bkw686 – Softbites

2020-03-112020-06-23

How to turn off cancer cell growth using mechanosensing

Original paper: Stopping transformed cancer cell growth by rigidity sensing

Our bodies are made up of cells that can sense and respond to their dynamic environment. As an example, pancreatic beta cells chemically sense increased blood sugar concentrations and respond by producing insulin. Scientists have found that cells can also mechanically sense their environment; “mechanosensing” determines whether a cell should grow or die. Cancer is characterized by uncontrolled cellular growth, where cells often contain mutations that inhibit the natural mechanisms of cell death. Because mechanosensing is one such mechanism, scientists have hypothesized that cancer cells keep growing because they lack the ability to probe their environments. In this week’s paper, published in Nature Materials, an international research team led by Bo Yang and Michael Sheetz from the National University of Singapore investigated that hypothesis by combining tools from soft matter physics and biology.

fig_1_bill_004 — **Figure 1.** (A) Normal and (B) cancer cells on fibronectin-coated polydimethylsiloxane (PDMS) pillars shown as black dots. The yellow and orange portions represent the cell locations after ten and twenty minutes of spreading, respectively. In the zoomed-in subfigures to the right, blue arrows pointing in the opposite direction of each other represent paired pillars. The randomly oriented red arrows are unpaired pillars. Normal cells pair pillars together (A), whereas cancerous cells do not (B).

The team of researchers measured how well normal and cancer cell lines were able to mechanically probe their environments. To test cellular mechanosensing, they placed cells on a substrate consisting of microscopic pillars (500 nm diameter) made of polydimethylsiloxane (PDMS), a common polymer used in contact lenses. The pillars were coated in fibronectin, a protein that acts like a glue to help the cells stick to the pillars. From prior work, the researchers anticipated that cells would probe the rigidity of the PDMS pillars by pulling pairs of them towards each other. By measuring how much the cells bent the pillars towards each other, the scientists determined the forces exerted by the cells. As shown by the red arrows in Figure 1, both cancer and normal cell types deformed the pillars. However, only normal cells probed the rigidity of the substrate by pulling pillars together (blue arrows in Figure 1A). To evaluate the universality of their findings, researchers studied a variety of tissues derived from humans, monkeys, and mice. They found that normal cells from multiple species and tissues produced significantly more paired pillars than cancer cells, which confirmed that normal cells are universally better at mechanically sensing their environments.

Next, the researchers correlated their findings with the ability of cells to tailor their growth patterns depending on the rigidity of their environment. They found that normal cells died when placed on soft surfaces, such as soft agar, but grew when seeded on more rigid ones. Contrarily, cancer cells divided, and eventually formed colonies no matter how soft the surface was. From these results, it was proven that the loss of mechanosensing ability is highly related to the uncontrolled growth of cancer cells.

fig_2_bill_004_corr — **Figure 2.** Schematic showing the connection between mechanosensing proteins and cell growth on soft and rigid substrates.

To confirm this finding, Yang and coworkers restored mechanosensing in cancer cells, expecting that doing so would also restore normal growth patterns. Normal cellular mechanical sensing is performed by protein complexes that can tug and pull on the cells’ external environment. The researchers noticed that cancer cells lacked proteins vital to the formation of these complexes. As outlined in Figure 2, when the activity of these proteins was restored, the cancer cells resumed growth patterns that were similar to normal cells. The researchers were able to subsequently cancel out the activity of other proteins, thereby reintroducing abnormal, cancer-like growth. By depleting and restoring proteins, researchers could turn cancer-like growth on and off reversibly and demonstrated that those proteins are vital in ensuring normal cellular growth.

In short, Yang and coworkers showed that normal and cancer cells differ in their ability to mechanically test their environments. Unlike normal cells, cancer cells were unable to differentiate between soft and rigid surfaces and kept growing in an uncontrolled fashion. However, upon restoring key sensing proteins, cancer cells grew and died similarly to normal ones. These findings may affect future cancer treatments; by restoring the ability of cancer cells to mechanically sense their environment using genetic tools, we may have one more method to limit or stop uncontrollable cell growth. Such a treatment would be invaluable in saving the lives of many.

2019-05-29

Switching miscibility: How to make polymer blends mix with electricity

Original paper: Jumping In and Out of the Phase Diagram Using Electric Fields:
Time Scale for Remixing of Polystyrene/Poly(vinyl methyl ether)
Blends

Many consumer products, such as clothes and food packaging, are made of blends of polymers, long molecules consisting of repeating chemical units. The attractiveness of using blends of different polymers arises from the engineers’ desire to combine multiple unique properties of each individual polymer, such as transparency, stretchability, and breathability, into a seamless whole. However, different polymers are not necessarily miscible, a term scientists use to describe whether two materials mix at the molecular level. Miscibility isn’t a one-and-done kind of deal: scientists and engineers have known for years how to make polymer blends mix by careful temperature control. What if there were conditions other than temperature to achieve polymer blend miscibility? This may ultimately help in industrial processing of polymer blends. In this week’s paper, Professors Annika Kriisa and Connie B. Roth from Emory University in Atlanta, Georgia, explore the mixing dynamics of two polymers by using a strong electric field.

fig_1_bill_003 — **Figure 1.** Miscibility diagram of a hypothetical polymer blend consisting of polymers A and B. The x-axis is the fraction of polymer A in the blend (Composition ?) and the y-axis is the temperature of the blend (T). The curves represent the temperature above which the blends are immiscible without an electric field (black curve) and with an applied electric field (blue curve). The presence of the electric field increases the miscibility of the blend (higher transition temperature) at a given fraction of polymer A. (Image adapted from original paper.)

Before we dive into the meat of the paper, it’s important to know how temperature affects the miscibility of a polymer blend. The black curve in Figure 1 is a representative miscibility diagram of two blended polymers, which shows the temperature at which a polymer blend transitions from being miscible (below the black curve) to immiscible (above the black curve) as a function of the fraction of one polymer (denoted Composition ?) of the blend itself. The polymer blend is considered more miscible if the miscibility curve is shifted upwards, so that the blend turns immiscible at a higher temperature (see blue curve in Figure 1).

Kriisa and Roth wanted to explore how the application of an electric field influences the mixing dynamics of polystyrene (PS) and poly(vinyl methyl ether) (PVME) polymers. You may be quite familiar with these materials: PS is the formal name of styrofoam, the main component in plastic cups, and PVME is typically used in glues and adhesives. In the past, Kriisa and Roth studied the effect of electric fields in blends of these materials, and found that the electric field enhances polymer blend miscibility: an electric field raises the temperature at which a PS/PVME blend becomes immiscible, similarly to the blue curve in Figure 1 [1]. What interested the authors the most in this today’s paper was the dynamics of mixing; in other words, how quickly do immiscible blends remix once they are exposed to an electric field? And what can we learn about the factors governing the remixing process?

fig2_bill_003 — **Figure 2.** Switching the miscibility of polystyrene/polyvinyl ethyl ether (PS/PVME) polymer blends as a function of time through the application of an electric field (E). The red curve is the intensity of the fluorescence of a molecule attached to polystyrene, which decreases with time. The blue curve is the imposed electric field, which is repeatedly switched on and off. (Image adapted from original paper.)

The authors showed that the dynamics of mixing a PS/PVME blend is highly sensitive to the application of an electric field. They demonstrated this by examining a PS/PVME blend at the temperature four Kelvin higher than the temperature at which it becomes immiscible. They repeatedly switched on and off an electric field regularly, causing the blend to switch from being miscible to immiscible (see blue curve in Figure 2). To determine how well mixed the blend was, they measured the intensity of the light emitted by a fluorescing molecule, which was chemically attached to the PS molecules (see red curve in Figure 2). When PS and PVME are fully mixed, the fluorescence intensity decreases to 0. After switching on the electric field, the blend starts mixing immediately, showing a high sensitivity to the presence of the electric field.

fig3_bill_003 — **Figure 3.** Remixing timescale (?) as a function of temperature (T) and applied electric field (E). The black symbols correspond to absence of electric field, red to electric field at E =12.8 MV/m, and blue at E=13.0 MV/m. *T_s* (shown by the dashed lines) is the temperature at which the blend becomes immiscible at the given electric field. The remixing timescales follow the same black curve, showing that they are largely independent of E. (Adapted from original paper.)

The authors repeated this experiment for a variety of temperatures and electric field strengths. From the fluorescence curves, they extracted the remixing timescale or the time it takes for the blend to remix, as shown in Figure 3. The black symbols correspond to absence of electric field, while the red correspond to E = 12.8 MV/m and the blue to 13.0 MV/m. One may notice that the time it takes for the polymer blend to remix is largely independent of the electric field strength at a given temperature, since all remixing timescales (?) follow the same black curve. Thus, the authors concluded that the rate of remixing is not affected by the electric field.

In short, Kriisa and Roth showed that the dynamics of remixing polymer blends are sensitive to electricity. They found that immiscible blends immediately begin to remix when exposed to an electric field and that the time it takes for the blend to completely remix is independent of the field’s strength. From an industrial perspective, this shows that the miscibility of polymer blends can be influenced by factors other than temperature. An important advantage is that an electric field can be applied uniformly and instantaneously, whereas changes in temperature take time to propagate through materials. Thus, engineers may be able to instantly tune the miscibility of polymer blends using electric fields; a discovery that may lead to future technological advances in devices and materials whose properties would be quickly ‘’switched’’ through electricity.

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[1] Kriisa, A.; Roth, C. B. “Electric Fields Enhance Miscibility of Polystyrene/Poly(vinyl methyl ether) Blends.” J. Chem. Phys. 2014, 141, 134908.

2018-12-26

Making Biomolecular Crystals Soft and Self-healing — Just Add Polymer

Original paper: Hyperexpandable, self-healing macromolecular crystals with integrated polymer networks

In the world of engineering, crafting a material that meets the needs of your application is challenging. Often, a given material may only provide a handful of the required properties for that application. Instead, you may choose to combine two or more materials, forming a composite with all of your desired properties. In this week’s paper, Zhang and coworkers from the University of California at San Diego took a similar approach in the world of biology by combining a biomolecular crystal with a flexible polymer. The crystal provides structure to the composite and the polymer contributes to its flexibility and expandability. They showed that the composite could reversibly expand to nearly 570% of its original volume and unexpectedly found that it was self-healing.

: Figure 1. A schematic of sodium chloride showing the repeating structure characteristic of an atomic crystal. Sodium and chloride ions are purple and green, respectively. [Image courtesy of Wikipedia]

Before we dive into the meat of this paper, let’s look at the properties of crystalline materials. An example is sodium chloride, also known as table salt, shown in Figure 1. You may immediately notice that the sodium (purple) and chloride (green) ions are precisely spaced apart from each other in a repeating pattern: a single sodium is surrounded by exactly six chlorides. This predictable structure is called a lattice. Many objects can form lattices if the interactions between neighboring objects can stabilize them. In the case of table salt, the crystal lattice is formed because sodium cations and chloride anions are oppositely charged, electrostatically attracting each other.

: Figure 2. (A) The ferritin crystal structure. Each sphere is a single ferritin molecule. (B) A schematic of the close contact interactions between neighboring ferritin molecules, mediated by calcium (Ca²⁺). (C) A cutaway of a ferritin crystal demonstrating the porosity of the crystal. Images adapted from Zhang and coworkers’ original paper.

As mentioned earlier, biomolecules are also capable of forming crystals under right conditions. Ferritin is a hollow, spherical protein that is slightly negatively charged. As shown in Figure 2A, a given ferritin molecule is in direct contact with six other ferritin molecules, forming a lattice similar to table salt. You can see in Figure 2B that this lattice is held together by neighboring ferritins strongly interacting with calcium ions at the point where they come closest together. Because of the particular packing of the ferritin molecules caused by these interactions, a ferritin crystal is quite porous. Indicated by the arrow in Figure 2C, the pores between ferritin molecules are approximately 6 nanometers wide, large enough to allow water, salt solutions, and other liquids to soak into the ferritin crystal. In fact, the close contact interactions that stabilize the crystal are easily weakened when pure water is introduced into the pores, washing out calcium ions and dissolving the crystal. Instead, Zhang and coworkers wanted the crystal to expand but remain intact in water. Thus, they needed some kind of “glue.”

[wpvideo wlaJ8h1m]

Movie 1. A video of a hybrid crystal expanding when placed in pure water, followed by contraction after being placed in sodium chloride and calcium chloride solutions (by Zhang and coworkers).

They solved this problem by introducing a positively charged polymer into the pores of the crystal lattice. These polymers are known as hydrogels, as they can absorb a large amount of water and swell to many times their dry volume without dissolving away. Note that the hydrogel can’t prevent water from breaking the close contact interactions between the negatively charged ferritin molecules. Instead, the hydrogel holds the lattice in place to prevent it from dissolving due to electrostatic attraction between the hydrogel and each ferritin molecule. The close contact interactions can then be restored when a calcium salt solution is added. As shown in Movie 1, the authors demonstrated that the hybrid crystal could be expanded to nearly 570% its starting volume in the presence of pure water and returned to its original state when exposed to salt.

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Movie 2. A video showing several examples of ferritin crystal cracking and healing upon expansion (by Zhang and coworkers).

Aside from the reversible expandability of this hybrid crystal, Zhang and coworkers unexpectedly found that it can self-heal. If the crystal expands too quickly, it tends to crack, as shown in Movie 2. Despite this, the authors noticed that the cracks often healed scarlessly over time. Hydrogels cannot typically self-heal on their own, unless explicitly designed to do so. In the case of the hybrid crystal, the hydrogel and ferritin molecules work in concert to heal cracks. The hydrogel does not allow ferritin molecules on each side of the crack to drift far away. Over time, these ferritin molecules then reform the reversible close contact interactions, thereby healing the crystal. However, this process seems to be somewhat imperfect, as the crystals tend to crack in the same spots upon repeated contraction and expansion.

In short, Zhang and coworkers were able to create a self-healing material with the structure of crystalline matter and the expandability typical of polymers. Further, these hybrid materials were unexpectedly self-healing after cracking during too-rapid expansion. Many crystals formed from proteins and other biomolecules are porous like ferritin and are stabilized by similar close contact interactions. These crystals could also be infiltrated with hydrogel and similarly made expandable and resilient. As Zhang and coworkers have done, rationally combining the properties of various classes of matter will allow the engineering of novel materials for a myriad of applications and with useful, and quite unexpected, properties.

2018-08-23

“Precise” Polymers Promote Fast Proton Transport

Original paper: Self-Assemble Highly Ordered Acid Layers in Precisely Sulfonated Polyethylene Produce Efficient Proton Transport

Global climate change has necessitated the development of ways to harvest electricity from renewable sources, such as the wind and the sun. However, because the wind isn’t always blowing and the sun isn’t always shining, we need to store some of the harvested energy for later use. We can store this energy by converting it into fuels such as methanol and hydrogen, but we need a way to convert it back into electricity when it’s needed. One device that allows us to do this is the fuel cell.

508px-Solid_oxide_fuel_cell_protonic — Figure 1: Example proton exchange membrane fuel cell. Hydrogen gas mixed with water vapor enters on the left-hand side of the device and reacts to produce protons and electrons. The protons drift rightward through the center of the device to react with oxygen to produce water. [1]

Fuel cells are electrochemical devices that can convert a stored simple fuel, like hydrogen or methanol, directly into electricity. Because these fuels can be stored and easily converted near homes, vehicles, and businesses, fuel cells can be used to produce energy on demand. One type of fuel cell is the proton exchange membrane (PEM) fuel cell, which uses hydrogen and oxygen gases to make electricity and water. Hydrogen gas reacts at the anode (the negatively charged electrode) to lose its electrons, forming protons (denoted H+ in Figure 1). The electrons flow first through the fuel cell’s load (e.g. a home or business), powering it, and subsequently to the fuel cell’s cathode. The protons must drift across the proton exchange membrane to meet the electrons and oxygen to form water.

One of the greatest challenges of making PEM fuel cells is designing the membrane after which they are named. Engineers would love to have a membrane that transports protons quickly and efficiently. Although there are polymeric materials (e.g. Nafion) that can do this fairly well, there is still a need for faster transport. This week’s paper investigates the role of a polymer’s “precise” structure in facilitating fast proton transport.

Designing polymers, which are large molecules consisting of repeating units called monomers, with a given role relies on interspersing functional groups along the polymer chain. These functional groups are atoms that may contain some important features, such as electrical charge, necessary for the role of polymer. Under most synthetic schemes, the spacing between these functional groups is random. In the case of a typical proton exchange membrane, such as Nafion, negatively charged groups known as sulfonic acids love to interact with each other and with water present in the fuel cell, forming channels that facilitate proton transport, as shown in Figure 2.

Figure 2: Structure of (left panel) disordered and (right panel) ordered polymer materials. Black lines are the polymer backbone, yellow spheres are sulfonic acid groups, blue spheres are water molecules, and green spheres are water molecules with protons attached. Adapted from current work.

However, because the locations of these groups along the polymer chain are random, each chain cannot organize uniformly, forming disordered channels (Figure 2 left panel). In contrast, Edward Trigg and coauthors were able to attach these sulfonic acids at controlled, repeating locations along the polymer chain. Because the sulfonic acid groups are uniformly dispersed along the polymer (every twenty one carbon atoms to be exact), the chains can fold precisely in the same way, forming a uniformly layered structure and ordered channels filled with protons and water and lined with sulfonic acid (Figure 2 right panel).

The authors of this work first compared this material with Nafion, a widely used PEM polymer that is currently one of the best performing materials available. Like Nafion, this material readily absorbs water into the channels formed by the sulfonic acid groups from the air. The water widens the channels and transports protons more quickly as a result. At high levels of humidity, the new polymer performs as well as Nafion. However, as mentioned earlier, Nafion’s structure is amorphous and disordered because of the random placement of its functional groups (see Figure 2 left panel). Why then is a membrane structure so different from Nafion able to transport protons just as well?

The authors used simulation to answer this question. Specifically, they examined the dynamics of water contained in channels. Faster proton transport is facilitated by faster water dynamics. They compared the ordered and disordered versions of their polymer. The water dynamics in the ordered structure were faster than those in the disordered structure, suggesting slower proton transport in the disordered material. They attributed the slower water dynamics to the nonuniform size and the poor connectivity of the water channels in the disordered structure. These findings suggest that PEM membrane materials like Nafion can be further improved by creating ordered, rather than disordered, channels.

In short, Edward Trigg and his colleagues opened a new potential path to better performing fuel cells through the design of a well-ordered polymeric proton exchange membrane. They demonstrated that the ordered polymeric structure within their PEM leads to faster proton transport than in a disordered version of the structure. With further refinement of the synthesis techniques, membranes like these may yield faster proton transport than is currently achievable, leading to exceptional performance in PEM fuel cells. With better performance, PEM fuel cells may be more readily available to quickly convert stored energy for use in domestic and industrial applications when renewable sources are not immediately available.

[1] https://en.wikipedia.org/wiki/Fuel_cell