Photoactuating Artificial Muscles of Motor Amphiphiles as an Extracellular Matrix Mimetic Scaffold for Mesenchymal Stem Cells

Mimicking the native extracellular matrix (ECM) as a cell culture scaffold has long attracted scientists from the perspective of supramolecular chemistry for potential application in regenerative medicine. However, the development of the next-generation synthetic materials that mimic key aspects of ECM, with hierarchically oriented supramolecular structures, which are simultaneously highly dynamic and responsive to external stimuli, remains a major challenge. Herein, we present supramolecular assemblies formed by motor amphiphiles (MAs), which mimic the structural features of the hydrogel nature of the ECM and additionally show intrinsic dynamic behavior that allow amplifying molecular motions to macroscopic muscle-like actuating functions induced by light. The supramolecular assembly (named artificial muscle) provides an attractive approach for developing responsive ECM mimetic scaffolds for human bone marrow-derived mesenchymal stem cells (hBM-MSCs). Detailed investigations on the photoisomerization by nuclear magnetic resonance and UV–vis absorption spectroscopy, assembled structures by electron microscopy, the photoactuation process, structural order by X-ray diffraction, and cytotoxicity are presented. Artificial muscles of MAs provide fast photoactuation in water based on the hierarchically anisotropic supramolecular structures and show no cytotoxicity. Particularly important, artificial muscles of MAs with adhered hBM-MSCs still can be actuated by external light stimulation, showing their ability to convert light energy into mechanical signals in biocompatible systems. As a proof-of-concept demonstration, these results provide the potential for building photoactuating ECM mimetic scaffolds by artificial muscle-like supramolecular assemblies based on MAs and offer opportunities for signal transduction in future biohybrid systems of cells and MAs.


Compound 8:
To a THF/methanol solution (v/v = 1/1, 6 mL) of compound 7 (158 mg, 0.155 mmol), an aqueous NaOH solution (4 M, 0.34 mL, 1.4 mmol) was added and the mixture was stirred at 90 °C for 1 h. The reaction mixture was allowed to cool down to room temperature. The solvent was removed under reduced pressure and then the residue was washed successively with water (50 mL) and ethyl acetate (20 mL). The combined organic layers were washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure and the residue was purified by column chromatography on SiO2 (methanol/dichloromethane; v/v = 1/19, Rf = 0.5) to obtain compound 8 (142 mg, 0.151 mmol, 98% yield) as a colorless oil. 1

Nile Red Fluorescence
Assay. [3][4][5] The self-assembly properties of MAP1 and MAS1 were analyzed by incorporation of the hydrophobic solvatochromic probe Nile Red (9-diethylamino-5benzo[α]phenoxazinone), which shows a blue shift of the emission wavelength when it is encapsulated in hydrophobic environments. Freshly prepared Nile Red ethanol solution (0.1 mM) was diluted into MA solutions to a final concentration of 0.25 μM, which means that the corresponding samples contain S7 only 0.25% ethanol, allowing to eliminate the effects of ethanol on the self-assembly behavior of MAP1 and MAS1. Subsequently, the mixture solutions containing MAP1 (or MAS1) and Nile Red solution (0.25 μM) were excited at 550 nm, and the fluorescence spectra with a wavelength range of 580-750 nm were recorded by using a JASCO FP6200 fluorometer. The blue shifts were calculated by subtracting the emission wavelength of Nile Red in Milli-Q water from the emission wavelength of the sample. Afterward, critical aggregation concentrations (CACs) of MAP1 and MAS1 were determined by plotting the obtained blue shifts values against the concentrations.

Cryogenic Transmission Electron
Microscopy. The MAP1 solution (or MAS1 solution) was prepared by direct dissolution into double deionized water, followed by heating at 80 °C for 30 min and then cooling to room temperature to obtain a clear solution (3.9 mM). To observe the self-assembly structures by cryo-transmission electron microscopy (cryo-TEM), 2.5 µL of MAP1 solution (or MAS1 solution in identical conditions) was placed on a glow-discharged holy carbon coated grid (Quantifoil 3.5/1, QUANTIFOIL Micro Tools GmbH, Großlöbichau, Germany). After blotting, the corresponding grid was rapidly frozen in liquid ethane (Vitrobot, FEI, Eindhoven, The Netherlands) and kept in liquid nitrogen until measurement. The grids were observed with a Gatan model 626 cryo-stage in a Tecnai T20 (FEI, Eindhoven, The Netherlands) cryo-electron microscope operating at 200 keV. Cryo-TEM images were recorded under low-dose conditions on a slow-scan CCD camera.

Standardized Preparation of Artificial Muscle of Motor Amphiphile in Water.
Artificial muscles of motor amphiphiles (MAs) were prepared with the standardized conditions as follows: by using a pipette, 3.5 µL of MA solution (55 mM) was manually drawn into an aqueous solution of CaCl2 (150 mM) in a cuvette by the shear-flow method, allowing for the formation of a noodle-like artificial muscle, with a diameter of 300 ± 30 μm and a length of 8.0 ± 2.0 mm ( Figure 6). The light source (Thorlabs, M365FP1, 0.7 A) was placed at a distance of 5.0 ± 0.5 mm toward the artificial muscle. The photoactuating speed of the MA artificial muscle was calculated from a saturated flexion angle, i.e., 90°, of bending depending on a particular time. All the photoactuation experiments were performed in triplicate and recorded with a Nikon Coolpix A900 Digital Camera to obtain an averaged value of actuation speed. On the basis of the 1 H NMR and UV-Vis absorption studies of MA solutions shown in Figure 2, Figure S2, Figure 3, and Figure S4, a ratio of metastable-MAs : stable-MAs at the photostationary state can be determined as 85:15, which was comparable to our previous report. 2,5 Therefore, a similar extent of switching process of MAs in the artificial muscles was expected when the MA artificial muscles reached the saturated flexion angle of 90 o . 7. Synchrotron Radiation X-ray Diffraction Analysis. 2,5,6 Through view X-ray diffraction (XRD) images of the MA artificial muscles were obtained using the BL45XU beamline at SPring-8 (Hyogo, Japan) equipped with an R-AXIS IV++ (Rigaku) imaging plate area detector or with a Pilatus3X 2M (Dectris) detector. The scattering vector, q = 4πsinθ/λ, and the position of incident X-ray beam on the detector were calibrated using several orders of layer reflections from silver behenate (d = 58.380 Å), where 2θ and λ refer to the scattering angle and wavelength of the X-ray beam (1.0 Å), respectively. The sample-to-detector distances for through-view XRD measurements were 2.02 m. The obtained diffraction patterns and images were integrated along the Debye-Scherrer ring to afford onedimensional (1D) intensity data using the FIT2D software. The lattice parameters were refined using the CellCalc ver. 2.10 software.

Scanning Electron Microscopy and Polarized
Optical Microscopy Analysis. 2,5,6 The MAP1 solution (or MAS1 solution) was prepared by heating at 80 °C for 30 min and then cooling to room temperature to obtain a clear solution (55 mM). By using a pipette, 3.5 µL of the MA solution was manually sheared into an aqueous solution of CaCl2 (150 mM) on a sapphire substrate to obtain a noodle-like MA artificial muscle with an arbitrary length. After the removal of CaCl2 solution, the MA artificial muscle was washed with deionized water (three times), and the resulting MA artificial muscle was used directly for polarized optical microscopy (POM, Nikon model Eclipse LV100POL) and XRD experiments. For scanning electron microscopy (SEM), the MA artificial muscle was dried in air for 48 h and subsequently observed under a Hitachi S-5500 Field Emission SEM (FE-SEM).
Freshly prepared artificial muscles of MAC1 and MAP1 were placed in 24-well plates, which were washed with PBS (0.5 mL) 3 times and subsequently washed by growth medium (0.5 mL) 3 times. Afterward, the hBM-MSCs with particular densities were evenly seeded into the growth medium containing MA artificial muscles. The samples were incubated in a 5% CO2 humidified atmosphere at 37 °C. The growth medium was changed every 3 d.

Cytotoxicity and Cell Viability Analysis.
The vitro cytotoxicity of MA artificial muscles was tested by using a direct contact method between hBM-MSCs and MA artificial muscles via a live/dead staining assay. 9 The hBM-MSCs with a density of 20,000 cells/well were evenly seeded into 24-well plates containing growth medium and MA artificial muscles, and incubated in a 5% CO2 humidified atmosphere at 37 °C. After incubation for 24 h, the cytotoxicity of hBM-MSCs was analyzed by the live/dead assay by using calcein-AM (2 µL, Molecular Probes, Invitrogen Detection Technologies) and ethidium homodimer-1 (4 µL, Molecular Probes, Invitrogen Detection Technologies) in PBS to stain the hBM-MSCs for 30 min. 7 After staining, the MA artificial muscles were moved out from the 24-well plates to a well plate (diameter: 60 mm, containing PBS) for being observed under a Leica fluorescence microscopy equipped with a 10× NA 0.30 objective and a Leica confocal laser scanning microscopy (CLSM) equipped with a 40× NA 0.80 objective.
Additionally, a deeper insight into the condition of hBM-MSCs cultured on the surface of the MA artificial muscles with prolonged incubation time is performed by F-actin and nuclei staining. The hBM-MSCs with a density of 2,500 cells/well were evenly seeded into 24-well plates containing growth medium and MA artificial muscles and incubated in a 5% CO2 humidified atmosphere at 37 °C for 1, 3, and 5 d. After incubation, the hBM-MSCs were fixed with 3.7% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature and then washed by PBS for three times. Subsequently, the cell membrane was permeabilized with a 0.5% TritonX-100 solution for 3 min, followed by washing three times with PBS. The obtained hBM-MSCs were stained with 4′,6diamidino-2-phenylindole (DAPI) and tetramethylrhodamine isothiocyanate(TRITC)-phalloidin for the cell nuclei and F-actin, respectively, which were observed under the Leica confocal laser scanning microscopy equipped with a 40× NA 0.80 objective. 7 The cell nuclei stained by DAPI were not shown because of the strong background fluorescence of the MA artificial muscles. The area of cell F-actin and the surface area of the MA artificial muscles were determined by the software of ImageJ. A spread cell shape with well-defined actin stress fibers of hBM-MSCs is commonly quantified as being in a viable state. Due to the overlapping fluorescence between the strong background of MA artificial muscles and the cell nuclei stained by DAPI, the change of cell F-actin area with prolonged incubation time, instead of the number of cell nuclei, was used to indicate the cell proliferation.

Post-Photoactuation Experiment of Artificial Muscles with Adhered hBM-MSCs.
The hBM-MSCs with a density of 20,000 cells/well were evenly seeded into 24-well plates containing growth medium and MA artificial muscles, and incubated in a 5% CO2 humidified atmosphere at 37 °C for 6 h. After incubation, hBM-MSCs were stained by using calcein-AM (2 µL) and ethidium homodimer-1 (4 µL) in PBS for 30 min. Subsequently, the MA artificial muscles were moved out from the 24well plates to a well plate (diameter: 60 mm, containing PBS) for observation under the Leica fluorescence microscopy equipped with a 10× NA 0.30 objective. The MA artificial muscles were irradiated by using a Thorlabs LED light (M365L2-C1, 120 mW). The photoactuation process upon irradiation was simultaneously recorded by fluorescence microscopy with cell visualization at a microlength scale and by an iPhone camera at a macroscopic scale, as shown in Figure S1.   The rate constants k of the first-order decay at different temperatures were obtained using the equation A/A0 = e -kt . Activation parameters were obtained by fitting to the linearized form of the Eyring equation using origin software. Analysis of these data using equation (Δ ‡ G = RT [ln (kB/h) -ln (k/T)], provides the Gibbs energy of activation (Δ ‡ G). The half-lives of metastable MAP1 and MAS1 at 25 °C (298.15 K) were obtained by a linear fitting of ln k/T and 1/T. The activation parameters and half-life of metastable MAP1 and MAS1 in acetonitrile are presented in Table S2.