DNA Tetrahedron-loaded Doxorubicin Modified By Aptamer AS1411 For Targeted Therapy Of Melanoma Cells

Background Malignant melanoma accounts for about 1% of all cutaneous malignancies.It is the most aggressive and lethal form of skin cancer.Clinically,the most effective treatment for melanoma is surgical resection and postoperative concurrent chemoradiotherapy,but the prognosis is poor,and a large number of patients relapse within 6-9 months after treatment.Doxorubicin is a widely used chemotherapeutic drug for the treatment of various cancers,such as sarcomas,skin tumors and hematologic malignancies.Doxorubicin can not be completely encapsulated in hydrophobic polymers because of its high water solubility.On the other hand,free doxorubicin solution has adverse side effects due to its wide distribution in normal tissues and tumors,further inducing doxorubicin resistance.Therefore,carrier drug delivery system is becoming a general trend in the research of chemotherapeutic drugs to reduce the side effects of drugs.In order to deliver drug molecules to cells effectively and safely,carriers must first be made of materials that will not cause damage to biological systems.Secondly,they must be good at loading drug molecules,targeting tumor cells and internalizing them into these cells.DNA nanocarriers modified by aptamers are one of the biological materials that have gradually attracted academic attention.However,there is still a lack of relevant research.Objective Aptamer AS1411 is a short DNA sequence,which can target nucleolin receptor protein overexpressed in many tumor cells.The aim of this study was to explore the efficacy of DNA tetrahedron loaded doxorubicin modified by aptamer AS1411 for targeted therapy of melanoma cells.Methods Firstly,the self-assembled AS1411-DNA tetrahedron(AS1411-Td)was physically characterized by agarose gel electrophoresis,dynamic light scattering and transmission electron microscopy.Subsequently,the fluorescence intensity of doxorubicin(Dox)was detected to determine the drug loading of DNA tetrahedron(Td)and AS1411-Td,the stability and cytotoxicity of AS1411-Td were also tested.Next,we evaluated the characteristics of this nanosystem in vitro.The expression of nucleolin in B16F10 and L929 cells was detected by RT-PCR.Laser scanning confocal microscopy(LSCM)was used to observe the cellular uptake of Td-Dox and AS1411-Td-Dox by the two kinds of cells.CCK-8 was used to detect the cytotoxicity of Td-Dox and AS1411-Td-Dox on normal L929 cells and tumor B16F10 cells.Results AS1411-DNA tetrahedron-Dox(AS1411-Td-Dox)targeted drug delivery system has been successfully synthesized.The advantages of AS1411-Td as a targeted drug delivery system have been fully demonstrated through the physical characterization of DNA carrier,drug loading test,stability test and cytotoxicity test.The AS1411-Td synthesis was confirmed by agarose gel electrophoresis;the average particle size of DNA tetrahedron(Td)was 10.4 nm and the average particle size of AS1411-Td was12.38 nm by dynamic light scattering;transmission electron microscopy showed that Td had a triangular structure of about 10 nm;the drug loading test results showed that each TD could carry Dox with a concentration ratio of 20 times,and each AS1411-Td could carry Dox with a concentration ratio of 25 times;stability results show that AS1411-Td could remain intact for 24 hours in a complete medium at 37℃,and stable for 7 hours in 50% fetal bovine serum;cytotoxicity test confirmed that normal human ovarian epithelial cells IOSE80 and AS1411-Td in the concentration range of 0-400 nm co-cultured for 24 h and 48 h had no obvious cytotoxicity.RT-PCR confirmed that nucleolin was highly expressed in mouse melanoma cell B16F10,while it was lowly expressed in normal mouse fibroblast L929;cell fluorescence imaging showed that the drug delivery system of self-assembled AS1411-Td tetrahedron carrying Dox had significantly targeting effect on melanoma cells;in addition,cytotoxicity tests showed that AS1411-Td-Dox was more cytotoxic to target cells than non-target cells.Conclusion AS1411-Td is a feasible targeted drug delivery system,and AS1411-Td-Dox is a potential therapeutic vector for tumor cells with positive expression of nucleolin receptor.