الملخص
Objective:To investigate the affinity and toxicity of epithelial cell adhesion molecule (EpCAM) targeted nucleic acid aptamer drug conjugate SYL3C-MMAE on human gastric epithelial cells GES-1 (hereinafter referred to as GES-1 cells) and human gastric cancer cells AGS and MKN45 (hereinafter referred to as AGS cells and MKN45 cells).Methods:The experimental study was conducted. The expression level of EpCAM in gastric cancer tissues was detected using immunohistochemistry. The mRNA expression level of EpCAM in gastric cancer tissues was detected using real-time fluorescence quantitative PCR (RT-PCR). The expression level of EpCAM protein in GES-1, AGS and MKN45 cells was detected using Western blot. The affinity of SYL3C on GES-1, AGS and MKN45 cells was detected using flow cytometry. SYL3C-MMAE was synthesized through a thiol-maleimide reaction. The toxicity of drugs on GES-1, AGS and MKN45 cells was detected using CCK-8 assay. The cell cycle condition of GES-1, AGS and MKN45 cells after drug treatment was detected using propidium iodide (PI) staining. Observation indicators: (1) expression of EpCAM in gastric cancer; (2) affinity of antibodies targeting EpCAM and SYL3C on GES-1, AGS and MKN45 cells; (3) situation of drug synthesis; (4) drug toxicity and inhibition of cell cycle. Measurement data with normal distribution were represented as Mean± SD. One-way ANOVA was used for comparison among multiple groups, and pairwise comparison was conducted using the least significant difference test. Comparison of unequal variances was conducted using the Welch' t test. Measurement data with skewed distribution were represented as M(IQR), and comparison between groups was conducted using the paired rank sum test. Count data were described as absolute numbers, comparison between groups was conducted using the paired chi-square test. Results:(1) Expression of EpCAM in gastric cancer. Results of immunohistochemistry on tissue microarrays showed that the positive rate of EpCAM was 82.9%(29/35) and 22.9%(8/35) in the 35 pairs of gastric cancer and its adjacent tissues (normal tissues), respectively, showing a significant difference between them ( P<0.05). Results of RT-PCR showed that the mRNA relative expression levels of EpCAM was 1.23 (4.13) and 4.04 (1.72) in 12 pairs of gastric cancer and its adjacent tissues respectively, showing a significant difference between them ( Z=-2.67, P<0.05). Results of Western blot showed that the relative expression levels of EpCAM protein in GES-1, AGS, and MKN45 was 0, 1.00, and 0.27, respectively, with the expression level of EpCAM protein in AGS cells as the standard. (2) Affinity of antibodies targeting EpCAM and SYL3C on GES-1, AGS and MKN45 cells. Results of flow cytometry showed that antibodies targeting EpCAM and SYL3C had good affinity on AGS and MKN45 cells but no affinity on GES-1 cells. (3) Situation of drug synthesis. Results of mass spectrometry showed that the drug solution of compound formed by connecting SYL3C with monomethylorestatin E (VcMMAE) exhibited a strong peak at the molecular weight position of 16 355, consistent with the expected molecular weight of the SYL3C-MMAE complex, indicating that SYL3C-MMAE was successfully synthesized. (4) Drug toxicity and inhibition of cell cycle. Results of CCK-8 assay showed that the half maximal inhibitory concentration (IC 50) of VcMMAE on GES-1, AGS and MKN45 cells was 123.00, 30.48 and 51.83 nmol/L, respectively. The IC 50 of SYL3C-MMAE on GES-1, AGS and MKN45 cells was 241.80, 20.66 and 27.64 nmol/L, respectively. Results of PI staining and flow cytometry showed that both VcMMAE and SYL3C-MMAE could induce G2/M phase blockage in the cell cycle of GES-1, AGS and MKN45 cells. Conclusion:The SYL3C-MMAE has a good affinity on gastric cancer cells. Compared with VcMMAE, SYL3C-MMAE exhibits efficient inhibition on gastric cancer cells, but less influence on normal cells.
الملخص
【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.
الملخص
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.
الموضوعات
Aptamers, Nucleotide , Drug Delivery Systems , Medicine, Chinese Traditional , Nucleic Acids , RNA, Small Interferingالملخص
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.
الملخص
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.
الملخص
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.
الملخص
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.