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 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 — polysiloxane-based and epoxy-based — 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 °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.
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.
1. Synthesis of Aligned Polysiloxane LCEs
2. Preparation of Electrically Responsive Polysiloxane LCE Nanocomposites
3. Preparation of Reversible Epoxy-based LCEs
4. Testing and Characterization of LCEs
5. Active Cell Culture through Electrical Stimulation of LCE Nanocomposites
6. Active Cell Culture with LCEs Using Direct Heating
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 °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 °C is observed and a smectic-to-isotropic transition temperature near 60 °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.
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 °C (B). LCE nanocomposites contract on the application of an electrical potential (C and D). The voltage applied is a 40 V AC signal. Please click here to view a larger version of this figure.
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). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 5. Fluorescence analysis of cardiomyocytes on an LCE nanocomposite surface. Cells are stained with Calcein AM, and live cells appear green. Please click here to view a larger version of this figure.
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 °C, and the materials can be re-shaped or re-aligned by applying a load at 160 °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.
The authors have nothing to disclose.
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's Hospital.
4-methoxyphenyl 4-(3-butenyloxy)benzoate | TCI America | M2106 | Reactive mesogen |
poly(methylhydrosiloxane) | Gelest | HMS-993 | Reactive polysiloxane |
1,4-di(10-undecenyloxybenzene) | N/A | N/A | see: Ali, S. A., Al-Muallem, H. A., Rahman, S. U. & Saeed, M. T. Bis-isoxazolidines: A new class of corrosion inhibitors of mild steel in acidic media. Corrosion Science 50 (11), 3070–3077, doi:10.1016/j.corsci.2008.08.011 (2008) |
(dichloro(1,5-cyclooctadiene)-platinum(II) | Sigma Aldrich | 244937 | Pt catalyst |
PTFE mold | N/A | N/A | fabricated at Rice machine shop |
carbon black nanoparticles | Cabot | VULCAN® XC72R | used in the synthesis of LCE nanocomposites |
polystyrene | Sigma Aldrich | 331651 | linear polystyrene |
4,4'-diglycidyloxybiphenyl | N/A | N/A | see: Giamberjni, M., Amendola, E. & Carfagna, C. Liquid Crystalline Epoxy Thermosets. Molecular Crystals and Liquid Crystals Science and Technology. Section A. Molecular Crystals and Liquid Crystals 266 (1), 9–22, doi:10.1080/10587259508033628 (1995). |
sebacic acid | Sigma Aldrich | 283258 | C8 linking group for epoxy-LCE synthesis |
hexadecanedioic acid | Sigma Aldrich | 177504 | C16 linking group for epoxy-LCE synthesis |
carboxydecyl-terminated polydimethylsiloxane | Gelest | DMS-B12 | Siloxane linking group for epoxy-LCE synthesis |
1,5,7-triazabicyclo[4.4.0] dec-5-ene | Sigma Aldrich | 345571 | catalyst for reversible LCEs |
carbon rods | Ladd Research | 30250 | used in cell culture experiments |
medical grade silicone adhesive | Silbione | MED ADH 4100 RTV | used to adhere carbon rods to vessel |