Membrane Interactions of Streptococcus agalactiae's CAMP factor

CAMP factor is an extracellular pore-forming toxin secreted by the group B streptococci Streptococcus agalactiae. In conjunction with the action of sphingomyelinase secreted by Staphylococcus aureus, which converts membrane sphingomyline to ceramide, CAMP factor kills susceptible cells by creating holes in them. Since the monomeric or oligomeric structure of CAMP factor is not yet known, no studies on the membrane-penetrating domain of this toxin have been done. In the present study, the interaction of a putative hydrophobic domain between residues T90 and V115 with the target membrane was examined by cysteine-scanning mutagenesis and site-selective fluorescent labeling. The combination of steady state and lifetime fluorescence measurements and collisional quenching experiments with nitroxide labeled fatty acids indicate that residues from T90 to V115 contact the membrane upon binding and oligomerization of CAMP factor on cell membranes. More importantly, all these individual assays indicate that the residues from N104C to F109C insert superficially into the membrane with a β-sheet conformation

Carbon Nanotubes as Versatile Devices for Detoxification and Cellular Entry

The ability to bypass most cellular barriers to gain access to intracellular compartments has great potential in cell biology. The possibilities range from efficient delivery of macromolecules such as plasmids to small proteins and oligonucleotides that are sensitive to degradation. In biomedicine, easy access means enhanced cellular imaging and delivery of many therapeutics currently hampered by poor stability and cellular uptake. Carbon Nanotubes (CNTs) are attractive in these applications due to their efficient cellular uptake. While mode of entry of CNTs into cells is debatable, possibly their natural shape allows for their selective penetration across biological barriers in a non-destructive way, making them versatile as membrane permeating particles. The present study explores the diverse functionalities of CNTs including: 1) Efficient delivery of DNA into HeLa cells using vertically aligned MWNT arrays, 2) The use of Single Walled Carbon Nanotubes (SWNTs) as nano detoxifiers and 3) the design of SWNTs for efficient cellular uptake. Generally, vertically aligned nanoneedles have been used to influence the behavior and differentiation of various cell types. In the first work described in chapter 2, periodic high-density array MWNT nanoneedles is shown to support cell growth and penetrate into HeLa cells, making it ideal for use in cellular imaging and the efficient delivery of plasmid DNA into cells. Most importantly, we show that transfection with the MWNT substrate exhibited more uniformity in comparison to the commercially available lipofection procedure. Lipofection involves the formation of a complex of DNA and cationic lipids that interact with the cell via electrostatic interactions, leading to internalization, DNA escape into the cytosol, and the eventual transport into the nucleus. Functionalized CNTS have demonstrated great biocompatibility and potential for drug delivery in vitro. In the work described in chapter 3, we synthesized acid-oxidized and non-covalently PEGlyated SWNTs, which were reported previously for drug delivery purposes, and explored their potential for detoxification in the bloodstream. We investigated the binding of SWNTs to a pore-forming toxin pyolysin. The SWNTs were found to prevent toxin-induced pore formation in the cell membrane of human red blood cells. Quantitative hemolysis assay and scanning electron microscopy were used to evaluate the inhibition of hemolytic activity of pyolysin. Unlike HeLa cells, human red blood cells did not internalize oxidized SWNTs according to Raman spectroscopy data. Molecular modeling and circular dichroism measurements were used to predict the 3D structure of pyolysin (domain 4) and its interaction with SWNTs. The Tryptophan-rich hydrophobic motif in the membrane-binding domain of pyolysin, a common construct in a large family of cholesterol-dependent cytolysins (CDCs), showed high affinity for SWNTs. In the final two chapters, chapters 4 and 5, we focused on shorter CNTs (<70 nm) that have less length variations. This enabled the determination of several length related characteristics such as cellular uptake and distribution of SWNTs within between cells. Here, cellular uptake of two water-soluble SWNTs, Short SWNTs (S_SWNTs) and Ultra-Short SWNTs (US_SWNTs), was evaluated against various mammalian cells. Cellular entry of S_SWNTs (chapter 4), similar in dimensions to those reported in the literature, is shown to be affected by their hydrophilic corona and exhibit time-dependent nuclear accumulation. In contrast, US_SWNTs show no dependence of cellular entry on their hydrophilic exterior (chapter 5). Furthermore, intracellular localization and excretion of the US_SWNTs is observed to be cell type-dependent. Results presented in this work show the potential of CNTs as nano detoxifiers. We also use CNTs as vertically aligned nanoneedles and as colloids to efficiently traverse the plasma membrane. While CNTs as nanoneedles show the potential as an efficient means of transfecting mammalian cells, the use of S_SWNTs and US_SWNTs highlight some key observations including the physical and chemical properties (size, surface functionality) and cell type influence on cellular uptake and intracellular trafficking. These findings contribute to the interpretation of SWNT-cell interactions by providing a correlation between CNT length and cellular uptake and also cell type on trafficking of internalized SWNTs. With the realization of the enhanced permeability and retention effects, tumor vascular leakiness resulting from increased angiogenesis and vasoactive factors enhancing permeability at the diseased site, nanoparticles that have long circulation time have higher chance of accumulating at the diseased sites.1 yea

<i>In Situ</i> Hybridization of Superparamagnetic Iron-Biomolecule Nanoparticles

The increase in interest in the integration of organic–inorganic nanostructures in recent years has promoted the use of hybrid nanoparticles (HNPs) in medicine, energy conversion, and other applications. Conventional hybridization methods are, however, often long, complicated, and multistepped, and they involve biomolecules and discrete nanostructures as separate entities, all of which hinder the practical use of the resulting HNPs. Here, we present a novel, in situ approach to synthesizing size-specific HNPs using Fe-biomolecule complexes as the building blocks. We choose an anticancer peptide (p53p, MW 1.8 kDa) and an enzyme (GOx, MW 160 kDa) as model molecules to demonstrate the versatility of the method toward different types of molecules over a large size range. We show that electrostatic interaction for complex formation of metal hydroxide ion with the partially charged side of biomolecule in the solution is the key to hybridization of metal-biomolecule materials. Electrochemical deposition is then used to produce hybrid NPs from these complexes. These HNPs with controllable sizes ranging from 30 nm to 3.5 μm are found to exhibit superparamagnetic behavior, which is a big challenge for particles in this size regime. As an example of greatly improved properties and functionality of the new hybrid material, <i>in vitro</i> toxicity assessment of Fe-GOx HNPs shows no adverse effect, and the Fe-p53p HNPs are found to selectively bind to cancer cells. The superparamagnetic nature of these HNPs (superparamagnetic even above the size regime of 15–20 nm!), their biocompatibility, and the direct integration approach are fundamentally important to biomineralization and general synthesis strategy for bioinspired functional materials