Application Of Aptamer-mediated DNA Cross Nanostructures In Colon Cancer Targeted Therapy

Objective:Colon cancer(CC)is a common malignancy.It is estimated that there are approximately 1.1 million new CC cases and 0.6 million CC-related deaths in 2018 worldwide.For early stage CC,surgery is the treatment of choice.For advanced colon cancer,however,chemotherapy is still the primary treatment modality.One of the main problems of chemotherapy is the frequently-occurring severe side effects,which greatly limit the duration and dosage of treatment,and therefore compromising the therapeutic efficacy.Hence,reducing the side effects of chemotherapy is critical for further improvement of clinical outcome.One strategy to reduce the side effects is targeted therapy Targeted therapy is often implemented with selective drug delivery system(SDDS),which typically are made of three components:anticancer drugs,drug carrier in the form of nanoparticle or nanostructure,and tumor-targeting ligand conjugated to the carrier.Because SDDS can target cancer cells but not healthy tissues,therapeutic efficacy is enhanced and off-target toxicity is reduced.Aptamers can be applied as tumor-targeting ligands.Aptamers are short,single-stranded oligonucleotides(DNA or RNA)that can form unique three-dimensional structures to serve as ligands.Aptamers not only can bind to their targets with high affinity and specificity,but have better penetration of tumor tissues vs.antibodies.What's more,aptamers can be chemically synthesized at low cost and easily conjugated with various functional molecules.Therefore,aptamer has great application prospect as a tumor targeting ligand.Holliday junction(HJ)is a DNA nanostructure with promising drug carrier potential.It is made of four single-strand DNA chains locked into the shape of a cross.HJ has excellent biocompatibility and can be easily constructed.Moreover,the size of HJ is relatively small,about 10-15 nm,allowing it to enter cancer tissues and lymph nodes more efficiently and serve as a better drug carrier.What's more,HJ has four open ends that can be linked to four tumor-targeting ligands,thereby improving its affinity to target cancer cells.To date,however,aptamer-modified HJ has not been used as a drug carrier of doxorubicin(Dox)to treat colon cancer.In this study,we designed and fabricated a novel SDDS consisting of a HJ modified by four AS 1411 aptamers,for targeted delivery of Dox to colon cancer cells.Methods:The SDDS was made of a self-assembled DNA nano-cross(Holliday junction,or HJ)functionalized by four AS 1411 aptamers(Apt-HJ)and loaded with Dox.The particle size and zeta-potential of Apt-HJ was determined by dynamic light scattering.Flow cytometry was performed to compare the cell binding capability of monovalent aptamer with Apt-HJ.To further improve the nuclease-resistance of Apt-HJ,Apt-HJ was modified with phosphorthioate.Electrophoresis was employed to detect serum stabilities of the DNA nanostructures.Doxorubicin was loaded into Apt-HJ and formed the Dox-loaded Apt-HJ complex(Apt-HJ-Dox).To evaluate the drug-loading capacity of Apt-HJ,Apt-HJ was mixed with Dox at increasing molar ratios,and mixture's fluorescence spectrum was analyzed.Confocal microscopy was used to detect whether Apt-HJ could selectively deliver Dox to target cancer cells.MTS assay was performed to evaluate Apt-HJ-Dox's cytotoxicity against CT26 cancer cells in vitro.To further characterize the pharmacological property of Apt-HJ-Dox,IC50 against CT2 was also evaluated with MTS assay.Animal experiments was performed to estimate the antitumor efficacy and the toxicity of Apt-HJ-Dox in vivo.Results:HJ containing four AS 1411 aptamers were constructed in a self-assembled way.Apt-HJ had an average size of 12.45 nm and a zeta potential of-11.6 mV.Compared with the monovalent AS 1411 aptamer,the quadrivalent Apt-HJ showed stronger binding to target cancer cells(CT26).A complex of Apt-HJ and doxorubicin(Apt-HJ-Dox)was formed by intercalating Dox into the DNA structure of Apt-HJ,with each complex carrying approximately 17 Dox molecules.Confocal microscopy revealed that Apt-HJ-Dox selectively delivered Dox into CT26 colon cancer cells but not the control cells.Moreover,Apt-HJ-Dox achieved targeted killing of CT26 cancer cells in vitro and reduced the damage to control cells.Importantly,compared with free Dox,Apt-HJ-Dox significantly enhanced the antitumor efficacy in vivo without boosting the adverse effects.Conclusion:In this study,we designed a novel complex of HJ with four AS 1411 aptamers for targeted therapy of colon cancer,and showed that such a nanostructure selectively deliver Dox to colon cancer cells in vitro and enhanced the antitumor efficacy in vivo.The results suggest that AS 1411-modified HJ has potential for targeted therapy of colon cancer.