Tumor Imaging Study At The Cell Level And In Vivo Based On Functionalized Nanomaterials And Aptamers

Due to its rising incidence and high mortality, the malignant tumor is a seriousthreat to human health and life. To explore novel diagnosis and prognostic monitoringmethods for early-stage tumors, especially to reveal the laws and mechanisms intumor occurrence and development through non-invasive, in-situ and real-timeimaging and characterization at the cell level and in vivo, holds great significance forthe improvement of its cure and survival rate. In recent years, the rapid progress ofnanotechnology and molecular engineering technology plus their continuousintercrossing with life science provides an opportunity for developing novel tumorimaging and characterization methods. On the one hand, a variety of functionalizednanomaterials with unique signal generation and enhancement properties afford agreat potential to overcome the background interference and realize the amplificationof targets. On the other hand, the tumor specific aptamer, a newly emerging andgradually enriching probe, provides an ideal kind of targeted recognition molecule tomeet the selectivity demand in clinical applications, resulting from a lot of advantagesover traditional biological antibodies. In this thesis, aiming at the problems andchallenges confronted in the exploration of novel tumor cell and in vivo imaging andcharacteriazation techniques including signal amplification, targeted recognition,signal activation and multi-functionality, a series of probes have been designed andconstructed by utilizing advantages of functionalized nanomaterials and aptamers. Onthis basis, a sensitive, specific, in-situ and real-time study on fluorescence tumorimaging and characteriazation has been systematically performed from the subcellularand cellular level to in vivo level. The detailed description is listed as follows:1. Fluorescent silica nanoparticles-based novel marker for lysosome location,tracking and imaging in tumor cellsIn order to overcome limitations of traditional fluorescent dyes, such as low signalintensity, easy photobleaching and short labeling life, and to satisfy demands forreal-time, in-situ, dynamic and long-time imaging and characterization of lifeactivities at the cell level, advantages of fluorescent silica nanoparticles (DSiNPs)have been utilized. On the basis of a systematic study on the passive-targeted locationand imaging of DSiNPs with different surface charges in tumor cells, a novellysosome characterization and tracking method was developed. Firstly, two kinds of DSiNPs with comparative diameters and opposite surface charges were successfullyprepared through a reverse microemulsion method using TAMRA as the core material.Utilizing the synchronous indication effect of NPs’ fluorescence combined withtraditional markers of organelles, a colocalization fluorescence imaging investigationwas conducted. It was revealed that due to a higher buffer capacity,TAMRA-NH2-DSiNPs with positive surface charge could enter into and then partiallyescape from lysosomes followed by the capture of endoplasmic reticulum. In contrast,TAMRA-DSiNPs with negative surface charge could locate in lysosomes for a longtime. After a further condition optimization using Hela cervix cancer cells as themodel, results showed that TAMRA-DSiNPs could specifically locate in lysosomes ofHela cells and the labeling pattern would not be affected by core materials, whichdisplays a potential application in multi-color staining of subcellular structures. Inparticular, compared with traditional markers, including big molecule Alexa488-dextran and small molecule LysoTracker Green, TAMRA-DSiNPs exhibitedseveral advantages like high photostability, long circulating-life and goodbiocompatibility. Moreover, TAMRA-DSiNPs-based lysosome marker also presentedexcellent tolerance to the cell fixation and permeabilization treatments. Subsequently,TAMRA-DSiNPs have been successfully applied to track lysosomes inchloroquine-treated cells and label lysosomes in different cell lines, which stronglysupported its further application in imaging and characterization of lysosomes-relatedlife activities in the tumor occurrence and development process.2. Annexin V-functionalized fluorescent silica nanoparticles-based novellabeling method for imaging of early-stage apoptotic tumor cellsTaking advantages of DSiNPs like strong fluorescence intensity, goodphotostability and low cell cytoxicity, and combining the “ligand-receptor”interaction, a novel, sensitive, specific, stable and facile method for early-stageapoptotic tumor cell imaging and characterization has been developed based on thespecific binding between the Annexin V-modified DSiNPs and the externalizedphosphatidylserine (PS) on the membrane of early-stage apoptotic tumor cells. Firstly,rhodamine B isothiocyanate (RBITC)-doped silica nanoparticles (RBITC-DSiNPs)were successfully prepared through a reverse microemulsion method using RBITC asthe core material. TEM characterization showed that RBITC-DSiNPs were uniformand well-dispersed with a diameter of50±5nm. By further modifying Annexin V ontoNP’s surface, an active-targeted recognition and labeling probe for early-stage apoptotic cells was successfully constructed. Through confocal fluorescence imaging,it was found that this probe not only effectively identified the paclitaxel-inducedearly-stage apoptotic MCF-7breast cancer cells from untreated MCF-7cells, but alsoachieved the in-situ characterization and tracking of PS’s externalization progress atthe apoptotic cell surface along with the extended induction time. In particular,compared with the pure Cy3-based labeling method, this probe exhibited much betterphotostability. Its fluorescent labeling signal could still be clear and bright after acontinuous laser irradiation as long as20minutes. This probe might play an importantrole in tumor cell apoptosis-related researches and the screening of anti-tumor drugs.3. Aptamers generated by cell-SELEX for in vivo fluorescence tumor imagingTo address the limitations of traditional biological antibodies and explore novelmolecular probes for in vivo tumor imaging, a series of tumor-targeted recognitionand labeling probes were constructed by adopting tumor specific aptamers evoledfrom cell-SELEX and directly modifying the near-infrared fluorescent dye Cy5. Then,a systematic in vivo fluorescence imaging study was performed for several tumorsincluding blood cancer and solid cancer. Ramos lymphoma and its aptamer TD05waschosen as the model firstly. Flow cytometry assays demonstrated that Cy5-TD05inmouse serum could well retain its recognition ability to both in vitro and ex vivocultured Ramos cells. In vivo fluorescence imaging results also showed thatCy5-TD05could achieve the targeted imaging of Ramos tumor inside mice withexcellent sequence dependence and tumor selectivity. On this basis, to further expandaptamers’ application in fluorescence imaging of other tumor types, both lung cancerand liver cancer were selected as the study objects. The selective imaging of differenttumor types in the same mouse body was successfully realized using thecorresponding Cy5-aptamers, which positively lays the foundation for aptamers to beapplied in in vivo imaging as a novel tumor-targeted recognition molecule in thefuture.4. Locked nucleic acid-modified aptamer probe for serum stabilityimprovement and in vivo fluorescence tumor imagingTo address aptamers’ drawbacks in complex biological systems, such asinsufficient stability, short residence time in tumor sites and narrow imaging timewindow, TD05was selected as the model to construct an imaging and detection probewith both the recognition ability against target tumor cells and good stability in bloodthrough locked nucleic acid (LNA) and3’-3’-thymidine (3’-3’-T) modifications. By investigating the effects of various modification strategies with different positions,numbers and combinations on TD05’s properties including stability, affinity andspecificity, it was found that the combined use of LNA and3’-3’-T had a synergisticeffect, and to a certain extent, the half-lives were gradually extended with the increaseof LNA incorporated. In particular, TD05.6with7-base pair-LNA substitution and3’-3’-T modification exhibited the significantly elevated detection stability affordinga half-life of5~6h to Ramos cells in serum, which was prolonged to be over ten timesof that before modification. Moreover, the in vivo tumor imaging window was alsogreatly extended from <150min of TD05to>600min of TD05.6. This strategy holdsa potential to be developed as a versatile method to provide more aptamer probes withclinical application merits for tumor imaging and detection studies.5. Hairpin activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration for in vivo fluorescence tumorimagingIn order to overcome the disadvantages of dye-labeled “always on” aptamerprobes, including long diagnosis time, low imaging contrast and limited sensitivity,the specific aptamer Sgc8c against CCRF-CEM leukemia cells was selected as themodel and a hairpin activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration was successfully constructed. This probewas mainly hairpin-shaped in its free state and displayed a quenched fluorescence inthe absence of targets as a result of the proximity between fluorescent molecule andquencher respectively at two terminus of the probe. However, upon interacting withtarget cells, this probe effectively underwent a conformational reorganization with anactivated fluorescence, thus indicating the presence of target tumor cells. Flowcytometry assays demonstrated that this probe not only had the highly specific signalactivation ability against target tumor cells, but also significantly improved theanalytical sensitivity in comparison with “always on” probes. It could efficaciouslydetected samples containing as low as118CCRF-CEM cells. In particular, whenapplied in in vivo tumor imaging, this probe sufficiently decrease the signalinterference from unbound probes in non-target tissues, thus greatly enhancing thetumor imaging contrast and shortening the diagnosis time from several hours of“always on” strategy to15minutes. In addition, this probe also displayed excellentsequence specificity and tumor selectivity. Considering the expansion of aptamerdiscovery for varying cancer targets, this probe design might be potentially used as a versatile method to develop highly sensitive and speicfic tumor imaging probes.6. Split activatable aptamer probe based on cell membrane surfaceprotein-triggered conformational alteration for tumor cell and in vivo imagingand detectionAlso using Sgc8c as the model, the split activatable aptamer probe wassuccessfully constructed by cutting Sgc8c into two nucleic acid fragments at theproper site. In the absence of targets, there was no obvious interaction between thesetwo fragments. But after introduncing target tumor cells, these two fragments couldbe induced by the target protein on cell membrane to form the recognitionconformation similar to the intact Sgc8c, thus binding to target cells. Through flowcytometry analysis, it was found that the specific affinity of this probe against targetcells was obviously sensitive to temperatures. Utilizing its targeted recognition abilityat low temperatures, a simple, convenient, washing-free, sensitive and specificimaging and detection technique for CCRF-CEM cells was successfully developed bycombining with the fluorescence enhancement effect based on the proximity of G-richsequence to silver nanoclusters. Utilizing the feature that the binding ability of thisprobe to target cells gradually decreaced with the temperature lifted from on ice to37℃, a selective capture and temperature-controlled release method was successfullyconstructed by using96-well microplates modified with this probe as the capturevessel. It was demonstrated that this method had good biocompatibility to cells andcould be effectively applied in the cyclic capture and release of target cells and theseparation and recovery of different tumor cells in mixed samples. Finally, throughfurther modification with a PEG linker to conjugate the two fragments of this probeand change the interaction into intramolecular interaction, its inactivation problem atphysiological conditions was successfully addressed. In addition, by integrated withthe Cy3-Cy5donor-acceptor pair-based FRET effect, this probe showed thepreliminary feasibility for tumor cell detection and imaging.7. pH-activatable aptamer probe stimulated by the acidic tumormicro-circumstance for tumor cell and in vivo fluorescence imagingAiming at the weak acidic feature of tumor tissues and tumor cell lysosomes (pH4-6), simultaneously combining the targeted recognition ability of aptamers, thespecific aptamer S6against A549lung cancer cells was selected as the model. ApH-activatable aptamer probe with both the detection specificity and “signal on”architecture was successfully constructed through conjugation of Cy5-aptamer and quencher BHQ3using an acid-labile linker ATU. At the neutral condition, this probeshowed a fluorescence quenching efficiency as high as about98%, and after incubatedin buffer with pH4.5for24hours, the signal nearly recovered to100%. Bycomparison with the hairpin activatable aptamer probe, it was found that this probeexhibited much better fluorescence stability no matter in serum or inside mice.Confocal microscope imaging results indicated that this probe not only held thesite-specific acid-activatable functionality in lysosomes of live cells, but alsoafforded a variety of advantages over “always on” probes, such as highly specific,high-contrast, simple, washing-free and so on. A further in vivo fluorescence tumorimaging study also demonstrated that this probe positively had the activatableimaging functionality inside tumor tissues of mice and the activation wassequence-dependent and tumor-targeted. This design might be developed as a versitilestrategy to construct probes for the characterization and imaging study of acidicdesease sites like tumors.8. Self-assembled aptamer-carbon nanotube activatable probe for in vivofluorescence tumor imagingBy integrating advantages of functionalized nanomaterials and aptamers as thesignal switch and the targeted recognition molecule respectively, Sgc8c was selectedas the model to construct an activatable probe for tumor imaging based on the strongadsorption and quenching effect of single-wall carbon nanotubes (SWNTs) tofluorophore-labeled DNA. In the absence of targets, Cy5-Sgc8c firmly assembled onthe surface of SWNTs and displayed a quenched fluorescence. However, with theaddition of target tumor cells, the binding of aptamer with the target protein on cellmembrane could force Cy5apart from SWNTs with an elevated fluorescence.Through flow cytometry assay and in vivo fluorescence imaging, it was found that nomatter in buffer or inside tumor-grafted mice, CCRF-CEM cells could effectivelyactivate the fluorescence signal of this probe and this activation was highlysequence-dependent and tumor-targeted. In particular, in comparison with “alwayson” probes, it was demonstrated that SWNTs could greatly reduce the nonspecificsignal background interference from unbound probes in non-target systems, thussignificantly improving the in vitro analysis sensitivity and in vivo imaging contrastfor target tumor cells. This probe design is simple and convenient, which held greatpotentials to be developed as a versatile tumor imaging method with low background,high contrast and high specificity. 9. Activatable aptamer probes-functionalized Au@Au/Ag nanoparticles for invivo fluorescence tumor imaging and the guided photothermal thaerapyOn the basis of above in vivo tumor imaging studies based on acitivatable aptamerprobes (AAPs), a preliminary study on the construction and application of theranosticprobes based on aptamer-nanomaterial functionalized assemblies was performed byfurther utilizing the antitumor properties of functionalized nanomaterials. Firstly,using Au nanorods as templates, Au@Au/Ag NPs were successfully synthesized bycoating Au nanorods with Ag layers and then etching with Au. The prepared NPsexhibited a broad and intense absorption from400to1100nm and a heatingefficiency4.5times higher than Au nanorods under a980nm laser irradiation.Thereafter, the acitivatable aptamer probe (AAP) sequence was designed by adoptingS6as the model and a theranostic probe based on AAP-modified Au@Au/Ag NPs wasthen successfully constructed after the self-assembly of DNA and NPs via “Au-S”bonding, in which Au@Au/Ag NPs served as both the “optical nano heater” and thefluorescence quencher. Through a series of in vitro and in vivo investigations, it wasdemonstrated that this probe could achieve not only the selective photothermaltherapy but also the acitvatable imaging and detection of target tumor cells. Byfurther intravenously injecting this probe into a mouse grafted with different tumors,the specific activatable imaging and the guided near-infrared photothermal therapy ofA549tumor was successfully realized. Considering the expansion of aptamerdiscovery for varying cancer targets and the broad and strong absorption spectra ofAu@Au/Ag NPs, this probe design might be explored to be a technique platform forthe synchronous imaging and therapy of multi-targets.