ALA-aracılı fotodinamik terapi ve genlerin özgün polimerler ile taşınması

Cancer is a challenging and complex disease to treat. Conventional therapeutic approaches, namely chemotherapy, radiotherapy and surgery, has limited success. Alternative treatment methods such as local phototherapies and gene therapy are highly promising in the treatment of different cancers as a single treatment modality or combination with conventional methods. In this thesis, in vitro studies for photodynamic therapy and its combination with chemotherapy for the treatment of colorectal cancer and bone targeting polyplexes made by novel copolymers for gene-therapy of osteosarcoma are described. Photodynamic therapy (PDT) is a local, approved and promising approach in the treatment of cancer and bacterial infections. It induces tumor cell death creating reactive oxygen species (ROS), such as singlet oxygen, by photosensitizer excitation at a specific wavelength. It can be applied alone or in combination with other therapeutic techniques such as photothermal therapy (PTT) or chemotherapy for the treatment of colorectal, head, neck and breast cancer. Combination of chemotherapy and photodynamic therapy enhances therapeutic efficiency overcoming multidrug resistance (MDR) at low drug doses. 5-Aminolevulinic acid (ALA), a well-known and FDA approved photosensitizer, is a promising prodrug with the strong fluorescence for diagnosis and fast accumulation in the tumor tissue. ALA is produced in mitochondria as a rate-limited step of the heme biosynthesis pathway and converted to protoporphyrin IX (PpIX), which is a photosensitizer. For efficient PDT, exogenous ALA administration is needed to increase the PpIX concentration to therapeutic levels in the tumor cells. In the first chapter of this thesis, ALA-mediated PDT and its combination with 5FU in the treatment of colorectal cancer is (HCT116 cells) described. Colorectal cancer is the 3rd most diagnosed tumor type and difficult to treat. Dose dependence of PDT, irradiation protocol and suitable cytotoxicity assays were first determined in 2D cell cultures and then applied to 3D early and late tumor models that are more realistic models to predict the efficiency of the treatment. Alamar Blue was determined as the more appropriate method to evaluate in vitro toxicity, 100 μM ALA concentration and 10 min irradiation with blue lamp were found as an effective treatment for effective PDT in 2D and 3D. Besides, PpIX accumulation was significantly increased with an increase in the complexity of cell structures from 2D to late 3D cultures. As a result, ALA-mediated PDT causing apoptotic/necrotic death of HCT116 colorectal cancer cells in 3D tumor models was demonstrated. It was also noticed that ALA to PpIX conversion is hindered with the co-existence of 5FU; therefore, the 1/0.15 mol ratio of ALA/5FU was suggested as a promising recipe for combination therapy. Osteosarcoma is aggressive bone cancer, whose treatment has not changed significantly for the past few decades. Although gene therapy has emerged as a potential treatment route, the need for efficient and non-toxic gene delivery systems targeting osteosarcoma cells remains a challenge. High molecular weight poly (ethyleneimine)s (PEIs), are used as universal transfection agents, however, cause significant cytotoxicity. On the other hand, poly (amidoamine)s (PAAs) are biocompatible, biodegradable Polymers with promising transfection efficiency. In the second chapter, in vitro transfection of osteosarcoma cells with PEI-PAA copolymers with (bis)phosphonic acid groups with the potential to target bone is discussed. A set of novel polymers (PAEI) comprised of low molecular weight branched PEI (Mw = 1800 Da) and PAA macromers which were functionalized in different ratios with 5-amino-1-pentanol (AP) and (bis)phosphonic acid groups were studied. The PAEI polymers synthesized by the group of Prof. Duygu Avcı Semiz were tested on an osteosarcoma cell line (U-2 OS cells), which is one of the hardest cell types to transfect, and on muscle cells (C2C12 cells) as the control. The cytotoxicity and transfection efficiency of PAEIs were adjusted by altering the ratios of phosphonic acid (via APA, aminophosphonic acid) or bisphosphonic acid (via ALE, an amino bisphosphonic acid) content. The highest transfection efficiency was obtained with ALE containing PAEIs. The presence of PAA, PEI and ALE seemed to play a synergistic effect in the transfection performance of PAEIs. The most efficient PAEI polymer, containing a 0.7: 0.3 AP: ALE ratio, displayed a transfection efficiency that was 5-times higher than 25 kDa bPEI. This novel set of polymers can be promising candidates for the gene therapy of osteosarcoma.