미토콘드리아 및 DNA 타겟 광역동 치료를 위한 술폰산벤조티오핀 포피린 합성

학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과 약품분석학전공, 2016. 2. 송준명.Porphyrins are rich in applications and it can be called as the colors of life. From the photosynthetic process in plants, to the red hemoglobin that carries oxygen to the cells in our bodies utilize porphyrin molecules. Thus, without any surprise porphyrins are actively explored as tools for wider applications to improve medicine and other biomedical fields. With the aim of improving photodynamic therapy (PDT) in mind, this thesis work examines new synthetic porphyrins for selective targeting of sub-cellular compartments in cancerous cells and execute apoptotic cell death upon PDT, and causing minimal destruction and irritation to normal tissue. PDT is a phototherapeutic cancer therapy in which a photosensitizer (PS)—light activated drug—absorbs light of specific wavelength and excites to the singlet state. From the excited singlet state, PSs can undergo an internal transition to the excited triplet state, a relatively long-lived and high-energy species that transfers its excess energy to molecular oxygen. Subsequently, molecular oxygen excites from the stable triplet state to the highly reactive singlet state. With no spin-state restriction, singlet oxygen is cytotoxic, readily reacting with electron-rich biomolecules such as unsaturated lipids, amino acids and DNA consequently destroying the tumor cell. Singlet oxygen has a limited range of diffusion. Therefore, the site of its generation is also the site of initial damage. Photosensitizers used in PDT are often classified as first, second and third generation PS. This classification is based on the historical development and conceptual approaches. Haematoporphyrin derivative (HpD) or Photofrin are otherwise called as first generation PS enjoyed its success in cancer therapy due to their tumor localizing and photophysical properties yet suffers from its drawbacks (mixture of compounds and less selectivity). Herein, second generation PSs with high purity and improved photophysical properties had been developed. Still there is always room for improver, and current research efforts are aimed at developing third generation PSs where additional biological criteria such as selective targeting of sub-cellular organelles are considered in the design principle. Herein, this thesis work focused in developing third generation PSs that can target selectively sub-cellular organelles such mitochondria and DNA and effectively induce apoptotic cell death after PDT. In Chapter I, a comprehensive background on history of photodynamic therapy, fundamental aspects, classification and requirements of PSs is presented. In chapter 2, synthesis, structural elucidation, photophysical properties and in vitro photokilling and mechanism of cell death of 5, 10, 15, 20–Tetrakis(7-sulfonatobenzo[b]thiophene)porphyrin (SBTP) is studied. Keywords: porphyrinphotosensitizersphotodynamic therapymitochondriaDNAapoptosisintrinsicextrinsichigh-content assay. Student number: 2010-31366Chapter I Introduction 1 1.0. Cancer 1 1.0.1. Causes of cancer 2 1.0.2. Cancer prevention 2 1.0.3. Cancer Treatments 3 1.2. Photodynamic Therapy 3 1.2.1. History of Photodynamic therapy 3 1.2.1. Basic principles of PDT 7 1.2.2. Mechanisms of tumour destruction 9 1.3. Sensitizers 10 1.3.1. Porphyrinoid photosensitizers 14 1.4. Classification of photosensitizers 17 1.4.1. First Generation Photosensitizers 18 1.4.2. The Second Generation of Photosensitizers 19 1.4.3. The Third Generation Photosensitizers 22 References 25 Chapter II Synthesis and Photodynamic Studies of Novel meso-substituted Benzo[b]thiophene Porphyrins 31 Abstract 31 2.0. Introduction 33 2.1. Results 37 2.1.1. Synthesis and structural characterization of 5, 10, 15, 20-Tetrakis(Benzo[b]thiophene)porphyrin (BTP) 38 2.1.2. Synthesis and structural characterization of 5, 10, 15, 20–Tetrakis(7-sulfonatobenzo[b]thiophene)porphyrin (SBTP) 41 2.1.3. Photophysical properties of BTP and SBTP 45 2.1.4. Cytotoxicity and photocytotoxicity of SBTP 49 2.1.5. Subcellular localization of SBTP 51 2.1.6. Cellular uptake property of SBTP 53 2.1.7. Intracellular ROS generation of SBTP under photodynamic action 55 2.1.8. High-content cell death dynamics 57 2.1.9. Studies on intrinsic and extrinsic apoptotic pathways 60 2.1.10. DNA fragmentation assay 62 2.2.0. Discussion 65 2.3.0. Conclusions 72 2.4.0. Materials and methods 73 2.4.1. General methods 73 2.4.2. Synthesis of 5, 10, 15, 20 – Tetrakis(benzo[b]thiophene) porphyrin (BTP) 75 2.4.3. Synthesis of 5, 10, 15, 20 – Tetrakis (3-sulfonatobenzo[b]thiophene) porphyrin (SBTP) 76 2.4.4. MTT assay 77 2.4.5. Intracellular uptake of SBTP by MCF-7 cells 78 2.4.6. Intracellular localization assay 78 2.4.7. Intracellular ROS generation 79 2.4.8. High-content screening assay 79 2.4.9. Intrinsic/extrinsic apoptotic pathway studies 80 2.5.0. DNA fragmentation assay 81 References 82Docto

The Application of Bactericidal Silver Nanoparticles in Wound Treatment

Even with the prevalence of wounds, the medical technol‐ ogy for efficiently managing skin damage is still primitive. The disruption of any of the numerous healing processes can lead to problems in the time-sensitive healing actions of the dermal and epidermal layers. Bacterial infection is one of the major obstacles to proper wound healing as it poses a danger of causing long-term negative effects. Keeping the wound free of bacteria is imperative to the proper and hasty repair of dermal wounds. Silver has been widely used to treat wounds for its bactericidal properties. Although the mechanism of silver’s antibacterial action is not fully understood, it exhibits a significant antimicrobial efficacy against a wide spectrum of bacterial species. A number of different approaches to the mechanism are reported and presented in this review. Silver nanoparticles (AgNPs) have been reported to exhibit enhanced antibac‐ terial activity due to their increased surface-area-to-volume ratio. AgNPs are capable of various modifications, signifi‐ cantly broadening the therapeutic properties of the mate‐ rial as a result. This review explores the different aspects of silver and silver nanoparticles, and their antibacterial properties, which can be applied in the field of wound treatments

Mitochondria and DNA Targeting of 5,10,15,20-Tetrakis(7-sulfonatobenzo[b]thiophene) Porphyrin-Induced Photodynamic Therapy via Intrinsic and Extrinsic Apoptotic Cell Death

Photodynamic therapy (PDT) selectively targets subcellular organelles and promises an excellent therapeutic strategy for cancer treatment. Here, we report the synthesis of a new water-soluble photosensitizer, 5,10,15,20-tetralds (7-sulfonatobenzo[b]-thiophene) porphyrin (SBTP). Rational design of the porphyrinic molecule containing benzo[b]thiophene moiety at the meso-position led to selective accumulation in both mitochondria and nucleus of MCF-7 cells. This multitarget ability of SBTP can cause damage to mitochondria as well as DNA simultaneously. FACS analysis showed rapid cellular uptake of SBTP. High-content cell-based assay was executed to concurrently monitor increase of cytosolic Ca2+ levels, mitochondrial permeability transition (MPT), and caspase-3/7/8 activation in MCF-7 cells under the pathological condition caused by PDT action of SBTP. The study of cell death dynamics showed that PDT action of SBTP caused an increase in the MPT followed by an increase in cytosolic Ca2+ level. The localization of SBTP in the mitochondria activated the intrinsic apoptotic pathway. Additionally, localization of SBTP in the nucleus led to DNA damage in MCF-7 cells. The DNA fragmentation that occurred by PDT action of SBTP was thought to be responsible for extrinsic apoptosis of MCF-7 cells. SBTP demonstrated effective PDT activity of 5 mu M IC50 value to MCF-7 cells by bitargeting mitochondria and DNA.N

Mitochondria and DNA Targeting of 5,10,15,20-Tetrakis(7-sulfonatobenzo[<i>b</i>]thiophene) Porphyrin-Induced Photodynamic Therapy via Intrinsic and Extrinsic Apoptotic Cell Death

Photodynamic therapy (PDT) selectively targets subcellular organelles and promises an excellent therapeutic strategy for cancer treatment. Here, we report the synthesis of a new water-soluble photosensitizer, 5,10,15,20-tetrakis (7-sulfonatobenzo­[<i>b</i>]­thiophene) porphyrin (SBTP). Rational design of the porphyrinic molecule containing benzo­[<i>b</i>]­thiophene moiety at the <i>meso</i>-position led to selective accumulation in both mitochondria and nucleus of MCF-7 cells. This multitarget ability of SBTP can cause damage to mitochondria as well as DNA simultaneously. FACS analysis showed rapid cellular uptake of SBTP. High-content cell-based assay was executed to concurrently monitor increase of cytosolic Ca²⁺ levels, mitochondrial permeability transition (MPT), and caspase-3/7/8 activation in MCF-7 cells under the pathological condition caused by PDT action of SBTP. The study of cell death dynamics showed that PDT action of SBTP caused an increase in the MPT followed by an increase in cytosolic Ca²⁺ level. The localization of SBTP in the mitochondria activated the intrinsic apoptotic pathway. Additionally, localization of SBTP in the nucleus led to DNA damage in MCF-7 cells. The DNA fragmentation that occurred by PDT action of SBTP was thought to be responsible for extrinsic apoptosis of MCF-7 cells. SBTP demonstrated effective PDT activity of 5 μM IC₅₀ value to MCF-7 cells by bitargeting mitochondria and DNA

Antibacterial Photodynamic Therapy in the Near-Infrared Region with a Targeting Antimicrobial Peptide Connected to a π-Extended Porphyrin

The increase of antimicrobial resistance to conventional antibiotics is worldwide a major health problem that requires the development of new bactericidal strategies. Antimicrobial photodynamic therapy (a-PDT) that generates reactive oxygen species acting on multiple cellular targets is unlikely to induce bacterial resistance. This localized treatment requires, for safe and efficient treatment of nonsuperficial infections, a targeting photosensitizer excited in the near IR. To this end, a new conjugate consisting of an antimicrobial peptide linked to a π-extended porphyrin photosensitizer was designed for a-PDT. Upon irradiation at 720 nm, the conjugate has shown at micromolar concentration strong bactericidal action on both Gram-positive and Gram-negative bacteria. Moreover, this conjugate allows one to reach a low minimum bactericidal concentration with near IR excitation without inducing toxicity to skin cells. Keywords: antimicrobial peptide; antimicrobial photodynamic therapy; bacteria; keratinocyte; near infrared; porphyrin