Regulation Of Pulmonary Arterial Endothelial Cell Proliferation With Noncanonical Self-assembled DNA Nanomaterials

Background:Pulmonary vascular remodeling（PVR）is a complicated and multi-factor process,among which the dysfunction of human pulmonary artery endothelial cells（HPAEC）is a crucial step in PVR development.HPAEC dysfunction can lead to the abnormal expression of regulatory factors,such as vasogenic active medias and inflammatory factors,resulting in disordered proliferation and epithelial-mesenchymal transition of HPVEC and smooth muscle cells.Therefore,the abnormal proliferation and apoptosis are key factors inducing PVR.Moreover,several researches demonstrated that autophagy is essential for modulating certain biological behaviors including PVR and the dysfunction of endothelial cells.The phosphorylation of Atg101,an important component of the autophagy initiation complex,cranks up autophagy assemblers and recruits downstream autophagy-related proteins,which could maintain the stability of autophagy precursor complexes.Recently,a few studies implied that a hypoxia environment increases the expression of Atg101.Moreover,inhibiting this gene expression can maintain endothelial cell stability and adjust the development of PVR through modulating cell proliferation disorders.DNA nanotechnology has attracted more and more attention in multiple research due to its intellectualization programmability,nontoxicity and no immunogenicity.More importantly,they can carry multiple functional molecules.Self-assembled DNA nanomaterials can be applied as a drug carrier for siRNA,CpG,protein and adriamycin delivery.However,DNA nanomaterials themselves are highly electronegative and need relatively high concentrations of salt solution to be synthesized,which are the big challenges.In addition,there are still some other problems in magnesium-mediated assembly,including easy degradation,the clearance of the mononuclear macrophage system and low cell uptake rate.New DNA-assembly strategies should be developed in nanobiology and nanomedicine.There are two types of assembly strategies:cation-mediated Self-assembled DNA nanostructures and nontraditional assembly of DNA nanostructures.The former contains positive ions,rather than metal ions,while the latter makes use of special substances,such as carbamide and formamide.Nitrogen-doped carbon quantum dots（NCDs）containing nitrogen on their surfaces has positive charge in neutral solution.Meanwhile,amine molecules such as spermidine which supply the necessary of life activities are featured with linear structures and protonated high-priced ionic compounds.Both NCDs and amine molecules have potential ability to induce their self-assembly into a well-defined structure,which is related with their electropositive and ability of interacting with DNA or RNA.Based on all the above,we hope to establish a new type of nontraditional DNA nanomaterials with electropositive small-molecule or nanometer-sized particles,which mediates magnesium-free self-assembled DNA nanomaterials.In this study,we utilized NCDs and Spermidine to structure the DNA nanosystem in combination with Atg101 si RNA,building a novel DNA composite nano-drug delivery system.After that,we estimated the cytotoxicity and biocompatibility of this new kind of nanocomposite in cancer cells and subsequently apply this material in PVR and other biomedical applications.Research Methods:1.Design and synthesis of DNA nanomaterialsAccording to the base pairing principle,we designed DNA Nano Prism and DNA Triangle by SEQUIN.These structures could conjugate with functional guest molecules,such as aptamers and si RNA,resulting in complex functional DNA nanomaterials.2.Synthesis and characterization of noncanonical self-assembled DNA nanostructures.The characterization of nitrogen-doped carbon quantum dots assembled DNA nanomaterials(NP^NCD)and Spermidine-assembled DNA nanomaterials was estimated by polyacrylamide gel electrophoresis（PAGE）and laser dynamic light-scattering（DLS）.Meantime,the effects of NCD or Spermidine on DNA nanomaterial assembly under various conditions were observed to obtain an optimum condition.3.Study on cytotoxicity and cellular uptake efficiency of noncanonical self-assembled DNA nanomaterials in tumor cells.（1）MTT-assay was used to detect the cytotoxicity of nitrogen-doped carbon quantum dots and Spermidine on cancer cells.（2）PAGE was performed to detect serum stability.In addition,we designed NP^NCD as a drug carrier combined with KRAS si RNA and assessed its synthesis yield by PAGE.（3）Through the confocal laser scanning microscopy（CLSM）and Fluorescence-activated Cell Sorting,we detected the cellular uptake of Spermidine or NCD assembled DNA nanomaterials in cancer cells.（4）Western blot was carried out to detect the effect of NP^NCD carrying KRAS si RNA on cancer-related protein expression levels.4.The mechanism of regulating pulmonary vascular endothelial barrier by noncanonical-assembled DNA nanomaterials.（1）Cellular uptake efficiency of the functional DNA nanotriangle carrying Atg101siRNA and aptamer was evaluated by CLSM.And this technology also was used to observe the targeting capability of DNT-APT nanomaterials to HPAECs.（2）CLSM was used to detect the influence of DNA-APT nanomaterials on the endothelial barrier function under hypoxia condition.（3）Western blot was carried out to detect the effects of DNA-APT nanomaterials on autophagy-related protein expression.（4）Monocrotaline was used to establish a pulmonary hypertension mouse model.Research Results:1.Design and synthesis of DNA nanostructures.We designed two types of the tiled-based DNA nanostructures by SEQUIN,including DNA Nanoprism and functional DNA Nanotriangle.Our results demonstrated these structures had relatively high yield and the particle size is consistent with the theoretical value of the structure.2.Nitrogen-doped carbon quantum dots or Spermidine mediated the self-assembly of DNA nanomaterials.（1）NCDs can mediate DNA Nanoprism self-assembly successfully.The results demonstrated that NCDs at a concentration of 30μg/mL could mediate the synthesis of DNA Nanoprism.Additionally,when the p H of NCDs ranged from 7.0 to 9.0,DNA Nanoprism could be synthesized effectively.Meanwhile,the results implied that after the nitrogen-doped carbon quantum dots mediated DNA nanoprism was placed at 4 ^oC,22 ^oC or37 ^oC for 2 h,PAGE showed that the yield of isothermal self-assembly is high.DLS showed that the-Nanoprisms diameter was 11.1±1.6 nm,which is in keeping with the theoretical value.（2）Our results showed that spermidine could mediate the synthesis of DNA Nanoprism and DNA nanotube.Furthermore,after this nanomaterial was placed at 37 ^oC,22 ^oC for 2 h,it could still mediate DNA nanotube assembly.In addition,spermidine of which the PH ranges from 6.5 to 7.5 could DNA nanotube assembly.3.The study of NCD or Spermidine assembled DNA nanomaterials on cancer cells:（1）We performed MTT assay to detect the cytotoxicity of these materials on cancer cells.The results showed that NCDs at 10 to 500μg/mL had no cytotoxicity on cancer cell proliferation.But,the spermidine at 20μM started to suppress cancer cells proliferation,which might be associated with the inhibited efficiency of spermidine on cancer cells.（2）The stability of NP^NCDCD was relatively higher than magnesium-mediated DNA nanoprism(NP^Mg).And even after 6 h,NP^NCD can maintain the integrity of DNA nanoprism.（3）The results from CLSM and flow cytometry showed that it is easier for NP^NCDCD to be taken up by cells than NP^Mg.Moreover,the uptake efficiency of NP^NCD in A549 cells and NCL-H23 cells was 6 or 2.6 times higher than that of NP^Mg.Meantime,spermidine-mediated DNA nanotube and nanoprism could be taken up by cells effectively.The results also demonstrated that the DNA nanotube situating in lysosome ultimately was taken up by cells in the way of clathrin-mediated endocytosis.（4）Through DNA nanoprism structural design,NP^NCD could carry KRAS si RNA(NP^NCDK),interacting with relative cancer cells.The results from MTT assay and Western blot implied that NP^NCDK inhibited the cancer cell proliferation and KARS expression more effectively,compared with NP^MgK.4.The effect of functional DNA nanotriangle on pulmonary endothelial barrier and PVR:（1）The results from PAGE showed that the synthesis yield of DNT-APT was very high.In addition,DLS results showed that the diameter of this structure was 16.88±2.33 nm,in consistent with the theoretical design value.（2）The results from CLSM also showed that HPAEC could take up DNT-APT in a time-dependent manner,and its uptake efficiency reached its highest at 48 h;anther result showed that Aptamer could bind to HPAECs specifically and DNA-APT be a be recognized and targeted taken up by HPAECs.（3）The results from WB and CLSM showed that the barrier function of pulmonary endothelium was apparently damaged and the level of ligandin decreased under hypoxia.But after these HPAECs taken up DNA-APT,the endothelial leakiness was attenuated.（4）The results from animal experiments showed that compared with the control group,the monocrotaline group could thicken the pulmonary vessel walls of mice,demonstrating our success in establishing a PVR mouse model.Conclusion:1.We have designed two types of DNA nanostructures successfully:DNA Nanoprism and DNA Nanotriangle.Furthermore,we modified the structures in a way that this DNA Nanoprism could conjugate with 6 KRAS si RNAs as well as the latter could carry 3 Atg101siRNAs and 3 Aptamers which can target the pulmonary artery endothelial cells.The results from PAGE、DLS and AFM showed that we synthesized these structures successfully.NCD and Spermidine mediated the Tile-based DNA nanostructures by the method of gradient annealing or at isothermal condition successfully;and these results implied that the assembled behavior of spermidine and NCD was closely linked with concentrations and p H of them.2.The DNA nanostructures mediated by NCDs and spermidine have the advanced prospect of function and application.（1）Compared with the structures synthesized with magnesium ions,NCD and Spermidine mediated the DNA nanostructures perform higher serum stability and cellular uptake than NP^Mg at the same condition.（2）NP^NCDCD captured with KRAS siRNA can effectively knockdown the KRAS gene and suppress cancer cell proliferation in A549 and NCL-H23.（3）Composite DNA nanomaterials show higher compatibility and nontoxicity.3.We optimized functional DNA Nano Triangle which can carry Atg101 si RNA and aptamer.It performs high internalization and targeting to HPACEs.The structure’s planarity and targeting make it possible to confirm the high selection and incorporation.Moreover,DNA-APT can effectively inhibit the HPAECs barrier impairment stimulated by hypoxia.At the same time,we studied the relationship between the DNT-APT mediated by NCDs and the pulmonary vascular remolding.