Kenneth A. Christensen

Multicolor Conjugated Polymer Dots for Biological Fluorescence Imaging

Highly fluorescent conjugated polymer dots were developed for demanding applications such as fluorescence imaging in live cells. These nanoparticles exhibit small particle diameters, extraordinary fluorescence brightness, and excellent photostability. Single particle fluorescence imaging and kinetic studies indicate much higher emission rates (approximately 10(8) s(-1)) and little or no blinking of the nanoparticles as compared to typical results for single dye molecules and quantum dots. Analysis of single particle photobleaching trajectories reveals excellent photostabilityas many as 10(9) or more photons emitted per nanoparticle prior to irreversible photobleaching. The superior figures of merit of these new fluorescent probes, together with the demonstration of cellular imaging, indicate their enormous potential for demanding fluorescence-based imaging and sensing applications such as high speed super-resolution single molecule/particle tracking and highly sensitive assays.

Ratiometric Single-Nanoparticle Oxygen Sensors for Biological Imaging

It makes sense: Conjugated polymer nanoparticles doped with a platinum porphyrin dye exhibit bright phosphorescence that is highly sensitive to the concentration of molecular oxygen. The small size, extraordinary brightness, excellent sensitivity, and ratiometric emission, together with the demonstration of single-particle sensing and cellular uptake, indicate the potential of the nanoparticle sensors for quantitative mapping of local molecular oxygen concentration.

Mechanism of Cellular Uptake of Highly Fluorescent Conjugated Polymer Nanoparticles

Conjugated polymer nanoparticles are formed by precipitation of highly fluorescent conjugated polymers to form small nanoparticles with extremely bright fluorescence. We characterized cellular uptake and cytotoxicity of 18 ± 5 nm PFBT conjugated polymer nanoparticles in J774A.1 cells. Significant nanoparticle uptake was observed, indicating efficient nanoparticle entry into cells, even for short (1 h) incubations. The high fluorescence of these nanoparticles allows extremely low loading concentrations; PFBT nanoparticle fluorescence in cells could be detected with loading concentrations of 155 pM (270 ppb). Cellular uptake slows at low temperature, consistent with endocytic entry. Nanoparticles colocalize with Texas Red dextran and are trafficked to lysosomes, as demonstrated by the location of nanoparticle fluorescence in perinuclear organelles that also stain with an anti-LAMP-1 antibody. Inhibition of uptake by phosphoinositide 3-kinase inhibitors implicates macropinocytosis as the operative endocytic mechanism. No significant cytotoxic or inflammatory effects could be observed, making PFBT nanoparticles attractive probes for live cell imaging.

Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles

Listeria monocytogenes (Lm) evade microbicidal defences inside macrophages by secreting a pore‐forming cytolysin listeriolysin O (LLO), which allows Lm to escape vacuoles. LLO also inhibits Lm vacuole fusion with lysosomes, which indicates LLO alters vacuole chemistry prior to release of Lm into cytoplasm. Using fluorescent probes to measure membrane permeability, calcium and pH, we identified small membrane perforations in vacuoles containing wild‐type but not LLO‐deficient (hly‐) Lm. The small membrane perforations released small fluorescent molecules and persisted for several minutes before expanding to allow exchange of larger fluorescent molecules. Macropinosomes and hly‐ Lm vacuoles acidified and increased their calcium content ([Ca2+]vac) within minutes of formation; however, the small perforations made by LLO‐expressing bacteria increased vacuolar pH and decreased [Ca2+]vac shortly after infection. Experimental increases in vacuolar pH inhibited Lm vacuole fusion with lysosomes. The timing of perforation indicated that LLO‐dependent delays of Lm vacuole maturation result from disruption of ion gradients across vacuolar membranes.

Surface-Enhanced Raman Scattering Detection of pH with Silica-Encapsulated 4-Mercaptobenzoic Acid-Functionalized Silver Nanoparticles

Sensors based upon surface-enhanced Raman spectroscopy (SERS) are attractive because they have narrow, vibrationally specific spectral peaks that can be excited using red and near-infrared light which avoids photobleaching, penetrates tissue, and reduces autofluorescence. Several groups have fabricated pH nanosensors by functionalizing silver or gold nanoparticle surfaces with an acidic molecule and measuring the ratio of protonated to deprotonated Raman bands. However, a limitation of these sensors is that macromolecules in biological systems can adsorb onto the nanoparticle surface and interfere with measurements. To overcome this interference, we encapsulated pH SERS sensors in a 30 nm thick silica layer with small pores which prevented bovine serum albumin (BSA) molecules from interacting with the pH-indicating 4-mercaptobenzoic acid (4-MBA) on the silver surfaces but preserved the pH-sensitivity. Encapsulation also improved colloidal stability and sensor reliability. The noise level corresponded to less than 0.1 pH units from pH 3 to 6. The silica-encapsulated functionalized silver nanoparticles (Ag-MBA@SiO(2)) were taken up by J774A.1 macrophage cells and measured a decrease in local pH during endocytosis. This strategy could be extended for detecting other small molecules in situ.

Membrane chromatography: Protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases

This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind D membrane adsorber and HiTrap DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind D membrane at equivalent volumetric throughput and higher capacities than the HiTrap DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap DEAE FF resin column and was the same for the new AEX membrane and Sartobind D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.

Selective interactions of sugar-functionalized single-walled carbon nanotubes with Bacillus spores.

It was reported previously that monosaccharide-functionalized single-walled carbon nanotubes (SWNTs) could interact with Bacillus anthracis (Sterne) spores with the mediation of a divalent cation such as Ca(2+) to result in significant spore aggregation and reduction in colony forming units. In this work a more systematic investigation was performed on interactions of the SWNTs functionalized with individual mannose and galactose moieties and their various dendritic configurations with B. anthracis and B. subtilis spores in the presence and absence of a divalent cation. Significant differences and selectivity between the Bacillus spores and between different sugars and their configurations were observed. The relevant results are presented, and their mechanistic implications are discussed.

Mutant Anthrax Toxin B Moiety (Protective Antigen) Inhibits Angiogenesis and Tumor Growth

Bacillus anthracis protective antigen (PA), the B subunit of the binary anthrax toxin, binds to the cellular receptors capillary morphogenesis gene 2 protein and tumor endothelial marker 8 with high affinity. Both receptors are expressed on endothelial cells during angiogenesis. We sought to determine whether one could inhibit angiogenesis by interfering with the binding of these receptors to their endogenous ligands. Here, we show that wild-type PA inhibits both vascular endothelial growth factor-induced and basic fibroblast growth factor-induced angiogenesis at moderate but statistically significant levels. Structure-activity studies identified a PA mutant that exhibited markedly enhanced inhibition of angiogenesis and also inhibited tumor growth in vivo. This mutant, PASSSR, is unable to undergo normal cellular processing and, thus, remains bound to the surface receptor. Further mutation of PASSSR so that it does not bind to these cell surface receptors abolished its ability to inhibit angiogenesis. We conclude that high-affinity anthrax toxin receptor (ATR) ligands, such as PA and PASSSR, are angiogenesis inhibitors and that ATRs are useful targets for antiangiogenic therapy. These results also suggest that endothelial cell-binding proteins from additional pathogens may inhibit angiogenesis and raise the question of the role of such inhibition in pathogenesis.

Quercetin, a fluorescent bioflavanoid, inhibits Trypanosoma brucei hexokinase 1

Hexokinases from the African trypanosome, Trypanosoma brucei, are attractive targets for the development of anti-parasitic drugs, in part because the parasite utilizes glycolysis exclusively for ATP production during the mammalian infection. Here, we have demonstrated that the bioflavanoid quercetin (QCN), a known trypanocide, is a mixed inhibitor of Trypanosoma brucei hexokinase 1 (TbHK1) (IC(50) = 4.1 ± 0.8μM). Spectroscopic analysis of QCN binding to TbHK1, taking advantage of the intrinsically fluorescent single tryptophan (Trp177) in TbHK1, revealed that QCN quenches emission of Trp177, which is located near the hinge region of the enzyme. ATP similarly quenched Trp177 emission, while glucose had no impact on fluorescence. Supporting the possibility that QCN toxicity is a consequence of inhibition of the essential hexokinase, in live parasites QCN fluorescence localizes to glycosomes, the subcellular home of TbHK1. Additionally, RNAi-mediated silencing of TbHK1 expression expedited QCN induced death, while over-expressing TbHK1 protected trypanosomes from the compound. In summary, these observations support the suggestion that QCN toxicity is in part attributable to inhibition of the essential TbHK1.

Head group-functionalized poly(ethyleneglycol)–lipid (PEG–lipid) surface modification for highly selective analyte extractions on capillary-channeled polymer (C-CP) fibers

Polypropylene (PP) capillary-channeled polymer (C-CP) fibers were modified by adsorption of a head group-functionalized lipid to generate analyte-specific surfaces for application as a stationary phase in high performance liquid chromatography (HPLC) or solid phase extraction (SPE). The aliphatic moiety of the lipid adsorbs strongly to the hydrophobic PP surface, with the hydrophilic active head groups orienting themselves toward the more polar mobile phase, thus allowing for interactions with the desired solutes. Initial proof-of-concept was achieved by adsorbing a biotin-poly(ethylene glycol)-functionalized lipid to the surface of the PP C-CP fibers. Surface modification and uniformity was evaluated by binding streptavidin labeled with Texas Red (SAv-TR) to the biotin moiety. Isolation of SAv-TR from a mixture in neat buffer and in cleared lysate demonstrated the capability of the modified fibers to extract an analyte of interest from a complex viscous mixture. It is believed that this surface modification approach is generally applicable to a diversity of selective protein immobilization applications, including clinical diagnostics and preparative scale HPLC on C-CP fibers as well as to other hydrophobic supports.