靶向寄生虫的核酸适配体应用研究进展

核酸适配体是人工合成的单链DNA或RNA寡核苷酸,经指数富集的配体系统进化技术体外筛选获得。核酸适配体可与病毒、细菌、寄生虫等生物体的蛋白质与其他小分子靶标进行高亲和力和特异性结合。与抗体相比,核酸适配体具有无免疫原性、制备简单、易于批量生产、便于修饰、性能稳定、价格低廉等优点,在疾病诊断和治疗领域具有重要的应用潜力。本文综述了靶向疟原虫、锥虫、利什曼原虫等寄生虫的核酸适配体的研究进展,以期为寄生虫感染的检测和防治提供新策略。     

Aptamer micelles targeting fractalkine-expressing cancer cells in vitro and in vivo

In this work we hypothesized that the chemokine fractalkine can serve as a cancer molecular target. We engineered aptamer micelles functionalized with an outer poly(ethylene glycol) (PEG) corona, and investigated the extent and efficacy of using them as a targeting tool against fractalkine-expressing colon adenocarcinoma cells. In vitro cell binding results showed that aptamer micelles bound and internalized to fractalkine-expressing cancer cells with the majority of the micelles found free in the cytoplasm. Minimal surface binding was observed by healthy cells. Even though partial PEGylation did not prevent serum adsorption, micelles were highly resistant to endonuclease and exonuclease degradation. In vivo biodistribution studies and confocal studies demonstrated that even though both aptamer and control micelles showed tumor accumulation, only the aptamer micelles internalized into fractalkine-expressing cancer cells, thus demonstrating the potential of the approach and showing that fractalkine may serve as a specific target for nanoparticle delivery to cancer cells.     

核酸适配体修饰聚酰胺-胺负载5-氟尿嘧啶纳米给药体系的制备表征及体外抗肝癌作用

目的研究核酸适配体(ZY1)修饰聚酰胺-胺(PAMAM)负载5-氟尿嘧啶-1-基乙酸(FUA)纳米给药体系(ZY1-PAMAM-FUA)的制备表征及其体外抗肝癌作用。方法将FUA、聚乙二醇(PEG)与PAMAM通过酰胺反应键合,然后将ZY1通过静电吸附与PEG-PAMAM-FUA缀合得到ZY1-PAMAM-FUA纳米给药体系。核磁共振氢谱仪(¹H-NMR)和傅里叶红外光谱仪(FT-IR)鉴定ZY1-PAMAM-FUA的化学结构,动态光散射技术(DLS)表征其粒径分布以及稳定性,紫外分光光度法检测其载药量和体外释药性能,流式细胞仪和荧光显微镜考察SMMC-7221肝癌细胞对Cy5标记的纳米给药体系的摄取能力;MTT法检测纳米给药体系对SMCC-7721肝癌细胞增殖活性的影响。结果ZY1-PAMAM-FUA粒径分布均匀,粒径为102.3 nm,其载药量为8.70%±0.43%,并具有良好的缓释性能。与PAMAM-FUA比较,ZY1-PAMAM-FUA可以更高效地靶向SMCC-7721细胞。ZY1-PAMAM-FUA具有良好的生物相容性且对SMCC-7721细胞具有良好的抗细胞增殖作用。结论ZY1-PAMAM-FUA制备方法简单稳定,具有良好的生物相容性,可以高效地将药物靶向SMCC-7721细胞发挥抗肝癌细胞增殖的作用。     

适配体传感器在病原菌检测中的研究进展

病原菌是各种感染性疾病的病原体之一,传统细菌培养鉴定方法耗时、操作繁琐,急需开发新的检测方法。基于纳米材料构建的适配体传感器具有灵敏度高、特异性好、操作简便、价格低廉等优势,有望用于病原菌检测。本文介绍了适配体传感器常用的纳米材料的特性和优势,以及不同检测方法适配体传感器检测各种病原菌的研究进展。     

Biomarker discovery in cardiac allograft vasculopathy using targeted aptamer proteomics

Cardiac allograft vasculopathy (CAV) limits long-term survival after heart transplantation. Non-invasive evaluation is challenging, and currently, there is no validated biomarker for CAV diagnosis or prognostication. To identify potential candidate CAV biomarkers, we utilized the Slow Off-rate Modified Aptamer (SOMAscan) assay, which evaluates over 1000 serum proteins, including many relevant to biological pathways in CAV. We evaluated three heart transplant patient groups according to angiographic ISHLT CAV grade: CAV_1-2 (mild-moderate CAV), CAV₃ (severe CAV), and CAV₀ (normal control). SOMAscan assays were performed and proteins quantitated. Comparisons of proteins between study groups were performed using one-way ANOVA (false discovery rate q-value < 0.10). Thirty-one patients (12 mild-moderate CAV, 9 severe CAV, 10 controls) were included: 81% male, median age 57 years and median 1.1 years post-transplant. Compared to controls, patients with mild-moderate CAV had similar characteristics, while patients with severe CAV had longer time from transplant and increased allosensitization. Statistical/bioinformatics analysis identified 14 novel biomarkers for CAV, including 4 specific for mild-moderate CAV. These proteins demonstrated important actions including apoptosis, inflammation, and platelet/coagulation activation. Upon preliminary receiver operating characteristics curve analysis, our protein biomarkers showed moderate-to-high discriminative ability for CAV (area under curve: 0.72 to 0.94). These candidate biomarkers are being validated in prospective studies.     

核酸适体在肿瘤诊治中的应用进展

恶性肿瘤作为全球重要的致死疾病,严重威胁人类健康与生命。实现肿瘤的早期发现和诊断,对肿瘤的治疗具有重要意义。核酸适体是采用SELEX技术进行特异性筛选的单链DNA或RNA分子,因具有高亲和力、无免疫原性、无毒性、易于合成和修饰、靶标广泛、分子量小等优势,在肿瘤的诊断和治疗中发挥了重大的作用。本文主要对核酸适体在常见恶性肿瘤的早期诊断和靶向治疗中的作用作一综述。     

Anti-Proliferative Effect of Doxorubicin-Loaded AS1411 Aptamer on Colorectal Cancer Cell

Background: Doxorubicin (Dox) inhibits DNA replication and causes DNA damage resulting in cell death. It is a common drug for treatment of many cancers. Treatment efficacy and side effects of Dox are critical issues in using it because the drug lacks of specificity. The objective of this study was to improve the specificity of Dox by the incorporation of this drug with AS1411 aptamer (ASA). Methods: Dox was intercalated into the duplex sites of ASA, a recognition molecule for a number of cancer cells, and formed Dox-loaded ASA. The recognition ability proceeded through specific binding between the aptamer and nucleolin overexpressed in the cancer cells. The tested cells were human colorectal adenocarcinoma cell line (SW480) and human normal colon cell CCD841 CoN (CCD841). Binding of ASA to the cells was tested using flow cytometer and fluorescence microscope. Intercalation of Dox into DNA duplex was confirmed by fluorescence spectrometry. Effect of ASA, Dox, and Dox-loaded ASA on cell viability was examined by cell proliferation assay. Caspase-3 activation was analyzed by western blotting. Results: ASA bound specifically to SW480 cells via interaction between the aptamer and nucleolin because the nucleolin was highly expressed in SW480 cells. ASA decreased the viability of SW480 cells in a dose-dependent manner. Dox was more toxic than ASA. Fluorescence quenching revealed that Dox was able to intercalate in base pairing sites of the aptamer. Dox-loaded ASA inhibited the proliferation of SW480 cells, because the aptamer facilitated the Dox uptake into these cells which caused the cell apoptosis, indicated by the significant decrease in procaspase-3, apoptosis marker protein. Conclusion: This study succeeded to prepare Dox-loaded ASA by intercalation of the drug that inherited the binding function from the aptamer and anti-cancer activity from Dox. Dox-loaded ASA showed promise for effective cancer treatment with lower level of side effects.     

Solubilization of flurbiprofen into aptamer-modified PEG–PLA micelles for targeted delivery to brain-derived endothelial cells in vitro

Abstract

Novel aptamer-functionalized polyethylene glycol–polylactic acid (PEG–PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer’s disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.     

ATP-Responsive Drug Delivery Systems

The combination of targeted drug delivery and controlled-release technology may pave the road for more effective yet safer chemotherapeutic options for cancer therapy. Drug-encapsulated polymeric nanoparticle–aptamer bioconjugates represent an emerging technology that can facilitate the delivery of chemotherapeutics to primary and metastatic tumours. Aptamers are short nucleic acid molecules with binding properties and biochemical characteristics that may make them suitable for use as targeting molecules. The goal of this review is to summarise the key components that are required for creating effective cancer targeting nanoparticle–aptamer bioconjugates. The field of controlled release and the structure and properties of aptamers, as well as the criteria for constructing effective conjugates, will be discussed.     

Man-Made Synthetic Receptors for Capture and Analysis of Ochratoxin A

Contemporary analytical methods have the sensitivity required for Ochratoxin A detection and quantification, but direct application of these methods on real samples can be rarely performed because of matrix complexity. Thus, efficient sample pre-treatment methods are needed. Recent years have seen the increasing use of artificial recognition systems as a viable alternative to natural receptors, because these materials seem to be particularly suitable for applications where selectivity for Ochratoxin A is essential. In this review, molecularly imprinted polymers, aptamers and tailor-made peptides for Ochratoxin A capture and analysis with particular attention to solid phase extraction applications will be discussed.