Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts

Background Post transcriptional gene silencing (PTGS) is a mechanism harnessed by plant biologists to knock down gene expression. siRNAs contribute to PTGS that are synthesized from mRNAs or viral RNAs and function to guide cellular endoribonucleases to target mRNAs for degradation. Plant biologists have employed electroporation to deliver artificial siRNAs to plant protoplasts to study gene expression mechanisms at the single cell level. One drawback of electroporation is the extensive loss of viable protoplasts that occurs as a result of the transfection technology. Results We employed fluorescent conjugated polymer nanoparticles (CPNs) to deliver siRNAs and knockdown a target gene in plant protoplasts. CPNs are non toxic to protoplasts, having little impact on viability over a 72 h period. Microscopy and flow cytometry reveal that CPNs can penetrate protoplasts within 2 h of delivery. Cellular uptake of CPNs/siRNA complexes were easily monitored using epifluorescence microscopy. We also demonstrate that CPNs can deliver siRNAs targeting specific genes in the cellulose biosynthesis pathway (NtCesA-1a and NtCesA-1b). Conclusions While prior work showed that NtCesA-1 is a factor involved in cell wall synthesis in whole plants, we demonstrate that the same gene plays an essential role in cell wall regeneration in isolated protoplasts. Cell wall biosynthesis is central to cell elongation, plant growth and development. The experiments presented here shows that NtCesA is also a factor in cell viability. We show that CPNs are valuable vehicles for delivering siRNAs to plant protoplasts to study vital cellular pathways at the single cell level.

Development of a Lab-on-a-Chip Device for Rapid Nanotoxicity Assessment In Vitro

Increasing useof nanomaterials in consumer products and biomedical applications creates the possibilities of intentional/unintentional exposure to humans and the environment. Beyond the physiological limit, the nanomaterialexposure to humans can induce toxicity. It is difficult to define toxicity of nanoparticles on humans as it varies by nanomaterialcomposition, size, surface properties and the target organ/cell line. Traditional tests for nanomaterialtoxicity assessment are mostly based on bulk-colorimetric assays. In many studies, nanomaterials have found to interfere with assay-dye to produce false results and usually require several hours or days to collect results. Therefore, there is a clear need for alternative tools that can provide accurate, rapid, and sensitive measure of initial nanomaterialscreening. Recent advancement in single cell studies has suggested discovering cell properties not found earlier in traditional bulk assays. A complex phenomenon, like nanotoxicity, may become clearer when studied at the single cell level, including with small colonies of cells. Advances in lab-on-a-chip techniques have played a significant role in drug discoveries and biosensor applications, however, rarely explored for nanomaterialtoxicity assessment. We presented such cell-integrated chip-based approach that provided quantitative and rapid response of cellhealth, through electrochemical measurements. Moreover, the novel design of the device presented in this study was capable of capturing and analyzing the cells at a single cell and small cell-population level. We examined the change in exocytosis (i.e. neurotransmitterrelease) properties of a single PC12 cell, when exposed to CuOand TiO2 nanoparticles. We found both nanomaterials to interfere with the cell exocytosis function. We also studied the whole-cell response of a single-cell and a small cell-population simultaneously in real-time for the first time. The presented study can be a reference to the future research in the direction of nanotoxicity assessment to develop miniature, simple, and cost-effective tool for fast, quantitative measurements at high throughput level. The designed lab-on-a-chip device and measurement techniques utilized in the present work can be applied for the assessment of othernanoparticles' toxicity, as well.

Development of a lab-on-a-chip device for rapid nanotoxicity assessment in vitro

Increasing useof nanomaterials in consumer products and biomedical applications creates the possibilities of intentional/unintentional exposure to humans and the environment. Beyond the physiological limit, the nanomaterialexposure to humans can induce toxicity. It is difficult to define toxicity of nanoparticles on humans as it varies by nanomaterialcomposition, size, surface properties and the target organ/cell line. Traditional tests for nanomaterialtoxicity assessment are mostly based on bulk-colorimetric assays. In many studies, nanomaterials have found to interfere with assay-dye to produce false results and usually require several hours or days to collect results. Therefore, there is a clear need for alternative tools that can provide accurate, rapid, and sensitive measure of initial nanomaterialscreening. Recent advancement in single cell studies has suggested discovering cell properties not found earlier in traditional bulk assays. A complex phenomenon, like nanotoxicity, may become clearer when studied at the single cell level, including with small colonies of cells. Advances in lab-on-a-chip techniques have played a significant role in drug discoveries and biosensor applications, however, rarely explored for nanomaterialtoxicity assessment. We presented such cell-integrated chip-based approach that provided quantitative and rapid response of cellhealth, through electrochemical measurements. Moreover, the novel design of the device presented in this study was capable of capturing and analyzing the cells at a single cell and small cell-population level. We examined the change in exocytosis (i.e. neurotransmitterrelease) properties of a single PC12 cell, when exposed to CuOand TiO2 nanoparticles. We found both nanomaterials to interfere with the cell exocytosis function. We also studied the whole-cell response of a single-cell and a small cell-population simultaneously in real-time for the first time. The presented study can be a reference to the future research in the direction of nanotoxicity assessment to develop miniature, simple, and cost-effective tool for fast, quantitative measurements at high throughput level. The designed lab-on-a-chip device and measurement techniques utilized in the present work can be applied for the assessment of othernanoparticles' toxicity, as well.^

Cancer therapy combining the modalities of hyperthermia and chemotherapy: In vitro cellular response after rapid heat accumulation in the cancer cell

Hyperthermia is usually used at a sub-lethal level in cancer treatment to potentiate the effects of chemotherapy. The purpose of this study is to investigate the role of heating rate in achieving synergistic cell killing by chemotherapy and hyperthermia. For this purpose, in vitro cell culture experiments with a uterine cancer cell line (MES-SA) and its multidrug resistant (MDR) variant MES-SA/Dx5 were conducted. The cytotoxicity, mode of cell death, induction of thermal tolerance and P-gp mediated MDR following the two different modes of heating were studied. Doxorubicin (DOX) was used as the chemotherapy drug. Indocyanine green (ICG), which absorbs near infrared light at 808nm (ideal for tissue penetration), was chosen for achieving rapid rate hyperthermia. A slow rate hyperthermia was provided by a cell culture incubator. The results show that the potentiating effect of hyperthermia to chemotherapy can be maximized by increasing the rate of heating as evident by the results from the cytotoxicity assay. When delivered at the same thermal dose, a rapid increase in temperature from 37°C to 43°C caused more cell membrane damage than gradually heating the cells from 37°C to 43°C and thus allowed for more intracellular accumulation of the chemotherapeutic agents. Different modes of cell death are observed by the two hyperthermia delivery methods. The rapid rate laser-ICG hyperthermia @ 43°C caused cell necrosis whereas the slow rate incubator hyperthermia @ 43°C induced very mild apoptosis. At 43°C a positive correlation between thermal tolerance and the length of hyperthermia exposure is identified. This study shows that by increasing the rate of heating, less thermal dose is needed in order to overcome P-gp mediated MDR.

Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Carbon nanostructure based electrodes for high efficiency dye sensitized solar cell

Synthesis and functionalization of large-area graphene and its structural, electrical and electrochemical properties has been investigated. First, the graphene films, grown by thermal chemical vapor deposition (CVD), contain three to five atomic layers of graphene, as confirmed by Raman spectroscopy and high-resolution transmission electron microscopy. Furthermore, the graphene film is treated with CF4 reactive-ion plasma to dope fluorine ions into graphene lattice as confirmed by X-ray photoelectron spectroscopy (XPS) and UV-photoemission spectroscopy (UPS). Electrochemical characterization reveals that the catalytic activity of graphene for iodine reduction enhanced with increasing plasma treatment time, which is attributed to increase in catalytic sites of graphene for charge transfer. The fluorinated graphene is characterized as a counter-electrode (CE) in a dye-sensitized solar cell (DSSC) which shows ~ 2.56% photon to electron conversion efficiency with ~11 mAcm−2 current density. Second, the large scale graphene film is covalently functionalized with HNO3 for high efficiency electro-catalytic electrode for DSSC. The XPS and UPS confirm the covalent attachment of C-OH, C(O)OH and NO3- moieties with carbon atoms through sp2-sp3 hybridization and Fermi level shift of graphene occurs under different doping concentrations, respectively. Finally, CoS-implanted graphene (G-CoS) film was prepared using CVD followed by SILAR method. The G-CoS electro-catalytic electrodes are characterized in a DSSC CE and is found to be highly electro-catalytic towards iodine reduction with low charge transfer resistance (Rct ~5.05 Ωcm 2) and high exchange current density (J0~2.50 mAcm -2). The improved performance compared to the pristine graphene is attributed to the increased number of active catalytic sites of G-CoS and highly conducting path of graphene. We also studied the synthesis and characterization of graphene-carbon nanotube (CNT) hybrid film consisting of graphene supported by vertical CNTs on a Si substrate. The hybrid film is inverted and transferred to flexible substrates for its application in flexible electronics, demonstrating a distinguishable variation of electrical conductivity for both tension and compression. Furthermore, both turn-on field and total emission current was found to depend strongly on the bending radius of the film and were found to vary in ranges of 0.8 - 3.1 V/μm and 4.2 - 0.4 mA, respectively.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating phosphatidylinositol 3 kinase localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. ^ It was initially found that comparing to wild type cells, gsk3 - cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. ^ I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.^

Multiparameter flow cytometric analysis of C -reactive protein binding to human-derived cell lines and peripheral blood monocytes

C-reactive protein (CRP), a normally occurring human plasma protein may become elevated as much as 1,000 fold during disease states involving acute inflammation or tissue damage. Through its binding to phosphorylcholine in the presence of calcium, CRP has been shown to potentiate the activation of complement, stimulate phagocytosis and opsonize certain microorganisms. Utilizing a flow cytometric functional ligand binding assay I have demonstrated that a monocyte population in human peripheral blood and specific human-derived myelomonocytic cell lines reproducibly bind an evolutionarily conserved conformational pentraxin epitope on human CRP through a mechanism that does not involve its ligand, phosphorylcholine. ^ A variety of cell lines at different stages of differentiation were examined. The monocytic cell line, THP-1, bound the most CRP followed by U937 and KG-1a cells. The HL-60 cell line was induced towards either the granulocyte or monocyte pathway with DMSO or PMA, respectively. Untreated HL-60 cells or DMSO-treated cells did not bind CRP while cells treated with PMA showed increased binding of CRP, similar to U-937 cells. T cell and B-cell derived lines were negative. ^ Inhibition studies with Limulin and human SAP demonstrated that the binding site is a conserved pentraxin epitope. The calcium requirement necessary for binding to occur indicated that the cells recognize a conformational form of CRP. Phosphorylcholine did not inhibit the reaction therefore the possibility that CRP had bound to damaged membranes with exposed PC sites was discounted. ^ A study of 81 normal donors using flow cytometry demonstrated that a majority of peripheral blood monocytes (67.9 ± 1.3, mean ± sem) bound CRP. The percentage of binding was normally distributed and not affected by gender, age or ethnicity. Whole blood obtained from donors representing a variety of disease states showed a significant reduction in the level of CRP bound by monocytes in those donors classified with infection, inflammation or cancer. This reduction in monocyte populations binding CRP did not correlate with the concentration of plasma CRP. ^ The ability of monocytes to specifically bind CRP combined with the binding reactivity of the protein itself to a variety of phosphorylcholine containing substances may represent an important bridge between innate and adaptive immunity. ^

Glycogen Synthase Kinase 3 Influences Cell Motility and Chemotaxis by Regulating Phosphatidylinositol 3 Kinase Localization in Dictyostelium discoideum

Glycogen Synthase Kinase 3 (GSK3), a serine/threonine kinase initially characterized in the context of glycogen metabolism, has been repeatedly realized as a multitasking protein that can regulate numerous cellular events in both metazoa and protozoa. I recently found GSK3 plays a role in regulating chemotaxis, a guided cell movement in response to an external chemical gradient, in one of the best studied model systems for chemotaxis - Dictyostelium discoideum. It was initially found that comparing to wild type cells, gsk3- cells showed aberrant chemotaxis with a significant decrease in both speed and chemotactic indices. In Dictyostelium, phosphatidylinositol 3,4,5-triphosphate (PIP3) signaling is one of the best characterized pathways that regulate chemotaxis. Molecular analysis uncovered that gsk3- cells suffer from high basal level of PIP3, the product of PI3K. Upon chemoattractant cAMP stimulation, wild type cells displayed a transient increase in the level of PIP3. In contrast, gsk3- cells exhibited neither significant increase nor adaptation. On the other hand, no aberrant dynamic of phosphatase and tensin homolog (PTEN), which antagonizes PI3K function, was observed. Upon membrane localization of PI3K, PI3K become activated by Ras, which will in turn further facilitate membrane localization of PI3K in an F-Actin dependent manner. The gsk3- cells treated with F-Actin inhibitor Latrunculin-A showed no significant difference in the PIP3 level. I also showed GSK3 affected the phosphorylation level of the localization domain of PI3K1 (PI3K1-LD). PI3K1-LD proteins from gsk3- cells displayed less phosphorylation on serine residues compared to that from wild type cells. When the potential GSK3 phosphorylation sites of PI3K1-LD were substituted with aspartic acids (Phosphomimetic substitution), its membrane localization was suppressed in gsk3- cells. When these serine residues of PI3K1-LD were substituted with alanine, aberrantly high level of membrane localization of the PI3K1-LD was monitored in wild type cells. Wild type, phosphomimetic, and alanine substitution of PI3K1-LD fused with GFP proteins also displayed identical localization behavior as suggested by the cell fraction studies. Lastly, I identified that all three potential GSK3 phosphorylation sites on PI3K1-LD could be phosphorylated in vitro by GSK3.