| DNA is the basic genetic material of living organisms,which was discovered as a double helix structure by Watson and Click in 1953.In the 1980 s,Seeman presented a new concept of "structural DNA nanotechnology".Since then,DNA has been used as a new material,and researchers can assemble nanostructures with different dimensions and rich structure morphology.This DNA nano-assembly structure has the advantages of suitable size,spatial addressability,strong biocompatibility,and easy to be functionally modified.As a result,the research focus of DNA self-assembly has been extended from structural design to practical application.In recent years,DNA assembly has become a new drug carrier due to its excellent function in cancer therapy.Nucleic acid aptmer was developed by the Systematic Evolution of Ligands by Exponential Enrichment(SELEX).It is essentially an oligonucleotide sequence,which can form stable secondary or tertiary structures through internal forces to bind specifically to target objects.Because of its high affinity,specificity and tumor permeability,it has attracted much attention in biomedical field in recent years.In this thesis,we used the long repetitive block ss DNA synthesized from RCA with a few staple strands to construction DNA assembly.Anticancer drug-hydrochloric acid doxorubicin(Dox)were used as drug models,and it can be readily incorporated double helix structure of the DNA assembly,to form DNA assembly-Dox complexes.The multivalent DNA assembly was promising for selective cancer cell recognition,bioimaging,and targeted anticancer drug delivery.The main research contents of this paper include the following aspects:(1)Apt-Assembly was constructed by the long repetitive block ss DNA synthesized from RCA with a few aptamer DNA strands through base pairing and the antitumor drug Dox can be readily incorporated double helix structure of it,to form Apt-Assembly-Dox.An Apt-Assembly can load up 200 Dox molecules.The results of fluorescence microscopy and microplate microscopy showed that the multivalent aptmer DNA assembly exhibited significantly higher binding ability to the receptor than the monovalent aptmer while retaining selectivity,and could achieve highly specifically targeting to B16 cells and promote internalization.In the cell scratch test,the wound healing degree of B16 cells was only 3% within 24 hours,indicating that AptAssembly-Dox could effectively inhibit the migration of B16 cells.The assay proved that the carriers release the drugs and induced selective cytotoxicity under the enzymolysis.The survival rate of the target cells was reduced by 58% at 500 n M Dox equivalent,compared with18% in the control cells.It can greatly reduced the adverse interactions of Dox to normal cells and reduced the side effects of chemotherapy.(2)Based on the theory of DNA origami,DNA Nanoladder was constructed by combining several staple chains with the periodicity and high molecular weight of RCA products.Compared with the traditional DNA origami structure,the number of staple chains was reduced and the sequence design was simplified.By means of agarose gel electrophoresis,atomic force microscope and transmission electron microscope characterization,the DNA origami structure composite preset was proved,and the width meets the preset value of 11 nm.Fluorescence microscopy and flow cytometry experiments showed that DNA Nanoladder could be used as drug carriers to enter target cells,but only a small amount of non-specific binding with non-target cells.CCK-8 assay was used to investigate the targeting killing ability of DNA Nanoladder to tumor cells.The survival rate of B16 cells treated with DNA Nanoladder-Dox decreased by 52%,while that treated with aptamer drug complex decreased by only 18%.The experimental results showed that due to the high molecular weight of DNA Nanoladder,their drug loading capacity was greatly improved,and compared with the monovalent aptamer,DNA Nanoladder were more effective in killing tumor cells. |
PDF Full Text Request