Synthesis, Characterization, and Biological Evaluation of Novel Phthalocyanine-Based Photosensitizers for Photodynamic Therapy

新型酞菁類光敏劑的合成,表徵及其在光動力治療中的應用

Student thesis: Doctoral Thesis

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Award date10 Sep 2018

Abstract

Photodynamic therapy (PDT) holds great potential for cancer treatment. My research work aims to develop novel phthalocyanine-based photosensitizers for enhanced PDT. The thesis describes the design, synthesis, characterizations, photophysical properties, and biological evaluation of a series of phthalocyanine-based photosensitizers.

Chapter 1 is the introduction part, which includes the basic principle of photodynamic reaction, the biological mechanisms of PDT in tumor ablation, and limitations of current PDT. Moreover, the recent development of phthalocyanine-based photosensitizers, which are thought to be ideal photosensitizers for PDT, has been reviewed in this chapter. It includes phthalocyanines conjugated with tumor homing ligands, activatable phthalocyanines, and chemotherapeutic drug-conjugated phthalocyanines for dual therapy. All the approaches aim to increase the tumor selectivity and therapeutic efficiency.

Short synthetic peptides, which render simple, cheap, and large-scale synthesis, are attracting vectors for targeted delivery of photosensitizers. Peptide GE11, a 12-amino acid peptide with a sequence of Tyr-His-Trp-Tyr-Gly-Tyr-Thr-Pro-Gln-Asn-Val-Ile, binds specifically to epidermal growth factor receptor (EGFR), which is overexpressed in a variety of human cancer cells. Chapter 2 describes the synthesis, characterization, photophysical properties, intracellular fluorescence emission, cellular uptake, in vitro cytotoxicity, and in vivo biodistribution of the zinc(II) phthalocyanine conjugated with GE11 peptide. This conjugate showed much higher tumor accumulation than the control compound conjugated with a random sequence peptide after intravenous administration in human epidermoid carcinoma A431 tumor bearing mice.

Activatable photosensitizers, which are specifically activated by tumor-associated stimuli, can minimize the damage to adjacent normal tissues. Glutathione (GSH), which is the most abundant intracellular thiol and is elevated in tumor tissues, is a well-documented stimulus for the design of activatable theranostics. Thus, we designed and synthesized a biotinylated GSH-activated zinc(II) phthalocyanine, in which the phthalocyanine is quenched by the dinitrobenzenesulfonyl (DNBS) moiety, but activated after cleavage of DNBS group by GSH. The coupling of biotin is designed to further increase the selectivity between tumor and normal tissues through active-targeting strategy. The photophysical properties, GSH-responsive behavior in fluorescence emission and singlet oxygen generation of this compound have been investigated. Its intracellular fluorescence emission, subcellular localization, cellular uptake, and cytotoxicity toward HepG2 human hepatocarcinoma cells have also been studied and presented in chapter 3. Besides, this conjugate exhibited preferential localization in endoplasmic reticulum (ER), the photo-induced ER-stress has also been examined.

The integration of multiple treatments can exhibit enhanced anticancer efficacy through the synergistic effect when compared with a single treatment. Chapter 4 presents a prodrug, which contains an anticancer drug 7-ethyl-10-hydroxy-camptothecin (SN-38) and a zinc(II) phthalocyanine, for combined chemo- and photodynamic therapy. This prodrug consists of two GSH sensitive moieties which are disulfide linker and DNBS group. The disulfide linker is used to conjugate the SN-38 and the phthalocyanine together, while the DNBS moiety is used to quench the fluorescence and singlet oxygen of the phthalocyanine. The photophysical properties, GSH-responsive behavior in fluorescence emission and singlet oxygen generation of this compound, as well as the release of SN-38, have been investigated. The cellular uptake, intracellular fluorescence emission, and cellular cytotoxicity of the prodrug have also been evaluated toward HeLa cells. As an extension of the work described in chapter 4, another chemo-photodynamic therapeutic agent, in which doxorubicin has been conjugated to the zinc(II) phthalocyanine via a disulfide linker, is reported in Chapter 5. The photosensitizing ability of the phthalocyanine is partially quenched by the doxorubicin and can be activated after the cleavage of disulfide linker. The doxorubicin can be released after thiol-triggered disulfide bond cleavage, followed by intramolecular immolative reaction. The thiol-responsive behavior in fluorescence emission and singlet oxygen generation of this compound, together with the release of doxorubicin, have been investigated. This conjugate exhibited higher phototoxicity toward HepG2 cells when compared with the the control compound without doxorubicin.

The infection of antibiotic-resistant pathogens has become a great threat to humans. Production of β-lactamases is a common way for bacteria to inhibit the action of antibiotics. Chapter 6 reports the design and synthesis of a series of β-lactamase-activated phthalocyanine-based photosensitizers, in which a zinc(II) phthalocyanine is conjugated to either a black hole quencher BHQ3 or another phthalocyanine unit through a cleavable β-lactam ring linker. The photosensitizing properties of these compounds are either self-quenched or quenched by the BHQ3 moiety. The photophysical properties, β-lactamase-responsive behavior in fluorescence emission, singlet oxygen generation and in vitro photodynamic antibacterial activity of these compounds have been investigated and presented in this chapter.

Chapter 7 presents the conclusion for the present study. Experimental details are shown in chapter 8. Nuclear magnetic resonance (NMR) spectra (1H and 13C) and mass spectra for related compounds are given in the Appendix.