Construction And Application Of Functionalized Nanoprobes In Tumor Hypoxic Microenvironment

Due to its rising incidence and high mortality,the malignant tumor is a serious threat to human health and life.According to report,90%mortality are caused by the tumor metastasis.All things happened for its intrinsic and extrinsic factor s.The event of cancer metastasis also depended on the origi nal tumor and the residual normal tissue.Tumor tissue includes two important parts:tumor cells and tumor microenvironment.Previous studies mainly focused on tumor cell s itself.However,modern research reveled that the tumor microenvironment also crucial to the tumor progression.Hypoxia is the common feature in all solid tumors.This result were mainly attributed to:?1?tumors grow in an exaggerated way and high metabolic rate leading to consumed O₂ more than available O₂;?2?aberrant vasculogenesis cause the microvascular obstruction making the O₂ not effective delivery to the intratumor.It is most obvious difference between tumor and normal cells that tumor cells survived in the hypoxic microenvironment,which will cause differences between the tumor cells and normal cells in the physiology and function.In this thesis,in order to provide reliable tools for hypoxic-related clinical studies and improve the screening or optimization of anticancer drugs,a series of functional nanoprobes have been designed and constructed by combining surface enhanced Raman scattering?SERS?,fluorescence imaging with advantages of nanomaterials to explore and detect the difference between hypoxic and normoxic cells in the physiology and function.The detailed description is listed as follows:?1?In the second chapter,we developed an activatable surface-enhanced Raman scattering nanoprobes for detection of pH in lung tumor cells under hypoxic conditions.We functionalized 4-NTP onto the surface of gold nanorods?AuNRs?with high SERS enhancement capacity via Au-S.Under hypoxic conditions,the nitroaromatic compounds have been proven to be superior substrates for nitroreductase?NTR?with reduced NADH serving as an electron donor.Thus,the nitro groups in 4-NTP could ultimately be reduced to an amino group termed4-aminothiophenol?4-ATP?.It is noteworthy that the as-generated 4-ATP can respond well as functions of different pH.This unique property can ensure the SERS monitoring of intracellular pH in living cells and tissues under hypoxic conditions.?2?Owing to mitophagy induced by various stresses,in the third chapter,we developed an azoreductase-responsive micelle?termed Micelle@Mito-rHP@TATp,MCM@TATp?by encapsulating cationic spiropyrane derivative?Mito-rHP?to realize specific imaging of mitophagy in living cells under hypoxia.Under hypoxic conditions,the azobenzene moiety-contained MCM@TATp would be disrupted by the highly expressed azoreductase,then the encapsulated Mito-rHP would be released.Since Mito-rHP is a mitochondria-targeted and pH-sensitive probe,thus it could target into mitochondria and displayed a desirable“off-on”fluorescence response to mitophagy during which mitochondria were regarded to undergo acidification.We expect this new strategy would be a powerful tool for evaluation the degree of mitophagy.?3?To realize convenient fluorescence imaging of mitophagy induced by hypoxia,in the forth chapter,we designed the hypoxia and mitophagy dual-activatable fluorescent probe molecules?MiAzoR?.The probe molecules which contain a spirolactam moiety to bind hydrogen protons and zao group to react with zaoreductase,display high sensitive and selective fluorescence response to mitophagy induced by hypoxia.The results showed that the probe had a very good effect on mitophagy imaging.?4?The disadvantages of conventional SERS approach are the instability of the SERS tags caused by the direct exposure of tag surface in complicated environments,and the issue of contaminants caused by reductants or surfactants still hinder its further application.In the fifth chapter,a new SERS analysis strategy combining stable SERS reporter element and duplex-specific nuclease?DSN?-assisted signal amplification for quantitative detection of exosomal miRNA extracted fro m hypoxic cancer cells is proposed.The SERS switch contains three imporant elements:a signal reporter element?ARANPs?,a recognition element?CP?,a separation element?SiMB?.Upon target miRNA binding,DNA in heteroduplexes formed by CP and exosome miRNA could be specifically cleaved by DSN,resulting in the release of ARANPs from the surface of SiMB.Meanwhile,target exosome miRNA remained intact and subsequently involved in the next round-recycling amplification.After cycling,number ARANPs were released to produce strong SERS signal and achieve the quantitative detection of exosomal miRNAs extracted from hypoxic tumor cells.Several physiological and functional changes of tumor cells have been studied under hypoxic conditions,which provides valuable guidance for the design of hypoxia-related probes.The changes of substances or components in living cell can be detected by activating probes with high expression of reductive substances in hypoxic cells.These design ideas also have a certain versatility and development prospects.