Application Of Fluorescent Dyes In Tumor Diagnosis And Therapeutics

Near-infrared dyes have absorption and emission wavelengths in the near-infrared (NIR) spectrum, between 650 and 900 nm. NIR dyes, as promising imaging and therapeutic agents, have been of great interests in detecting and treating tumors recently. They can absorb NIR light with a specific wavelength to reach an excited singlet state. Part of the energy of the excited singlet state would be dissipated in the form of light with a longer wavelength, called fluorescence. Therefore, NIR dyes can be applied for in vivo tumor imaging effectively. And it has high specificity because targeted NIR molecules can distinguish the molecular changes between tumor and normal tissues. Furthermore, NIR imaging shows high sensitivity owing to extremely low absorption and autofluorescence from organic tissue in the NIR spectral range, which can minimize background interference and improve tissue penetration.In addition, some energy of the excited singlet state can be transited through vibronic relaxation or other nonradiative transitions pathways, which will be converted into heat. If the rate of heat production within the tissue can exceed that of tissue heat dissipation, the temperature of tissue would increase gradually. When temperature reaches up to 41.5℃, tumor cellular cytotoxicity occurs. And temperatures above 43℃ can induce vascular destruction within tumor tissue. Therefore, NIR dyes can also be utilized as promising theranostic agents for photothermal therapy (PTT) while detecting tumors. Apart from the above-mentioned two kinds of energy transition way, the excited singlet state can move to a lower-energy-excited triplet state via intersystem crossing. In the excited triplet state, NIR dyes can induce reactive species generation, for example, free radical or reactive singlet oxygen. They induce oxidation reaction with nearby biomacromolecules and destruct organic tissues effectively. In which, generated singlet oxygen is more responsible for the destruction of targeted tissue. Thus, NIR dyes could also act as excellent photodynamic agents. In addition, photochemical internalization (PCI) was developed as a method for photodynamic-enhanced cytosolic release of membrane-impermeable therapeutic molecules entrapped in endocytic vesicles. Briefly, the therapeutic molecules, such as protein or oligonucleotides, colocalize with a photosensitizer in endocytic vesicles. Light-activation of the photosensitizer results in singlet oxygen mediated damage of the endocytic membranes with the subsequent release of the therapeutic molecules into cytosol. This thesis mainly includes the following three parts:(1) IR-780, a representative hydrophobic near-infrared (NIR) fluorescence dye, is capable of fluorescently imaging and photothermal therapy in vitro and in vivo. However, insolubility in all pharmaceutically acceptable solvents limits its further biological applications. To increase solubility, we developed a novel self-assembled IR-780 containing micelle (PEG-IR-780-C13) based on the structural modification of IR-780. Briefly, a hydrophilic PEG2000 was modified on the one side of IR-780, and the hydrophobic carbon chain on the other side was extended from C3 to C16 (additional C13 carbon chain). The modification provides a better self-assemble capability, improved water solubility and higher stability. In addition, PEG-IR-780-C13 micelles are specifically targeted to the tumor after intravenous injection and can be used for tumor imaging. The in vitro cell viability assays and in vivo photothermal therapy experiments indicated that CT-26 cells or CT-26 xenograft tumors can be effectively ablated by combining PEG-IR-780-C13 micelles with 808nm laser irradiation. More importantly, no significant toxicity can be observed after intravenous administration of the therapeutic dose of generated micelles. Overall, our micelles may have the least safety concern while showing excellent treatment efficacy, and thus may be a new photothermal agent potentially useful in clinical applications.(2) Application of photodynamic therapy for treating cancers has been restrained by suboptimal delivery of photosensitizers to tumor cells. Nanoparticle-based delivery has become an important strategy to improve tumor delivery; however, the success is still limited. One problem for many nanoparticles is poor penetration into tumors, and thus the photokilling by photodynamic therapy is not complete. We aimed to use chemical conjugation strategy to engineer small nanoparticles for targeted delivery of photosensitizers to cancer cells. Thus, to improve the 2D cellular internalization and 3D multicellular penetration of a photosensitizer, Chlorin e6 (Ce6), was covalently conjugated to PAMAM dendrimer that was also modified by tumor-targeting RGD peptide. With several Ce6 molecules in a single nanoconjugate molecule, the resultant targeted nanoconjugates RGD-P-Ce6 showed uniform and monodispersed size distribution with a diameter of 28 nm. The singlet oxygen generation efficiency and fluorescence intensity of RGD-P-Ce6 in aqueous media were significantly higher when compared with free Ce6. The targeted nanoconjugates demonstrated approximately 16-fold enhancement in receptor-specific cellular delivery of Ce6 into tumor cells compared to free Ce6 and thus were able to cause massive cell killing under photoirradiation at low nanomolar concentrations without causing cytotoxicity in dark. Due to their small size, the targeted nanoconjugates could penetrate deeply into 3-D tumor spheroids. As a result of their greater cellular delivery, smaller size, and lack of cytotoxicity compared to conventional nanoparticles, the multivalent nanoconjugates may provide an effective tool for targeted photodynamic therapy to tumors.(3) Recently, therapeutic oligonucleotides, such as splice switching oligonucleotides (SSOs) have great potential for gene therapies. However, the development of SSOs as therapeutic agents has progressed slowly, because difficult cytosolic delivery of SSOs into cytosol and nucleus remains a major barrier. Photochemical internalization (PCI), a promising strategy for endosomal escape, was introduced to disrupt the endosomal membrane using light and photosensitizer. Here we constructed Poly(amido amine) (PAMAM) conjugates to simultaneously deliver oligonucleotides and photosensitizers into endo/lysosomal compartments. After photoirradiation, considerable oligonucleotides were observed to diffuse to cytosol and accumulate in nucleus. Furthermore, the PCI mediated cytosolic delivery of SSOs effectively enhanced their nuclear splice switching activity.