Name: preparation of monodomain liquid crystal elastomers and liquid crystal elastomer nanocomposites
Uploaded: 2016-02-06T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites at JoVE.com

This work was supported by the National Career Foundation (CBET-1336073 to RV), the ACS Petroleum Research Fund (52345-DN17 to RV), the American Heart Association (BGIA to JGJ), the National Science Foundation (CAREER CBET-1055942 to JGJ), the National Institutes of Health/ National Heart, Lung and Blood Institute (1R21HL110330 to JGJ), Louis and Peaches Owen and Texas Children&#39;s Hospital.

1. Synthesis of Aligned Polysiloxane LCEs
<ol>
	<li>Combine 166.23 mg of reactive mesogen (4-methoxyphenyl 4-(3-butenyloxy)benzoate), 40 mg of poly(hydromethylsiloxane), and 12.8 mg of crosslinker (1,4-di(10-undecenyloxybenzene)30 with 0.6 ml of anhydrous toluene in a small vial (approximately 13 mm in diameter and 100 mm in length) charged with a stirring bar. Stir the solution at 35 &#176;C for 25 min to dissolve.</li>
	<li>In a separate vial, prepare a solution of 1 wt % dichloro(1,5-cyclooctadiene)-plati...

<ol>
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In order to produce monodomain LCEs, the LCEs need to be uniaxially loaded during crosslinking. This is challenging in practice because the LCE is loaded when it is only partially crosslinked, and therefore is not mechanically robust and can easily break or tear. The procedure described above (steps 1.1 - 1.4) can produce monodomain LCEs consistently. One critical step is the removal of the LCE from the PTFE mold for loading at the appropriate time. If the LCE is removed too quickly, it will easily break or tear. On the...

Materials that can exhibit fast, reversible, and programmable shape changes are desirable for a number of emerging applications1-9. Shape-responsive stents can assist with wound healing and treatment7. Artificial robots can aid in exploration or in carrying out tasks in environments that are harmful or unsafe for humans10. Shape-responsive elastomers are desirable for use in active cell culture, in which cells are cultured in an active environment.11-14 Other applications include packaging, sensing, and drug delivery.
Liquid crystal elastomers (LCE) are polymer networks with liquid crystal ordering15-20. LCEs are made by combining a flexible polymer network with liquid crystal molecules known as mesogens. The responsiveness of LCEs is derived from the coupling of liquid crystal order to strains in the polymeric network, and stimuli that influence the ordering of mesogens will generate network strains, and vice versa. In order to achieve large and reversible shape-changes in the absence of an external load, the mesogens must be aligned in a single direction in the LCE. A common practical challenge in working with LCEs is generating monodomain LCEs. Another challenge is generating shape changes in response to stimuli other than direct heating. This can be done through the addition of nanoparticles or dyes to LCE networks21-28.
Here, we demonstrate the preparation of monodomain LCEs and LCE nanocomposites. First, we demonstrate the preparation of monodomain LCEs using the two-step method first reported by Kupfer et al.29 This is still the most popular and well-known method for preparing monodomain LCEs, but achieving uniform alignment and consistency between samples can be challenging. We demonstrate an approach that can be easily implemented using standard lab equipment, including full details on sampling handling and preparation. Next, we show how conductive carbon black nanoparticles can be added to LCEs to produce conductive, electrically responsive LCEs. We then demonstrate the synthesis and alignment of epoxy-based LCEs. These materials exhibit exchangeable network bonds and can be aligned by heating to elevated temperatures and applying a uniform load. All LCEs are characterized through macroscopic sample imaging, X-ray diffraction measurements, and dynamic mechanical analysis. Finally, we demonstrate one potential application of LCEs as shape-responsive substrates for active cell culture.

<table border="0" cellpadding="0" cellspacing="0" width="859">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:379px;">4-methoxyphenyl 4-(3-butenyloxy)benzoate</td>
			<td style="width:167px;">TCI America</td>
			<td style="width:145px;">M2106</td>
			<td style="width:168px;">Reactive mesogen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">poly(methylhydrosiloxane)</td>
			<td style="width:167px;">Gelest</td>
			<td style="width:145px;">HMS-993</td>
			<td>Reactive polysiloxane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,4-di(10-undecenyloxybenzene)</td>
			<td style="width:167px;">N/A</td>
			<td style="width:145px;">N/A</td>
			<td>see: Ali, S. A., Al-Muallem, H. A., Rahman, S. U. &#38; Saeed, M. T. Bis-isoxazolidines: A new class of corrosion inhibitors of mild steel in acidic media. Corrosion Science 50 (11), 3070&#8211;3077, doi:10.1016/j.corsci.2008.08.011 (2008)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(dichloro(1,5-cyclooctadiene)-platinum(II)&#160;</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">244937</td>
			<td>Pt catalyst</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">PTFE mold</td>
			<td style="width:167px;">N/A</td>
			<td>N/A</td>
			<td>fabricated at Rice machine shop</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">carbon black nanoparticles</td>
			<td style="width:167px;">Cabot</td>
			<td>VULCAN&#174;&#160;XC72R</td>
			<td>used in the synthesis of LCE nanocomposites</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">polystyrene</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td>331651</td>
			<td>linear polystyrene&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">4,4&#39;-diglycidyloxybiphenyl</td>
			<td style="width:167px;">N/A</td>
			<td style="width:145px;">N/A</td>
			<td>see:&#160; Giamberjni, M., Amendola, E. &#38; Carfagna, C. Liquid Crystalline Epoxy Thermosets. Molecular Crystals and Liquid Crystals Science and Technology. Section A. Molecular Crystals and Liquid Crystals 266 (1), 9&#8211;22, doi:10.1080/10587259508033628 (1995).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">sebacic acid</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">283258</td>
			<td>C8 linking group for epoxy-LCE synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">hexadecanedioic acid</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">177504</td>
			<td>C16 linking group for epoxy-LCE synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">carboxydecyl-terminated polydimethylsiloxane</td>
			<td style="width:167px;">Gelest</td>
			<td style="width:145px;">DMS-B12</td>
			<td>Siloxane linking group for epoxy-LCE synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">1,5,7-triazabicyclo[4.4.0] dec-5-ene</td>
			<td style="width:167px;">Sigma Aldrich</td>
			<td style="width:145px;">345571</td>
			<td>catalyst for reversible LCEs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">carbon rods</td>
			<td style="width:167px;">Ladd Research&#160;</td>
			<td style="width:145px;">30250</td>
			<td>used in cell culture experiments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">medical grade silicone adhesive</td>
			<td style="width:167px;">Silbione</td>
			<td style="width:145px;">MED ADH 4100 RTV</td>
			<td>used to adhere carbon rods to vessel</td>
		</tr>
	</tbody>
</table>

preparation of monodomain liquid crystal elastomers and liquid crystal elastomer nanocomposites

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and as soft robots. Here, we demonstrate the preparation of shape-responsive liquid crystal elastomers (LCEs) and LCE nanocomposites along with characterization of their shape-responsiveness, mechanical properties, and microstructure. Two types of LCEs &#8212; polysiloxane-based and epoxy-based &#8212; are synthesized, aligned, and characterized. Polysiloxane-based LCEs are prepared through two crosslinking steps, the second under an applied load, resulting in monodomain LCEs. Polysiloxane LCE nanocomposites are prepared through the addition of conductive carbon black nanoparticles, both throughout the bulk of the LCE and to the LCE surface. Epoxy-based LCEs are prepared through a reversible esterification reaction. Epoxy-based LCEs are aligned through the application of a uniaxial load at elevated (160 &#176;C) temperatures. Aligned LCEs and LCE nanocomposites are characterized with respect to reversible strain, mechanical stiffness, and liquid crystal ordering using a combination of imaging, two-dimensional X-ray diffraction measurements, differential scanning calorimetry, and dynamic mechanical analysis. LCEs and LCE nanocomposites can be stimulated with heat and/or electrical potential to controllably generate strains in cell culture media, and we demonstrate the application of LCEs as shape-responsive substrates for cell culture using a custom-made apparatus.

Monodomain LCEs are shape-responsive due to coupling of network conformation with liquid crystal ordering. Heating LCEs results in a decrease in the liquid crystal order parameter, producing a contraction of the polymeric network along the primary alignment direction. This is easily visualized by placing an LCE on a hotplate, as shown in Figure 1A and 1B. In heating up from RT, the LCE contracts along the length of the sample, and above the isotropic tran...

Watch this Scientific Journal Video about Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites at JoVE.com

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

We demonstrate the preparation of siloxane-based and epoxy-based liquid crystal elastomers (LCEs) and LCE nanocomposites. The LCEs are characterized with respect to reversible strain, liquid crystal ordering, and stiffness. As a potential application, we demonstrate their use as shape-responsive substrates in a custom device for active cell culture.

LCEs are shape-responsive materials with fully reversible shape change and potential applications in medicine, tissue engineering, artificial muscles, and ...

The overall goal of this procedure is to prepare aligned, shape responsive, liquid crystal elastomers and nanocomposites. So liquid crystal elastomers are stimuli responsive materials that change shape in response to stimuli in the environment. And they can be used in a variety of applications, such as tissue engineering, where we know cells respond to stresses and strains in the environment.In this procedure that we're gonna demonstrate, we rely on materials that are low cost. The method we demonstrate is reliable and it can be implemented in any lab without the need for expensive or specialized equipment. These materials can be applied to the development of cardiac cell sheets, where we know that stresses and strains can affect the differentiation, the maturation and the function of cardiac muscle cells.Visual demonstration of this procedure is important because the process can be difficult to learn and the samples especially delicate when we're hanging, loading and crosslinking it. So, demonstrating the procedure will be Hojin Kim, Bohan Zhu and Huiying Chen, students from our laboratory and the Jeff Jaycot Laboratory. Begin by combining 166.23 milligrams of a reactive mesogen, 40 milligrams of siloxane and 12.8 milligrams of crosslinker with 0.6 milliliters of anhydrous toluene in a small vial charged with a stirring bar.Stir the solution at 35 degrees Celsius for 25 minutes for the reagents to completely dissolve. In a separate vial, prepare a one weight percent solution of a platinum catalyst in dichloromethane. Then, add 30 microliters of the catalyst solution to the previously prepared solution and stir the mixture with the stir bar.Next, pour the solution into a custom made, rectangular polytetrafluoroethylene mold. Cover the mold loosely with a glass slide and place it into an oven at 60 degrees Celsius for 30 minutes. Shake the mold periodically to remove bubbles during the first 15 minutes.Remove the mold from the oven and cool it quickly with liquid nitrogen by pouring liquid nitrogen into a small container and contacting the bottom of the PTFE mold with the liquid nitrogen for 2 seconds. Once the mixture has cooled, set down a PTFE sheet and use a metal spatula to carefully remove the elastomer from the mold and place it on top of the sheet. Next, use a razor blade to trim the edges of the liquid crystal elastomer and cut it along its length into three equal sized pieces.Hang each piece to a horizontal rod and hold the elastomer in place using tape. Add one paper clip at a time to the end of the elastomers in 10 minute increments. Allow them to hang for seven days at room temperature and note any changes in length or uniformity.Discard any sample that tears or breaks. To prepare an electrically responsive, liquid crystal elastomer nanocomposite, add 4.38 milligrams of carbon black nanoparticles to the reaction solution containing the reactive mesogen, the crosslinker and siloxane. During the hanging step of the fabrication, use a total of five paperclips instead of 10 to load the sample.Add additional carbon black nanoparticles to the liquid crystal elastomer surface. Prepare a 1%weight per volume solution of carbon black nanoparticles in toluene. Sonicate the solution for 20 minutes to disperse carbon nanoparticles and then pour the dispersion into a petri dish.Then immerse the liquid crystal elastomers into the nanoparticle dispersion for six hours. Once removed from the solution, use the pipette to withdraw excess solution from the petri dish and allow the elastomer to dry in air. Gently clean excess carbon particles on the surface using tape or a cotton swab.To prepare reversible epoxy-based liquid crystal elastomers, mix 246.15 milligrams of four four prime diglycidyl diloxy biphenyl, 101 milligrams of sebacic acid, 71.6 milligrams of hexadecanedioic acid and 76 milligrams of carboxydecyl terminated PDMS in the rectangular PTFE mold. Heat the samples by placing them on a hotplate at 180 degrees Celsius. Then, add 11.48 milligrams of catalyst and use metal tweezers preheated to 180 degrees Celsius to stir the mixture.Continue to stir the mixture periodically to remove bubbles generated by the reaction. Continue reacting the mixture for approximately 20 minutes. Once gelled, remove the PTFE boat from the hotplate and allow the mixture to cool to room temperature.Then, use a razor blade to separate the elastomer from the PTFE mold. Next, place two PTFE sheets in a polymer press at 180 degrees Celsius. Place the elastomer between the PTFE sheets and compress the sample to a thickness of 0.3 to 0.5 millimeters.Continue heating the elastomer at 180 degrees Celsius for four hours. After four hours, remove the sample and cool it to room temperature. Then, cut the sample into rectangular pieces, 2.5 centimeters in length and 0.5 centimeters in width.Hang the sample at one end using polyimide tape inside a heating oven. Attach 12 paper clips with a total weight of 8.88 grams to the free end of the sample. Then set the temperature of the oven to 165 degrees Celsius and heat the samples overnight.Remove the elastomer from the oven and note the change in length. Next, heat the sample to 80 degrees Celsius on a hotplate to remove residual stress, then cool back to room temperature. Measure reversible strain by placing the samples onto a hotplate and heating the plate back up to 120 degrees Celsius.Image the sample at room temperature after heating to 120 degrees Celsius and after cooling back to room temperature. Use the images to measure the sample length at each step. For thermomechanical measurements, use a razor blade to manually cut samples to dimensions of two centimeters by 0.3 centimeters and carefully fasten them, one at a time, in between the tension clamps.Apply a force of one millinewton to the sample in order to first remove any slack. Then, thermally equilibrate samples at 30 degrees Celsius, followed by heating and cooling cycles. Heat the sample at five degrees Celsius per minute from 30 degrees Celsius up to 120 degrees Celsius.Record the changes in the length and width of the sample during the temperature change. Treat one surface of liquid crystal elastomer nanocomposites under oxygen plasma for 30 seconds. Then, spin cast 300 microliters of a 1%weight per volume solution of polystyrene in toluene at 3, 300 rpm for one minute on top of the plasma cleaned surface.Dry the elastomer under vacuum for 12 hours to remove toluene. And then treat the polystyrene coated surface of the LCE nanocomposite using oxygen plasma for 30 seconds. Sterilize the plasma treated elastomer nanocomposites in 70%ethanol for 30 minutes.And then wash them with phosphate buffered saline. Transfer the washed elastomer nanocomposites to a dry petri dish with a polystyrene coated side facing up. Next, add five milliliters of type one collagen solution to a dish and coat the entire surface of the samples by immersing them in the collagen solution.Then, cover the samples and incubate them at 37 degrees Celsius and 5%carbon dioxide for at least 30 minutes. Plate isolated neonatal rat ventricular cardiomyocytes on top of the substrates at a density of 100, 000 to 600, 000 cells per square centimeter. Culture the cells as described in the accompanying text protocol.Once the cells have firmly attached, transfer the seeded liquid crystal elastomer nanocomposites to a custom, 3D printed vessel. Next, insert a rectangular plastic piece through the notches in the 3D vessel to hold the liquid crystal elastomer in place at one or both ends. Place the sample across the carbon rods and fix it on one end to ensure a good electrical contact.Ensure that the vessel is filled with cell culture maintenance media and is equipped with parallel conductive carbon rods connected to an electric source. Electrically stimulate liquid crystal elastomers with a 40 volt A/C electrical potential, which has a five second on off time for a total of 24 hours. Liquid crystal elastomers contract and elongate reversibly when heated from room temperature to above the nematic to isotropic transition temperature, roughly 80 degrees Celsius.Dynamic mechanical analysis provides a quantitative measure of the liquid crystal elastomer shape change as a function of temperature. The sample described by the graph shown here was heated and cooled through four cycles. Once heated, the samples display about a 35%shortening in the direction it was hung during fabrication.The sample also expands 25 to 30%in the perpendicular direction. Liquid crystal elastomer nanocomposites contract on the application of an electrical potential. When exposed to 40 volt A/C, the liquid crystal elastomer nanocomposites shorten about 22 1/2%in the direction they were hung during fabrication.Here, the samples were exposed to an on off cycle every 15 seconds. So once the procedure is mastered, it can be completed in two hours and the entire synthesis will be complete after one week after hanging the sample. While attempting this procedure, it is important that you remember to hang the LC with correct amount of weight.Otherwise, you may not align the sample properly or break it during the fabrication. Following this procedure, you can add other types of additives to LCE like carbon nanotubes to understand what stimuli response it may have. We hope that this research will pave the way for biologists and bioengineers to shape responsive substrates to investigate the effect of stress and strain on cell growth, cell alignment and cell behavior.So after watching this video, you should have a good understanding of how to make aligned, shape responsive liquid crystal elastomers and liquid crystal elastomer nanocomposites.

preparation-monodomain-liquid-crystal-elastomers-liquid-crystal

Research

JoVE Journal

Bioengineering

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

In situ TEM of Biological Assemblies in Liquid

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an additional application for these instruments- viewing viral assemblies in a liquid environment. This exciting and novel method of visualizing biological structures utilizes a recently developed microfluidic-based specimen holder. Our video article demonstrates how to assemble and use a microfluidic holder to image liquid specimens within a TEM. In particular, we use simian rotavirus double-layered particles (DLPs) as our model system. We also describe steps to coat the surface of the liquid chamber with affinity biofilms that tether DLPs to the viewing window. This permits us to image assemblies in a manner that is suitable for 3D structure determination. Thus, we present a first glimpse of subviral particles in a native liquid environment.

In situ TEM of Biological Assemblies in Liquid

Here we describe a procedure to image viral complexes in liquid at nanometer resolution using a transmission electron microscope.

Researchers regularly use Transmission Electron Microscopes (TEMs) to examine biological entities and to assess new materials. Here, we describe an ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...