Construction Of Two-photon Fluorescence Nanoprobe With High Biocompatibility And Its Application In Biomedical Imaging

In recent years, two-photon fluorescence confocal microscopy imaging techniques(TPM), as a new type of optical imaging technology is increasingly a cause for greatconcern. Compared to the traditional single-photon excited fluorescence microscopyimaging (OPM), two-photon excitation (TPE) with near infrared (NIR) photons as theexcitation source has the advantages of lower tissue autofluorescence andself-absorption, reduced photodamage and photobleaching, higher spatial resolutionand deeper penetration depth (>500m). Of particular importance for TPEapproaches is the construction of the two-photon (TP) probes. To date, a variety ofTPE fluorescent probes have been developed for applications in chemical sensors andbiological imagings. These probes, however, are mainly focused on organictwo-photon absorption (TPA) molecules, and are arguably the lack of cell permeabilityand poor photobleaching resistance, as well as weak ability of cellular localization.Moreover, for biomolecules detection, design and synthesis of the organicmolecule-based TPA probes are still great challenges.With the rapid advancement of nanotechnology and desirable properties ofnanostructures with including antienzyme action, carrier properties, a tunablecirculation lifetime, enhanced permeability and retention (EPR) effect, the TPAnanoprobes would provide an alternative for the TPE-based imaging protocols. At thesame time,-cyclodextrin polymer (CDP) is a high mocecular weight derivativecontaining CD units based on physically or chemically mixed method. CDP as a newcarrier with excellent biocompatible and low toxicity, which not only maintain aninclusion capacity for slow release, catalytic and recognition, but also combine goodmechanical strength and chemical adjustability of polymer, due to the formation ofthree-dimensional spatial network structure consisting of a plurality of pivot in thepolymerization process.Combining the unique properties of nanostructures (such as PMMA-co-MAA,AuNPs and CDP), and the deeper penetration depth and higher spatial resolution ofTPM technique, in this thesis, small organic molecules with two-photon properties arechosed as research subjects, and a series of new two-photon fluorescence nanoprobeswith high biocompatibility are fabricated for quantitative detection of bioactivemolecules (such as ions and proteases) and targeting biomedical imaging. The thesis has done the following works:(1) We chose the trans-4-[4-N,N-diethylamino)benzyl]-4’-(4-N, N-diethylamino)phenyl(DEAS) as TPA organic molecule, and design a new TPA fluorescencenanoprobe (DEAS@PMMA-co-MAA) based on co-precipitation and self-assemblymethod, which improve the solubility,biocompatibility and two-photon properties ofTPA organic molecules. This novel TPA nanoprobe is successfully used to TPEimaging in living cells and deeper tissues.(2) We chose the TPdye as fluorophores and AuNPs as nanocarrier, and design anovel TPA fluorescence nanoprobe (AuNPs@CDGRG-TPdye). This nanoprobe issuccessfully used to collagenase activation imaging in deeper tissues and CN-detection in complex systems.(3) A facile strategy for TPA fluorescent nanomicelle preparation has beendeveloped through the inclusion interaction between CD andtrans-4-[p-(N,N-diethylamino)styryl]-N-methylpyridinium iodide (DEASPI). Thisnanomicelle exhibits desirable two-photon-sensitized fluorescence properties, highphotostability, high cell-permeability, high stability and excellent biocompatibility. Asa result, by anchoring the RGD peptide on the nanomicelle‘s surface via the highbinding affinity of labeled adamantine with CD, the targeted TPE imaging has beensuccessfully achieved in cancer cells and tumor tissues. The material design approachreported here also opens up new perspectives for developing a wide range of uniquesensing schemes and will have great potential in cancer diagnosis and biomedicalresearch, which are compatible with the benefits of multiphoton imaging techniques.(4) On the basis of above chapter, we have successfully developed a TPAnanomicelle-based two-photon fluorescent nanoprobe for enzymatic activities assay inliving cells and tissues with high sensitivity and selectivity. In vitro assays revealedthat the DEASPI/βCDP nanomicelle-based TPA fluorescent nanoconjugate provided arobust, sensitive, and selective sensor for quantitative detection of caspase-3even ifunder complex biological conditions. TPM experiments with HeLa cells and tissue ssuggested that the βCDP nanomicelle-based conjugate was efficiently delivered intolive cells and acted as a―signal-on‖fluorescent sensor for specific, high-contrastimaging of target biomolecules.