New Multifunctional Nanosystems For Biological Diagnosis Based On Functional Nucleic Acids And Nanomaterials

With the development of life science and medicine,human are requiring higher life quality.Efficient access to biological information to explore all kinds of life phenomena and the realization of disease diagnosis and treatment has become the important development direction of the biomedical science and life science.Therefore,the rapid and sensitive detection of life-related biomolecules and the construction of new disease diagnosis and treatment systems is an important research topic of modern biochemical analysis.In this field,functional nucleic acids and nanomaterials are getting more and more attention by virtue of their excellent physical and chemical properties.They can combine with each other to construct a series of new multi-functional nanosystems for biological diagnosis and treatment.Functional DNA are DNA molecules with functions beyond genetic storage.They are short single-stranded DNA molecules with enzymatic function(called DNAzymes or deoxyribozymes),biorecognition function(called aptamers)or both(called aptazymes).All the functional nucleic acids are obtained through artificial synthesis.DNAzymes are DNA molecules with catalytic activity.They are obtained by an in vitro selection method from a large pool of random DNA molecules.Another class of functional DNA molecules is aptamers that are single stranded DNA molecules isolated from random-sequence DNA libraries similar to the selection of DNAzymes.They can bind to a broad range of targets,from metal ions and organic molecules to proteins and cells,with high specificity and affinity.The isolation of the DNA aptamers is done through a combinatorial biology technique called systematic evolution of ligands by exponential enrichment(SELEX).The third class of functional DNA is allosteric DNAzymes or aptazymes.They are designed by combination of the functional regions of DNAzymes and aptamers in one system.They have a ligand binding motif for a specific small molecule and a catalytic fragment which can catalyze a chemical reaction as described above.Functional nucleic acids can identify a variety of specific targets,even those that are poorly immune to produce antibodies or molecules with high toxicity.Compared with small molecule probes,the functional nucleic acids have better water solubility and biocompatibility.Compared with antibodies or other proteins,the functional nucleic acids have several advantages:a)after the sequence of the functional nucleic acid is identified,it can be synthesized with automated DNA synthesis at a low cost and at a large scale;b)chemical modifications of DNA with various reactive and reporting functionalities can be done without interfering with their functions;c)they can be denatured and renatured without loss of function;d)Functional nucleic acids do not require the construction of target molecule binding sites through complex engineering or design knowledge;e)their ability to bind to the targets can be adjusted by different screening conditions.All of these unique advantages make functional nucleic acids a compelling,universal platform for the detection of biomolecules in cells and biomedical diagnosis.Nanomaterials are ultrafine particles with special effects within 1-100 nm.Compared to other conventional materials,nanomaterials have many unique properties,including quantum size effects,surface plasmon resonance(SPR),quantum tunneling and magnetism.Nanomaterials,such as gold nanoparticles(AuNP),semiconductor nanocrystals(quantum dots,QDs),upconversion nanoparticles(UCNPs),graphene,polymeric nanoparticles,DNA nanostructures,MnO2 nanosheets and mesoporous silica nanoparticles(MSNs)have undergone many advances in synthesis and characterization in the past few years.These nanomaterials normally possess large surface area-to-volume ratio,strong load capacity,unique shape,as well as composition-dependent physical and chemical properties.For example,some nanomaterials have their own fluorescence or magnetic effects and can be used for biological imaging such as fluorescence imaging and magnetic resonance imaging.Some nanomaterials themselves can produce photothermal effects or react with biological molecules and can be used for biological therapy such as photothermal therapy.In addition,nanomaterials can be combined with each other or in combination with small molecules,nucleic acid molecules,proteins or other biological materials,using their respective advantages to play important roles in the field of biological imaging and biological therapy.Based on the advantages of functional nucleic acid and nanomaterials,we have developed a series of new multi-functional nanosystems for biological diagnosis and treatment.The major contents are as follows:(1)Manganese is one of the trace elements in human body,essential for the constitution of a number of enzymes that have important physiological effects.Nevertheless,manganese deficiency or manganese poisoning will cause damage to the body's nervous system and secretion system.Therefore,monitoring manganese content is very important for human health.For the detection of metal ions,DNAzyme is of great concern for its rapid,sensitive and efficient advantages.In chapter 2,in vitro selection of manganese-dependent DNAzyme was carried out.Manganese(?)-dependent DNAzyme was isolated,and its activity and selectivity was demonstrated.(2)DNAzymes hold promise for gene-silencing therapy,but the lack of sufficient cofactors in the cell cytoplasm,poor membrane permeability,and poor biostability have limited the use of DNAzymes in therapeutics.In chapter 3,we report a DNAzyme-MnO2 nanosystem for gene-silencing therapy.MnO2 nanosheets adsorb chlorin e6-labelled DNAzymes(Ce6),protect them from enzymatic digestion,and efficiently deliver them into cells.The nanosystem can also inhibit 1O2 generation by Ce6 in the circulatory system.In the presence of intracellular glutathione(GSH),MnO2 is reduced to Mn2+ ions,which serve as cofactors of 10-23 DNAzyme for gene silencing.The release of Ce6 generates 1O2 for more efficient photodynamic therapy.The Mnin ions also enhance magnetic resonance contrast,providing GSH-activated magnetic resonance imaging(MRI)of tumor cells.The integration of fluorescence recovery and MRI activation provides fluorescence/MRI bimodality for monitoring the delivery of DNAzymes.(3)Molecular imaging is a powerful tool for early detection and management of malignant tumors.Synergistic combination of two or more imaging techniques provides solutions able to address multiple issues of sensitivity,resolution,and tissue penetration in tumor diagnosis.In chapter 4,a novel dual-activatable fluorescence/MRI bimodal platform is designed for cell imaging by using a redoxable dioxide(MnO2)-aptamer nanoprobe.The redoxable MnO2 acts as a DNA nanocarrier,fluorescence quencher,and(GSH)-activated MRI contrast agent.In the absence of target cells,neither fluorescence signaling nor MRI contrast of the nanoprobe is activated.In the presence of target cells,the of aptamers to their targets weakens the adsorption of aptamers on the MnO2,causing partial fluorescence recovery,illuminating the target cells,and also facilitating the of nanoprobes into target cells.After,the reduction of MnO2 by GSH further activates the fluorescence signals and generates large amounts of Mn2+ ions suitable for MRI.This platform should facilitate the development of various dual-activatable fluorescence/MRI bimodalities for use in cells or in vivo.(4)Photodynamic therapy(PDT)has been applied in cancer treatment by utilizing reactive oxygen species to kill cancer cells.However,a high concentration of glutathione(GSH)is present in cancer cells and can consume reactive oxygen species.To address this problem,in chapter 5,we report the development of a photosensitizer-MnO2 nanosystem for highly efficient PDT.In our design,MnO2 nanosheets adsorb photosensitizer chlorin e6(Ce6),protect it from self-destruction upon light irradiation,and efficiently deliver it into cells.The nanosystem also inhibits extracellular singlet oxygen generation by Ce6,leading to fewer side effects.Once endocytosed,the MnO2 nanosheets are reduced byintracellular GSH.As a result,the nanosystem is disintegrated,simultaneously releasing Ce6 and decreasing the level of GSH for highly efficient PDT.Moreover,fluorescence recovery,accompanied by the dissolution of MnO2 nanosheets,can provide a fluorescence signal for monitoring the efficacy of delivery.(5)Photodynamic therapy(PDT)mainly utilizes reactive oxygen species(ROS)generated through the reaction between photosensitizer and oxygen presented in tissues to achieve effective treatment.However,the therapeutic effect is seriously hindered in cancer cells because of their hypoxic environment and strong defense system against ROS.Many strategies have been developed to overcome hypoxia,but few reports pay attention to disarm the defense system directly.In chapter 6,we firstly developed a strategy to attack cancer cell defense system against ROS to improve PDT efficacy with a Ce6@MSN@MTH1 siRNA nanosystem.Once endocytosed,this nanosystem could inhibit the expression of MTH1 protein,which is not essential for normal cells against ROS but is highly required for cancer-cell survival.This protein hydrolyzes oxidized dNTP pools caused by ROS and protects cancer cells from damage of ROS.Our strategy significantly enhances cancer cell sensitivity to ROS and improves PDT efficacy in vivo.This work,therefore provides a simple and efficient method that may possess a good promising prospect in cancer therapy.