Construction Of Peptides/Gold Nanoclusters-Functional Nucleic Acids Nanohybrids And Their Applications In Tumor Theranostics

Gold nanocluster(AuNC),an ultra-small(<2 nm)and biosafe nanomaterial with unique optical properties,has attracted much attention in the field of tumor diagnosis and treatment in recent years.On the one hand,the photoluminescence property and catalytic activity of AuNCs endowed them with exhibit unique advantages for tumor fluorescence imaging and biomarker detection.On the other hand,due to its photosensitivity and X-ray attenuation properties,AuNCs could be applied for tumor inhibition by such means as photodynamic therapy.Taking advantage of these properties,developing a novel strategy integrated diagnostic and therapeutic functions based on AuNCs is of great significance for improving diagnosis precision and reducing side effects.However,the current theranostic nanoplatforms fabricated with single AuNCs still stay in an infant stage and challenged by three aspects:non-specific photoluminescence property,limited drug loading capacity,and uncontrollable drug release.In recent years,the emerging functional nucleic acid molecules,such as aptamers,DNAzymes and antisense oligonucleotides(ASO),have been successively developed and exhibited special biological functions of high specificity and recognition ability,which make up for the shortcomings of AuNCs and provide an important molecular tool for the construction of an accurate and efficient tumor theranostic platform.AuNCs-functional nucleic acids based nanosystems for theranostic applications are rarely reported so far,as well as the problems of in vivo nonspecific imaging and uncontrollable drug release need to be solved urgently.In this thesis,focusing on the improvement of specificity,stability,precision and efficiency,a series of AuNCs-functional nucleic acid nanohybrids have been developed for cancer theranostic applications by combining the inherent properties of AuNCs and advantages of functional nucleic acids.The detailed description is listed as follows:1.Aptamer functionalized gold nanocluster-antisense oligonucleotides nanohybrid for contrast-enhanced tumor imaging and gene silencingIn view of the insufficient imaging specificity and drug loading delivery of single gold nanoclusters(AuNCs)-based theranostic nanoplatform,multifunctional AuNCs endowed with positive charge,legumain-specific domains and nanosurface energy transfer(NSET)effect were synthesized by utilizing the functional peptides as bioligands.Based on the non-covalent selfassembly of AuNCs and functional nucleic acids(Apt-M-Cy5),a highly specific nanohybrid integrated with activated imaging unit and gene drug loading unit was constructed.Apt-M-Cy5 is a single strand DNA composed of an aptamer for specific targeting and an antisense oligonucleotide(ASO)of mRNA for gene silencing,which can be assembled with positive AuNCs via electrostatic interaction to form the Apt-M-Cy5/AuNCs nanohybrid.The fluorescence of Cy5 was absolutely quenched on the NSET effect of AuNCs.With the aid of the aptamer,Apt-M-Cy5/AuNCs nanohybrid could target and bind to cell surface receptors specifically,leaving part of Apt-M-Cy5 away from the surface of AuNCs and realizing the specific imaging on the cell membrane.The partially disassembled Apt-M-Cy5/AuNCs then entered into the target cells and were further disassembled by the specific hydrolysis of overexpressed legumain in the lysosome,achieving legumain-responsive cell imaging and gene silencing of MnSOD mRNA.The results showed that the affinity of Apt-M-Cy5/AuNCs nanohybrid to target cells was significantly enhanced(Kd≈83 nM)compared to Apt-M-Cy5(Kd≈132 nM),and the cell internalization efficiency was significantly improved.Furthermore,the imaging contrast of tumor cells was significantly improved with the NSET of AuNCs.Cytotoxicity assay showed that the protease activation strategy based on Apt-M-Cy5/AuNCs nanohybrid achieved efficient gene therapy with good biocompatibility.Moreover,the in vivo experiment revealed that the Apt-M-Cy5/AuNCs was specifically activated by MCF-7 tumors inside mice.As a multifunctional integrated system with strong specificity,high stability and biosafety,the AuNCs-functional nucleic acids nanohybrid developed in this work provides a new idea for the development of a new cancer diagnosis and treatment platform.2.Endogenous protease responsive gold nanocluster-DNAzy me nanohybrid for activated tumor imaging and gene-chemo combination therapyThe work in the previous chapter has obtained the multifunctional AuNCs with positive charge,legumain-responsive domain and NSET effect.In order to further improve the precision and efficiency of tumor theranostic and broaden the application of AuNCs in this area,functional nucleic acid-Dzs containing DNAzyme was introduced to construct AuNCs/Dzs-Dox nanohybrid in this work for legumain activated tumor imaging and gene-chemo therapy.The flurophore of TAMRA modified on PL2 was used as a signal molecule.The AuNCs sheltered the therapeutic cargos of DNAzyme and Dox(Dzs-Dox)via electrostatic interaction to aggregate larger nanoparticles(AuNC s/Dzs-Dox),which could be selectively internalized into cancer cells by integrin-mediated endocytosis and in turn locally hydrolyzed in the lysosome with the aid of legumain.An explosion behavior including“spark-like”appearance(fluorescence on)derived from the diminished NSET effect of AuNCs and disaggregation(cargo release)was subsequently monitored.The results showed that the cellular imaging contrast of AuNCs/Dzs was improved by 7.4 times compared with monomeric AuNCs.In contrast to Dzs,AuNCs/Dzs-Dox nanohybrid exhibited higher cellular uptake efficiency,significantly improved stability and cleavage specificity for intracellular target mRNAs.Meanwhile,cytotoxicity assay revealed that the activation strategy based on AuNCs/Dzs-Dox readily achieved efficient gene-chemo combination therapy.Moreover,the specific activation of fluorescence imaging and super therapeutic efficacy(9 8.9%)was further demonstrated by treating a xenografted MDA-MB-231 tumor model in vivo.We envision that our multipronged design of AuNCs/Dzs-Dox nanosystem integrated protease-activated imaging and dual therapeutic mode provides a novel strategy and great promise in the application of high contrast imaging and on-demand drug delivery for precise cancer theranostics.3.Multistimuli responsive AuNCs-DNA tetrahedra nanohybrid for programmed tumor imaging and gene-chemo combination therapyIn the last two chapters,we found that the assembly of flexible singlestranded nucleic acid with AuNCs is non-directional,which limits the uniformity and ordering of AuNCs-nucleic acid nanohybrids.In order to further explore and optimize the structure and performance of the nanohybrid,the rigid DNA tetrahedra(DT)with controllable spatial structure was introduced with the response units of up-regulated biomarkers(GSH and TK1 mRNA)into the DNA framework.Herein,we reported a well-defined DNA tetrahedra-AuNCs nanohybrid with programmable self-assembly/disassembly formulations for stimuli-responsive imaging and sequentially site-specific cargo release.The framework of DT was well-designed with disulfide bonds in three edges and TK1 mRNA antisense as bottom edges.Benefiting from the positively charged and NSET properties,AuNCs could be served as activated imaging units and guarded on the rigid DT frame via electrostatic attraction to guarantee the stability of the formed nanohybrids.After being internalized by tumor cells,the Dox loaded DT/AuNCs nanohybrids displayed a sequentially response to multistimuli in the order of legumain-GSH/TK1 mRNA,leading to a two-step disassembly process:1)legumain triggered ligands hydrolysis of AuNCs for activated cancer imaging and denudation of DT;2)GSH/TK1 mRNA triggered fragmentation of DT for concentrated drug release and gene silencing at the tumor site.The in vivo results showed that the nanohybrid realized specific tumor imaging and an excellent tumor inhibition efficacy about 98.4%but negligible body weight loss.Additionally,DT/AuNCs can be metabolized by the kidney and liver after tumor-specific disassembly and significantly cleared one week after injection.Based on the self-assembly/programmed disassembly mechanism of DT and AuNCs,this work provides an effective strategy for optimization of structural performance and site-specific enhancement of nanohybrid,which is of great significance for the development of precise and efficient tumor diagnosis and treatment platforms.