In Vitro Optimization of EtNBS-PDT against Hypoxic Tumor Environments with a Tiered, High-Content, 3D Model Optical Screening Platform
ABSTRACT Hypoxia and acidosis are widely recognized as major contributors to the development of treatment resistant cancer. For patients with disseminated metastatic lesions, such as most women with ovarian cancer (OvCa), the progression to treatment resistant disease is almost always fatal. Numerous therapeutic approaches have been developed to eliminate treatment resistant carcinoma, including novel biologic, chemo, radiation, and photodynamic therapy (PDT) regimens. Recently, PDT using the cationic photosensitizer EtNBS was found to be highly effective against therapeutically unresponsive hypoxic and acidic OvCa cellular populations in vitro. To optimize this treatment regimen, we developed a tiered, high-content, image-based screening approach utilizing a biologically relevant OvCa 3D culture model to investigate a small library of side-chain modified EtNBS derivatives. The uptake, localization, and photocytotoxicity of these compounds on both the cellular and nodular levels were observed to be largely mediated by their respective ethyl side chain chemical alterations. In particular, EtNBS and its hydroxyl-terminated derivative (EtNBS-OH) were found to have similar pharmacological parameters, such as their nodular localization patterns and uptake kinetics. Interestingly, these two molecules were found to induce dramatically different therapeutic outcomes: EtNBS was found to be more effective in killing the hypoxic, nodule core cells with superior selectivity, while EtNBS-OH was observed to trigger widespread structural degradation of nodules. This breakdown of the tumor architecture can improve the therapeutic outcome and is known to synergistically enhance the antitumor effects of front-line chemotherapeutic regimens. These results, which would not have been predicted or observed using traditional monolayer or in vivo animal screening techniques, demonstrate the powerful capabilities of 3D in vitro screening approaches for the selection and optimization of therapeutic agents for the targeted destruction of specific cellular subpopulations.
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ABSTRACT: The benzophenothiazinium dye EtNBS has previously been tested as a photosensitizer to mediate photodynamic therapy (PDT). It has been employed to kill cancer cells and microbial cells in vitro and to treat tumors and infections in vivo. We synthesized a panel of derivatives substituted at the 1-position of the benzene ring with electron donating or electron withdrawing groups (amino, acetamido and nitro) and tested their production of reactive oxygen species (ROS) and light-mediated killing of two species of Gram-positive and two species of Gram-negative bacteria. All three compounds showed lower fluorescence, lower yield of ROS and less microbial killing than parent EtNBS, while the order of activity (nitro > amino > acetamido) showed that an electron withdrawing substituent was better than electron donating. To test the hypothesis that 1-substitution distorts the planar structure of the conjugated rings we compared two compounds substituted with N-ethylpropylsulfonamido either at the 1-position or at the 4-position. The 4-isomer was significantly more photoactive than the 1-isomer. We also prepared an EtNBS derivative with a guanidinium group attached to the 5-amino group. This compound had high activity against Gram-negative bacteria due to the extra positive charge. Cellular uptake of the compounds by the four bacterial species was also measured and broadly correlated with activity. These results provided three separate pieces of structure-activity relationship data for antimicrobial photosensitizers based on the EtNBS backbone.European Journal of Medicinal Chemistry 02/2014; 75C:479-491. DOI:10.1016/j.ejmech.2014.01.064 · 3.43 Impact Factor
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ABSTRACT: For well over a decade, RNA interference (RNAi) has provided a powerful tool for investigators to query specific gene targets in an easily modulated loss-of-function setting, both in vitro and in vivo. Hundreds of publications have demonstrated the utility of RNAi in arrayed and pooled-based formats, in a wide variety of cell-based systems, including clonal, stem, transformed, and primary cells. Over the years, there have been significant improvements in the design of target-specific small-interfering RNA (siRNA) and short-hairpin RNA (shRNA), expression vectors, methods for mitigating off-target effects, and accurately interpreting screening results. Recent developments in RNAi technology include the Sensor assay, high-efficiency miR-E shRNAs, improved shRNA virus production with Pasha (DRGC8) knockdown, and assessment of RNAi off-target effects by using the C9-11 method. An exciting addition to the arsenal of RNA-mediated gene modulation is the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas) system for genomic editing, allowing for gene functional knockout rather than knockdown.Journal of Biomolecular Screening 08/2014; DOI:10.1177/1087057114548414 · 2.01 Impact Factor
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ABSTRACT: We report the synthesis and anticancer photodynamic properties of two new decacationic fullerene (LC14) and red light-harvesting antenna-fullerene conjugated monoadduct (LC15) derivatives. The antenna of LC15 was attached covalently to C60> with distance of only <3.0 Ǻ to facilitate ultrafast intramolecular photoinduced-electron-transfer (for type-I photochemistry) and photon absorption at longer wavelengths. Because LC15 was hydrophobic we compared formulation in CremophorEL micelles with direct dilution from dimethylacetamide. LC14 produced more (1)O2 than LC15, while LC15 produced much more HO· than LC14 as measured by specific fluorescent probes. When delivered by DMA, LC14 killed more HeLa cells than LC15 when excited by UVA light, while LC15 killed more cells when excited by white light consistent with the antenna effect. However LC15 was more effective than LC14 when delivered by micelles regardless of the excitation light. Micellar delivery produced earlier apoptosis and damage to the endoplasmic reticulum as well as to lysosomes and mitochondria.Nanomedicine: nanotechnology, biology, and medicine 12/2013; 10(4). DOI:10.1016/j.nano.2013.11.014 · 5.98 Impact Factor