Screening Of Novel OFA/iLRP Nucleic Acid Aptamers And Construction Of AML Targeted Therapy System

Objective:Acute myeloid leukemia(AML)is a serious threat to human health and causes more deaths than other types of leukemia.The standard treatment for most AML is chemotherapy,which is often associated with severe adverse effects.One strategy to reduce the adverse effects and improve the therapeutic efficacy is targeted therapy,in which the anticancer drugs are selectively delivered to tumor cells,thus reducing the damage to normal tissue.Immature laminin receptor protein(OFA/iLRP)is a potential target for AML treatment,because it is over-expressed on the surface of AML cells but under-expressed in normal tissue.Aptamers may serve as tumor-targeting ligands because they can bind to molecular target with high affinity and specificity.Moreover,aptamers have certain technical advantages,including low immunogenicity,better tumor penetration,easy chemical modification,and low production cost.To date,no OFA/iLRP aptamer has been reported in literature.In this study,we developed the first OFA/iLRP aptamer and construct an Apt-Dox complex as an AML-targeted drug delivery system.Methods:OFA/iLRP aptamer was selected employing the standard SELEX technique.Flow cytometry was used to evaluate the binding of the aptamer to OFA/iLRP peptide,BSA,OVA,and trypsin.Flow cytometry and confocal microscopy were utilized to monitor the binding of the aptamer to OFA/iLRP-positive and-negative cells.Apt-Dox complex was formed with a self-assembly process.The uptake of Apt-Dox by the cells was evaluated by confocal microscopy.MTS assay was performed to analyze the cytotoxic effects of Apt-Dox against OFA/iLRP-positive or-negative cells.Results:The selected aptamer(AB3)was a 59-base DNA oligonucleotides.The sequence of AB3 was 5'-TGCGTGTGTAGTGTGTCTGTTGTTTGTATTGTTGTCTAT CCTCTTAGGGATTTGGGCGG-3'.It bound to OFA/iLRP structure with a Kd of 101 nM and had minimal cross-reactivity to albumin,trypsin,or ovalbumin.Moreover,AB3 could bind to OFA/iLRP-positive AML cells but not the OFA/iLRP-negative control cells.An aptamer-doxorubicin(Apt-Dox)complex was formed by intercalating doxorubicin into the DNA structure of AB3.Apt-Dox selectively delivered Dox to oFA/iLRp-positive AML cells but notably decreased the drug intake by OFA/iLRP-negative control cells.In addition,cytotoxicity study revealed that Apt-Dox efficaciously destroyed the OFA/iLRP-positive AML cells,but signifcantly reduced the damage to control cells.Conclusion:OFA/iLRP is over-expressed on the surface of AML cells but under-expressed in normal tissue.In this work,we selected the first OFA/iLRP aptamer(AB3),which could bind to OFA/iLRP structure with high affinity and specificity.AB3-based Apt-Dox complex could achieve targeted killing of OFA/iLRP-positive AML cells,while reducing the damage to control cells.The results indicate that the OFA/iLRP aptamer may have application potential in targeted therapy against AML.