Absorption And Clearance Of Intracellular RNA Using DNA Nanosponge For Anti-tumor Adjuvant Chemotherapy

The development of DNA nanotechnology provides important programmable modular units for the construction of various functional nanoparticles.DNA nanostructures are of simple synthesis,great biocompatibility,easiness for functional modification and so forth,which have great potential in biomedical and biotechnological applications.The DNA sequence can be designed as a variety of biologically functional structure such as aptamers or DNAzyme for targeted delivery,specific silencing genes and bioimaging.Our work realized the adsorption and clearance of intracellular RNA molecues for anti-tumor adjuvant chemotherapy drugs by designing the DNA nanoflowers with biological functions.1.Absorption of intracellular RNA molecues using DNA nanosponge for the sensitization of adjuvant anti-tumor chemotherapy drugWe constructed a multifunctional DNA nanosponge delivery system.Repetitive antisense nucleotides of miRNA-21 in DNA nanosponge could efficiently capture miRNA-21 in the cytoplasmic microenvironment to realize gene silencing;The MUC1 aptamer enabled targeted delivery to mucin-overexpressing cancer cells;The linker containing GC repeats could hybridize with complementary strands to form duplex domains to efficiently load chemotherapeutic agents doxorubicin(DOX).Drug release experiments showed that the cumulative release rate of the therapeutic group in the mildly acidic(pH 5.0)was 83.75%,which was higher than that of the PBS group(56.79%).This feature indicated the therapeutic group could realize the accurate drug release in tumor cells,enhancing the anti-tumor effect and reducing the adverse reaction of DOX.We systematically investigated the remarkable stability of DNA nanosponge in different conditions.DNA nanosponge that has several copies of the complement of the miRNA-21 could effectively adsorb small molecules miRNA-21.It significantly down-regulated the expression of the anti-apoptotic protein Bcl-2 which is a downstream target of miRNA-21 and up-regulated the expression of Caspase family protein.Therefore,it is expected to prevent the protective mechanism of tumor cells and increase sensitivity of the chemotherapy drug DOX,which can facilitate cell apoptosis.Cell uptake and endocytosis pathway study demonstrated DNA nanosponge could actively target tumor cells through the endocytosis mediated by MUC1.We also verified the selective recognition ability of the DNA nanosponge to target MCF-7 cells but not to nontarget Hs578 Bst cells.Meanwhile,we came to conclusion that antisense nucleotides of miRNA-21 in DNA nanosponge could effectively regulate apoptosis by RT-PCR and Western Blot.The cell inhibition rate and apoptosis percentage of the therapeutic group reached up to 80.69% and 65.28%,respectively.We indicated it had a significantly synergetic effect for the combination of miRNA-21 antisense nucleotides and DOX.2.Autophagy gene silencing using DNA nanoflowers for anti-tumor adjuvant chemotherapyIn this study,we obtained multifunctional DNA nanostructures that were decorated with a series of aptamers.It loaded with AS1411 aptamer,ATG5 DNAzyme and drug loading sites for DOX with payload capacity,which achieved target recognition to nucleolin-overexpressing tumor cells and autophagy gene silencing to chemotherapy sensitization.We built monodisperse DNA nanomaterials via rolling circle replication(RCA),whose diameter was 200 nm and surface structure was petal-like.The drug loading rate was 97.45%(n(NFs/AS1411/ATG5):n(DOX)= 1:400).Drug release experiments showed that the cumulative release rate of the therapeutic group in the mildly acidic(pH 5.0)was 81.85%,which was higher than that of the PBS group(62.16%).We found DNA nanostructures showed the pHresponsive collapsed behaviors in acidic tumor environment.DNA nanostructures were composed of a long single-stranded DNA that contains ATG5 DNAzyme and magnesium pyrophosphate.The magnesium ions could work as a cofactor of the DNAzyme to enhance their catalytic capacity for silencing ATG5 mRNA which modulate autophagy related function,and the decrease of mRNA could enhance cancer therapy efficiency of chemotherapy drug.The cell uptake study verified the selective recognition ability of the degradable therapeutic system with high affinity and specificity to target MCF-7 cells.And most importantly,RT-PCR study verified synthetic DNA nanostructures could catalytically cleave autophagy-related mRNAs(ATG5 mRNA)with high efficiency.In the end,we found the inhibition rate of the therapeutic group for 48 h on MCF-7 cells reached up to 85.79%.The cell apoptosis rate of the treatment group was 1.41 times greater than that of the control group.We verified that DNA nanostructures had targeted therapeutic effect on MCF-7 cells.As a result,the degradable multifunctional therapeutic system showed significantly enhanced anti-tumor effects through the combination of gene therapy and chemotherapy.