M. Tech. / Photodynamic therapy (PDT) is a type of phototherapy which is based on the interaction of photosensitizer with visible light in the presence of oxygen resulting in the production of reactive oxygen species (Pfaffel-Schubart et al., 2008). PDT has rapidly matured in the past 6 years and is an accepted standard treatment for various cancerous diseases. The main advantages of PDT include significant drug selectivity in tumour cells, absence of toxicity in the dark, possibility to treat only cancer cells and the ability to retreat a tumour in order to improve the prognosis (Pfaffel-Schubart et al., 2008). Photofrins are the most studied photosensitizers and their disadvantages is the inability of these photosensitizers to localize specifically in tumour cells and are retained in normal cells for prolonged periods (Nowis et al., 2005). This factor has stimulated the development of second generation photosensitizers with improved physical, chemical and spectral properties (Sharman et al., 1999). Phthalocyanine compounds are second generation photosensitizers which have a potential as photosensitizers in PDT treatment of many cancers. Traditional treatments of cancer show limited efficiency and other treatment modalities need to be explored. PDT has shown promising results in the treatment of many cancers using phthalocyanine compounds as photosensitizers but little is known about Zn sulfophthalocyanine on lung cancer cells. This study investigated the effects of Zn sulfophthalocyanine photosensitizer and laser irradiation on a lung cancer cell line (A549). Lung cancer cells were cultured in RPMI medium 1640 supplemented with 10% foetal bovine serum and antibiotics and incubated at 37 °C with 5% CO2 and 85% humidity. Cells were divided into 4 groups. Group 1 was an unirradiated control not treated with a photosensitizer. Group 2 was photosensitized at a concentration of 15.8 μM but not irradiated. Group 3 was irradiated but not photosensitized while Group 4 was irradiated and photosensitized at a concentration of 15.8 μM. Laser irradiations were done using a diode laser emitting 636 nm with an output of 110 mW at 5.3 J/cm2. Cell morphology was evaluated using the light inverted microscope. Cell viability was assessed using adenosine triphosphate (ATP) luminescence assay and Trypan blue exclusion test while cell proliferation was measured using the alamarBlue assay. Cytotoxicity was evaluated by assessing membrane permeability for lactate dehydrogenase and DNA damage was evaluated by comet assay. The mode of cell death was assessed by annexin V-FITC apoptosis detection kit using flow cytometry. In addition, expression of Bcl-2/Bax was monitored by western blot analysis and to determine the levels of induced Heat shock protein 70, Hsp 70 ELISA was used. Cells treated with photosensitizer at 15.8 μM and irradiated with 5.3 J/cm2 showed changes in cell morphology, decrease in cellular viability, proliferation and Hsp 70 expression and an increase in cytotoxicity and DNA damage. This indicated that the metalated phthalocyanine was effective in inducing cell death and the analysis of cell death by annexin FITC-V revealed an apoptotic cell death pattern. Furthermore, loss of the antiapoptotic Bcl-2 protein suggested that there was no inhibition of apoptosis while the absence of proapoptotic Bax suggested that other proapoptotic protein might have played a role in the induction of apoptosis. Untreated cells, irradiated cells and cells treated with photosentitizer alone showed no changes in morphology, increase in cellular viability, proliferation, expression of Bcl2/Bax, and Hsp 70 and a decrease in DNA damage and cell membrane damage. However, treatment of cell with photosensitizer alone caused DNA damage. These results indicate that irradiation or photosensitizer alone has no effect on the A549 cells but photosensitizer alone induces DNA damage.

D.Tech. (Biomedical Technology) / Photodynamic therapy (PDT) is an alternative treatment modality for malignant tumours based on the photodamage to tumour cells through a photochemical reaction (Ahn et al., 2013). PDT utilizes a light sensitive photosensitizer (PS) that selectively localizes in tumour cells and is excited by light of a specific wavelength in the presence of molecular oxygen. The excited PS leads to the generation of singlet oxygen or other reactive oxygen species(ROS) which induces cytotoxic damage to cellular organelles and eventually cell death. Singlet oxygen has a very short life and its generation is controlled by the presence of the PS and the laser light (Senge and Radomski, 2013).The subcellular localization site of the PS plays a vital role in determining the effectiveness and the extent of cellular damage as well as the mechanism involved in cell death. Lung cancer is the leading cause of cancer death worldwide in both males and females, with an estimated 1.4 million deaths each year (American Cancer Society, 2011). Therapeutic modalities used in the treatment of lung cancer such as chemotherapy, radiotherapy and immunotherapy have rarely yielded a good prognosis and effective treatment remains a challenging problem to date. An alternative treatment modality with minimal complications such as PDT needs to be explored. Most in vitro PDT experiments are conducted on monolayer cultures and the cellular environment of these cultures does not correspond to that of in vivo studies. Multicellular tumour spheroids (MCTSs) serves as an important model in cancer research for the evaluation of therapeutic interventions since they mimic different aspects of the human tumour tissue environment.

Thesis (MS)--Montana State University--Bozeman, 2008. / Typescript. Chairperson, Graduate Committee: Jean Starkey. Includes bibliographical references (leaves 70-76).

This work focuses on the development of red light-activated porphycene-based photosensitizers for anti-tumor photodynamic therapy (PDT) under both normoxic and hypoxic conditions. A total of seven water-soluble porphycenes have been designed, synthesized and evaluated as potential PDT agents in terms of their photophysical and photobiological properties using principally the human nasopharyngeal carcinoma (HK-1) cells. Among the porphycenes synthesized, two were neutral amphiphilic aryl porphycenes, TDEGPPo and Zn(II) TDEGPPo, with relatively weak photo-cytotoxic activities even under normoxic condition. Two cationic porphycenes, TPyBPo and TriPyPPo, exhibited strong photo-cytotoxic activities, with LD50 of 0.3 mM at a light dose of 3 J/cm2, under normoxic condition. However, much lower photo-cytotoxicity was observed under hypoxic condition for TPyBPo and TriPyPPo, with LD50 of 3 mM and 3.5 mM, respectively, obtained at high light doses (>10 J/cm2). Two alkyl porphycenes with one and two sulfonoamide diglycol functionalities, TBPoS-OH and TBPoS-2OH, were synthesized and shown to exhibit very potent photo-cytotoxic activities, with respective LD50 of 53 nM and 20 nM (light dose 8 J/cm2) under normoxic conditions. Most importantly, comparably potent photo-cytotoxicity was also observed for these porphycenes under hypoxic conditions, with respective LD50 of 65 nM and 50 nM (light dose 8 J/cm2). In addition, these porphycenes were taken up by the HK-1 cells very rapidly, with >90% accumulated inside the cells after only 1 h of incubation. Confocal microscopy revealed that these porphycenes were localized at the lysosomes, mitochondria as well as endoplasmic reticulum. Furthermore, the predominant mode of cell death caused by the PDT action of these porphycenes was shown to be apoptosis. In an attempt to effect mitochondria localization to enhance apoptotic cell death for these porphycenes, TBPoS-OH was conjugated with rhodamine B to produce the TBPoS-Rh B conjugate. This porphycene-Rh B conjugate also displayed very potent photo-cytotoxicity under both normoxic and hypoxic conditions, with LD50 of 52 nM and 85 nM, respectively, at a light dose of 8 J/cm2. However, confocal microscopy revealed its principal subcellular localization was at the lysosomes, not the mitochondria. The PDT activities of these porphycenes were compared to a well-known patented PDT agent, EtNBS, which is active under both normoxic and hypoxic conditions, with LD50 of 58 nM and large than 1000 nM, respectively, towards the HK-1 cells. This comparison clearly shows that our sulfonoamido-porphycenes, TBPoS-OH, TBPoS-2OH and TBPoS-Rh B conjugate, display a 15- to 25-fold stronger hypoxic PDT activity relative to EtNBS, thus making these porphycenes excellent candidates for hypoxic anti-tumor photodynamic therapy.

M.Tech. (Biomedical Technology) / The uncontrolled growth of cells in the body is often associated with cancer. It constitutes a major health problem and is one of the leading causes of death in the world. Cancers of the lung, breast, colon/rectum and prostate are no longer only associated with developed countries but are the most common occurring cancers worldwide. Breast cancer is the leading cancer faced by women in South Africa as well as in the world. Conventional cancer therapies often result in uncertain outcomes with numerous side effects and may be associated with limited therapeutic advantage. This has led to the development of safer and better treatment regimes with improved therapeutic outcomes. Photodynamic therapy (PDT) is a treatment used for a wide range of conditions, including cancer. This treatment utilises a photosensitiser (PS), a light activated chemotherapeutic agent, and light of a specific wavelength and power density. It is based on the selective tumour localisation of the PS and the ability to generate high levels of reactive oxygen species (ROS) in the presence of light. The generation of ROS causes permanent damage to the tumour cells resulting in cancer cell death. The distinctive criteria when comparing different PDT modalities is the choice of PS as the treatment outcomes are greatly influenced by the light dependent properties of the chemotherapeutic agent. Phthalocyanines are second generation PSs used in PDT. Effects of members of this PS family have been studied and they exhibited good photosensitising properties including lack of cytotoxicity in the absence of light, extended retention times in the tumour and high triplet lifetime of singlet oxygen species.

The combination of chemotherapy and photodynamic therapy was investigated by synthesis and characterization of octacarboxy phthalocyanine covalent conjugates with platinum complexes. This work presents the synthesis, characterization and photophysicochemical properties of aluminium (diaquaplatinum) octacarboxyphthalocyanine and aluminium (diammine) octacarboxyphthalocyanine. The conjugates were prepared by conjugating aluminium octacarboxy phthalocyanine with potassium tetrachloro platinate to yield aluminium tetrakis and trikis (diaquaplatinum) octacarboxy phthalocyanine. The aluminium octacarboxy phthalocyanine was also conjugated with cis-diamminedichloroplatinum to yield aluminium bis and tris (diaquaplatinum) octacarboxy phthalocyanine. From the characterization of the conjugates it was discovered that the aluminium (diaquaplatinum) octacarboxy phthalocyanine had formed platinum nanoparticles with the Pc acting as a capping agent. The triplet lifetimes decreased with the increasing number of platinum complexesconjugated to the Pc. The heavy atom effect improved the overall photophysicochemical properties.

Leung, Ching Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 101-110). / Abstracts in English and Chinese. / Acknowledgements --- p.i / 摘要（Abstract in Chinese) --- p.iii / Abstract --- p.v / List of Abbreviations --- p.vii / List of Figures and Tables --- p.ix / Table of Content --- p.xi / Chapter CHAPTER 1 --- Introduction / Chapter 1.1 --- History and development of photodynamic therapy --- p.1 / Chapter 1.2 --- Basic principle of photodynamic therapy: the beauty of the treatment --- p.3 / Chapter 1.3 --- "Photosensitizers: From discovery, synthesis to modifications" --- p.6 / Chapter 1.4 --- Enhancement of selective retention of PS in cancerous tissue --- p.10 / Chapter 1.5 --- Development of silicon (IV) phthalocyanine derivatives --- p.14 / Chapter 1.6 --- Death mechanisms in photodynamic therapy --- p.17 / Chapter 1.7 --- Objectives of the present study --- p.18 / Chapter CHAPTER 2 --- Materials and Methods / Chapter 2.1 --- Synthesis of BAM-SiPc --- p.20 / Chapter 2.2 --- Preparation of BAM-SiPc solution for photodynamic treatment --- p.20 / Chapter 2.3 --- Cell line and culture conditions --- p.21 / Chapter 2.4 --- Animal tumor model --- p.23 / Chapter 2.5 --- PDT laser source --- p.23 / Chapter 2.6 --- In vitro photodynamic activity assay --- p.23 / Chapter 2.6.1 --- Preparation of cells for photodynamic treatment / Chapter 2.6.2 --- In vitro photodynamic treatment / Chapter 2.6.3 --- Cell viability assay / Chapter 2.7 --- "Determination of reactive oxygen species production by 2',7'- dichlorofluorescein diacetate (DCFDA) assay" --- p.28 / Chapter 2.8 --- Analysis of cell cycle arrest --- p.28 / Chapter 2.9 --- Biodistribution of BAM-SiPc --- p.29 / Chapter 2.10 --- In vivo photodynamic treatment --- p.30 / Chapter 2.11 --- Assay for plasma enzyme activities --- p.30 / Chapter 2.12 --- Determination of cellular uptake of BAM-SiPc --- p.31 / Chapter 2.13 --- Metabolism of BAM-SiPc --- p.31 / Chapter 2.14 --- Histochemical staining --- p.32 / Chapter 2.14.1 --- Preparation of paraffin-embedded tissue section / Chapter 2.14.2 --- Haematoxylin and Eosin (H & E) staining / Chapter 2.14.3 --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay / Chapter 2.15 --- Conjugation of BAM-SiPc with LDL --- p.34 / Chapter 2.15.1 --- Analysis of the phototoxicity and cellular uptake of BAM- SiPc in the presence of LDL / Chapter 2.15.2 --- Gel filtration analysis of the mixture of LDL and BAM- SiPc / Chapter 2.16 --- Statistical analysis --- p.35 / Chapter CHAPTER 3 --- Results / Chapter 3.1 --- In vitro photodynamic activity assays --- p.36 / Chapter 3.2 --- Tissue distribution of BAM-SiPc in HepG2- bearing nude mice --- p.39 / Chapter 3.3 --- Anti-tumor activities of in vivo PDT with BAM-SiPc --- p.42 / Chapter 3.3.1 --- In vivo effect of PDT treatment with BAM-SiPc on HepG2 and HT29 tumor growth / Chapter 3.3.2 --- Dosage effect on anti-tumor activities by BAM-SiPc mediated PDT / Chapter 3.4 --- Analysis of intrinsic toxicity induced by BAM-SiPc mediated PDT --- p.48 / Chapter 3.4.1 --- H & E staining of liver sections of nude mice after in vivo PDT / Chapter 3.4.2 --- Plasma enzyme activity assays of PDT treated mice / Chapter 3.5 --- BAM-SiPc metabolism in in vitro culture cells and liver homogenate --- p.53 / Chapter 3.5.1 --- Cellular uptake of BAM-SiPc / Chapter 3.5.2 --- BAM-SiPc metabolism in cultured normal liver cells and cancer cells / Chapter 3.5.3 --- BAM-SiPc metabolism by mice liver homogenate / Chapter 3.6 --- Death mechanism induced by BAM-SiPc mediated PDT --- p.62 / Chapter 3.6.1 --- Events related to cell death induced by in vitro BAM-SiPc mediated PDT / Chapter 3.6.2 --- Death mechanism exerted by in vivo BAM-SiPc mediated PDT / Chapter 3.7 --- Effect on phototoxicity of BAM-SiPc in the presence of LDL --- p.70 / Chapter 3.7.1 --- Effect on phototoxicity of BAM-SiPc after mixing BAM- SiPc with LDL / Chapter 3.7.2 --- Gel filtration for analysis of the LDL-BAM-SiPc mixture / Chapter CHAPTER 4 --- Discussion / Chapter 4.1 --- Anti-cancer effect of BAM-SiPc on different cancer cell lines --- p.76 / Chapter 4.2 --- Tissue distribution of BAM-SiPc in HepG2 bearing nude mice --- p.77 / Chapter 4.3 --- In vivo effect of BAM-SiPc mediated PDT on HepG2 and HT29 tumor growth --- p.80 / Chapter 4.4 --- Analysis of the safety of using BAM-SiPc as a potential agent in PDT --- p.83 / Chapter 4.5 --- Metabolism of BAM-SiPc --- p.84 / Chapter 4.6 --- Mechanism of the apoptosis triggered by BAM-SiPc mediated PDT --- p.88 / Chapter 4.7 --- Death mechanism induced by in vivo PDT with BAM-SiPc --- p.93 / Chapter 4.8 --- Phototoxicity of BAM-SiPc in the presence of LDL --- p.94 / Chapter CHAPTER 5 --- Conclusion and Future perspective / Chapter 5.1 --- Conclusion --- p.97 / Chapter 5.2 --- Future perspective --- p.98 / References

This work reports on the synthesis, characterization and the physicochemical properties of novel unsymmetrically substituted zinc phthalocyanines: namely tris{11,19, 27-(1,2- diethylaminoethylthiol)-2-(captopril) phthalocyanine Zn ((ZnMCapPc (1.5)), hexakis{8,11,16,19,42,27-(octylthio)-1-(4-phenoxycarboxy) phthalocyanine} Zn (ZnMPCPc(1.7)) and Tris {11, 19, 27-(1,2-diethylaminoethylthiol)-1,2(caffeic acid) phthalocyanine} Zn ((ZnMCafPc (1.3)). Symmetrically substituted counterparts (tetrakis(diethylamino)zinc phthalocyaninato (3.8), octakis(octylthio)zinc phthalocyaninato (3.9) and tetrakis (carboxyphenoxy)zinc phthalocyaninato (3.10) complexes) were also synthesized for comparison of the photophysicochemical properties and to investigate the effect of the substituents on the low symmetry Pcs. The complexes were successfully characterized by IR, NMR, mass spectral and elemental analyses. All the complexes showed the ability to produce singlet oxygen, while the highest triplet quantum yields were obtained for 1.7, 1.5 and 3.9 (0.80, 0.65 and 0.62 respectively and the lowest were obtained for 1.3 and 3.10 (0.57 and 0.47 respectively). High triplet lifetimes (109-286 μs) were also obtained for all complexes, with 1.7 being the highest (286 μs) which also corresponds to its triplet and singlet quantum yields (0.80 and 0.77 respectively). The photosensitizing properties of low symmetry derivatives, ZnMCapPc and ZnMCafPc were investigated by conjugating glutathione (GSH) capped silver nanoparticles (AgNP). The formation of the amide bond was confirmed by IR and UV-Vis spectroscopies. The photophysicochemical behaviour of the novel phthalocyanine-GSH-AgNP conjugates and the simple mixture of the Ag NPs with low the symmetry phthalocyanines were investigated. It was observed that upon conjugation of the phthalocyanines to the GSH-AgNPs, a blue shift in the Q band was induced. The triplet lifetimes and quantum yields improved upon conjugation as compared to the phthalocyanines (Pc) alone. Complex 1.5 triplet lifetimes increased from 109 to 148 and triplet quantum yield from 0.65 to 0.86 upon conjugation. Fluorescence lifetimes and quantum yields decreased for the conjugates compared to the phthalocyanines alone, due to the quenching caused by the Ag NPs. The antimicrobial activity of the zinc phthalocyanines (complexes 1.3 and 1.5) and their conjugates against Escherichia coli was investigated. Only 1.3 and 1.5 complexes were investigated because of the availability of the sample. In general phthalocyanines showed increase in antibacterial activity with the increase in phthalocyanines concentration in the presence and absence of light. The Pc complexes and their Ag NP conjugates showed an increase in antibacterial activity, due to the synergistic effect afforded by Ag NP and Pcs. Improved antibacterial properties were obtained upon irradiation. 1.5-AgNPs had the highest antibacterial activity compared to 1.3-AgNPs conjugate; these results are in agreement with the photophysical behaviour. This work demonstrates improved photophysicochemical properties of low symm

According to the results, Pa-PDT showed inhibitory effect on MDA-MB-231 cells in vitro with an IC50 value of 0.5 muM at 24 h. Pa-PDT was demonstrated to activate intracellular mitogen activated protein kinases (MAPK) pathways via reactive oxygen species (ROS) production. Pa-PDT IS also believed to induce extracellular signal-regulated kinase (ERK)-mediated autophagy and endoplasmic reticulum stress. Pa-PDT in combination with Tamoxifen is demonstrated to exert a synergetic effect in inhibiting cancer growth. The combination treatment induces both intrinsic and extrinsic apoptosis. Regarding the direct cancer cell killing activity, two dimensional gel electrophoresis screening revealed that Pa-PDT regulates proteins which involve in human leukocyte antigen (HLA) class I-restricted antigen-processing machinery. This activation of antigen presentation was confirmed by Western blot analysis and immunostaining. Furthermore, a cross-presentation of antigen with HLA class I proteins and 70-kDa heat shock protein was found in Pa-PDT-treated cells, as shown by the fluorescent microscopic observation and immunoprecipitation assay. Moreover, the immunogenicity of breast cancer cells was increased by Pa-PDT treatment that triggered phagocytic activity by human macrophages. Our findings provide the first evidence that Pa-PDT can trigger both apoptosis and anti-tumour immunity. / Cancer is one of the most lethal diseases worldwide. Treatments of cancer comprise surgical intervention, radiotherapy or chemotherapy; however, their side effects are still need to be overcome. In order to search for anti-cancer treatments with milder side effects and higher efficiency, traditional Chinese medicine (TCM) has been investigated. Previous study in our laboratory reported that pheophorbide a (Pa), an active compound purified from Scutellaria barbata, combined with photodynamic therapy (PDT) approach produces anti-tumour effect in a wide range of human cancers. Because of the lack of protocols for curing late phase breast cancer, my project is to investigate the therapeutic potential of Pa-PDT and its action mechanism on human breast cancer. A human breast cancer cell line MDA-MB-231, which is estrogen receptor nude and resistant to a conventional breast cancer drug tamoxifen, was used as an in vitro tumour model in my study to mimic the late stage of breast cancer. / Pheophorbide a (Pa) has been proposed to be a potential photosensitizer for the photodynamic therapy of human cancer. However, the immunomodulatory effect of Pa, in the absence of irradiation, has not yet been investigated. The present study revealed that Pa possessed immunostimulating effect on a murine macrophages cell line RAW 264.7. Pa could stimulate the growth of RAW 264.7 cells with the maximal effect at 0.5 muM after 48 h of treatment, where MAPK family including c-Jun N-tenninal kinase (JNK), ERK and p38 MAPK were activated by Pa treatment in a dose-dependent manner. Moreover, the induction of interleukin-6 and tumour necrosis factor-a secretion, and the enhancement of phagocytic activity were observed in Pa-treated RAW 264.7 cells. The results were similar in Pa-treated human immune competent cells (e.g. CD4+ and CD14+ cells) at higher Pa concentrations (from 1 to 10 muM). The present work is the first report to demonstrate the potential immunomodulatory effects of Pa on immune competent cells, apart from its well-known anti-tumour activity. / Bui Xuan, Ngoc Ha. / "December 2010." / Advisers: Fung Kwok Pui; Wong Chun Kwok. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.

Hoi, Wan Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 212-245). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.