ABSTRACT
Targeted drug delivery vehicles make it possible to deliver anti-cancer drugs to the cells or tissues of interest. Aptamers are peptide or oligonucleotide molecules that can serve as targeting elements of drug carriers. In the current study, we evaluated the capacity of an aptamer-based drug carrier to deliver Paclitaxel (PTX) to cancer cells. After being synthesized, SPIONs@PTX-SYL3C aptamer was characterized using different methods, including differential light scattering (DLS), infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), X-ray diffraction (XRD), Thermal gravimetric analysis (TGA), and vibrating sample magnetometer (VSM). Encapsulation efficiency (EE) and loading efficiency (LE) were also evaluated. The carrier was applied on 4T1, MCF 7, and MCF-10A breast cell lines to evaluate its drug delivery potency and specificity. EE and LE were calculated to be 77.6% and 7.76%, respectively. MTT results revealed that aptameric SPIONs@PTX was more toxic than non-aptameric SPIONs@PTX. Flowcytometry analysis and DAPI staining confirmed that SPIONs@PTX-Aptamer had higher cell internalization rate when compared to non-targeted SPIONs@PTX. Our results indicate that aptamer-conjugated SPIONs@PTX has a good capacity in recognizing its target cells and inhibiting their growth and division.
Subject(s)
Aptamers, Nucleotide/chemistry , Breast Neoplasms/drug therapy , Magnetite Nanoparticles/chemistry , Molecular Targeted Therapy , Paclitaxel/therapeutic use , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Survival , Drug Liberation , Endocytosis , Female , Fluorescence , Humans , Inhibitory Concentration 50 , Magnetite Nanoparticles/ultrastructure , Mice , Particle Size , Reproducibility of Results , Spectroscopy, Fourier Transform Infrared , Static Electricity , Thermogravimetry , X-Ray DiffractionABSTRACT
BACKGROUND: The effect of the growth hormone on target cells is mediated by the insulin-like growth factor 1 (IGF-1). IGF-1 binds to the insulin-like growth factor binding proteins (IGFBPs) in blood and biological fluids. Considering the important application of IGBP3 as a drug component, in this research we cloned and expressed the full-length IGFBP3 in the pET-11a vector and BL21 (DE3) expression host. MATERIALS AND METHODS: First the sequence encoding of IGFBP3 was designed based on the amino acid sequence of the protein and then by codon optimization, in order to ensure the maximum expression in Escherichia coli. In the next step, the synthetic DNA encoding IGFBP3 was inserted into the pUC57 vector, at the appropriate restriction sites and then subcloned in the pET-11a expression vector in the same restriction sites. The constructed vector was transformed to E. coli BL21 as an expression host and induced in the presence of IPTG for expression of the IGFBP3 protein. Protein expression was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Double digestion of the new plasmid (pET-11a -IGBP3) with NdeI and BamHI showed two bands in 873 bp and 5700 bp. To study the accurate cloning procedure, the plasmid was sequenced and its authenticity was confirmed. Also the expected protein band (31.6 kDa) was observed in SDS-PAGE analysis. CONCLUSION: DNA fragment encoding the full-length IGFBP3 protein was accurately cloned in the pET-11a expression vector and the recombinant plasmid transformed to E. coli BL21 (DE3) expression host. Results of the SDS-PAGE analysis verified that recombinant IGFBP3 (31.6 kDa) are successfully expressed under the control of T7 promoter. As we shown pET-11a can be successfully used for expression of the IGFBP3 protein.
