大肠杆菌O157：H7荧光纳米颗粒试纸条的研制

建立大肠杆菌O157：H7荧光纳米颗粒免疫层析法。方法：首先将荧光纳米颗粒与大肠杆菌O157：H7单抗共价偶联,制成大肠杆菌O157：H7单抗-荧光纳米颗粒偶联物。用该偶联物代替金标颗粒,以双抗体夹心作为反应模式来制备大肠杆菌O157：H7免疫层析试纸条。在紫外灯下观察免疫层析试纸条上经免疫反应而产生的质控线和检测线上的荧光信号,并利用荧光强度来半定量检测大肠杆菌O157：H7。结果：用所制备的试纸条对11属24种54株菌进行检测,所有27株大肠杆菌O157：H7的检测结果呈阳性．其它非大肠杆菌O157菌株检测结果呈阴性。用该试纸条检测人工污染的鸡肉样品。灵敏度为2．7×10^4CFU／mL。通过观察检测线的有无。可对大肠杆菌O157：H7进行半定量检测。分别用所制备的大肠杆菌O157：H7免疫层析试纸条和标准法检测57份样品,2种方法的总符合率为80．7％。结论：大肠杆菌O157：H7荧光纳米颗粒检测试纸条的研制成功,为大肠杆菌O157：H7的快速检测提供了一个极好的检测方法．便于野外检测的开展．具有广阔的应用前景。     

迷迭香精油壳聚糖纳米粒的制备及其对冷藏草鱼保鲜效果研究

为探讨迷迭香精油壳聚糖纳米粒对冷藏(4℃)草鱼肉的保鲜效果,利用离子交联法制备迷迭香精油壳聚糖纳米制剂(CS-NP-REO)并优化其制备工艺。通过超滤离心法测定纳米粒包封率和载药量,在扫描电镜下观察其粒径大小和外观形态,进一步测定其对冷藏草鱼肉的保鲜效果。结果表明:在壳聚糖(CS)、三聚磷酸钠(TPP)和迷迭香精油(REO)质量浓度分别为2、1.5和2mg/mL,体积比5∶2∶1,pH 4的条件下制备的CS-NP-REO纳米粒包封率为84.52%,载药量为10.56%,粒径约200nm,颗粒饱满,大小均一;用CS-NP-REO处理过的冷藏草鱼肉片,在贮藏第7天时菌落总数为3.55×10~6 CFU/g、硫代巴比妥酸值(TBARS)为0.61×10~(-3) mg/g、挥发性盐基氮值(TVB-N)为18.74mg/hg,货架期为8d以上,相比对照组延长3～4d,其保鲜效果优于迷迭香精油和壳聚糖纳米粒的单独使用。说明迷迭香精油壳聚糖纳米粒具有抑制微生物生长及脂肪氧化、延长冷藏草鱼肉货架期的作用。     

Nanoparticle for siRNA delivery and its pancreatic cancer targeting ability

AIM: To synthesize a safe, efficient and targeted nanoparticulate carrier for siRNA delivery to pancreatic cancer cells. METHODS: Iron oxide nanocrystal with carboxylic acid group-polyethyleneimine (IONP-PEI) was synthesized and investigated as a nonviral carrier of siRNA to the pancreatic cells. The size, surface and charge using zeta potential were characterized. The perfect charge ratio between amino groups of IONP-PEI and phosphate groups of siRNA (N/P) was determined by the transfection efficiency detection, gel retardation assay and MTS assay. An antibody-directed nonviral vector, scFvCD44v6-IONP-PEI nanoparticle attaching to the cancer-associated CD44v6 single-chain variable fragment, was constructed as a cancer-targeting nanocarrier for siRNA delivery. Prussian blue staining and immunofluorescent staining were performed to detect the distribution of scFvCD44v6-IONP-PEI/siRNA complexes in the cells. The transfection efficiency, fluorescence intensity and the expression of KRAS at mRNA and protein levels in the cells transfected by IONP-PEI/siRNA and scFvCD44v6-IONP-PEI/siRNA were detected by flow cytometry, fluorescence microscopy, real-time PCR and Western blotting, respectively. RESULTS：The mass ratio of IONP to PEI was 0.75. The suitable ratio of N/P was 20. The averaged size and surface zeta potential of IONP-PEI/siRNA in deionized water were (51.3±2.2)nm (diameter) and (21.73±8.07)mV,respectively. Red fluorescence was seen in both targeting and nontargeting groups, which clearly revealed the intracellular distribution of siRNA and delivery agents. Transfection efficiencies in targeting and nontargeting groups were (89.75±1.81)% and (59.87±4.52)%, respectively. Down-regulation of the KRAS mRNA in Panc-1 cells transfected with siKRAS by scFvCD44v6-IONP-PEI and IONP-PEI was up to (34.02± 6.15)% and (51.09±6.70)%, respectively. The protein level of KRAS was lower in targeting group than that in nontargeting group. CONCLUSION：scFvCD44v6-IONP-PEI is a safe and efficient nanoparticulate carrier for gene delivery. It is more effective to transfer siRNA into the cells and mediate gene silencing effect in vitro than the nontargeting group.     

Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida

Nanoparticles (NPs) of TiO₂ and ZnO are receiving increasing attention due to their widespread applications. To evaluate their toxicities to the earthworm Eisenia fetida (Savigny, 1826) in soil, artificial soil systems containing distilled water, 0.1, 0.5, 1.0 or 5.0 g kg⁻¹ of NPs were prepared and earthworms were exposed for 7 days. Contents of Zn and Ti in earthworm, activities of antioxidant enzymes, DNA damage to earthworm, activity of cellulase and damage to mitochondria of gut cells were investigated after acute toxicity test. The results from response of the antioxidant system combined with DNA damage endpoint (comet assay) indicated that TiO₂ and ZnO NPs could induce significant damage to earthworms when doses were greater than 1.0 g kg⁻¹. We found that Ti and Zn, especially Zn, were bioaccumulated, and that mitochondria were damaged at the highest dose in soil (5.0 g kg⁻¹). The activity of cellulase was significantly inhibited when organisms were exposed to 5.0 g kg⁻¹ of ZnO NPs. Our study demonstrates that both TiO₂ and ZnO NPs exert harmful effects to E. fetida when their levels are higher than 1.0 g kg⁻¹ in soil and that toxicity of ZnO NPs was higher than TiO₂.     

Ethylene control in cut flowers: Classical and innovative approaches

Ethylene-mediated premature floral senescence and petal or flower abscission affect postharvest longevity of several species used as cut flowers. Exposure to exogenous or endogenously produced ethylene can be controlled in several ways. These include the use of ethylene biosynthesis inhibitors or ethylene action inhibitors, and ethylene removal technologies. In addition, genetic modification can be very effective in controlling ethylene synthesis and perception. We review here the potential for applications of nanotechnology to control ethylene levels and postharvest management in the flower industry. Already, nanosponges have been shown to enhance efficacy of the ethylene inhibitor 1-MCP in several flower species. In carnation, 1-MCP included in nanosponges also allowed better control of Botrytis cinerea damage. However other applications are also considered based on successes in the use of this technology to increase agricultural production and decrease postharvest waste. Nano-metal based sensors could be used for detection of ethylene in the store and to label the product along the distribution chain. Furthermore, nanocomposites could be included as scavengers for ethylene removal in active packaging, and nanocatalysts could promote ethylene catalytic degradation in the warehouse. Nanoparticles could also be introduced into a new generation of packaging to control effects of gases and UV, and increase strength, quality and packaging appearance. This review highlights recent results on the use of nanotechnology sensu lato and potential application for cut flower vase life improvement, focusing on ethylene control strategies.     

氯霉素荧光纳米颗粒免疫层析法的建立

本研究将荧光纳米颗粒与氯霉素单克隆抗体共价偶联在一起,用该颗粒代替常用的金标颗粒标记氯霉素单克隆抗体,以竞争法作为反应模式来制备免疫层析试纸条。在紫外灯下观察免疫层析试纸条上经免疫反应而产生的质控线和检测线上的荧光信号,利用荧光强度来半定量检测动物源性食品中的氯霉素残留。所制备的检测氯霉素的免疫层析试纸条只与氯霉素有交叉反应,与非目标检测物之间没有交叉反应。该试纸条的肉眼检测灵敏性为0.5ng/mL,满足相关检测标准要求。通过比较检测线和质控线之间的亮度,能够对氯霉素进行半定量检测。本研究建立的用于氯霉素残留检测的免疫层析法,具有简便、快速、灵敏度高的特点,有广阔的应用前景。     

恩诺沙星纳米粒的制备及稳定性研究

[目的]优化恩诺沙星纳米粒制备参数,考察其稳定性。[方法]以壳聚糖为辅料,采用离子交联法和冷冻干燥法制备恩诺沙星壳聚糖纳米粒,以包封率为参考指标设计正交试验,确定优化制备参数,以透射电镜观察其表观特征,并对其热稳定性及紫外稳定性进行考察。[结果]以优化参数制备的恩诺沙星壳聚糖纳米粒平均粒径237．4nm平均包封率（71．9±2．2）％,平均载药量（12．4±0．6）％。与原料药相比,4h热降解率和紫外降解率分别减少了38．4％和13．3％。[结论]离子交联法和冷冻干燥法适用于制备恩诺沙星壳聚糖纳米粒,其产品具有良好的热稳定性和紫外稳定性。     

Experimental study of 5-fluorouracil loaded polylactic acid nanoparticles control-releasing preparation on tumor

AIM: To investigate the preparation techniques and anti-tumor effects both in vitro and in vivo of a novel nanoparticles control-releasing preparation of 5-fluorouracil (5-FU) by intravenous injection.METHODS: With polylactic acid (PLA) as marix materials,we adopted ultrasound emulsification method to prepare PLA enveloped 5-FU nanoparticles (5-FU-NPs).Scanning electricity microscopy was used to observe the morphology of 5-FU-NPs and laser optical scattering experiment was conducted to determine its diameter distribution.The drug-carrying capacity (ratio) of the nanoparticles was determined by means of high-power liquid chromatography(HPLC) and MTT test was used to observe cytotoxicity in vitro.The anti-tumor effects were determined at different dosages,frequencies of taking drugs in vivo.RESULTS: Scanning electron microscopy showed that the 5-FU-NPs were globular particles with smooth surface in an average particle diameter of 191.9 nm with a normal distribution,and the drug-carrying capacity of 5-FU-NPs was 15.2%.5-FU-NPs had the same anti-cancer effect as unenveloped drug in vitro and showed typical dose-effect relationship.Compared to naked 5-FU,5-FU-NPs presented significant difference (P<0.05) in anti-cancer effect.CONCLUSION: Nanoparticles may serve as effective carrier for controlled release of 5-FU,which lead to reasonable administration of 5-FU with less toxicity.     

Establishment of human pancreatic tumor xenograft mouse model for evaluating tumor-homing and gene-silencing effects of siRNA-loading nanoparticles

AIM：To establish an effective and rapid method to develop transplanted subcutaneous pancreatic carcinoma by inducing PANC-1 cells into nude mice, and then use this mouse model to evaluate the tumor-homing and gene-silencing effects of siRNA-loading nanoparticles in vivo. METHODS：Different numbers of PANC-1 cells in 100 μL or 300 μL PBS were inoculated subcutaneously into the right flank of BALB/c (nu/nu) mice. When the tumor volume reached 100 mm3, siRNACY5.5 nanoparticles were injected through the mouse tail vein to perform in vivo imaging assay. Besides, the mice were randomly divided into 3 treatment groups treated with PBS, scrambled control RNA nanoparticles and siKras nanoparticles, respectively. The protein expression of Kras was detected by Western blotting and immunohistochemical staining. RESULTS：After inoculated with 1×107 PANC-1 cells in 300 μL PBS, all mice developed tumors within 2 weeks. The in vivo results showed that siRNA-loading nanoparticles accumulated in the tumor tissues and exerted gene silencing effect. CONCLUSION:In the present study, an effective and rapid method was established for PANC-1 cells to induce transplanted subcutaneous pancreatic carcinoma in nude mice within 2 weeks, which is suitable for in vivo imaging and treatment evaluations as a reproducible and reliable way for the further experiments.     

Preventing Effects of Nano-Selenium Particles on Serum Concentration of Blood Urea Nitrogen,Creatinine, and Total Protein During Intense Exercise in Donkey

The present study was aimed to determine the effects of oral administration of selenium nanoparticles (Se NPs) on blood urea nitrogen (BUN), creatinine, and total protein changes during intense exercise in donkey. Eight female donkeys, 2-5 years of age, weighing 130-190 kg, were randomly divided in two groups: treated and control groups receiving Se NPs 0.5 mg/kg and normal saline for 10 consecutive days, respectively. Blood samples were taken at the beginning of the experiment (before supplementation), closely after Se NPs supplementation (before exercise), and at 2-, 24-, and 72-hour postexercise recovery times. Results showed that serum selenium concentration was significantly increased in response to Se NPs supplementation and intense exercise. Creatinine concentration of both groups was significantly increased at 2-hour postexercise recovery time and sharply decreased in treated group at 72-hour postexercise recovery time (P < .05). A similar pattern was obtained for BUN changes in control group; as such its concentration was significantly increased at 2-hour postexercise recovery time in comparison with the Se NPs-supplemented group (P < .05). These findings may explain the positive effects of Se NPs supplementation on serum BUN and creatinine changes in response to intense exercise in donkey. The positive effect of Se NPs might be related to the incorporation of Se into proteins such as selenocysteine and its preventive role on tissue oxidative damages.