Metabolic response of lung cancer cells to radiation in a paper-based 3D cell culture system.

This work demonstrates the application of a 3D culture system-Cells-in-Gels-in-Paper (CiGiP)-in evaluating the metabolic response of lung cancer cells to ionizing radiation. The 3D tissue-like construct-prepared by stacking multiple sheets of paper containing cell-embedded hydrogels-generates a gradient of oxygen and nutrients that decreases monotonically in the stack. Separating the layers of the stack after exposure enabled analysis of the cellular response to radiation as a function of oxygen and nutrient availability; this availability is dictated by the distance between the cells and the source of oxygenated medium. As the distance between the cells and source of oxygenated media increased, cells show increased levels of hypoxia-inducible factor 1-alpha, decreased proliferation, and reduced sensitivity to ionizing radiation. Each of these cellular responses are characteristic of cancer cells observed in solid tumors. With this setup we were able to differentiate three isogenic variants of A549 cells based on their metabolic radiosensitivity; these three variants have known differences in their metastatic behavior in vivo. This system can, therefore, capture some aspects of radiosensitivity of populations of cancer cells related to mass-transport phenomenon, carry out systematic studies of radiation response in vitro that decouple effects from migration and proliferation of cells, and regulate the exposure of oxygen to subpopulations of cells in a tissue-like construct either before or after irradiation.

Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel).

A paper-based invasion assay: assessing chemotaxis of cancer cells in gradients of oxygen.

This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.

Polymer-based mesh as supports for multi-layered 3D cell culture and assays.

Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system-Cells-in-Gels-in-Mesh (CiGiM)-that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells-layer-by-layer-within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis-(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format.

Noncovalent polymerization of mesogens crystallizes lysozyme: correlation between nonamphiphilic lyotropic liquid crystal phase and protein crystal formation.

Crystallization of proteins is important for fundamental studies and biopharmaceutical development but remains largely an empirical science. Here, we report the use of organic salts that can form a class of unusual nonamphiphilic lyotropic liquid crystals to crystallize the protein lysozyme. Certain nonamphiphilic organic molecules with fused aromatic rings and two charges can assemble into stable thread-like noncovalent polymers that may further form liquid crystal phases in water, traditionally termed chromonic liquid crystals. Using five of these mesogenic molecules as additives to induce protein crystallization, we discover that molecules that can form liquid crystal phases in water are highly effective at inducing the crystal formation of lysozyme, even at concentrations significantly lower than that required for forming liquid crystal phases. This result reveals an example of inducing protein crystallization by the molecular assembly of the additives, and is consistent with a new mechanism by which the strong hydration of an assembly process provides a gradual means to compete for the water molecules to enable solvated proteins to form crystals.

Noncovalent polymerization and assembly in water promoted by thermodynamic incompatibility.

This work studies the phase separations between polymers and a small molecule in a common aqueous solution that do not have well-defined hydrophobic-hydrophilic separation. In addition to poly(acrylamide) (PAAm) and poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP) also promotes liquid crystal (LC) droplet formation by disodium cromoglycate (5'DSCG) solvated in water. In the presence of these polymers, the concentration of 5'DSCG needed for forming LC droplets is substantially lower than that needed for forming an LC phase by 5'DSCG alone. To define the concentration ranges that 5'DSCG molecules form liquid crystals (either as droplets or as an isotropic-LC mixture), we constructed ternary phase diagrams for 5'DSCG, water, and a polymer - PVA, PVP, or PAAm. We discovered that PAAm with high molecular weight promotes LC droplet formation by 5'DSCG more effectively than PAAm with low molecular weight. At the same weight percentage, long-chain PAAm can cause 5'DSCG to form LC droplets in water, whereas short-chain PAAm does not. Poly(vinyl pyrrolidone) (PVP), which has functional groups that are more dissimilar to 5'DSCG than PVA and PAAm, promotes LC droplet formation by 5'DSCG more effectively than either of the other two polymers. Additionally, small angle neutron scattering data revealed that the assembly structure of 5'DSCG promoted by the presence of PVA is similar to the thread structure formed by 5'DSCG alone. Together, these results reveal how noncovalent polymerization can be promoted by mixing thermodynamically incompatible molecules and elucidate the basic knowledge of nonamphiphilic colloidal science.

Nonamphiphilic assembly in water: polymorphic nature, thread structure, and thermodynamic incompatibility.

Self-assembly of large quantities of entirely water-soluble molecules is entropically challenging. In this work, we describe the design and synthesis of water-soluble aromatic (dichromonyl) molecules that can form nonamphiphilic assemblies and the so-called chromonic liquid crystal phase in water. We discover a new molecule, 5'DSCG-diviol, that exhibits a large birefringent phase, and we show that the formation of this unique class of nonamphiphilic lyotropic liquid crystal shares enormous similarity to the polymorphism observed for crystal formation. Small-angle neutron scattering (SANS) revealed a concentration-independent rod-shaped assembly at concentrations below and above the formation of liquid crystal phase. Adding a small percentage of monoanionic aromatic molecules to the liquid crystal resulted in the elimination of the liquid crystal phase, but addition of dianionic aromatic molecules retained the liquid crystal phase. Together, these results suggest a new assembly structure for nonamphiphilic molecules in water, which is comprised of long threads of small molecules connected by salt bridges stacked over aromatic groups, with the molecular threads heavily hydrated with solvent water. Furthermore, mixing molecules with different structures can result in new liquid crystalline materials, or in segregation of the molecules into different solvation volumes, each of which contains only one type of molecule. The unusual thermodynamic incompatibility of entirely water-soluble molecules also supports the model of molecular threads, in which two polymer-like assemblies do not mix.

Molecular gradients of bioinertness reveal a mechanistic difference between mammalian cell adhesion and bacterial biofilm formation.

Chemical gradients play an important role in guiding the activities of both eukaryotic and prokaryotic cells. Here, we used molecularly well-defined chemical gradients formed by self-assembled monolayers (SAMs) on gold films to reveal that mammalian cell adhesion and bacterial biofilm formation respond differently to a gradient of surface chemistry that resists cell attachment. Gradient self-assembled monolayers (SAMs) consisting of two mixed alkanethiols were fabricated by differential exposure of the gold film to one alkanethiol, followed by soaking in another alkanethiol solution. A gradient in bioinertness that resisted cell attachment was created on SAMs from a gradient in the surface density of HS(CH2)11(OCH2CH2)3OH, backfilled with either HS(CH2)11OH or HS(CH2)11CH3. Measurements of the amounts of mammalian cells and bacterial biofilms on these gradient surfaces reveal that, for mammalian cells, a critical density of adhesion ligands from absorbed proteins on surfaces exists for supporting maximum adhesion and proliferation, whereas for the bacterium Escherichia coli , the amount of biofilm formed on surfaces increased linearly with the surface density of adhesive groups (methyl or hydroxyl groups) in different media. These results are consistent with mammalian cell adhesion requiring an anchorage via specific molecular recognitions and suggest that biofilms can form by immobilization of bacteria via nonspecific interaction between bacteria and surfaces.

Identifying the important structural elements of brominated furanones for inhibiting biofilm formation by Escherichia coli.

A collection of structurally closely related furanones was synthesized to identify the most important structural elements in brominated furanones for inhibiting the formation of bacterial biofilms. The results suggest that a conjugated exocyclic vinyl bromide on the furanone ring is the most important structural element for the non-toxic but inhibition activity for Escherichia coli biofilm formation. Furanones bearing monosubstituted bromide groups on saturated carbons were found to have a toxic effect that attenuates the bacterial growth.

Inhibition of Escherichia coli biofilm formation by self-assembled monolayers of functional alkanethiols on gold.

Bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. To further understand bacterium-surface interactions and to develop efficient control strategies, self-assembled monolayers (SAMs) of alkanethiols presenting different functional groups on gold films were analyzed to determine their resistance to biofilm formation. Escherichia coli was labeled with green florescence protein, and its biofilm formation on SAM-modified surfaces was monitored by confocal laser scanning microscopy. The three-dimensional structures of biofilms were analyzed with the COMSTAT software to obtain information about biofilm thickness and surface coverage. SAMs presenting methyl, L-gulonamide (a sugar alcohol tethered with an amide bond), and tri(ethylene glycol) (TEG) groups were tested. Among these, the TEG-terminated SAM was the most resistant to E. coli biofilm formation; e.g., it repressed biofilm formation by E. coli DH5alpha by 99.5% +/- 0.1% for 1 day compared to the biofilm formation on a bare gold surface. When surfaces were patterned with regions consisting of methyl-terminated SAMs surrounded by TEG-terminated SAMs, E. coli formed biofilms only on methyl-terminated patterns. Addition of TEG as a free molecule to growth medium at concentrations of 0.1 and 1.0% also inhibited biofilm formation, while TEG at concentrations up to 1.5% did not have any noticeable effects on cell growth. The results of this study suggest that the reduction in biofilm formation on surfaces modified with TEG-terminated SAMs is a result of multiple factors, including the solvent structure at the interface, the chemorepellent nature of TEG, and the inhibitory effect of TEG on cell motility.

Water-in-water emulsions stabilized by non-amphiphilic interactions: polymer-dispersed lyotropic liquid crystals.

Emulsion systems involving surfactants are mainly driven by the separation of the hydrophobic interactions of the aliphatic chains from the hydrophilic interactions of amphiphilic molecules in water. In this study, we report an emulsion system that does not include amphiphilic molecules but molecules with functional groups that are completely solvated in water. These functional groups give rise to molecular interactions including hydrogen bonding, pi stacking, and salt bridging and are segregated into a dispersion of droplets forming a water-in-water emulsion. This water-in-water emulsion consists of dispersing droplets of a water-solvated biocompatible liquid crystal--disodium cromoglycate (DSCG)--in a continuous aqueous solution containing specific classes of water-soluble polymers. Whereas aqueous solutions of polyols support the formation of emulsions of spherical droplets consisting of lyotropic liquid crystal DSCG with long-term stability (for at least 30 days), aqueous solutions of polyamides afford droplets of DSCG in the shape of prolate ellipsoids that are stable for only 2 days. The DSCG liquid crystal in spherical droplets assumes a radial configuration in which the optical axis of the liquid crystal aligns perpendicular to the surface of the droplets but assumes a tangential configuration in prolate ellipsoids in which the optical axis of the liquid crystal aligns parallel to the surface of the droplet. Other classes of water-soluble polymers including polyethers, polycations, and polyanions do not afford a stable emulsion of DSCG droplets. Both the occurrence and the stability of this unique emulsion system can be rationalized on the basis of the functional groups of the polymer. The different configurations of the liquid crystal (DSCG) droplets were also found to correlate with the strength of the hydrogen bonding that can be formed by the functional groups on the polymer.

A biocompatible surfactant with folded hydrophilic head group: enhancing the stability of self-inclusion complexes of ferrocenyl in a beta-cyclodextrin unit by bond rigidity.

This work reports a new biocompatible surfactant structure, of which the hydrophilic head group is composed of a folded, stable self-inclusion complex of a ferrocenyl substituted beta-cyclodextrin (betaCD). While multiple intra- or intermolecular complexes can exist for this amphiphile, the molecule folds into a unique intramolecular complex with well-defined conformation, in which part of the aliphatic chain and the ferrocene group are both included in the annular cavity of betaCD. Study of different isosteric covalent linkages indicates that this folded structure is stable against displacement by the presence of other small guest molecules. Furthermore, in contrast to ferrocene-CD conjugates that are without the aliphatic chain, the presence of small guest molecules in solution does not influence at all the induced circular dichroism signal of this amphiphile, indicating a sterically congested, but stable, folded conformation of the inclusion complex. This new amphiphile is surface active and, more importantly, does not denature the membrane protein bacteriorhodopsin. Finally, because this surfactant forms self-assembled aggregates, this work introduces a folded structure into soft matters formed by amphiphiles in water.