Construction And Investigaton Of Targeting DNA Nanocarriers

DNA nanotechnology is an emerging and exciting field,and represents a forefront frontier for the biomedical field.The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two-and three-dimensional nanostructures called DNA origami.Due it its exceptional properties of linear structure,self-recognizing ability and nanoscale,DNA origami can optimize the therapeutic effects of drugs when being applied to the drug delivery system(DDS),realizing the function that traditional DDS can not reach currently.Doxorubicin(DOX)is an anti-tumor drug that has anthracene structure,it can be intercalated into DNA duplexes and form stable compounds.We modified the DNA origami on the basic of its properties,in order to achieve the chemical therapy and biological therapy at the same time,and improve the therapeutic action of DOX.Objective:Make the single-stranded scaffold DNA(M13mp18)self-assembled into rectangular flakes by PCR,modify the C2 NP onto the RE to build a tumor-targeting drug delivery system,and improve the therapeutic action of DOX and lower its side-effect.After the DOX was loaded onto the RE,the new DDS can exert both chemical therapy and biological therapy.Methods:The single-stranded scaffold and over 200 short oligonucleotide ‘staple strands' were self-assembled spontaneously into rectangular flakes by PCR.And the aptamer C2 NP was modified onto the RE according to the principles of Watson-Crick base pairing.Change the amount and position of C2 NP by controlling the corresponded staple strands.Then the RE was characterized by atomic force microscope(AFM)and gel electrophoresis.Optimized the incubation conditions with DOX,and confirm the drug loading capacity and the release feature of the DDS in vitro.The K299 cells' proliferation inhibition rate was measured by CCK8,in order to evaluate the bioactivity and chemotherapy of the DDS.Using the flow cytometry and confocal laser scanning microscope to study the qualitative and quantitative cellulor uptake of the DDS,determined the location where the DDS went in the cell.A series of cell experiments were done by Annexin V-FITC Apoptosis Detection Kit,DNA Content Quantitation Assay and Reactive Oxygen Species Assay Kit,for further investigating the drug delivery system.Results:We synthesized a noncytotoxic,stable and tumor-target DNA origami carrier successfully.After loading the DOX,it can improve the stability of DOX as well as reduce its release rate.Each base pair can hold one DOX molecular approximately,but the RE can not enter into cell nucleus due to its measure.After incubating with K299 cells 2 hours,the cellular uptake of DDS-16 Apt and DDS-4Apt are 30.23% and 56.84%,respectively.The anti-tumor activity was improved significantly comparing to the dissociative DOX(4.43%).In terms of the biological therapies,the RE modified with C2 NP improved the DOX' inhibition rate.When at the low concentration of DOX,the DDS-Apt showed the biological effect and chemotherapy at the same time,and the chemical therapy was dominant when increase the DOX' concentration.Comparing to the DOX,the DDS can enhance the apoptosis rate from 37.4% to 62.1%;and improve the level of reactive oxygen level in the cells,to further promote the apoptosis.Meanwhile,the DDS can also display the S phase arrest to block the cell proliferation.Conclusion:The tumor-targeted drug delivery system we built can enhance the anti-tumor activity of DOX.The bioactivity of C2 NP was also improved by modified onto the RE.The DDS showed biological effect and chemotherapy at the same time.Targeted DNA origami carrier and the controllability of distance and amount about the aptamer provided a new way for the precise treatment against tumor.