A. Lale Doğan

Designing excited states: theory-guided access to efficient photosensitizers for photodynamic action.

Photodynamic therapy (PDT) is a treatment modality for certain malignant (skin, head and neck, gastrointestinal, gynecological cancers), premalignant (actinic keratosis), and nonmalignant (psoriasis) indications. Broader acceptance by the medical community and applicability is hampered, at least in part, by the less than optimal photophysical characteristics of the porphyrin derivatives. This situation sparked a worldwide search for novel sensitizers leading to new compounds, some holding more promise than others. The primary cytotoxic agent involved in the photodynamic action is singlet oxygen (Dg), the efficient generation of which is linked invariably to the intersystem crossing (ISC) efficiency of the excited sensitizer. Most organic dyes have low triplet quantum yields, and in many recent candidates for photodynamic sensitizers, heavy atoms are incorporated into the structure as a strategy to improve spin–orbit coupling leading to facilitated intersystem crossing. While this approach seems fail-safe, incorporation of heavy atoms such as bromine, iodine, selenium, and certain lanthanides very often leads to increased “dark toxicity”. Unlike traditional chemotherapy agents, in principle, photodynamic therapy sensitizers themselves can be nontoxic, either at cellular or organ levels, even at relatively high concentrations. We have been interested in trying to find alternative ways of achieving increased intersystem crossing without the use of heavy atoms to minimize dark toxicity, turning our attention to the excitedstate properties of the sensitizers. Designing efficient photoinduced O2 generators requires that any existing operative fluorescence cycle of the fluorophore, which is through the S0!S1!S0 states, has to be perturbed so as to minimize or shut down the S1!S0 deactivation, and switch to the triplet surface once S1 is accessed. A general design principle for a favorable S1!T1 transition from an electronic structure viewpoint would in principle require the structural and electronic compatibility of the S1 and T1 states to surpass that of the S1–S0 pair. Once multiple electronic states come into play, quantum mechanical calculations providing a detailed understanding of the electronic structure are extremely helpful. Multi-configurational self-consistent field (MCSCF) techniques are the stateof-the-art computational chemistry approaches, when near degeneracies and excited states are considered. These methods may not reach chemical accuracy ( 2–3 kcalmol ) for computing total energies, but they are crucial for a qualitatively correct description of the excited states and are capable of providing a conceptually complete picture of the photophysics taking place. Therefore, we mainly employed a popular variant of MCSCF techniques; the complete active space SCF (CASSCF) method in combination with relatively large basis sets and different active spaces. Details of CASSCF calculations are provided in the Supporting Information. Our calculations on the parent Bodipy (4,4-difluoro-4-bora-3a,4adiaza-s-indacene, Scheme 1) showed that natural orbital occupancies of the S1 state describe an open-shell singlet with essentially double (> 1.9) or zero (< 0.1) electrons for all orbitals except the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) that are singly occupied (see the Supporting Information, Figure S1). It is no surprise to observe a fluorophore with low triplet quantum yield to have an excited state that possesses only two orbitals with single occupancy. Hence, to achieve our goal of efficient switching to the triplet manifold, we have to access excited states that differ from the ones that arise from simple HOMO!LUMO transitions. Among multiply excited configurations, doubly substituted ones are particularly important in enhancing S1–T1 coupling as shown by the seminal work of Salem and Rowland and the following work by Michl. Thus, the substitutions should invoke a simultaneous two-electron excitation from the Scheme 1. Structure and numbering of the parent Bodipy compound. [*] Y. Cakmak, S. Kolemen, B. Kilic, Prof. Dr. E. U. Akkaya UNAM-Institute of Materials Science and Nanotechnology Bilkent University, Ankara, 06800 (Turkey) E-mail: eua@fen.bilkent.edu.tr

Intravesical cationic nanoparticles of chitosan and polycaprolactone for the delivery of Mitomycin C to bladder tumors

Cationic nanoparticles of chitosan (CS), poly-epsilon-caprolactone coated with chitosan (CS-PCL) and poly-epsilon-caprolactone coated with poly-L-lysine (PLL-PCL) were developed to encapsulate intravesical chemotherapeutic agent Mitomycin C (MMC) for longer residence time, higher local drug concentration and prevention of drug loss during bladder discharge. Nanoparticle diameters varied between 180 and 340 nm depending on polymer used for preparation and coating. Zeta potential values demonstrated positive charge expected from cationic nanoparticles. MMC encapsulation efficiency depended on hydrophilicity of polymers since MMC is water-soluble. Encapsulation was increased by 2-fold for CS-PCL and 3-fold for PLL-PCL as a consequence of hydrophilic coating. Complete drug release was obtained with only CS-PCL nanoparticles. On the other hand, CS and PLL-PCL nanoparticles did not completely liberate MMC due to strong polymer-drug interactions which were elucidated with DSC studies. As far as cellular interaction was concerned, CS-PCL was the most efficient formulation for uptake of fluorescent markers Nile Red and Rhodamine123 incorporated into nanoparticles. Especially, CS-PCL nanoparticles loaded with Rhodamine123 sharing hydrophilic properties with MMC were selectively incorporated by bladder cancer cell line, but not by normal bladder epithelial cells. CS-PCL nanoparticles seem to be promising for MMC delivery with respect to anticancer efficacy tested against MB49 bladder carcinoma cell line.

Comparative evaluation of polymeric and amphiphilic cyclodextrin nanoparticles for effective camptothecin delivery

Camptothecin (CPT) is a potent anticancer agent. The clinical application of CPT is restricted by poor water solubility and instability under physiological conditions. Solubilization and stabilization of CPT were realized through nanoparticulate systems of amphiphilic cyclodextrins, poly(lactide-co-glycolide) (PLGA) or poly-epsilon-caprolactone (PCL). Nanoparticles were prepared with nanoprecipitation technique, whereas cyclodextrin nanoparticles were prepared from preformed inclusion complexes of CPT with amphiphilic cyclodextrins. Polymeric nanoparticles, on the other hand, were loaded with CPT:HP-beta-CD inclusion complex to solubilize and stabilize the drug. Mean particle sizes were under 275 nm, and polydispersity indices were lower than 0.2 for all formulations. Drug-loading values were significantly higher for amphiphilic cyclodextrin nanoparticles when compared with those for PLGA and PCL nanoparticles. Nanoparticle formulations showed a significant controlled release profile extended up to 12 days for amphiphilic cyclodextrin nanoparticles and 48h for polymeric nanoparticles. Anticancer efficacy of the nanoparticles was evaluated in comparison with CPT solution in dimethyl sulfoxide (DMSO) on MCF-7 breast adenocarcinoma cells. Amphiphilic cyclodextrin nanoparticles showed higher anticancer efficacy than PLGA or PCL nanoparticles loaded with CPT and the CPT solution in DMSO. These results indicated that CPT-loaded amphiphilic cyclodextrin nanoparticles might provide a promising carrier system for the effective delivery of this anticancer drug having bioavailability problems.

Evaluation of the cytotoxicity of calcium phosphate root canal sealers by MTT assay.

The purpose of this study was to evaluate the cytotoxicity of some calcium phosphate-based sealers (Sankin apatite root canal sealers (SARCS) types 1 to 3) in comparison with currently used sealers (CRCS, Ketac Endo, AH26, and Endomethasone) by using MTT assay on L929 cells. Monolayer cell cultures were prepared on 96-well plates. After incubation at 37 degrees C in a humidified 5% CO2-containing air atmosphere for 24 h in the presence of each sealer extracts, 25 microliters of 5 mg/ml of MTT in saline were added into each well and incubated a further 3 h at 37 degrees C. A solubilization buffer consisting of 23% sodium dodecyl sulfate in 50% N,N-dimethylformamide (pH 4.7) was used to dissolve formazan precipitate. The optical densities of the plates were then read by a microplate spectrophotometer at 570 nm. Greater magnitude of optical density due to intense blue coloring is regarded as showing a higher percentage of cell viability. Among the different types of sealers, SARCS types 1 to 3 and CRCS did not exert any cytotoxic effects, whereas AH26, Ketac Endo, and Endomethasone produced some cytotoxicity.

Preparation and in vitro evaluation of meloxicam-loaded PLGA nanoparticles on HT-29 human colon adenocarcinoma cells

Context: The inhibitors of cyclooxygenase (COX)-2 play an important role in cancer chemoprevention. Certain COX-2 inhibitors exert antiproliferative and pro-apoptotic effects on cancer cells. Objective: In this study, meloxicam, which is an enolic acid-type preferential COX-2 inhibitor, was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) to maintain local high concentration, and its efficacy was determined. Methods: NPs were prepared by using salting-out and emulsion-evaporation steps. Meloxicam-loaded NP formulations were evaluated with respect to the drug loading, particle size, polydispersity index, zeta potential, drug release rate, and residual poly(vinyl alcohol) (PVA) percentage. The effects of PLGA and PVA molecular weight variations on the physicochemical properties of NPs were investigated. Stability of meloxicam in NPs was assessed over 3 months. COX-2 expressing human colon adenocarcinoma cell line HT-29 was used in cellular uptake and viability assays. Results: NPs had a spherical shape and a negative zeta potential, and their size ranged between 170–231 nm with a lower polydispersity index. NPs prepared with high molecular weight PLGA were shown to be physically stable over three months at 4°C. The increase in molecular weight of the polymer and emulsifier reduced the in vitro release rate of meloxicam from NPs. Meloxicam-loaded NPs showed cytotoxic effects on HT-29 cells markedly at 800 µM. Cancer cells had high uptake of coumarin-6-loaded NPs. Conclusion: The PLGA NPs developed in this study can be a potentially effective drug delivery system of meloxicam for the treatment of colon cancer.

Effect of fludarabine on leukocyte functions.

Background: Fludarabine induces leukemic cell apoptosis and is highly efficient in chronic lymphocytic leukemia. However, fludarabine therapy causes severe leukopenia. Leukocyte myeloperoxidase (MPO) catalyzes the formation of HOCl, and this is the main microbicidal function in phagocytes. The aim of our study was to evaluate the effect of fludarabine on leukocytes, i.e. their degranulation capacity, MPO activity and HOCl production. Methods: Peripheral blood leukocytes were incubated for 48 h with fludarabine. Degranulation was measured using a flow-cytometric method. MPO activity and HOCl production were measured spectrophotometrically. Results: The degranulation capacity of fludarabine-treated leukocytes was significantly elevated compared to untreated controls. MPO activity and HOCl production were also increased in parallel. A possible direct activating effect of fludarabine was tested on the MPO activity of HL60 cells. Fludarabine did not affect MPO activity at concentrations ranging from 10 µM to 2 mM. Conclusion: Fludarabine had no inhibitory effect on the microbial killing of leukocytes.

Nanosized multifunctional liposomes for tumor diagnosis and molecular imaging by SPECT/CT

Among currently used cancer imaging methods, nuclear medicine modalities provide metabolic information, whereas modalities in radiology provide anatomical information. However, different modalities, having different acquisition times in separate machines, decrease the specificity and accuracy of images. To solve this problem, hybrid imaging modalities were developed as a new era, especially in the cancer imaging field. With widespread usage of hybrid imaging modalities, specific contrast agents are essentially needed to use in both modalities, such as single-photon emission computed tomography/computed tomography (SPECT/CT). Liposomes are one of the most desirable drug delivery systems, depending on their suitable properties. The aim of this study was to develop a liposomal contrast agent for the diagnosis and molecular imaging of tumor by SPECT/CT. Liposomes were prepared nanosized, coated with polyethylene glycol to obtain long blood circulation, and modified with monoclonal antibody 2C5 for specific tumor targeting. Although DTPA-PE and DTPA-PLL-NGPE (polychelating amphilic polymers; PAPs) were loaded onto liposomes for stable radiolabeling for SPECT imaging, iopromide was encapsulated into liposomes for CT imaging. Liposomes [(DPPC:PEG2000-PE:Chol:DTPA-PE), (PL 90G:PEG2000-PE:Chol:DTPA-PE), (DPPC:PEG2000-PE:Chol:PAPs), (PL 90G:PEG2000-PE:Chol:PAPs), (60:0.9:39:0.1% mol ratio)] were characterized in terms of entrapment efficiency, particle size, physical stability, and release kinetics. Additionally, in vitro cell-binding studies were carried out on two tumor cell lines (MCF-7 and EL 4) by counting radioactivity. Tumor-specific antibody-modified liposomes were found to be effective multimodal contrast agents by designating almost 3–8 fold more uptake than nonmodified ones in different tumor cell lines. These results could be considered as an important step in the development of tumor-targeted SPECT/CT contrast agents for cancer imaging.

Injectable biodegradable polymeric system for preserving the active form and delayed-release of camptothecin anticancer drugs

One of the most challenging problems for camptothecin (CPT) family anticancer drugs (i.e. topotecan (TPT)) is the conversion of the active lactone ring into an inactive toxic carboxylate form under physiological conditions (pH = 7.4) in the body. Therefore, a simple platform based on thermosensitive PLLA-mPEG gels was designed to maintain TPT and CPT in lactone form, especially for brain tumor therapies. A high stabilization of the lactone species CPT and TPT within gel (>95%), efficient versatile homogenous drug loadings at 0.015%, 1%, and 10%, and the sustained-release of CPT and TPT over three weeks were all successful. The stabilization mechanism of drugs with gel was elucidated by ATR-FTIR, confocal and light microscopy. The cytotoxic efficacy of TPT in the PLLA-mPEG platform (PLLA-mPEG-TPT) was evaluated on LLC-1 and 4T1 cancer cell lines. In vivo, the administration of PLLA-mPEG-TPT to mice with breast tumors resulted in a significant reduction in tumor size and better survival percentages.

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia.

Biology and Genetics of Breast Cancer

Breast cancer is the most common cancer among women, and it has a complex genetic basis for susceptibility. Understanding the mechanisms of DNA alterations leading to carcinogenesis can provide crucial insights for resolving the development of malignant processes such as growth, invasion, and metastasis. This chapter reviews hereditary and somatic genetic alterations, epigenetic misregulations, and miRNA signatures associated with breast cancer. The chapter also emphasizes the molecular profiles of breast cancer and the critical signaling pathway alterations.