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)-platinum(II) catalyst in dichloromethane. Add 30 &#181;l of catalyst solution to the reagents from step 1.1 via pipette, stir to mix, and pour the solution into a custom-made (3 cm x 2 cm x 1 cm) rectangular polytetrafluoroethylene (PTFE) mold. Cover the mold loosely with a glass slide and place in heating oven at 60 &#176;C for 30 min while shaking periodically to remove bubbles during the first 15 min.</li>
	<li>Remove mold from heating oven and cool 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 sec.
	<ol>
		<li>Once the mixture has cooled, carefully remove elastomer from mold using a metal spatula and place on top of a PTFE sheet. Trim the edges of the LCE using a razor blade and cut the LCE along its length into three equal sized pieces (approx. 2.7 cm length and 0.5 cm width).</li>
	</ol>
	</li>
	<li>Hang each piece by one end to a horizontal rod and attach 10 paperclips (4.4 g) to the other end of the LCE. Hold the LCE in place using tape, and add paperclips one at a time in 10 min increments. Hang the LCE for 7 days at RT, noting changes in length and uniformity. Discard any sample that tears or breaks. Remove samples and store at ambient.</li>
</ol>
2. Preparation of Electrically Responsive Polysiloxane LCE Nanocomposites
<ol>
	<li>To prepare LCE nanocomposites with carbon black dispersed through the bulk of the sample, first repeat steps 1.1 - 1.4 above. Add 4.38 mg carbon black nanoparticles to the reaction solution containing reactive mesogen, crosslinker, and siloxane. Use a total of 5 paperclips instead of 10 for loading.</li>
	<li>In order to add additional carbon black nanoparticles to the LCE surface, prepare 1% w/v solution of carbon black nanoparticles in toluene. Sonicate for 20 min to disperse nanoparticles and then pour the dispersion into a Petri dish. Immerse the LCEs from step 2.1 in the nanoparticle dispersion for 6 hr.</li>
	<li>After 6 hr, use a pipette to withdraw the 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.</li>
</ol>
3. Preparation of Reversible Epoxy-based LCEs
<ol>
	<li>Mix 246.15 mg of 4,4&#39;-diglycidyloxybiphenyl31, 101 mg sebacic acid, 71.6 mg of hexadecanedioic acid, and 76 mg of carboxydecyl-terminated polydimethylsiloxane in a custom-made (3 cm x 2 cm x 1 cm) rectangular PTFE mold. Heat the samples by placing on a hotplate at 180 &#176;C.
	<ol>
		<li>Add 11.48 mg of (1,5,7-triazabicyclo[4.4.0] dec-5-ene) catalyst and stir using metal tweezers pre-heated to 180 &#176;C. Continue reacting until the mixture forms a gel, after approximately 20 min, and stir periodically to remove bubbles generated by the reaction.</li>
	</ol>
	</li>
	<li>Remove the PTFE boat from the hotplate and allow to cool to RT. Use a razor blade to separate the elastomer from the PTFE mold.</li>
	<li>Place two PTFE sheets in a polymer press at 180 &#176;C. Place the elastomer from step 3.2 between the PTFE sheets and compress the sample to a thickness of 0.3 - 0.5 mm. Continue heating at 180 &#176;C for 4 hr.</li>
	<li>Remove the sample and cool to RT. Cut the sample into rectangular pieces (approximately 2.5 cm length and 0.5 cm width). Hang the sample at one end using polyimide tape inside a heating oven. Attach 12 paperclips (8.88 g) to the free end of the sample. Set the temperature of the heating oven to 165 &#176;C O/N, or for 12 - 16 hr.</li>
	<li>Remove the elastomer from the heating oven and note the change in length. Heat the sample to 80 &#176;C on a hotplate to remove residual stress then cool back to RT.</li>
</ol>
4. Testing and Characterization of LCEs
<ol>
	<li>Measure reversible strain by heating the samples on a hotplate to 120 &#176;C and imaging with a camera. Note the initial sample length at RT, the sample length after heating to 120 &#176;C, and the length after cooling back to RT. LCEs should contract by approximately 30% and return to their initial length on cooling. See example images shown in Figure 1A and 1B.</li>
	<li>Analyze phase transition temperature and glass transition by differential scanning calorimetry (DSC) by cutting a small piece from each LCE and scanning from 0 &#176;C to 150 &#176;C at a heating/cooling rate of 10 &#176;C/min32,33.</li>
	<li>Quantify the degree of liquid crystal alignment by X-ray diffraction measurements. Place samples in an X-ray diffractometer with 2D imaging capabilities.33 See example diffraction images shown in Figure 2. 
	Note: The diffraction image should be anisotropic, reflecting the alignment of the LCE33. Polysiloxane LCEs are nematic and epoxy-based LCEs exhibit a smectic phase.</li>
	<li>Measure stiffness of LCE and change in length and width using dynamic mechanical analysis (DMA). Record length and stiffness changes as a function of temperature for LCEs and as a function of electrical potential for the LCE nanocomposites.
	<ol>
		<li>For thermo-mechanical measurements, use a razor blade to manually cut samples to dimensions of 2 cm x 0.3 cm and carefully fasten in between tension clamps. Apply a force of 1 mN to remove any slack.
		<ol>
			<li>Thermally equilibrate samples at 30 &#176;C followed by heating and cooling cycles at 5 &#176;C/min. Heat sample from 30 &#176;C up to 120 &#176;C. Changes in temperature produce changes in the length and width of the sample, which are recorded during the DMA measurement. See Figure 3A for thermomechanical measurements of an LCE sample.</li>
		</ol>
		</li>
		<li>For electromechanical measurements, manually cut LCE nanocomposite samples to dimensions of 2 cm x 0.3 cm and glue a copper wire at opposite ends of the LCE nanocomposites using a silver epoxy. Fasten the LCE nanocomposite using tension clamps with 1 mN tension.
		<ol>
			<li>Apply an electrical potential through the copper wires at a voltage ranging from 0 - 60 V, a frequency of 60 Hz, and an on/off pulse duration ranging from 0.1 sec - 30 sec.</li>
			<li>Record shape changes in response to the electrical potential. Apply a fixed force of 1 mN to remove slack. The change in position of the clamps corresponds to shape changes in the sample. See Figure 3B for electromechanical measurements of an LCE nanocomposite sample.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Active Cell Culture through Electrical Stimulation of LCE Nanocomposites
<ol>
	<li>Treat one surface of LCE nanocomposites under oxygen plasma for 30 sec. Spin cast 300 &#181;l of a solution of polystyrene in toluene (1% w/v) at 3,300 rpm for 1 min on top of the plasma cleaned surface. Dry the elastomer under vacuum for 12 hr to remove toluene, and treat the polystyrene-coated surface of the LCE nanocomposite using oxygen plasma for 30 sec.</li>
	<li>Place LCE nanocomposites in 70% ethanol solution for 30 min to sterilize the surface.
	<ol>
		<li>Wash the LCE nanocomposite with phosphate buffered saline and transfer the LCE to a dry Petri dish with the polystyrene-coated side facing up. Coat the entire surface of the LCE by immersing in 5 ml of a rat tail collagen type I solution (50 &#181;g/ml in 0.02 N acetic acid). Incubate the LCE nanocomposite at 37 &#176;C and 5% CO2 for at least 30 min.</li>
	</ol>
	</li>
	<li>Isolate neonatal rat ventricular cardiomyocytes and suspend in high-serum plating media as previously reported11.
	<ol>
		<li>Plate cells on top of LCE substrates described as above at a density of 100,000 - 600,000 cells/cm2. Around 24 hr later, transfer the cells to low serum maintenance media (DMEM, 18.5% M199, 5% HS, 1% FBS and antibiotics). Allow cardiomyocytes to attach and proliferate on the surface of the LCE for 4 days.</li>
	</ol>
	</li>
	<li>Design and fabricate a custom vessel using a 3-D printer and using the schematic of the vessel shown in Figure 4 using manufacturer&#39;s protocol. 
	Note: The 3D printed vessel is a rectangular container with outer dimensions of 60 mm x 40 mm x 20 mm and inner dimensions of 50 mm x 30 mm x 15 mm. On two lateral surfaces, there are two sets of 5 mm holes used for inserting conductive carbon rods. Notches around the holes and up to the top edge of the container allow for placing a rectangular plastic piece (dimensions of 52.5 mm x 12 mm x 4 mm) across the vessel to hold the LCE in place at both ends. The distance between the holes is 3 mm on one side of the vessel, and notches are located around the holes as shown in Figure 4. This is designed to be compatible with the size of the LCE substrates described above. Conductive carbon rods are obtained through a commercial supplier as shown in the Materials Supplement.
	<ol>
		<li>Insert carbon rods through the holes across the vessels and hold in place using medical grade silicone adhesive. Cure the adhesive O/N.</li>
	</ol>
	</li>
	<li>Transfer LCE nanocomposites with cardiomyocytes to a custom 3D-printed vessel filled with cell culture maintenance media and with parallel conductive carbon rods connected to an electric source. Place the LCE across the carbon rods and fix on one end to ensure electrical contact.
	<ol>
		<li>Insert a rectangular plastic piece through the notches in the 3-D vessel to hold the LCE in place at one or both ends, but place this loosely over the LCE sample. Electrically stimulate LCE through the application of a 40 V AC electrical potential with a 5 sec on/off time for a total of 24 hr.</li>
	</ol>
	</li>
	<li>Stain the membrane of living cells using Calcein AM as described previously11.</li>
	<li>For nuclei staining, cover the cells with DAPI-containing mounting medium before imaging under an inverted fluorescent microscope. Use ImageJ to count the number of living cells and determine the angle of cell alignment using the best fit function.</li>
</ol>
6. Active Cell Culture with LCEs Using Direct Heating
<ol>
	<li>Repeat steps 5.1 - 5.3 above using a pure LCE without carbon black nanoparticles added. This procedure is also described in detail in a prior publication.11</li>
	<li>Transfer the LCE with cardiomyocytes to a Petri dish with cell culture maintenance media and a 0.5&#34; x 2&#34; Kapton resistive heater. Supply heat to the LCE by turning on the resistive heater with heating power of 12 W. Cycle on and off the heat with 5 sec intervals for at least 24 hr.</li>
</ol>

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<li>Krause, S., Dersch, R., Wendorff, J. H., Finkelmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photocrosslinkable+Liquid+Crystal+Main-Chain+Polymers%3A+Thin+Films+and+Electrospinning%5BTitle%5D)+AND+%22Macromol.+Rapid+Commun%22%5BJournal%5D)">Photocrosslinkable Liquid Crystal Main-Chain Polymers: Thin Films and Electrospinning.</a> Macromol. Rapid Commun. 28 (21), 2062-2068 (2007).</li>
<li>Liu, D., Broer, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+controlled+friction+at+a+liquid+crystal+polymer+coating+with+switchable+patterning%5BTitle%5D)+AND+%22Soft+Matter%22%5BJournal%5D)">Light controlled friction at a liquid crystal polymer coating with switchable patterning.</a> Soft Matter. 10 (40), 7952-7958 (2014).</li>
</ol>

The authors have nothing to disclose.

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 other hand, waiting too long before loading results in poor alignment of the final LCE. Another important step is the loading of the LCEs. Loading too much weight or loading the weight too quickly can result in the sample breaking or falling. While the protocol gave a specific load for aligning an LCE, the load can be adjusted for larger or thicker LCE samples. An important limitation of the procedure is the continuous, large-scale production of aligned LCEs. Each LCE substrate is aligned by hanging and loading. 
LCE nanocomposites are produced by introducing conductive carbon black nanoparticles in two separate steps. This is important for achieving materials that are conductive and exhibit a stable electro-mechanical response for several days of continuous electrical stimulation. Prior work explored only surface infiltration of carbon black nanoparticles, but this produced LCE nanocomposites with an electromechanical response that degraded quickly.21 LCE nanocomposites are prepared and aligned similarly to the pure LCEs, but are more delicate than the pure LCEs due to a lower network content. A reduced load is used in the preparation of LCE nanocomposites. 
In the case of epoxy-based LCEs, alignment is more straightforward. At elevated temperatures, epoxy-LCEs exhibit reversible network crosslinking. Thus, the network synthesis and alignment can be carried out in separate steps. If done properly, the LCE will be significantly longer along the loading direction after alignment. The alignment in these materials can also be erased by heating the samples above 160 &#176;C, and the materials can be re-shaped or re-aligned by applying a load at 160 &#176;C or higher temperatures. 
Implementation of LCEs for active cell culture presents a number of practical challenges. First, the LCEs must be sterilized. This is typically done using ethanol or exposing the sample to UV light. The surface of the LCEs is of low surface-energy, and therefore the LCE surface has to be modified to promote cellular attachment. One approach presented here is to deposit a thin layer of polystyrene by spin casting followed by rat tail collagen I. UV-ozone treatment is used to promote adhesion between the layers and remove organic contaminants. A common problem is ensuring uniformity of the top LCE surface in the case of the LCE nanocomposites. The surface becomes significantly rougher during the preparation of LCE nanocomposites. The surface can be cleaned by immersing in solvents to remove adhered nanoparticle and gently rubbing the surface with a spatula or cotton swab. Scotch tape is also an effective method to remove carbon black aggregates on the surface but has been found to be detrimental to cellular attachment and proliferation. 
Liquid crystal elastomers are unique in that they exhibit a fully reversible shape response to a variety of external stimuli. One potential application is for dynamic cell culture, and the fabrication of 3-D LCE constructs34 or electrospun fibers35,36 could extend this work to scaffolds for cellular growth and engineering. LCEs also have applications in sensing, responsive surfaces,37 and robotics.

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><tbody><tr><td>4-methoxyphenyl 4-(3-butenyloxy)benzoate</td><td>TCI America</td><td>M2106</td><td>Reactive mesogen</td></tr><tr><td>poly(methylhydrosiloxane)</td><td>Gelest</td><td>HMS-993</td><td>Reactive polysiloxane</td></tr><tr><td>1,4-di(10-undecenyloxybenzene)</td><td>N/A</td><td>N/A</td><td>see: Ali, S. A., Al-Muallem, H. A., Rahman, S. U. &amp;amp; Saeed, M. T. Bis-isoxazolidines: A new class of corrosion inhibitors of mild steel in acidic media. Corrosion Science. 50 (11), 3070&amp;ndash;3077, doi:10.1016/j.corsci.2008.08.011 (2008)</td></tr><tr><td>(dichloro(1,5-cyclooctadiene)-platinum(II)&amp;nbsp;</td><td>Sigma Aldrich</td><td>244937</td><td>Pt catalyst</td></tr><tr><td>PTFE mold</td><td>N/A</td><td>N/A</td><td>fabricated at Rice machine shop</td></tr><tr><td>carbon black nanoparticles</td><td>Cabot</td><td>VULCAN&amp;reg;&amp;nbsp;XC72R</td><td>used in the synthesis of LCE nanocomposites</td></tr><tr><td>polystyrene</td><td>Sigma Aldrich</td><td>331651</td><td>linear polystyrene&amp;nbsp;</td></tr><tr><td>4,4&#039;-diglycidyloxybiphenyl</td><td>N/A</td><td>N/A</td><td>see:&amp;nbsp; Giamberjni, M., Amendola, E. &amp;amp; Carfagna, C. Liquid Crystalline Epoxy Thermosets. Molecular Crystals and Liquid Crystals Science and Technology. Section A. Molecular Crystals and Liquid Crystals. 266 (1), 9&amp;ndash;22, doi:10.1080/10587259508033628 (1995).</td></tr><tr><td>sebacic acid</td><td>Sigma Aldrich</td><td>283258</td><td>C8 linking group for epoxy-LCE synthesis</td></tr><tr><td>hexadecanedioic acid</td><td>Sigma Aldrich</td><td>177504</td><td>C16 linking group for epoxy-LCE synthesis</td></tr><tr><td>carboxydecyl-terminated polydimethylsiloxane</td><td>Gelest</td><td>DMS-B12</td><td>Siloxane linking group for epoxy-LCE synthesis</td></tr><tr><td>1,5,7-triazabicyclo[4.4.0] dec-5-ene</td><td>Sigma Aldrich</td><td>345571</td><td>catalyst for reversible LCEs</td></tr><tr><td>carbon rods</td><td>Ladd Research&amp;nbsp;</td><td>30250</td><td>used in cell culture experiments</td></tr><tr><td>medical grade silicone adhesive</td><td>Silbione</td><td>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 transition temperature the contraction is a maximum. The sample will also become optically clear above the isotropic transition temperature, while some haziness is observed for even perfectly aligned LCEs below the isotropization temperature. LCE nanocomposites will also exhibit shape-changes in response to heating, as shown in Figure 1C and 1D. LCE nanocomposites can be heated either on a hotplate (not shown) or by applying an electrical potential across the sample. The sample will contract when the voltage is turned on. If little or no shape change is observed, this is likely a reflection of poor alignment of the liquid crystal director and the synthesis of the LCE should be repeated. As a check, the birefringence of pure LCE samples can be tested using a polarized optical microscope. Aligned samples should exhibit maximum birefringence when oriented at 45 degrees relative to crossed polararizers and should appear dark when oriented along or perpendicular to either the analyzer or polarizer.
Direct information on liquid crystal ordering can be obtained through X-ray diffraction33. As shown in Figure 2, an aligned LCE exhibits anisotropic liquid crystal diffraction peaks due to alignment of the mesogens. Peaks at wide angles are due to intermolecular spacing along the width of the molecule. In the case of epoxy-LCEs with smectic ordering, additional peaks are observed at low angles reflecting the smectic layer spacing. In all samples, the diffraction is anisotropic in the liquid crystal phase and disordered above the isotropization temperature. As shown in Figure 2, the siloxane LCE will exhibit nematic XRD peaks along the alignment direction while the epoxy-LCEs are main chain LCEs and exhibit wide-angle XRD peaks perpendicular to the alignment direction and low-angle peaks along the alignment direction corresponding to the smectic layer spacing.
Differential scanning calorimetry (DSC) provides phase transitions in the LCEs32. Silicone based LCEs have a glass-transition temperature (Tg) well below RT and below the resolution of our DSC, but a clear peak is observed near 90 &#176;C corresponding to the nematic-to-isotropic transition. A similar peak is observed in the LCE nanocomposites. In the case of the epoxy-based LCEs presented, a glass transition temperature near 20 &#176;C is observed and a smectic-to-isotropic transition temperature near 60 &#176;C. It is important to note that the glass and isotropic transition temperature can be modified by changing the composition of the elastomers and the linking group.
Dynamic mechanical analysis provides a quantitative measure of LCE shape change as a function of temperature and, in the case of LCE nanocomposites, as a function of applied voltage (Figure 3). The sample contracts with increasing temperature, up to the transition to the isotropic phase. In the case of a pulsed electrical voltage, LCE nanocomposites exhibit cyclic strain in phase with the electrical potential.
Active cell culture experiments are performed using a custom, 3-D printed vessel (Figure 4). The through-holes allow for placement of conductive carbon rods, and the vessel is filled with cell culture media. An example of cell attachment on an LCE nanocomposite surface is shown in Figure 5 for a non-stimulated sample after 3 days of culturing. Cardiomyocytes show good attachment and viability.
<img alt="Figure 1" src="/files/ftp_upload/53688/53688fig1.jpg" /> 
Figure 1. Shape-response of LCEs and LCE Nanocomposites. LCEs contract and elongate reversibly when heated from RT (A) to above the nematic-to-isotropic transition temperature, roughly 80 &#176;C (B). LCE nanocomposites contract on the application of an electrical potential (C and D). The voltage applied is a 40 V AC signal. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53688/53688fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53688/53688fig2.jpg" /> 
Figure 2. 2-D X-ray diffraction from aligned LCEs. Aligned LCEs exhibit anisotropic diffraction patterns due to liquid crystal alignment. The alignment direction is in the vertical direction as indicated by the white arrow in frames (B and D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53688/53688fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53688/53688fig3.jpg" /> 
Figure 3. Dynamic Mechanical Analysis (DMA) of shape-responsiveness in LCEs. (A) thermomechanical measurements of a siloxane LCE for 4 heating and cooling cycles. The maximum contraction is 35% along the sample length. (B) Electromechanical strain measured in an LCE nanocomposite with a 40 V AC electrical potential turned on and off every 15 sec. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53688/53688fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53688/53688fig4.jpg" /> 
Figure 4. Schematic of custom vessel for active cell culture. Through holes allow for insertion of conductive carbon rods, which are secured and sealed at the edges using a silicone, bio-grade adhesive. The two plates are used to secure the LCE on one or both ends. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53688/53688fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53688/53688fig5.jpg" /> 
Figure 5. Fluorescence analysis of cardiomyocytes on an LCE nanocomposite surface. Cells are stained with Calcein AM, and live cells appear green. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53688/53688fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.6K Views.  Rice University. 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.

Video: Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

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 ...

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline ...

Chemistry

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 ...

Preparation of Liquid Crystal Networks for Macroscopic Oscillatory Motion Induced by Light

A strategy based on doped liquid crystalline networks is described to create mechanical self-sustained oscillations of plastic films under continuous light irradiation. The photo-excitation of dopants that can quickly dissipate light into heat, coupled with anisotropic thermal expansion and self-shadowing of the film, gives rise to the self-sustained deformation. The oscillations observed are influenced by the dimensions and the modulus of the film, and by the directionality and intensity of the light. The system developed offers applications in energy conversion and harvesting for soft-robotics and automated systems.
The general method described here consists of creating free-standing liquid crystalline films and characterizing the mechanical and thermal effects observed. The molecular alignment is achieved using alignment layers (rubbed polyimide), commonly used in the display manufacturing industry. To obtain actuators with large deformation, the mesogens are aligned and polymerized in a splay/bend configuration, i.e., with the director of the liquid crystals (LCs) going gradually from planar to homeotropic through the film thickness. Upon irradiation, the mechanical and thermal oscillations obtained are monitored with a high-speed camera. The results are further quantified by image analysis using an image processing program.

The goal of the protocol is to create liquid crystalline polymer films that can mechanically oscillate under continuous light irradiation. We describe in great detail the conception of free-standing films, from the liquid crystal alignment method to the photo-actuation. The experimental protocol applied to prepare this material is broadly applicable.

A strategy based on doped liquid crystalline networks is described to create mechanical self-sustained oscillations of plastic films under continuous ...

Engineering

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Microfluidic Preparation of Liquid Crystalline Elastomer Actuators

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation usually consists in the formation of droplets containing low molar mass liquid crystals at elevated temperatures. Subsequently, these particle precursors are oriented in the flow field of the capillary and solidified by a crosslinking polymerization, which produces the final actuating particles. The optimization of the process is necessary to obtain the actuating particles and the proper variation of the process parameters (temperature and flow rate) and allows variations of size and shape (from oblate to strongly prolate morphologies) as well as the magnitude of actuation. In addition, it is possible to vary the type of actuation from elongation to contraction depending on the director profile induced to the droplets during the flow in the capillary, which again depends on the microfluidic process and its parameters. Furthermore, particles of more complex shapes, like core-shell structures or Janus particles, can be prepared by adjusting the setup. By the variation of the chemical structure and the mode of crosslinking (solidification) of the liquid crystalline elastomer, it is also possible to prepare actuating particles triggered by heat or UV-vis irradiation.

This article describes the microfluidic process and parameters to prepare actuating particles from liquid crystalline elastomers. This process allows the preparation of actuating particles and the variation of their size and shape (from oblate to strongly prolate, core-shell, and Janus morphologies) as well as the magnitude of actuation.

This paper focuses on the microfluidic process (and its parameters) to prepare actuating particles from liquid crystalline elastomers. The preparation ...

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 ...

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 ...

Preparation of Monodomain Liquid Crystal Elastomers and Liquid Crystal Elastomer Nanocomposites

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