Accurate Diagnosis And Treatment Of Liver Cancer Based On In Situ Biosynthesis Of Au-miRNA Nanoclusters

MicroRNAs（miRNAs）are short,endogenous non-codingRNAs with the function of regulating gene expression and have important applications in disease diagnosis,treatment,and prognosis.Numerous data have shown that miRNAs were involved in tumorigenesis and development.Tumor therapy with miRNAs,especially synthetic mimics,will be a new development direction in tumor therapy.However,after the injection of miRNA mimics therapy,it will be hindered in many ways,such as not being easily taken up by cells,not long enough in the tissue,easy to be degraded,and ineffective and unsafe delivery in the body.RNA interference（RNAi）is a transient gene silencing mechanism found endogenously in eukaryotic cells.Small interferingRNA（siRNA）is effector molecules in the process ofRNAi.An exogenously introduced expressing short hairpinRNA（shRNA）has similar functions to siRNA and can also exertRNAi effects.Alternatively,the shRNA can be converted into siRNA in the cell to exert a gene silencing effect and achieve long-term knockdown of the targeted gene.However,this method achieves tissue specificity in vivo,and its effective and safe delivery also faces similar challenges as miRNA.As a new type of fluorescent nanomaterials,fluorescent metal nanoclusters are compared with traditional fluorescent dyes and quantum dots.It has the advantages of easy preparation,easy modification,ultra-small size,large Stokes shift,good biocompatibility,and adjustable fluorescence spectrum,which has a wide range of applications in biochemical analysis,environmental detection,medical imaging and tumor treatment.Moreover,polymer nanocarriers have a long-term and promising existence in drug delivery.Increasing research shows that polymer-based nanocarriers have the ability to capture active ingredients,improve their water solubility,prevent their degradation and deliver them to the desired tissues in vivo.Biomacromolecules DNA andRNA molecules are rich in various functional groups,which can combine with different metal ions,so they are also ideal templates for the synthesis of nanoclusters.It is well-known that tumor cells/tissues spontaneously produce large amounts of oxidizing substances（such as reactive oxygen species or reactive nitrogen species）,and the occurrence and development of inflammatory diseases and tumors are usually related to the overexpression of these species.Based on the oxidative stress state of tumor cell microenvironment which is completely different from that of normal cells,this study proposes a novel strategy for in situ biosynthesis of Au-miRNA/shRNA nanocomplexs（Au-miRNA/shRNA NCs）.Through related in vivo and in vitro experiments,the results showed that the in situ biosynthetic Au-miRNA/shRNA NCs not only have fluorescence and enhanced Raman information imaging and other multi-functional high biocompatibility,good tumor targeting in vitro,but also it have no significant liver and kidney toxicity.Therefore,it can be effectively used in tumor treatment.The specific results are as following.（1）Construction and characteristics of in situ biosynthetic Au-miRNA NCs delivery systemFirstly,laser confocal fluorescence imaging of target cancer cells was explored through in-situ self-assembly of Au-miRNA NCs.After co-culturing miRNA mimics（such as miRNA-34a,let-7a and miRNA-200a）with HAu Cl₄solution,bright intracellular green fluorescence from in situ self-assembled fluorescent Au-miRNA NCs was clearly observed in tumor cells（Hep G2 and SMMC-7721）.However,L02cells（human liver cell line）have no intracellular fluorescence under all experimental conditions.Transmission electron microscope（TEM）and atomic force microscope（AFM）characterization demonstrated various conformational states（2D and/or 3D）of the in situ self-assembled fluorescent Au-miRNA NCs.In addition,surface-enhanced Raman scattering and Fourier transform infrared spectroscopy provided characteristic fingerprint peaks of related miRNAs,and the remarkable changes in the UV-vis absorption and fluorescence spectra also support the presence of Au-miRNA NCs in cell extracts.Observation from a biological point of view showed that MTT cell proliferation experiment,cell scratches and q RT-RCR analysis proved the effect of in situ self-assembled Au-miRNA NCs to transfect miRNA.In a word,from the physical and chemical properties,optical properties and biological characteristics,it is proved that the biological response self-assembled Au-miRNA NCs delivery system is feasible.（2）Bio responsive self-assembly of Au-miRNA NCs for imaging and targeted cancer theranosticsSecondly,let-7a is taken as an example to investigate how in situ self-assembled Au-miRNA NCs can inhibit tumor cell growth.Based on mirror-loaded cell culture microcavity,long-term culture of tumor cells（i.e.,SMMC-7721）was carried out to trace the temporospa-tial processes of cell division blockade and cell death by Au-let-7a NCs.In the following 6-h of observation,we were surprised to find that the cell cycle of SMMC-7721 was arrested and division was delayed due to let-7a transfection.In addition,the protein and mRNA levels of let-7a’s downstream target c-myc were significantly decreased after co-incubation of let-7a with HAu Cl₄solution compared with the control group.These results provide a basis for the self-assembled Au-miRNA NCs transfected miRNA and the corresponding anti-tumor efficiency.The combination of Au-miRNA NCs-based gene therapy and photothermal therapy（PTT）can improve the anti-tumor effect.After 8 minutes of 640 nm laser irradiation,flow cytometry displayed that PTT and gene therapy resulted in a significant increase in the necrosis and apoptosis of SMMC-7721 cells.After constructing tumor-bearing mice for intervention,it was shown that PTT and gene therapy combined with in situ self-assembled Au-let-7a NCs can effectively inhibit tumor growth in a short time.In vivo nude mouse studies have indicated that self-assembled Au-let-7a NCs can not only quickly and specifically target tumor tissues,but also can effectively bioimaging and inhibit tumor growth,and Au-let-7a NCs had no obvious toxicity or side effects to tumor mice.In conclusion,the new strategy of in situ self-assembly Au-miRNA NCs can be used to precisely target cancer cells/tissues for accurate biological imaging and treatment,which may provide new insights for understanding and effective tumor treatments such as hepatocellular carcinoma（HCC）.（3）Systemic shRNA delivery via bio-responsive fluorescent Au-shRNA complexes for lncRNA-regulated autophagy and effective cancer theranosticThirdly,based on the study of Au-miRNA NCs delivery system,we further explored the possibility of constructing an in-situ bio-responsive self-assembled Au-shRNA nanocomplexs（Au-shRNA NCs）delivery system for precise HCC treatment by co-incubating gold salt and shRNA.Metastasis-associated lung adenocarcinoma transcript 1（MALAT1）is the star molecule of long non-codingRNA（lncRNA）.It was found that MALAT1 is highly expressed in HCC tissues and cell lines.Two specific shRNAs were constructed for the target gene MALAT1,which can be used for subsequentRNAi experiments.Next,we utilized bio-self-assembled Au-shRNA NCs to silence MALAT1 and observed the resulting biological effects.Confocal imaging shows that in situ bio-responsive self-assembled Au-shRNA NCs delivery system can not only promote the uptake of Au-shRNA NCs by tumor cells,but also can successfully complete endosomal escape to achieve efficient transfection.Moreover,they effectively silenced target gene MALAT1,and several molecules involved in cellular autophagic flux were modulated following the down regulation of MALAT1（including LC3 and p62）.Furthermore,the combination of TEM and AFM bioimaging technology and the fluorescence properties of these complexes provided consistent and strong evidence for in situ self-assembly of Au-shRNA1 NCs.In vitro and tumor-bearing mouse model experiments demonstrated that Au-shRNA NCs silenced MALAT1 expression in tumor tissue,significantly regulated autophagy-related pathway molecules,and inhibited the level of autophagy,thus exerting a tumor suppressor effect.Overall,our study revealed a novel strategy of lncRNA-regulated autophagy in HCC and casts new light on understanding the relevant process and mechanism of shRNA-based tumor nano-therapeutics.