓度、时间依赖性;其中以SGC7901细胞（来源于转移淋巴结）表达最高,推测胃癌细胞中PD-L1的表达与肿瘤分化程度无关,与组织来源和淋巴结转移有关;IFN-γ上调胃癌细胞PD-L1,表明IFN-γ并非都是抑制肿瘤增殖、生长,可能参与介导肿瘤免疫逃逸,故临床运用IFN-γ辅助治疗胃癌时应慎用。     

PD-L1 在胃癌患者手术前后外周血T 细胞和单核细胞上的表达及意义

目的:检测胃癌患者术前术后外周血细胞表面PD-L1的表达,探讨其表达改变及临床意义。方法:收集44例胃癌患者术前术后和18名健康志愿者外周血标本,流式细胞术检测CD3⁺ CD4⁺ T、CD3⁺ CD8⁺ T 细胞表面PD-1 和PD-L1 以及CD14⁺单核细胞表面PD-L1 的表达。结果:与健康志愿者相比,胃癌患者外周血CD4⁺ T 和CD8⁺ T 细胞表面PD-L1 表达无显著差异[(11.1±6.4)% vs (9.8±5.6)%,P =0.453 2;(13.9±12.0)% vs (12.0±7.1)%,P =0.558 9],而CD14⁺ 单核细胞表面PD-L1 表达水平显著增加[(29.2±16.7)% vs (17.5±9.7)%,P=0.007 3]。但胃癌患者术后CD4+ T 细胞和CD8⁺ T 细胞表面PD-L1 的表达均显著高于术前水平[(15.8±8.2)% vs (11.1±6.4)%,P= 0.001 5;(22.5±13.3)% vs (13.9±12.0)%,P =0.000 2],而术后外周血CD14+单核细胞PD-L1 表达水平并无显著变化[(33.8±17.3)% vs (29.2±16.7)%,P=0.082 8]。同时,胃癌患者手术前后外周血CD4⁺T 和CD8⁺T 细胞表面PD-1的表达无统计学差异[(25.6±9.9)% vs (26.9±8.9)%,P=0.505 5;(26.5±14.6)% vs (29.9±10.4)%,P=0.118 7]。结论:PD-L1 可作为监测原发性胃癌患者的免疫功能和预后评估的有效指标。     

Cardiomyocyte-targeted siRNA delivery by prostaglandin E(2)-Fas siRNA polyplexes formulated with reducible poly(amido amine) for preventing cardiomyocyte apoptosis

A cardiomyocyte-targeted Fas siRNA delivery system was developed using prostaglandin E(2) (PGE(2))-modified siRNA polyplexes formed by a reducible poly(amido amine) to inhibit cardiomyocyte apoptosis. PGE(2), which was used as a specific ligand for cardiomyocyte targeting, was conjugated to the terminal-end of the sense siRNA (PGE(2)-siRNA). The reducible cationic copolymer, synthesized via Michael-type polyaddition of 1,6-diaminohexane and cystamine bis-acrylamide (poly(DAH/CBA)), tightly condensed the PGE(2)-siRNA conjugate to form nanosize polyplexes having a diameter of 100-150nm. The PGE(2)-siRNA/poly(DAH/CBA) polyplexes decomplexed to release PGE(2)-siRNA in a cytosolic reducing environment due to the degradation of the reducible poly(DAH/CBA). The cellular uptake of the PGE(2)-siRNA/poly(DAH/CBA) polyplex was increased in rat cardiomyocytes (H9C2 cells) due to PGE(2) receptor-mediated endocytosis. When H9C2 cells were transfected with siRNA against Fas, a key regulator of ischemia-induced apoptosis, the PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex delivery system led to a significant increase in Fas gene silencing, resulting in inhibition of cardiomyocyte apoptosis. The PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex did not induce interferon-alpha in peripheral blood mononuclear cells. These results suggest that the PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex formulation may be clinically applicable as a cardiomyocyte-targeted Fas siRNA delivery system to inhibit apoptosis in cardiovascular disease.     

Octa-functional PLGA nanoparticles for targeted and efficient siRNA delivery to tumors

Therapies based on RNA interference, using agents such as siRNA, are limited by the absence of safe, efficient vehicles for targeted delivery in vivo. The barriers to siRNA delivery are well known and can be individually overcome by addition of functional modules, such as conjugation of moieties for cell penetration or targeting. But, so far, it has been impossible to engineer multiple modules into a single unit. Here, we describe the synthesis of degradable nanoparticles that carry eight synergistic functions: 1) polymer matrix for stabilization/controlled release; 2) siRNA for gene knockdown; 3) agent to enhance endosomal escape; 4) agent to enhance siRNA potency; 5) surface-bound PEG for enhancing circulatory time; and surface-bound peptides for 6) cell penetration; 7) endosomal escape; and 8) tumor targeting. Further, we demonstrate that this approach can provide prolonged knockdown of PLK1 and control of tumor growth in vivo. Importantly, all elements in these octa-functional nanoparticles are known to be safe for human use and each function can be individually controlled, giving this approach to synthetic RNA-loaded nanoparticles potential in a variety of clinical applications.     

Surface functionalized hollow manganese oxide nanoparticles for cancer targeted siRNA delivery and magnetic resonance imaging

Multifunctional hollow manganese oxide nanoparticles (HMON) were produced by a bio-inspired surface functionalization approach, using 3,4-dihydroxy-L-phenylalanine (DOPA) as an adhesive moiety, for cancer targeted delivery of therapeutic siRNA and simultaneous diagnosis via magnetic resonance imaging (MRI). Cationic polyethylenimine-DOPA conjugates were stably immobilized onto the surface of HMON due to the strong binding affinity of DOPA to metal oxides, as examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. These nanoparticles were subsequently functionalized with a therapeutic monoclonal antibody, Herceptin, to selectively target cancer cells. Confocal microscopy and MR imaging studies revealed that the surface functionalized HMON enabled the targeted detection of cancer cells in T(1)-weighted MRI as well as the efficient intracellular delivery of siRNA for cell-specific gene silencing. These nanomaterials are expected to be widely exploited as multifunctional delivery vehicles for cancer therapy and imaging applications.     

Enhanced silencing and stabilization of siRNA polyplexes by histidine-mediated hydrogen bonds

Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine–lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively ¹⁵N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1–H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5–1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.     

Efficient RNAi-based gene family knockdown via set cover optimization

OBJECTIVE: We address the problem of selecting an efficient set of initiator molecules (siRNAs) for RNA interference (RNAi)-based gene family knockdown experiments. Our goal is to select a minimal set of siRNAs that (a) cover a targeted gene family or a specified subset of it, (b) do not cover any untargeted genes, and (c) are individually highly effective at inducing knockdown. METHODS AND MATERIAL: We give two formalizations of the gene family knockdown problem. First, we show that the problem, minimizing the number of siRNAs required to knock down a family of genes, is NP-Hard via a reduction to the set cover problem. Second, we generalize the basic problem to incorporate additional biological constraints and optimality criteria. To solve the resulting set-cover variants, we modify the classical branch-and-bound algorithm to include some of these biological criteria. We find that in many typical cases these constraints reduce the search space enough that we are able to compute exact minimal siRNA covers within reasonable time. For larger cases, we propose a probabilistic greedy algorithm for finding minimal siRNA covers efficiently. We apply our methods to two different gene families, the FREP genes from Biomphalaria glabrata and the olfactory genes from Caenorhabditis elegans. RESULTS AND CONCLUSION: Our computational results on real biological data show that the probabilistic greedy algorithm produces siRNA covers as good as the branch-and-bound algorithm in most cases. Both algorithms return minimal siRNA covers with high predicted probability that the selected siRNAs will be effective at inducing knockdown. We also examine the role of "off-target" interactions: the constraint of avoiding covering untargeted genes can, in some cases, substantially increase the complexity of the resulting solution. Overall, we find that in many common cases our approach significantly reduces the number of siRNAs required in gene family knockdown experiments, as compared to knocking down genes independently.     

Targeted gene delivery by polyplex micelles with crowded PEG palisade and cRGD moiety for systemic treatment of pancreatic tumors

Adequate retention in systemic circulation is the preliminary requirement for systemic gene delivery to afford high bioavailability into the targeted site. Polyplex micelle formulated through self-assembly of oppositely-charged poly(ethylene glycol) (PEG)-polycation block copolymer and plasmid DNA has gained tempting perspective upon its advantageous core–shell architecture, where outer hydrophilic PEG shell offers superior stealth behaviors. Aiming to promote these potential characters toward systemic applications, we strategically introduced hydrophobic cholesteryl moiety at the ω-terminus of block copolymer, anticipating to promote not only the stability of polyplex structure but also the tethered PEG crowdedness. Moreover, M_w of PEG in the PEGylated polyplex micelle was elongated up to 20 kDa for expecting further enhancement in PEG crowdedness. Furthermore, cyclic RGD peptide as ligand molecule to integrin receptors was installed at the distal end of PEG in order for facilitating targeted delivery to the tumor site as well as promoting cellular uptake and intracellular trafficking behaviors. Thus constructed cRGD conjugated polyplex micelle with the elevated PEG shielding was challenged to a modeled intractable pancreatic cancer in mice, achieving potent tumor growth suppression by efficient gene expression of antiangiogenic protein (sFlt-1) at the tumor site.     

基质细胞衍生因子1靶向超声对比剂在体内可与血管内皮细胞紧密结合

背景：基质细胞衍生因子1是心肌梗死区域微环境中效力最强的趋化因子,在趋化干细胞修复梗死心肌以及在促进血管新生方面起到重要的作用。微泡和声学活性物质携带靶向配基,可制备成超声成像靶向对比剂并与活体细胞结合,用于分子成像,超声分子成像的关键是寻找“成像靶点”,并成功制备能与“成像靶点” 特异、高效结合的靶向超声对比剂。 目的：实验制备和评价携基质细胞衍生因子1单克隆抗体的靶向微泡超声对比剂。 方法：采用“生物素-亲和素”桥接法构建携基质细胞衍生因子1单克隆抗体的靶向微泡超声对比剂,并从外观、pH值、粒径测定、光镜及荧光显微镜下观、流式细胞仪检测等多个方面对靶向对比剂进行评价。4头中华小型猪均结扎左冠状动脉前降支第一对角支制备心肌梗死模型,2头开胸但不结扎左冠状动脉前降支第一对角支,均注入靶向超声对比剂,心肌组织冰冻切片后采用免疫荧光法检测靶向微泡的体内稳定性。 结果与结论：通过生物素-亲和素桥接法可将基质细胞衍生因子1抗体和超声微泡两者结合。体外实验中对比剂外观：表现为半透明的淡黄或绿色,静置后分层。非靶向对比剂pH值为7.02±0.12,靶向微泡对比剂的pH值为6.10±0.19。荧光显微镜下观察靶向微泡明亮且呈指环状绿色荧光环绕外壳周边,剧烈震荡后表面荧光无明显改变。靶向对比剂在携带基质细胞衍生因子1抗体之后微泡粒径大小为(2 422.62±238.82) nm。流式细胞仪检测显示,靶向对比剂在不同时间段的基质细胞衍生因子1携带率稳定,静置1 h后携带率稳定且剧烈震荡前后差异无显著性意义。在体内实验中可见靶向微泡在心梗部位血管内皮细胞处聚集。结果证实,经生物素-亲和素桥接法制备的携基质细胞衍生因子1单克隆抗体靶向微泡超声对比剂体内可与血管内皮细胞结合,在体外结合率高而且结合稳定。