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Coumarinyl-Caged Ceramides, a New Tool for Assessing the Biological Effects of Ceramide In CellsDay, Jenna January 2015 (has links)
Ceramide, a sphingolipid, is an important lipid second messenger that is involved in regulating a number of cellular processes, including programmed cell death, cell growth and differentiation, as well as cellular responses to stress stimuli. Many of the biological effects of ceramide are linked to its ability to modulate the biophysical properties of membranes and cause clustering of signalling molecules in ceramide-rich domains, which allows for more efficient signal transmission in the cell. However, the specific roles of different ceramide species in these signaling pathways have yet to be clearly established. Assessing the effects of long N-acyl chain ceramides in cells involves some limitations due to their poor solubility and their low membrane permeability. Caging these molecules with photolabile protecting groups allows for their delivery into cells where photochemical uncaging of the biologically active compound can be achieved with spatial and temporal control.
A series of coumarinyl-caged ceramides has been prepared in order to probe the biological effects of ceramide in cells. This unique series of compounds was used to investigate the dependence of these cellular effects on N-acyl chain length. Hereafter, I describe the photophysical and photochemical characterization of these novel caged ceramides, assess their uptake and measure the biological effects of the different ceramides which are generated photochemically in HeLa cells. The caged ceramides were shown to be taken up by the cells and to cause a decrease in viability, with UV irradiation, that can be detected after 24 hours of treatment. An investigation of the mechanism of cell death induced by coumarinyl-caged ceramides in HeLa cells revealed that cell death proceeds in a caspase-independent manner and involves the mitochondria. The role of the mitochondria in this cell death pathway, however, remains to be studied further. RIP1 kinase activity, which was also probed in the cells, was determined to not be implicated in cell death caused by photochemically generated ceramide. Intracellular ROS generation, however, was shown to occur in this system, but results primarily from UV irradiation of the free coumarin. Overall, the results from this study have provided insight into the signalling pathways triggered by treatment of HeLa cells with the bioactive lipid ceramide using coumarin photocages.
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Design of Photocage Ligands for Light-Activated Changes in Coordination of d-block Transition MetalsCiesienski, Katie Lynnann January 2010 (has links)
<p>The concept of light-activated "caged" metal ions was first introduced for Ca2+. These high affinity coordination complexes are activated by UV light to release calcium ions intracellularly and have found widespread use in understanding the many roles of calcium in biological processes. There is an unmet need for photocaging ligands for biologically relevant transition metal ions. Described here are the first examples of uncaging biologically important d-block metal ions using photoactive ligands. </p>
<p>New nitrogen-donor ligands that contain a photoactive nitrophenyl group within the backbone have been prepared and evaluated for their metal binding affinity. Exposure of buffered aqueous solutions of apo-cage or metal-bound cage to UV light induces cleavage of the ligand backbone reducing the denticity of the ligands. Characterization of several caging compounds reveals that quantum efficiency and metal binding affinity can be tuned by modifications to the parent structure. The change in reactivity of caged vs. uncaged metal for promoting hydroxyl radical formation was demonstrated using the in vitro deoxyribose assay. The function of several of these compounds in vivo pre- and post-photolysis has been validated using MCF-7 cells. This strategy of caging transition metals ions is promising for applications where light can trigger the release of metal ions intracellularly to study metal trafficking and distribution, as well as, selectively impose oxidative stress and/or metal toxicity on malignant cells causing their demise.</p> / Dissertation
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Light Mediated Drug Delivery Using Photocaged Molecules and Photoswitchable PeptidesMitra, Deboleena 01 January 2014 (has links)
There are many different types of targeted therapy for cancer treatment. The method of light mediated targeted therapy that we have developed uses photocaged molecules and photoswitchable peptides.
In photocaging, a biologically active molecule is made inactive by the attachment of a photocleavable blocking group. On exposure to UV radiation the photocleavable entity is removed and the biologically active molecule is released. Using this concept we have designed a prodrug that consists of a cell impermeable hydrophilic molecule attached to a photocaged doxorubicin. Upon irradiation with UV light the photosensitive group is removed and cytotoxic doxorubicin is released at the tumor site. This concept has been further modified by attaching receptor binding molecules to the photocaged entity to increase its specificity.
A peptide which consists of an azobenzene photoswitch has been used which, in the dark state is randomly coiled and cell impermeable but upon illumination becomes helical and cell permeable and can be used to deliver drugs into the cells. Upon illumination with UV light of suitable wavelength the azobenzene linker will change from a trans to a cis form and this will convert the randomly coiled cell impermeable peptide into an α helical permeable form. Thus a series of peptides have been designed with different arginine mutations which develop an arginine patch in the helical form. This arginine patch would help in cell permeability by interacting with cell surface glycans. The method could potentially be used to deliver drugs into cells in presence of light.
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Development of a Dynamic Cell Patterning Strategy on a Hyaluronic Acid HydrogelGoubko, Catherine A. 15 January 2014 (has links)
Cell behavior is influenced to a large extent by the surrounding microenvironment. Therefore, in the body, the cellular microenvironment is highly controlled with cells growing within well-defined tissue architectures. However, traditional culture techniques allow only for the random placement of cells onto culture dishes and biomaterials. Cell micropatterning strategies aim to control the spatial localization of cells on their underlying material and in relation to other cells. Developing such strategies provides us with tools necessary to eventually fabricate the highly-controlled microenvironments found in multicellular organisms. Employing natural extracellular matrix (ECM) materials in patterning techniques can increase biocompatibility. In the future, with such technologies, we can hope to conduct novel studies in cell biology or optimize cell behavior and function towards the development of new cell-based devices and tissue engineering constructs.
Herein, a novel cell patterning platform was developed on a hydrogel base of crosslinked hyaluronic acid (HA). Hydrogels are often employed in tissue engineering due to their ability to mimic the physicochemical properties of natural tissues. HA is a polymer present in all connective tissues. Cell-adhesive regions on the hydrogel were created using the RGDS peptide sequence, found within the cell-adhesive ECM protein, fibronectin. The peptide was bound to a 2-nitrobenzyl “caging group” via a photolabile bond to render the peptide light-responsive. Finally, this “caged” peptide was covalently bound to the hydrogel to form a novel HA hydrogel with a cell non-adhesive surface which could be activated with near-UV light to become adhesive. In this way, we successfully formed chemically patterned cell-adhesive regions on a HA hydrogel using light as a stimulus to form controlled cell patterns.
While the majority of cell patterning strategies to date are limited to patterning one cell population and cannot be changed with time, our strategy was novel in using small, adhesive, caged peptides combined with multiple, aligned light exposure steps to allow for dynamic chemical cell patterning on a hydrogel. Multiple cell populations, even held apart from one another, were successfully patterned on the same hydrogel. Furthermore, cell patterns were deliberately modified with time to direct cell growth and/or migration on the hydrogel base.
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Development of a Dynamic Cell Patterning Strategy on a Hyaluronic Acid HydrogelGoubko, Catherine A. January 2014 (has links)
Cell behavior is influenced to a large extent by the surrounding microenvironment. Therefore, in the body, the cellular microenvironment is highly controlled with cells growing within well-defined tissue architectures. However, traditional culture techniques allow only for the random placement of cells onto culture dishes and biomaterials. Cell micropatterning strategies aim to control the spatial localization of cells on their underlying material and in relation to other cells. Developing such strategies provides us with tools necessary to eventually fabricate the highly-controlled microenvironments found in multicellular organisms. Employing natural extracellular matrix (ECM) materials in patterning techniques can increase biocompatibility. In the future, with such technologies, we can hope to conduct novel studies in cell biology or optimize cell behavior and function towards the development of new cell-based devices and tissue engineering constructs.
Herein, a novel cell patterning platform was developed on a hydrogel base of crosslinked hyaluronic acid (HA). Hydrogels are often employed in tissue engineering due to their ability to mimic the physicochemical properties of natural tissues. HA is a polymer present in all connective tissues. Cell-adhesive regions on the hydrogel were created using the RGDS peptide sequence, found within the cell-adhesive ECM protein, fibronectin. The peptide was bound to a 2-nitrobenzyl “caging group” via a photolabile bond to render the peptide light-responsive. Finally, this “caged” peptide was covalently bound to the hydrogel to form a novel HA hydrogel with a cell non-adhesive surface which could be activated with near-UV light to become adhesive. In this way, we successfully formed chemically patterned cell-adhesive regions on a HA hydrogel using light as a stimulus to form controlled cell patterns.
While the majority of cell patterning strategies to date are limited to patterning one cell population and cannot be changed with time, our strategy was novel in using small, adhesive, caged peptides combined with multiple, aligned light exposure steps to allow for dynamic chemical cell patterning on a hydrogel. Multiple cell populations, even held apart from one another, were successfully patterned on the same hydrogel. Furthermore, cell patterns were deliberately modified with time to direct cell growth and/or migration on the hydrogel base.
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Drug Delivery Strategies Using Light Sensitive MoleculesDcona, Martin 12 March 2012 (has links)
Cancer remains one of the most dreaded diseases due to inevitable suffering and possible fatality. Only cardiac disease has caused more deaths than cancer. Present day cancer treatment involves radiation, surgery or chemotherapy. In chemotherapy, an anti-tumoral drug is used to treat the tumor either by killing or stalling the growth of the tumor cells. In certain types of cancer, for e.g. metastatic breast cancer, the first line of therapy is often chemotherapy. But the inability of current clinically approved drugs to selectively target tumor cells, ultimately results in side effects. To reduce these side effects, prodrug therapies have been developed. A prodrug is defined as a drug molecule inactivated by a temporary cap or carrier, subsequently removed by an external intra or extracellular stimulus. Several prodrug strategies such as ADEPT (Antibody–Directed Enzyme Prodrug Therapy) have been tested in clinical trials but have thus far met with limited success. In the wake of these limitations, development of photo-activatable prodrugs may be particularly desirable for minimizing the adverse side effects associated with current cancer chemotherapeutics. Photodynamic therapy (PDT) is a light dependent tumor treatment modality that has existed for many years. PDT involves a photosensitizer which is administered to the patient and later activated using the light of wavelengths between 650-800 nm. The activated photosensitizer creates singlet oxygen, which acts as cytotoxic agent to the tumor cells. But this approach has several drawbacks including slow uptake of the photosensitizer by the tumor cells and the dependence on molecular oxygen that is not always present at even moderate levels in the tumor tissues. To address these limitations of PDT, we developed a new prodrug concept called ‘Photocaged Permeability’ in our first project, and demonstrated drug delivery using this approach. The basis of this concept is that, by attaching a hydrophilic molecule to the drug via a photosensitive linker, the permeability of the drug could be restrained. But the drug could be released at the site of the tumor after irradiating with UV light. To achieve this goal, we designed and synthesized a photosensitive drug conjugate that was comprised of doxorubicin attached to a negatively charged, cell impermeable molecule, EDANS (5-((2-Aminoethyl) amino) naphthalein-1-sulfonic acid) via a photosensitive nitroveratryl linker. Later, we performed MTT (cell viability) assays using esophageal adenocarcinoma (JH-EsoAd1) cells to determine the efficiency of our drug conjugate to induce cell death. As expected our drug conjugate was able to induce cell death, but only in presence of light. But in the dark, the cells remained unaffected. Also, we did several control studies to substantiate the fact that the cell death was actually due to drug release but not due to light or other entities. Further, we performed FACS (Fluorescence Assisted Cell Sorting) and confocal assays to show that in dark, the drug conjugate did not permeate cells. But upon irradiation with UV light, the drug was released from the conjugate, permeated the cells and induced cell death. A weakness of the above mentioned approach is that the drug is “decaged” or photo-released from the conjugates only under UV light; which cannot be translated to physiological conditions. This is because the UV light cannot penetrate deeper than 5 mm into the human skin. As a result, tumor cells that are deeply embedded in the human body cannot be treated using these approaches. To address this problem, Near Infrared (NIR) light could be used as it penetrates deeper than UV. Recently, several groups have reported using Upconverting Nanoparticles (UCNP) for the purpose of drug activation. The basis of this phenomenon is that the incidence of NIR light on these particles initiates multi-photon processes, eventually emitting UV/VIS wavelengths. The advantage of the NIR is that it deeply penetrates into the human skin. In our latest project, we have designed a drug conjugate that would be attached to UCNPs. We envision that after grafting the drug conjugate onto the nanoparticles and irradiating it with NIR drug release will occur as a result of upconversion. The above two systems describes novel methodologies for controlled release of the drug. To further improve the efficacy of the drug action, we designed new photosensitive systems based on the concept of targeted drug delivery. Targeted drug delivery is a treatment methodology in which the modified chemotherapeutic drug with higher tumor affinity could be concentrated in the tumor tissues. In certain cases, the receptors of tumor cells are targeted for the purpose of therapy. Receptors are cell surface proteins that are expressed on their plasma membrane. A select few of them such as Folic Acid Receptor (FAR) and PSMA (Prostate Specific Membrane Antigen) are overexpressed in malignant cells. In our new designs, we attached folic acid and urea based (DUPA) ligand, which were previously reported to bind to FAR and PSMA receptors respectively. Cell studies are currently underway to determine the specificity of these drug conjugates in targeting tumor cells. Once we demonstrate the above drug delivery strategies in vitro and later in vivo, we will have established novel drug delivery systems that could potentially be applied towards chemotherapeutic treatment.
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