纳米粒子包载反义雷帕霉素靶蛋白基因转染对移植静脉内膜增生的影响

目的观察纳米粒子包载的反义雷帕霉素靶蛋白（mTOR）基因局部转染对移植静脉内膜增生的影响。方法应用聚乳酸聚乙醇酸共聚物（PLGA）和聚乙烯醇（PVA）包载mTOR基因。建立自体静脉移植模型，随机分成转基因组、空载体组、对照组。转基因组移植静脉转染纳米粒子包载的反义mTOR基因，空载体组单纯转染纳米粒子包载的空载体，对照组不予特殊处理。分别于移植3、7、14、28d取材，常规苏木素．伊红（HE）、Verhoeff染色，检测mTOR基因的mRNA及蛋白产物表达的变化，TUNEL法观察血管平滑肌细胞（VSMC）凋亡的动态变化。结果转基因组内膜中mTOR基因的mRNA及蛋白产物表达明显减少（P〈0．01）；转基因组内膜增生程度在术后7、14、28d较其他组明显减少（P〈0．01）；转基因组凋亡细胞明显增多（P〈0．01）。结论纳米粒子可以作为基因载体，反义mTOR基因的表达能够抑制移植静脉内膜的增生，促进VSMC凋亡。     

Nano-C60 cytotoxicity is due to lipid peroxidation

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C₆₀, a fullerene aggregate that readily forms when pristine C₆₀ is added to water. Nano-C₆₀ was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses 50 ppb (LC₅₀=2–50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C₆₀ colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C₆₀ inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C₆₀ was completely prevented.     

阿糖胞苷纳米粒的制备及其释药规律研究

采用乳化聚合法制备阿糖胞苷纳米粒，研究其体内外释药特性。结果表明阿糖胞苷纳米粒体外释药规律符合双指数方程，有明显的缓释作用。在家兔体内的药物动力学过程符合二室模型，与阿糖胞苷注射剂相比，t1／2β和MRT延长，CL降低，表明阿糖胞苷纳米粒可显著延长阿糖胞苷在体内存留时间，具有明显的缓释特征。     

Applications of platform technologies in veterinary vaccinology and the benefits for one health

The animal-human interface has played a central role in advances made in vaccinology for the past two centuries. Many traditional veterinary vaccines were developed by growing, attenuating, inactivating and fractioning the pathogen of interest. While such approaches have been very successful, we have reached a point where they have largely been exhausted and alternative approaches are required. Furthermore, although subunit vaccines have enhanced safety profiles and created opportunities for combined discrimination between vaccinated and infected animal (DIVA) approaches, their functionality has largely been limited to diseases that can be controlled by humoral immunity until very recently. We now have a new generation of adjuvants and delivery systems that can elicit CD4 + T cells and/or CD8 +  T cell responses in addition to high-titre antibody responses. We review the current vaccine platform technologies, describe their roles in veterinary vaccinology and discuss how knowledge of their mode of action allows informed decisions on their deployment with wider benefits for One Health.     

Factors controlling the pharmacokinetics,biodistribution and intratumoral penetration of nanoparticles

Nanoparticle drug delivery to the tumor is impacted by multiple factors: nanoparticles must evade clearance by renal filtration and the reticuloendothelial system, extravasate through the enlarged endothelial gaps in tumors, penetrate through dense stroma in the tumor microenvironment to reach the tumor cells, remain in the tumor tissue for a prolonged period of time, and finally release the active agent to induce pharmacological effect. The physicochemical properties of nanoparticles such as size, shape, surface charge, surface chemistry (PEGylation, ligand conjugation) and composition affect the pharmacokinetics, biodistribution, intratumoral penetration and tumor bioavailability. On the other hand, tumor biology (blood flow, perfusion, permeability, interstitial fluid pressure and stroma content) and patient characteristics (age, gender, tumor type, tumor location, body composition and prior treatments) also have impact on drug delivery by nanoparticles. It is now believed that both nanoparticles and the tumor microenvironment have to be optimized or adjusted for optimal delivery. This review provides a comprehensive summary of how these nanoparticle and biological factors impact nanoparticle delivery to tumors, with discussion on how the tumor microenvironment can be adjusted and how patients can be stratified by imaging methods to receive the maximal benefit of nanomedicine. Perspectives and future directions are also provided.     

Nervous system effects in rats on subacute exposure by lead-containing nanoparticles via the airways

Context and objective: Lead (Pb) is a heavy metal harmful for human health and environment. From leaded gasoline (still used in certain countries), and in Pb processing and reprocessing industries, airborne particles are emitted which can be inhaled. In such exposure, the size of particles entering the airways is crucial. The nervous system is a primary target for Pb, and consequences like occupational neuropathy and delayed mental development of children are well-known. The aim of this work was to investigate the neurotoxicity of Pb nanoparticles (NPs) applied into the airways of rats.

Methods: Nano-sized lead oxide particles (mean diameter ca. 20?nm) were suspended in distilled water and instilled into the trachea of adult male Wistar rats (in doses equivalent to 2 and 4?mg/kg Pb), 5 times a week for 3 and 6 weeks. At the end, open field motility was tested, then central and peripheral nervous activity was recorded in urethane anesthesia.

Results and conclusion: The treated rats’ body weight gain was significantly lower than that of the controls from the 3rd week onwards, and the weight of their lungs was significantly increased. Horizontal motility increased while vertical motility decreased. Spontaneous cortical activity was shifted to higher frequencies. The somatosensory cortical evoked potential showed increased latency and decreased frequency-following ability, and similar alterations were seen in the tail nerve. Significant Pb deposition was measured in blood, brain, lung and liver samples of the treated rats. The experiments performed seem to constitute an adequate model of the human effects of inhaled Pb NPs.     

Effect of microfluidization parameters on the physical properties of PEG-PLGA nanoparticles prepared using high pressure microfluidization

The objective of this work was to develop uniformly distributed poly(ethylene glycol) grafted poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles of mean size range ～100–200 nm using ethyl acetate as the solvent. In the multiple emulsion solvent evaporation method a high pressure microfluidization process was adopted to produce the W/O/W multiple emulsion. Non-toxic ethyl acetate was used to solubilize PEG-PLGA. The mean size of nanoparticles obtained was less than 180 nm. The particle size and size distribution were dependent on the microfluidization conditions applied. Mean particle size steadily increased from 121 nm at three passes to 172 nm at 20 passes of the microfluidizer, indicating that over-processing may be detrimental to PEG-PLGA nanoparticles prepared using this technique. There was no significant alteration of the PEG-PLGA matrix, as evidenced from the differential scanning calorimetric studies.     

Delivery of an inactivated avian influenza virus vaccine adjuvanted with poly(D,L-lactic-co-glycolic acid) encapsulated CpG ODN induces protective immune responses in chickens

In poultry, systemic administration of commercial vaccines consisting of inactivated avian influenza virus (AIV) requires the simultaneous delivery of an adjuvant (water-in-oil emulsion). These vaccines are often limited in their ability to induce quantitatively better local (mucosal) antibody responses capable of curtailing virus shedding. Therefore, more efficacious adjuvants with the ability to provide enhanced immunogenicity and protective anti-AIV immunity in chickens are needed. While the Toll-like receptor (TLR) 21 agonist, CpG oligodeoxynucleotides (ODNs) has been recognized as a potential vaccine adjuvant in chickens, poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles, successfully tested as vaccine delivery systems in other species, have not been extensively explored. The present study, therefore, assessed both systemic and mucosal antibody-mediated responses following intramuscular vaccination (administered at 7 and 21 days post-hatch) of chickens with PLGA encapsulated H9N2 AIV plus encapsulated CpG ODN 2007 (CpG 2007), and nonencapsulated AIV plus PLGA encapsulated CpG 2007 vaccine formulations. Virus challenge was performed at 2 weeks post-secondary vaccination using the oculo-nasal route. Our results showed that chickens vaccinated with the nonencapsulated AIV vaccine plus PLGA encapsulated CpG 2007 developed significantly higher systemic IgY and local (mucosal) IgY antibodies as well as haemagglutination inhibition antibody titres compared to PLGA encapsulated AIV plus encapsulated CpG 2007 vaccinated chickens. Furthermore, chickens that received CpG 2007 as an adjuvant in the vaccine formulation had antibodies exhibiting higher avidity indicating that the TLR21-mediated pathway may enhance antibody affinity maturation qualitatively. Collectively, our data indicate that vaccination of chickens with nonencapsulated AIV plus PLGA encapsulated CpG 2007 results in qualitatively and quantitatively augmented antibody responses leading to a reduction in virus shedding compared to the encapsulated AIV plus PLGA encapsulated CpG 2007 formulation.     

相变型纳米粒在动脉血栓的溶栓效果与靶向深度相关性的实验研究

目的 探讨相变型（PT）纳米粒（NPs）靶向动脉血栓深度及其与体外溶栓效果的相关性。方法 通过双乳化法制备一种靶向纤维蛋白的PLGA-PFH-CREKA NPs，验证其理化特性。采集新西兰大白兔的动脉血制作动脉血栓，分别置于荧光标记的靶向PT、非靶向PT、靶向载双蒸水（NPT）的NPs中，并用1 W/cm2声功率密度的低强度聚焦超声（LIFU）辐照，记录前后的质量，计算溶栓率，同时将处理后的血凝块制成切片，通过共聚焦显微镜观察、测量穿透深度，线性回归分析穿透深度与溶栓率的相关性。建立SD大鼠腹主动脉血栓模型，采用靶向PT、非靶向PT NPs评价体内靶向能力。结果 制备的靶向纤维蛋白的PT NPs平均粒径（297.8±11.82）nm，表面电位（1.44±0.22）mV，结构呈均匀球形，分散性好；体外溶栓实验发现靶向PT组与非靶向PT组、NPT组的溶栓率差异有统计学意义（F=108.508，P<0.001）；靶向PT组对血栓的穿透性相较于非靶向PT组、NPT组差异有统计学意义（F=96.187，P<0.001），线性回归发现靶向PT组对血栓穿透深度与溶栓率呈正相关（R2=0.818，P<0.05）。在SD大鼠腹主动脉血栓模型观察到靶向相变组较非靶向相变组对血栓的靶向性更好。 结论 靶向纤维蛋白的相变型NPs，对血栓纤维蛋白有良好的靶向性，有较好的溶栓效果，对血栓有很好的穿透性，并且相变所致溶栓率与血栓穿透深度呈正相关。