Study Of Cascaded Drug Delivery System Basing On DNA Tetrahedron Reversing Drug-resistance Of Human Breast Cancer Cell

Malignant tumors are serious threat to lives and health of human beings,but so far chemotherapy is known to be important way for cancer therapy.One of the important problems that chemotherapy brought is drug-resistance.Furthermore,there are several shortcomings of chemotherapy drugs,such as poor targeting and poisonous side effect.Therefore,it is high time to construct drug delivery systems(DDSs)that not only can reverse drug resistance of malignant tumors but also improve tumor targeting effects and reduce the side effects.DNA nanotechnology,as a new member of nanomaterials technology,has drawn much attention because of its strong advantages can be designed and predictability and unique morphology features since its coming out.Among them,DNA tetrahedron has stable structure and properties.It also has multiple molecular sites can be modified.So DNA tetrahedron can be used as an ideal drug carrier.Liposomes with nanometer size are often used to construct drug delivery systems because of their good biocompatibility and bio-safety properties.The cell nucleus is the main target of most chemotherapy drugs,but due to the cancer cell's chemotherapy drug efflux,the majority of chemotherapeutic drugs are difficult to reach the nucleus to play a therapeutic role.The diameter of the nucleus nuclear pore is about 9 nm,and the self-assembled DNA tetrahedron's diameter is nearly 5 nm,DNA tetrahedron can go through the nuclear pore into the nucleus efficiently.So we embedded clinical first-line chemotherapy drug DOX into DNA tetrahedron to form DOX@Td,and then encapsulate the system DOX@Td into immune liposome efficiently to form a DNA tetrahedron-based DOX@Td@Lipo efficient nuclear drug delivery system.The drug delivery system constructed in this experiment has the following characters: 1.Using immune liposome to transfer DOX@Td efficiently into cancer cells;2.Using DNA tetrahedron's small size and nuclear degradable properties to transfer DOX efficiently into nucleus and it can not only avoid the tumor cell's drug efflux pathway but also reverse the drug-resistance of tumor cells.The system significantly improves the efficiency of DOX in drug-resistant breast cancer and reduces the side effects of DOX.We carried out the following three studys using MCF-7 and MCF-7/ADR cells as the cell model and BLAB/C nude mices as the animal model.1.Preparation and characterization of DOX@Td@Lipo cascaded drug delivery system: single stranded DNA(ssDNA)can renature and then self-assemble by base-pairing reactions to form DNA tetrahedron.We used Automatic Force Microscope(AFM)and gel-imaging to investigate DNA Tetrahedron,the diameter was nearly 5 nm,and the distribution was uniform.DOX is easy to insert into the DNA double helix structure to prepare DOX@Td conjugation,The results showed that the tetrahedron after the insertion of DOX still maintains the tetrahedral basic structure,and the gal-imaging result was just the same as DNA tetrahedron.So the DOX@Td is stable enough;finally,we prepared the DOX@Td@Lipo by the reverse evaporation method,and the Transmission Electron Microscope(TEM)results showed that the diameter of DOX@Td@Lipo was about 147 nm,it had a dense phospholipid bilayer surface and the particle size distribution was uniform.2.The evaluation of DOX@Td@Lipo cascaded drug delivery system's anti-tumor ability in-vitro and study of DOX@Td's drug resistance reversion: The antitumor activity of DOX@Td@Lipo on MCF-7 and MCF-7/ADR cells was investigated systematically.The results showed that DOX,DOX@Td and DOX@Td@Lipo had no significant difference in the proliferation of MCF-7 cells(70.55%,69.55%,65.08%)for 48 h.However,the inhibitory effect of DOX on ADR cells was significantly lower than that of DOX@Td and DOX@Td@Lipo groups(21.23%,25.73%,79.83%)for 48 h.It indicated that at the cellular level,DOX@Td and DOX@Td@Lipo succeeded in reversing the resistance of MCF-7/ADR cells to DOX and thus enhanced the ability of DOX to kill tumor cells.In addition,this study also validated the nucleus targeting ability of the constructed system by fluorescence imaging.The results showed that,compared with DOX group,the Td-FAM group,the DOX@Td group and the DOX@Td@Lipo group showed out green fluorescence of FAM and the red fluorescence of DOX in the MCF-7/ADR cell's nucleus,and DOX group did not appear the red fluorescence in the nucleus.It was demonstrated that DOX can not enter the nucleus of the drug-resistant cell but the DNA tetrahedron can enter the nucleus of the drug-resistant cell,and the DOX@Td@Lipo is able to transfer DOX into the drug-resistant cell and release the DOX in cell nucleus.3.The evaluation of DOX@Td@Lipo cascaded drug delivery system's anti-tumor ability in-vivo: We used the ADR tumor bearing BLAB/C nude mices as models to evaluate the constructed anti-tumor activity of the drug delivery system.The distribution of the constructed delivery system in nude mice was investigated by near-infrared light imaging.The results showed that DOX@Td@Lipo entered tumor tissue at 1 hour after intravenous injection.In addition,the results also showed that encapsulation of immune liposomes enhances the circulatory time of the drug in nude mice.Pharmacodynamics results showed that the DOX@Td@Lipo group's inhibition is 66.67%,and the DOX@Lipo is 41.67%,the Td@Lipo group had no obviously inhibility after 2 weeks of treatment.It indicated that the DOX@Td@Lipo group has significantly anti-tumor activity to drug-resistance breast tumor.Pathological results confirmed the treatment effect of DOX@Td@Lipo furtherly,and compared with the normal tissues and organs,DOX@Td@Lipo reduced the toxic side effects of DOX obviously.