MIL-101-NH2介导的核酸分子递送与基因沉默研究

核酸是DNA和RNA的总称,能够参与生物体内基因表达的调控.然而,核酸分子极易被降解,如果直接通过口服或静脉注射给药,生物利用率极低,因此需要合适的载体进行核酸的递送.选取了生物兼容性较好的铁基金属-有机框架（MOF）MIL-101-NH₂,分别负载单链DNA和siRNA来探究其细胞学行为.MIL-101-NH₂能够有效递送单链DNA和siRNA进入细胞,且siRNA在细胞内能够发生溶酶体逃逸,并发挥基因沉默的效应.结果表明：基于MIL-101-NH₂的纳米复合物是一种具有潜力的核酸递送与基因调控策略.     

PHBV/葡聚糖纳米药物载体的制备及表征

两亲性聚合物纳米颗粒作为疏水性抗肿瘤药物载体因其能够增强化疗效率并降低毒副作用而受到广泛关注.采用双乳液溶剂挥发法制备了聚(3-羟基丁酸酯-co-3-羟基戊酸酯)(PHBV)/葡聚糖纳米颗粒,测得平均粒径为205.0±6.9nm,Zeta电势为-1.59±0.12mV,纳米颗粒具有明显的壳核结构,粒径均一,分散性良好.将疏水性化疗药物顺铂包载后,其粒径及电势均无明显变化,载药量达19.3±2.9%.顺铂在模拟肿瘤细胞环境pH=5.5的磷酸盐缓冲液(PBS)中比正常细胞环境pH=7.4时释放更快,且累计释放周期均长达7d以上,表明该药物载体具有一定的pH响应性以及优异的缓释性能.细胞集落形成实验表明PHBV/葡聚糖纳米药物载体具有良好的生物相容性,而载药纳米颗粒对肿瘤细胞的毒性明显高于正常细胞,表明该纳米颗粒对肿瘤细胞具有更强的杀伤作用.综上所述,PHBV/葡聚糖纳米颗粒具有两亲性分子结构,合适的粒径及Zeta电势,显著的缓释效果,对肿瘤细胞具有pH响应性及更强的杀伤作用等优势,有望成为一种新型纳米药物载体,在癌症化疗中显著提高药物利用率并降低毒副作用.     

p-Hydroxybenzoic acid (p-HA) modified polymeric micelles for brain-targeted docetaxel delivery

Chemotherapies for brain diseases have been hampered due to the inability of transport of drug across the blood-brain barrier (BBB). In order to overcome the barrier, p-hydroxybenzoic acid (p-HA), a small molecule of benzamide analogue, was used as a ligand for brain-targeted drug delivery. The p-HA was conjugated to PEG-DSPE to form p-HA-PEG-DSPE. Docetaxel-loaded polymeric micelles were prepared by a thin-film hydration method using methoxy-poly(ethylene glycol)-distearoylphosphatidyl- ethanolamine (mPEG 2000 -DSPE) as a carrier and the p-HA-PEG-DSPE as a brain targeted material. The prepared micelles showed spherical with a mean diameter of (18±3) nm. Encapsulation efficiency and drug loading were (83.49±1.3)%, (7.7±1.2)% for un- modified micelles and (80.65±1.6)%, (7.47±1.8)% for p-HA-modified micelles, respectively. In vitro cellular uptake experiments showed that the p-HA-modified micelles increased BCECs cellular uptake by 1.2 times compared to the unmodified micelles. Ex vivo near-infrared fluorescence imaging showed that brain uptake of the p-HA-modified micelles was 1.3-1.8 times higher than that of the unmodified micelles. In vitro cytotoxicity assay against glioblastoma cell U87 MG showed that inhibition rate of the p-HA-modified micelles increased by 1.2 times compared to that of the unmodified micelles and 1.7 times compared to that of DTX. Survival time of nude mice bearing intracranial glioblastoma showed that the lifetime of saline group, Taxotere group, mPEG-DSPE/DTX micelles group and p-HA-PEG-DSPE/DTX micelles group was 22, 27, 32 and 45.8 d, representively, which indicated that anti-glioblastoma activity of DTX could be significantly enhanced by the p-HA-modified polymeric micelles. These results demonstrated that the p-HA-modified micelles could be a promising brain-targeted drug delivery system for hydrophobic drugs against glioblastoma.     

阳离子脂质体递送STAT3 siRNA抑制黑色素肿瘤细胞探究

利用阳离子脂质体作为载体,用其搭载鱼精蛋白与STAT3 siRNA复合物,通过一系列实验证实该复合体系可显著抑制STAT3基因在黑色素瘤细胞B16中的表达,促进肿瘤细胞的凋亡。首先对载体材料进行了一系列表征测定,检测了不同复合比例下载体和siRNA复合物的粒径和电位。利用载体和siRNA的复合物对B16细胞进行转染并测定转染效率,随后对复合材料的毒性进行了检测。此外还进行了细胞凋亡、平板克隆、荧光定量PCR以及Western Blot等一系列实验来进一步确定载体复合物的有效性。实验结果表明,阳离子脂质体搭载复合了鱼精蛋白的STAT3siRNA表现出了良好的靶向治疗性及优秀的递送效率,且复合体系稳定性良好,毒性低。     

Transvascular delivery of small interfering RNA to the central nervous system

A major impediment in the treatment of neurological diseases is the presence of the blood-brain barrier, which precludes the entry of therapeutic molecules from blood to brain. Here we show that a short peptide derived from rabies virus glycoprotein (RVG) enables the transvascular delivery of small interfering RNA (siRNA) to the brain. This 29-amino-acid peptide specifically binds to the acetylcholine receptor expressed by neuronal cells. To enable siRNA binding, a chimaeric peptide was synthesized by adding nonamer arginine residues at the carboxy terminus of RVG. This RVG-9R peptide was able to bind and transduce siRNA to neuronal cells in vitro, resulting in efficient gene silencing. After intravenous injection into mice, RVG-9R delivered siRNA to the neuronal cells, resulting in specific gene silencing within the brain. Furthermore, intravenous treatment with RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Repeated administration of RVG-9R-bound siRNA did not induce inflammatory cytokines or anti-peptide antibodies. Thus, RVG-9R provides a safe and noninvasive approach for the delivery of siRNA and potentially other therapeutic molecules across the blood-brain barrier.     

Preparation and cellular uptake of PLGA particles loaded with lamivudine

Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles loaded with lamivudine and coated with bovine serum albumin (BSA) were prepared via a double emulsion method. The influences of experiments parameters such as volume of inner aqueous phase, concentration of organic phase and ultrasonication time on the particle size and drug entrapment efficiency were investigated, obtaining PLGA particles with a diameter of ~260 nm and drug entrapment efficiency of ~35%. The particles were observed by scanning electron microscopy and transmittance electron microscopy, showing a core-shell structure. BCA assay found that 58 mg BSA was present on/in 1 g LPB particles. The loaded lamivudine showed a burst release at beginning and sustained release until 24 h in physiological conditions. Low pH could accelerate the release of lamivudine from PLGA particles, making the PLGA particles potential intelligent intracellular drug carriers. The PLGA particles were readily internalized into the human liver cells within a short time and increased gradually with the prolongation of incubation time regardless of the loading of lamivudine. The particles either resided within lysosomes or transferred to cytoplasm, but could not enter into the cell nucleus. The cell viability was not significantly influenced in the presence of the particles regardless of lamivudine encapsulation, suggesting that this kind of particles may be a good candidate for the intracellular anti-hepatitis B drug delivery.     

BGC-823细胞中慢病毒途径针对HIF-1α基因的RNAi实验

目的：构建人HIF-1α基因的shRNA慢病毒载体、包装生产重组慢病毒颗粒,病毒途径高效感染胃癌细胞株BGC-823,并验证基因沉默效率。方法：在NCBI数据查找人HIF-1α基因序列,然后使用siRNA在线设计软件,设计针对基因CDS区的3条siRNA序列及Negative序列。根据设计好的siRNA序列设计双链互补的shRNA-Oligo DNA,退火形成双链后与线性化载体链接,构建shRNA重组表达载体。经由293TN细胞包装shRNA重组慢病毒颗粒,随后使用重组病毒感染BGC-823,通过荧光标记蛋白GFP确定感染效率后收集细胞样本,分别采用Real-time PCR和Western blot方法检测靶基因在mRNA和蛋白质水平的沉默效率。结果：shRNA重组表达载体测序结果与设计序列完全一致,包装病毒后滴度达到1×104 ifu/μL。慢病毒感染BGC-823细胞取得了极高的基因转导效率。mRNA检测结果显示,siRNA3对于对与目的基因的沉默效果最好,较阴性对照序列组相比较,mRNA的表达量下降了92%,Western blot检测的结果与mRNA检测结果完全相符合。结论：通过慢病毒途径,可以在BGC-823细胞中高效的进行HIF-1α基因的沉默。     

MDR1-targeted siRNA delivery with cationic dendritic conjugated polymers

A cationic dendritic polyfluorene (PFP) is examined as a siRNA delivery vector. This material was designed to facilitate the nucleic acid binding, encapsulation and efficient cellular uptake. PFP can effectively protect siRNA against nuclease degradation, which is necessary for gene carriers. PFP can be used for multidrug resistance gene-targeted siRNA delivery in doxorubicin (Dox)-resistant human breast cancer cells (MCF7) cells. As a siRNA transfection agent, PFP can efficiently achieve the reversal of drug resistance and enhance the drug sensitivity. These new features and capabilities represent a major step toward conjugated polymers that can function for therapeutic application.     

羧甲基壳聚糖修饰阿霉素纳米脂质体的制备及体外细胞实验

以羧甲基壳聚糖(CMCT)为修饰剂,采用薄膜-pH梯度法制备具有pH敏感性的阿霉素纳米脂质体(CMCT-DOX-NL),以增加抗癌药物在肿瘤部位的蓄积,同时增强抗癌药物向肿瘤细胞内的传递。结果表明:制备的CMCT-DOX-NL粒子形貌圆整,粒径分布均匀为(38±22.1)nm,药物包封率为88.83%;相比传统的阿霉素纳米脂质体(DOX-NL),CMCT-DOX-NL与Hela细胞的结合和摄取均有所提高,对细胞的杀伤作用更强;CMCT-DOX-NL的体外药物释放具有明显的pH敏感性,比普通的阿霉素脂质体更能促进阿霉素(DOX)向肿瘤细胞内的传递。     

用于高效磁转染的鱼精蛋白修饰的铁氧磁性纳米粒研究

 功能化铁氧磁性纳米粒在生物医学中应用广泛,可用于肿瘤磁感应热疗、磁共振成像（Magnetic Resonance Imaging,MRI）、药物输送及磁转染等方面。为了探讨鱼精蛋白功能化修饰的铁氧磁性纳米粒的制备及其作为基因载体在体外磁转染中的可行性,采用共沉淀法制备Fe3O4磁性纳米粒,经表面氨基化修饰后在其表面偶联鱼精蛋白。利用透射电镜、傅里叶红外光谱仪、zeta电位与粒度分析仪等,对磁性纳米粒进行形态、粒径及zeta电位分析等表征检测。共聚焦显微镜观察磁转染方法转染报告基因绿色荧光蛋白质粒pEGFP-N1进入HepG2细胞的表达,以真核转染试剂vigofect为对照。结果显示,实验中制备的磁性纳米粒粒径10nm左右,在交变磁场下具有良好的升温性能。鱼精蛋白功能化修饰磁性纳米粒后,其zeta电位进一步增大,更利于与DNA有效结合,在HepG2细胞系,其转染pEGFP-N1质粒的效率高于vigofect。研究表明,鱼精蛋白功能化修饰的铁氧磁性纳米粒可作为磁转染的有效载体,由于其同时具备在交变磁场下升温的性能,在基因治疗联合热疗的研究领域具有一定的应用价值。     

基于小热休克蛋白-细胞穿膜肽蛋白纳米粒的siRNA递送系统

利用大肠杆菌表达系统原核表达并纯化小热休克蛋白-细胞穿膜肽融合蛋白(Hsp-Tat);用动态光散射和透射电镜成像分析Hsp-Tat纳米粒的粒径和形貌;用噻唑蓝(MTT)法和活/死细胞染色法研究蛋白纳米粒的细胞相容性;用凝胶阻滞实验检测纳米粒的小干扰RNA(siRNA)复合特性;用激光共聚焦荧光显微镜成像研究蛋白纳米粒的...     

Application of immobilized psychrotrophs in ICCBR to treat domestic wastewater and its microbiological investigation

To guarantee the efficiency of biotreatment of domestic wastewater in chilly season, six high efficient cold-adapted microorganisms were isolated, and identified as Zoogloea, Aeromonas, Flarobacterium, Micrococcus, Bacillus, Pseudomonas. These cold-adapted microorganisms with 63.67% Chemical Oxygen Demand (COD) removal efficiency higher than that of mesophiles at low temperature were immobilized on soft polyurethane foams and applied to internal circulation compound bioreactor (ICCBR) to treat domestic wastewater. In an experiment period of 12 months, the treatment efficiency, ecological factors, sludge physicochemical properties, and microbiology were studied. Results showed that the average COD removal efficiency in the ICCBR was 85.79% between 4℃ and 10℃ in winter, and 86.66% in the whole year, and COD of systemic effluent was below 60 mg•L⁻¹ which could achieve the first-degree B discharge standard of pollutants in China for municipal wastewater treatment plant. The reduction in microfauna and biomass on carriers could be associated with the seasonal temperature transients, the concentrations of protozoa and metazoan decreased from 110000±30000 microorganisms/mL sludge in summer to 35000±20000 microorganisms/mL sludge in winter, and biomass on carriers increased in the beginning and slightly reduced in the end.     

RNAi-mediated gene silencing in non-human primates

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.     

Immobilization of arsenic in soils by stabilized nanoscale zero-valent iron, iron sulfide (FeS), and magnetite (Fe3O4) particles

Arsenic is a widespread contaminant in soils and groundwater. While various iron-based materials have been studied for immobilizing arsenic in contaminated soils, the feasibility of stabilized iron-based nanoparticles has not been reported. This study investigates the effectiveness of using three types of starch-stabilized iron-based nanoparticles, including zero-valent iron (ZVI), iron sulfide (FeS), and magnetite (Fe₃O₄), for immobilization of arsenic in two representative As-contaminated soils (an orchard soil and a fire range soil). To test the effect of the nanoparticles on the arsenic leachability, As-contaminated soils were amended with the nanoparticles at various Fe/As molar ratios (5:1–100:1) and contact time (3 and 7 d). After three days’ treatments of a field-contaminated sandy soil, the PBET-based bioaccessibility of As decreased from an initial (71.3±3.1)% (mean±SD) to (30.9±3.2)% with ZVI, (37.6±1.2)% with FeS, and (29.8± 3.1)% with Fe₃O₄ at an Fe/As molar ratio of 100:1. The TCLP-based leachability of arsenic in a spiked fire range soil decreased from an initial (0.51±0.11)% to (0.24±0.03)%, (0.27±0.04)% and (0.17±0.04)% by ZVI, FeS, and Fe₃O₄ nanoparticles, respectively. The Fe₃O₄ nanoparticles appeared to be more effective (5% or more) than other nanoparticles for immobilizing arsenic. When the two soils were compared, the treatment is more effective on the orchard soil that has a lower iron content and higher initial leachability than on the range soil that already has a high iron content. These results suggest that these innocuous iron-based nanoparticles may serve as effective media for immobilization of As in iron-deficient soils, sediments or solid wastes.     

Water-soluble chitosan nanoparticles as a novel carrier system for protein delivery

High MW chitosan (CS) solutions have already been proposed as vehicles for protein delivery. The aim of the present work is to investigate the potential utility of water-soluble chitosan (WSC) as vehicles to load and deliver proteins. WSC nanoparticles (WSC NP) with various formations were prepared based on ionic gelation of WSC with pentasodium tripolyphosphate (TPP) anions. Bovine serum albumin (BSA) was used as a model protein drug incorporated into the WSC nanoparticles. Blank and BSA-loaded WSC nanoparticles were examined and determined to have a spherical shape with diameters between 35―190 nm, and zeta potential between 35―42 mV. FTIR confirmed that the tripolyphosphoric groups of TPP linked to the ammonium groups of WSC in the nanoparticles. Some factors affecting delivery properties of BSA have been investigated. Altering the concentration of BSA from 0.05 to 1 mg/mL enhanced the loading capacity of BSA but decreased loading efficiency simultaneously. Also, with the introduction of poly ethylene glycol (PEG), BSA release accelerated. Nanoparticle preparation from WSC with various deacetylation degrees (DDs) from 72.6% to 90% and MWs ranging from 3.5 to 15.8 kDa promoted loading efficiency and decreased the release rate. These results indicate that WSC nanoparticles are promising carriers for protein delivery.     

磷硫代siRNA的抗肿瘤基因沉默活性研究

由于目前尚无高效合成立体特异性磷硫代siRNAs(PS-siRNAs)的化学体系,本研究针对潜在的癌症治疗靶点PLK1,用a-磷硫代三磷酸腺苷(ATPaS)、a-磷硫代三磷酸胞苷(CTPaS)和a-磷硫代三磷酸尿苷(UTPaS)通过T7RNA聚合酶转录合成了部分磷硫代修饰的Rp-磷硫代siRNAs(Rp-PS-siRNAs),探究了nat-siRNAs和PS-siRNAs血清稳定性和基因沉默活性的差异性。发现酶促合成的磷硫代siRNA几乎不影响siRNA的基因沉默效率,但却显著提高siRNA的血清稳定性。因此,酶促转录合成的Rp-PS-siRNA可望作为siRNA的修饰形式,以延长siRNA的生物活性半衰期,使siRNA广泛应用于生物医学临床研究领域。     

RNAi作用机制及其应用研究进展

在Dicer酶作用下,dsRNA形成siRNA,组装成具有活性的蛋白质复合物RISC,触发染色质固缩,DNA甲基化、基因沉默和干扰蛋白质合成等。RNAi基因沉默高效、特异和简捷等优点,为细胞内基因表达研究提供了新思路、新方法,为临床治疗遗传性基因扩增型疾病提供极具潜力的全新策略。文章就RNAi机制及其应用研究做一综述。     

壳寡糖纳米载体细胞内递送EGFR脱氧核酶的研究

设计靶向EGFR mRNA的脱氧核酶(EGFR DRz),以壳寡糖(COS)为材料,建立了一种有效的纳米基因细胞内传递体系,并研究其介导的靶向EGFR的脱氧核酶在Hela细胞内的生物学效应.流式结果表明COS-EGFR DRz复合体转染效率为88.7%,与脂质体转染试剂的89.7%相比无显著差异.半定量RT-PCR结果显示,经壳寡糖纳米载体递送的EGFR DRz能有效地靶向切割Hela细胞内的EGFR mRNA,使其表达下降.进一步的流式分析显示细胞被阻滞在G0～G1期,并且出现凋亡现象,其中COS组的凋亡率为19.3%,大于对照组脂质体的凋亡率13.0%.研究表明,COS较脂质体有相似的转染效率和更低的毒性,是一种潜在的、有效的脱氧核酶递送载体.     

聚乙二醇-聚乙烯亚胺负载超顺磁纳米Fe3O4的合成及其基因转染应用

采用单甲基醚聚乙二醇改性聚乙烯亚胺,得到水溶性接枝共聚物聚乙二醇接枝支化聚乙烯亚胺(mPEG-g-PEI),并利用离子交换法制备了PEG-PEI-SPIO. 同时,还合成了mal-PEG-COOH,并用其制备了多功能负载SPIO的抗体-聚乙二醇-聚乙烯亚胺. 负载SPIO的抗体-聚乙二醇-聚乙烯亚胺与pDNA复合后形成的复合物粒径约为105 nm,体外实验结果显示T细胞能特异性的吸收抗体-聚乙二醇-聚乙烯亚胺负载的SPIO,靶向组在基因转染实验中有较好的效果,可用磁共振手段进行实时无创观测.