Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells

The A₃B-type Lactosome comprised of poly(sarcosine)₃-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A₃B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT.     

Toxicogenomics of Non-viral Vectors for Gene Therapy: A Microarray Study of Lipofectin- and Oligofectamine-induced Gene Expression Changes in Human Epithelial Cells

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.     

siRNA抑制人乳腺癌SK-BR-3细胞株VEGF基因表达的实验研究

目的研究siRNA(small interfering,RNA)对乳腺癌细胞SK-BR-3的VEGF基因表达的抑制作用,为RNAi技术在肿瘤生物治疗中的应用提供实验基础。方法体外合成一条针对VEGF基因的siRNA,使用脂质体转染的方法导入细胞,观察转染后乳腺癌细胞SK-BR-3的增殖变化,MTT法检测细胞存活率,RT-PCR检测转染后VEGFmRNA表达水平的变化,ELISA检测蛋白表达的下降效果。结果所设计的siRNA能有效抑制乳腺癌细胞的生长;降低了VEGFmRNA的表达;蛋白表达水平也显著降低。作为阴性对照的错义序列组siRNA则没有这种效果,不起作用。结论 siRNA可以有效抑制细胞株SK-BR-3中VEGF的表达,从而抑制细胞生长。应用RNA干扰技术可以有效抑制肿瘤细胞的增殖。     

Survivin基因沉默对裸鼠人卵巢癌细胞移植瘤凋亡的影响

目的探讨survivin基因沉默对人卵巢癌细胞株SKOV3裸鼠移植瘤凋亡的影响。方法将重组survivin shRNA plasmid转染人卵巢癌细胞株SKOV3细胞,通过裸鼠移植瘤实验比较survivin基因沉默对裸鼠移植瘤的生长抑制作用,荧光定量聚合酶链反应(PCR)和蛋白印迹法检测与凋亡密切相关的cytochrome-c、caspase-9和caspase-3基因的表达。结果裸鼠移植瘤实验显示,与对照组(空载体组和未转染组)相比,survivin shRNA plasmid转染组人卵巢癌细胞株SKOV3细胞生长速度减慢,肿瘤体积缩小(P<0.05);荧光定量PCR和蛋白印迹法结果都显示,与对照组相比,survivin shRNA plasmid转染组cytochrome-c、caspase-9和caspase-3的表达明显升高(P<0.05)。结论 survivin基因沉默可通过促进肿瘤细胞的凋亡而抑制裸鼠移植瘤生长。     

靶向Akt1小干扰RNA对人喉癌细胞增殖和凋亡的影响

目的：研究Akt1基因沉默对人喉癌细胞系（Hep-2）增殖和凋亡的影响。方法：设计并合成针对Akt1的特异性小干扰RNA（siRNA）序列,利用转染试剂转入体外培养人Hep-2细胞,48h后收集细胞,RT-PCR和Westernblot验证siRNA的沉默效应,MTT法检测细胞增殖活性、台盼蓝细胞染色法计数细胞存活率、AnnexinV/PI检测细胞凋亡和坏死。结果：设计的Akt1siRNA能够有效抑制体外培养Hep-2细胞内Akt1表达实现其基因沉默效应;Akt1siRNA干扰组Akt1mRNA转录和蛋白表达水平均低于空白和阴性对照组;Akt1siRNA干扰组细胞增殖活性下降,细胞存活率下降,Akt1siRNA干扰后细胞凋亡率和坏死率增加。结论：RNA干扰Akt1基因沉默具有抑制喉癌细胞增殖,促进细胞凋亡的作用,Akt1可作为喉癌基因治疗的后选新靶点。     

Enhanced transfection of polyplexes based on pluronic-polypropylenimine dendrimer for gene transfer

Third generation cationic dendritic polymeric polypropyleneimine (PPI) was modified by Pluronic P123 and investigated for gene delivery. The cytotoxicity of P123-PPI was evaluated by the MTT assay and shown to be much lower than that of PPI alone. P123-PPI and PPI can both condense plasmid DNA into nanoparticles with a size of approximately 100 nm and a zeta potential of about 15 mV at the N/P ratio 20:1. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 0.4 U/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 75 μg/mL sodium heparin. The transfection efficiency of SPC-A1 cells using P123-PPI/DNA nanoparticles was much higher than the transfection utilizing PPI/DNA nanoparticles. The addition of free P123 during the preparation of P123-PPI/DNA nanoparticles could significantly enhance the transfection efficiency in the presence of 10% fetal bovine serum. Therefore, P123-PPI/DNA complex nanoparticles may be a safe, efficient and promising cationic conjugate for gene delivery.     

Development of a high-content screening method to evaluate the cellular population-based biological activity of new siRNA delivering reagent

Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-content screening (HCS) strategy to evaluate a novel delivery system (named CLD) of siRNA therapeutics, with which both the content of intracellular siRNAs and changes in protein expressing levels have been quantified in group cells and cellular population. We also observed that with the better cell uptake, CLD provided siRNA therapeutics (siBraf) better antitumor capability. This novel strategy was proved to be with efficiency, accuracy and high competency to adherent cell lines, thus making siRNA research more simplified     

特异沉默人核糖核苷酸还原酶M2基因对人骨肉瘤细胞生物学特性的影响

目的 探讨小干扰RNA(siRNA)沉默核糖核苷酸还原酶M2(RRM2)基因表达对入骨肉瘤细胞Saos-2生物学影响及分子机制.方法 利用小干扰RNA技术,观察沉默Saos-2细胞中RRM2基因的表达,用实时荧光定量PCR(Real time-PCR)及Western blot技术检测人骨肉瘤细胞Saos-2和人正常成骨细胞hFOB1.19的mRNA及蛋白的表达;用CCK-8方法检测si-RRM2对Saos-2细胞增殖的影响;用Transwell细胞迁移系统检测si-RRM2对Saos-2细胞迁移能力的影响;用酶联免疫吸附试验(ELISA法)检测Saos-2细胞凋亡;采用Western blot技术检测细胞周期调控因子细胞周期素D1(Cyclin D1)和抗凋亡蛋白B细胞淋巴瘤/白血病-2(Bcl-2)蛋白水平变化.结果 Saos-2细胞RRM2 mRNA和蛋白比hFOB1.19细胞高表达;用si-RRM2转染Saos-2细胞后,Real time-PCR结果证实能时间及剂量依赖性下调RRM2基因表达;CCK-8方法结果显示下调RRM2基因会时间及剂量依赖性抑制Saos-2增殖,而人正常成骨细胞增殖抑制没有显著变化;Transwell细胞迁移系统检测显示下调RRM2基因后,显著抑制了Saos-2的迁移能力;si-RRM2联合化疗药物阿霉素促进Saos-2细胞凋亡;Western blot结果显示沉默RRM2后细胞周期调控因子Cyclin D1和抗凋亡蛋白Bcl-2均下调.结论 RRM2的过表达与人骨肉瘤细胞Saos-2的高增殖和迁移能力有关,抑制RRM2的功能是治疗人骨肉瘤的一个潜在的治疗策略.     

Synchronous conjugation of i-motif DNA and therapeutic siRNA on the vertexes of tetrahedral DNA nanocages for efficient gene silence

The functionality of DNA biomacromolecules has been widely excavated, as therapeutic drugs, carriers, and functionalized modification derivatives. In this study, we developed a series of DNA tetrahedron nanocages (Td), via synchronous conjugating different numbers of i-(X) and therapeutic siRNA on four vertexes of tetrahedral DNA nanocage (aX-Td@bsiRNA, a+b = 4). This i-motif-conjugated Td exhibited good endosomal escape behaviours in A549 tumor cells, and the escape efficiency was affected by the number of i-motif. Furthermore, the downregulating mRNA and protein expression level of epidermal growth factor receptor (EGFR) caused by this siRNA embedded Td were verified in A549 cells. The tumor growth inhibition efficiency of the 2X-Td@2siRNA treated group in tumor-bearing mice was significantly higher than that of non-i-motif-conjugated Td@2siRNA (3.14-fold) and free siRNA (3.63-fold). These results demonstrate a general strategy for endowing DNA nanostructures with endosomal escape behaviours to achieve effective in vivo gene delivery and therapy.     

CTLA-4 siRNA对慢性乙型肝炎患者外周血Th1/Th2细胞因子的影响

目的:观察化学合成的小干扰RNA(small interfering RNA,siRNA)对慢性乙型肝炎(chronic hepatitis B,CHB)患者外周血淋巴细胞细胞毒T淋巴细胞相关抗原4(cytotoxic T-lympho-cyte antigen 4,CTLA-4)表达的抑制作用及干扰后对细胞因子IFN-γ、IL-2和IL-4分泌的影响,探讨CTLA-4对慢性乙型肝炎的T细胞免疫调节作用。方法:检测CHB患者外周血淋巴细胞CTLA-4,观察其与HBV-DNA的相关性(P<0.05);根据人淋巴细胞CTLA-4的基因序列,设计合成CTLA-4 siRNA及阴性对照siRNA(siR-NA-co),电穿孔法转染CHB患者外周血淋巴细胞,采用实时荧光定量PCR(real-time Q-PCR)方法检测淋巴细胞CTLA-4 mRNA的表达,采用Western Blotting检测淋巴细胞CTLA-4蛋白表达,采用酶联免疫吸附试验(enzyme-linkedim-muno sorbent assay,ELISA)检测IFN-γ、IL-2和IL-4分泌。结果:CHB患者外周血淋巴细胞CTLA-4表达量与血清HBV-DNA载量有关;CTLA-4 siRNA转染CHB患者外周血淋巴细胞后,CTLA-4 mRNA及CTLA-4蛋白表达均受到抑制,IFN-γ、IL-2分泌增加,与阴性对照相比差异具有统计学意义(P<0.01)。而IL-4分泌没有变化,与阴性对照相比差异没有统计学意义(P>0.05)。结论:CHB患者外周血淋巴细胞CTLA-4表达一定程度抑制免疫反应,利于HBV-DNA的复制;利用siRNA在mRNA水平抑制CHB患者外周血淋巴细胞CTLA-4的表达,能够诱导Th1型细胞因子IL-2、IFN-γ分泌增加,对Th2型细胞因子IL-4无影响。说明抑制CTLA4有助于慢性乙型肝炎患者T细胞免疫的增强。     

单克隆抗体G250修饰的基因载体携带Ki-67小干扰RNA作用Hela细胞体外实验研究

目的：评价携带肿瘤增殖基因Ki-67小干扰RNA（siRNA）的表达质粒pSliencer-Ki-67对人宫颈癌细胞株Hela细胞的基因治疗功效。方法将构建的携带肿瘤增殖基因Ki-67 siRNA表达质粒pSliencer-Ki-67,用G250单克隆抗体（G250 mAb）与非病毒基因载体聚乙烯亚胺（PEI）偶联制成新型基因转染载体感染Hela细胞以及人肝癌细胞株HepG2细胞、小鼠胚胎成纤维细胞株NIH3T3细胞,通过四甲基偶氮唑蓝比色法（MTT法）检测其抑制肿瘤细胞增殖的效能,应用膜联蛋白Ⅴ-碘化丙啶（AnnexinⅤ-PI）法、4′,6-二脒基-2-苯基吲哚（DAPI）法检测pSliencer-Ki-67对细胞早、晚期凋亡的影响,并应用逆转录-聚合酶链反应（RT-PCR）、蛋白质印迹（Western blotting）法检测Ki-67 mRNA和Ki-67蛋白的表达。结果在RT-PCR与Western blotting检测中,用G250 mAb-PEI/pSliencer-Ki-67转染细胞后,Ki-67 mRNA、Ki-67蛋白表达较对照组明显降低;MTT法检测发现,G250 mAb/PEI-pSliencer-Ki-67对Hela细胞（G250抗原阳性）有明显抑制作用,而对HepG2细胞、NIH3T3细胞（G250抗原阴性）无明显抑制作用。通过荧光显微镜检测也发现,在早、晚期凋亡实验中,G250 mAb/PEI-pSliencer-Ki-67转染Hela细胞后,可诱导细胞凋亡,但对正常细胞的凋亡诱导作用非常弱。结论pSliencer-Ki-67能够诱导Hela细胞（G250抗原阳性）凋亡,而对HepG2细胞及NIH3T3细胞（G250抗原阴性）无明显抑制作用,pSliencer-Ki-67在Hela细胞的基因治疗方面有潜在应用前景。     

Sulfhydryl Based Cationic Surfactants and the Impact of Polyanions on Disulfide Bond Formation: Implications for Gene Transfer Vectors

Compacting plasmid DNA (pDNA) into a small size is a fundamental necessity for the efficient in vivo transfer of nucleic acids to somatic cells. An approach for accomplishing this is to condense pDNA using cationic detergents with sulfhydryl groups, near their critical micelle concentration. In this study, a model surfactant was used to study how the rate of disulfide bond formation relates to environmental factors. It was shown that the thiol detergent had the ability to form a disulfide bond when oxidized and the presence of polyanions was significantly increased. The addition of a reducing agent disrupted the disulfide bonds initially, but this was followed by disulfide bond reformation in a short time period.     

siRNA沉默ASIC1a基因对佐剂性关节炎大鼠关节软骨细胞凋亡的影响研究

目的:探讨小分子干扰RNA(siRNA)技术诱导佐剂性关节炎(adjuvant-induced arthritis,AA)大鼠关节软骨细胞中ASIC1a表达沉默对细胞凋亡的影响。方法:通过化学合成法合成特异性荧光短链ASIC1asiRNA-FAM,使用Lipo-fectamine 2000转染试剂盒将ASIC1asiRNA转染入关节软骨细胞,采用荧光显微镜、流式细胞术、实时荧光定量PCR(q-RT-PCR)及WesternBlot法检测siRNA转染效率及其对ASIC1amRNA和蛋白表达的抑制作用。同时采用An-nexin-V/PI流式细胞术检测各组细胞凋亡情况。结果:ASIC1asiRNA能成功转入软骨细胞,转染后AA大鼠关节软骨细胞中ASIC1amRNA表达显著低于对照组(P<0.01),最大抑制率为85.4%;Western Blot结果显示,转染特异性siRNA后ASIC1a蛋白表达明显低于对照组(P<0.01)。Annexin-V/PI流式细胞术结果表明,与模型组相比,siRNA-3转染引起ASIC1a表达沉默后AA大鼠软骨细胞凋亡明显减少。结论:siRNA介导的AA大鼠关节软骨细胞ASIC1a表达沉默模型是研究酸敏感离子通道对软骨细胞代谢影响的可靠模型,siRNA-3转染对胞外酸化刺激条件下AA大鼠关节软骨细胞凋亡的保护作用可能与其调节的表达有关。     

Delivery of magic bullets: on the still rocky road to gene therapy

In this issue, the promises, problems and current progress towards gene therapy are examined in a themed set of six reviews. These cover the major methodologies deployed over the last twenty to thirty years to deliver a gene or other potentially therapeutic molecules into an organism. Initial enthusiasm and optimism concerning the prospects for gene therapy and more generally, the delivery of magic bullets, arose after the pioneering discoveries of monoclonal antibodies and retroviral infection during the 1970''s and were fuelled by strategies to make synthetic viruses and the advent of chemical vectors over the succeeding twenty years. However, despite significant advances, to date, the early hopes of widespread gene therapy still remain largely unfulfilled.This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

基于RNA干扰技术探讨消岩汤对肺腺癌A549细胞凋亡的影响

目的 分析消岩汤干预下sh-survivin对人肺腺癌A549细胞增殖及凋亡的影响。方法 选择人肺腺癌A549细胞, 采用中药复方进行干预及shRNA进行转染, 将转染后的细胞分组, 通过MTT法观察消岩汤对A549细胞生长的影响, 免疫组化法检测survivin蛋白的表达, 流式细胞仪检测细胞凋亡率。结果 消岩汤能有效地抑制人肺腺癌A549的细胞增殖, 运用消岩汤作用细胞24 h后, 侵袭的细胞数明显减少, 该方作用48 h后可以诱导肿瘤细胞凋亡;消岩汤中剂量组与转染sh-survivin组抑制率相当, 不同剂量的消岩汤均可协同sh-survivin使肺癌细胞凋亡率增加。结论 消岩汤可以通过干预肿瘤细胞凋亡抑制蛋白的表达而有效抑制肿瘤的发展。     

Transfection of primary human nasal epithelial cells using a biodegradable poly (ester amine) based on polycaprolactone and polyethylenimine as a gene carrier

The purpose of this study was to prepare and characterize poly (ester amine) (PEA)/pGL3 complexes and investigate their transfection efficiency in human nasal epithelial (HNE) cells. Particle size, zeta potential, and gel retardation characteristics of PEA /pGL3 complexes were also measured. After treatment of DNase-I, protection and release assay of PEA/pGL3 complexes were performed. To assess the transfection efficiency and cytotoxicity, measurement of relative luciferase activity and MTS assay were performed. PEA/pGL3 complexes showed effective and stable DNA condensation with the particle sizes below 200 nm, implicating their potential for intracellular delivery. PEA/pGL3 complexes successfully transfected into the HNE cells with higher viability of the cells. These results suggested that, the PEA can be used as an efficient cationic polymeric vehicle which provides a versatile platform for further investigation of structure property relationship along with the controlled degradation, significant low cytotoxicity, and high transfection efficiency of the primary HNE cells.     

Synthesis of a novel biodegradable poly(ester amine) (PEAs) copolymer based on low-molecular-weight polyethyleneimine for gene delivery

In this paper, a novel biodegradable poly(ester amine) (PEA) copolymer was successfully prepared from low-molecular-weight polyethyleneimine (PEI, Mn = 1800) and poly(-caprolactone)–Pluronic–poly(-caprolactone) (PCFC) copolymers. According to the results of agarose gel electrophoresis, particle sizes and zeta potential measurement and transfection efficiency, these PEA copolymers showed great ability to condense plasmid DNA effectively into nano-complexes with small particle size (≤200 nm) and moderate zeta potential (≥12 mV) at proper polymeric carrier/DNA weight ratio. Compared with low-molecular-PEI (Mn = 1800), the obtained PEAs exhibited higher transfection efficiency as well as lower cytotoxicity. These results indicated that such PEAs might have great potential application in gene delivery system.