Preliminary study of ssDNA aptamer cytotoxicity on RhD+ red blood cells / 中国输血杂志

【Objective】 To investigate the effect of ssDNA aptamer of RhD blood group antigen on erythrocyte toxicity. 【Methods】 Two full-length ssDNA aptamers(82 bp) of RhD blood group antigen were obtained by gene synthesis.Five samples of whole blood with EDTA anticoagulant were collected to prepare red blood cell suspensions (4×107/mL), which were split into 10 tubes(100 μL/tube), corresponding to 5 experimental groups and 5 controls.Two monospecific full-length ssDNA sequences (100 pmol/μL, 5μL each) were added into the experimental group, while the same amount of normal saline into the control.After treatment, the experimental group and the control were incubated for 60 min at 37℃.After washing, they were suspended in LISS solution and stored at 4℃.The experimental group and the control were set according to different time point during storage (1 h, 1 d, 3 d, 10 d and 17 d), with 5 tubes in each group.For erythrocytes in LISS suspension at different storage time, Annexin V labeled with FITC was used as a probe to label the phosphatidylserine (PS) content and Fluo-4 to label Ca2+ .The eversion of PS and the change of Ca2+ concentration in red blood cells in LISS suspensions were determined by flow cytometry. 【Results】 After incubation, all groups were examined under the light microscope.No agglutination occurred in the experimental group, while agglutination occurred in the control.Flow cytometry showed that the number of Annexin V-FITC staining cells of suspended erythrocytes at the same storage time-point was similar between the experimental group and the control, with no significant differences.In the experimental group, apoptosis rate of Annexin V cells at 10-day storage(6.06±1.38) was significantly higher than that at 1-hour storage(P<0.05), so as at 17-day storage(7.77±1.23) than 1-hour, 1-day and 3-day storage(P<0.05). The apoptosis rate of Fluo-4 AM cell in suspended RBCs at the same storage time-point was similar between the two groups(P>0.05). In the experimental group, the apoptosis rate of Fluo-4 AM cell at the 3-day, 10-day and 17-day storage was 20.84±4.16, 22.35±3.37 and 27.06±2.81, respectively(P<0.05). 【Conclusion】 ssDNA aptamer was not found to have any cytotoxic effects on red blood cells, and RhD ssDNA aptamer may be used as a material for the detection and preparation of universal blood.

Nucleic acid aptamer-based traditional Chinese medicine application:therapy,targeting and diagnosis / 中国中药杂志

Nucleic acid aptamers, broad-spectrum target-specific single-stranded oligonucleotides, serve as molecules in targeted therapy, targeted delivery and disease diagnosis for the treatment of tumor or microbial infection and clinical detection. Due to the existence of components in the use of traditional Chinese medicine(TCM), the target is difficult to concentrate and the specificity of treatment is poor. The effective components of TCM are toxic components, so a highly sensitive detection method is urgently needed to reduce the toxicity problem at the same time. The combined application of TCM and modern medical treatment strategy are difficult and cannot improve the therapeutic effect. Aptamers, advantageous in biosensors, aptamer-nanoparticles for targeted drug delivery, and aptamer-siRNA chimeras, are expected to connect Chinese medicinals with nanotechnology, diagnostic technology and combined therapies. We summarized the preparation, screening, and modification techniques of nucleic acid aptamers and the biomedical applications and advantages in therapy, targeting, and diagnosis, aiming at providing a reference for the in-depth research and development in TCM.

Current Research Status of Aptamer-mediated Liposome Targeted Delivery of Antitumor Drugs / 中国实验方剂学杂志

Tumor has become the second most serious disease that threatens human health and life. Treating with chemical drugs (referred to as chemotherapy) is the most basic treatment, but most chemotherapeutic drugs cause damage to normal tissues. It is a difficult problem in the field of biomedical research that how to deliver anti-tumor drugs more efficiently, increase the concentration of drugs in tumor tissues, enhance the anti-tumor effect, and decrease the drug distribution in normal tissues to weaken the damage to normal tissues. In order to achieve the goals of accurate delivery of anti-tumor drugs and synergism and attenuation, the researchers used systematic evolution of ligands by exponential enrichment technology (SELEX technology) to screen aptamers that can specifically target tumor markers or tumor cells, and designed the novel liposome targeting drug delivery system with aptamers as targeting molecules (ligands). This paper briefly introduced nucleic acid aptamer technology and common tumor markers, and reviewed the research advances on the antitumor effect of aptamer-liposome drug delivery system. It will provide references for the selection of appropriate tumor markers as targets and the application of aptamer technology in the research and development of high-efficiency and low-toxicity liposome targeting agents of anti-tumor traditional Chinese medicine. Meanwhile, it is of great significance for promoting the application of aptamer technology in targeted drug delivery systems.

Targeting effect of TLS9a nucleic acid aptamer on mice hepatic cancer cells / 重庆医学

Objective To investigate the targeting effect of TLS9a nucleic acid aptamer on mice hepatic cancer cells.Methods The liposome modified with maleimide and loading doxorubicin(DOX) was prepared,then TLS9a nucleic acid aptamer modified by FITC fluorescence and sulfydryl was synthesized,which was coupled to the liposome surface.The entrapment efficiency of DOX was detected by UV spectrophotometry.The dynamic light scattering(DLS) was applied to measure the particle size of nanoparticles and the potential distribution.The uptake of DOX in mice hepatic cancer cells was detected by the Nikon inverted microscope and the mean fluorescence intensity of liposome/DOX and TLS9a-liposome/DOX was detected by flow cytometry.The cells activity was detected by MTT.Results Flow cytometry assay showed that the binding rate of TLS9a nucleic acid aptamer with BNL.1ME.A.7R.1 mice hepatic cancer cells was 54.1％.TLS9a-liposome particle size distribution was in (116.0 ± 5.0)nm.TLS9a-liposome/DOX released DOX quickly at pH 5.0,and the release amount in 72 h was more than 70 ％ of the total release amount.TLS9a-liposome/DOX effectively inhibited the growth of mice hepatic cancer cells BNL.1ME.A.7R.1.Conclusion TLS9a nucleic acid aptamer could specifically combined with mice hepatic cancer cells BNL.1ME.A.7R.1,which could be used to detect mice hepatic cancer cells.

Targeting effect of TLS9a nucleic acid aptamer on mice hepatic cancer cells / 重庆医学

Objective To investigate the targeting effect of TLS9a nucleic acid aptamer on mice hepatic cancer cells.Methods The liposome modified with maleimide and loading doxorubicin(DOX) was prepared,then TLS9a nucleic acid aptamer modified by FITC fluorescence and sulfydryl was synthesized,which was coupled to the liposome surface.The entrapment efficiency of DOX was detected by UV spectrophotometry.The dynamic light scattering(DLS) was applied to measure the particle size of nanoparticles and the potential distribution.The uptake of DOX in mice hepatic cancer cells was detected by the Nikon inverted microscope and the mean fluorescence intensity of liposome/DOX and TLS9a-liposome/DOX was detected by flow cytometry.The cells activity was detected by MTT.Results Flow cytometry assay showed that the binding rate of TLS9a nucleic acid aptamer with BNL.1ME.A.7R.1 mice hepatic cancer cells was 54.1％.TLS9a-liposome particle size distribution was in (116.0 ± 5.0)nm.TLS9a-liposome/DOX released DOX quickly at pH 5.0,and the release amount in 72 h was more than 70 ％ of the total release amount.TLS9a-liposome/DOX effectively inhibited the growth of mice hepatic cancer cells BNL.1ME.A.7R.1.Conclusion TLS9a nucleic acid aptamer could specifically combined with mice hepatic cancer cells BNL.1ME.A.7R.1,which could be used to detect mice hepatic cancer cells.

Applications of nucleic acid aptamers in cancer diagnosis and treatment / 国际肿瘤学杂志

Due to their small molecular weight,strong penetrating power,wide target range,strong ability of binding targets,stable quality,little immunogenicity,easiness to be synthesized and modified,and functional roles in molecular recognition and signal transduction,nucleic acid aptamers are now used as tools for molecular recognition and drugs delivery for the diagnosis and treatment of many diseases.