Supporting Information

Transcription

1 Supporting Information Janus Nanocomposite Hydrogels for Chirality-Dependent Cell Adhesion and Migration Andisheh Motealleh, a and Nermin Seda Kehr* a a Physikalisches Institut and CeNTech,Westfälische Wilhelms-Universität Münster, Heisenbergstraße 11, D Münster, Germany seda@uni-muenster.de S-0

2 1. Supplementary Experimental Procedures 1.1. Synthesis of disc shape zeolites: 2.76 g of potassium hydroxide (Fluka, pallets 86%), 1.74 g of sodium hydroxide (Merck, pallets GR for analysis), and 0.62 g of aluminium hydroxide (Riedel-de Haën, pulver, purum) were added to g of doubly distilled water and refluxed for 3 h in an oil bath at 120 o C g of Ludox (Ludox HS-40, colloidal silica 40 wt%, suspension in water) was added after the solution cooled to room temperature and shaked strongly with hand for a short time. As soon as the opaque gel is formed, gel was transferred to pressure teflon vessel for crystallization at 160 o C for 48 h under dynamic conditions (rotation at 40 rpm). The composition of gel is 5.40 K2O-5.50 Na2O-1.00 Al2O SiO H2O. After crystallization the teflon vessel was cooled in ice for 1 h before opening. The product was centrifuged (4500 rpm, 10 min) and washed with boiling doubly distilled water until the ph of the supernatant becomes neutral. The crystals were dried overnight at 70 o C, yielding about 1.75 g material Synthesis of OH: mg of CTAB was dissolved in 90 ml of H2O, 33 ml of ethanol, and a 28 wt% ammonia (0.075 g) solution. The reaction mixture was stirred at room temperature for 1 h before the addition of BTME (1.27 g). The above reaction mixture was continuously stirred for an additional 72 h at room temperature. The CTAB mesoporous template was removed by stirring the sample in ethanol (50 ml) with a 36 wt% aqueous solution of HCl (1.5 g) at 50 o C for 6 h. The resulting solid was recovered by centrifugation, washed with ethanol and acetone several times, and dried at 60 o C in vacuum loading of OH: OH (100 mg) was suspended in 10 ml toluene and mixed with (0.8 mg). This reaction mixture was refluxed at 120 o C for 2 days. The final product ( OH) was obtained by centrifugation, washed with toluene x 2, ethanol x 2, and dried at room temperature loading of OH: OH (100 mg) was mixed with (0.5 mg) in a glass ampoule and dehydrated at mbar for 8 h and sealed. The mixture, in a sealed glass ampoule, was kept in an oven at 290 C for 18 h to obtain insertion in zeolite channels. Thereafter, the ampoule was opened and the loaded zeolites ( OH) were obtained by centrifugation, washed with toluene until the supernatant did not show any fluorescence and dried at room temperature Poly-L(D)-Lysine coating of OH ( PLL and PDL): OH (20 mg) was suspended in water (20 ml) and mixed with PLL or PDL (MW: ) (10 mg). The reaction mixtures were stirred for 1 day at room temperature. The final product PLL and PDL was obtained by centrifugation of the resulting suspension, followed by washing of the residue with water two times and drying at room temperature Poly-L(D)-Lysine coating of OH ( PLL and PDL): OH (20 mg) was suspended in water (20 ml) and mixed with PLL or PDL (MW: ) (10 mg). The reaction mixtures were stirred for 1 day at room temperature. The final product PLL and PDL S-1

3 were obtained by centrifugation of the resulting suspension, followed by washing of the residue with water two times and drying at room temperature Determination of the quantitative amount of PLL and PDL on PMO/PLL and PMO/PDL: UV/Vis spectrophotometry was used to determine the quantitative amount of PLL and PDL on PMO/PLL and PMO/PDL. The different concentrations of PDL/PLL was suspended in water. The absorption spectrum of the different concentrations of PDL/PLL in water was measured against water between 190 nm and 300 nm using a quartz cell with a path length of 1 cm. The absorbance at λmax = 210 nm was plotted as a function of the PDL/PLL concentration and its slope is evaluated. The y = 2,3629x + 0,1379 and y = 2,0257x + 0,2131 formula were obtained from the plotted graph of PDL and PLL respectively (y is absorbance and x is the concentration of PDL/PLL). Then solution of PLL and PDL (ca. 0,5 mg) in water (1 ml) were prepared and the absorption spectrum of these solutions were measured. The quantitative amount of PDL/PLL in water was measured using the formula of the plotted graph of PDL/PLL and called as C1PLL and C1PDL.Thereafter PMO/OH (ca. 1 mg) was suspended in these water solutions of PLL and PDL. The reaction mixtures were stirred for 1 day at room temperature. The final product PMO/PLL and PMO/PDL was removed from the supernatant by centrifugation. PMO/PLL and PMO/PDL were further washed with water two times, centrifuged and the all supernatants in each step were collected. The absorption spectrum of the supernatant was measured against water between 190 nm and 300 nm using a quartz cell with a path length of 1 cm. The quantitative amount of PDL/PLL in supernatant was measured using the formula of the plotted graph of PDL/PLL and called as C2PLL and C2PDL. The quantitative amount of PLL and PDL adsorbed on PMO/OH (C3PLL and C3PDL) was calculated (Table S2) using this formula: C3PLL = C1PLL C2PLL and C3PDL = C1PDL C2PDL 1.8. Determination of the quantitative amount of OH, PLL, PDL in OH-, PLL-, PDL-, respectively: The amount of PMOs in each NC hydrogel was determined by using a fluorescence intensity calibration curve of loaded PMOs (y=17282x+3616) (Table S3) Determination of the swelling ratio of alginate and NC hydrogel scaffolds: The weight of dried scaffolds were first measured then they were immersed in cell culture media for 1 day and 4 days at physiological temperature (37 C). The swollen scaffolds were removed from cell culture media and weighed again. The swelling ratio (SR) was calculated using the following equation: SR= Ws-Wd/Wd where Ws is the mass of the swollen scaffolds and Wd is the dry mass of them and all experiments were carried out in triplicate. S-2

4 1.10. Determination of the equilibrium water content of alginate and NC hydrogel scaffolds: The mass of the scaffolds at the swollen (Ws) and total weight of the dry samples (Wd) were also used to determine the equilibrium water content (EWC) of the nanocomposite scaffolds using the following equation: EWC (%) = Ws/Wd* Degradation behavior of alginate and NC hydrogel scaffolds: Nanocomposite hydrogel scaffolds were immersed within cell culture media and incubated for 1 day and 4 days at 37 C in presence of cells, after each incubation time the scaffolds were taken out and dried at 37 C. The degradation behavior [weight loss (%)] was found out by measuring the weight of the samples before and after immersing in cell culture media according to the following equation: Wloss (%) = [W1-W2/W1] * 100 Where W1 and W2 are the weight before and after degradation, respectively. The results reported are averages of three measurements and standard deviations were calculated Pore size and porosity measurement of alginate and NC hydrogel scaffolds: Pore size of samples were carried out by using ImageJ software and for porosity measurement, the ethanol replacement method was used. Dried nanocomposite hydrogel scaffolds were immersed in absolute ethanol overnight and weighed after blotting the excess ethanol on the surface. The porosity was calculated from the following equation: Porosity (%) = ((M2 M1) / ρv)*100 Where, M1 and M2 are the mass of nanocomposite hydrogel scaffolds before and after the immersion in absolute ethanol, respectively; ρ is the density of absolute ethanol and V is the volume of the samples Characterization: The morphology of the OH, PLL, and PDL, OH, PLL, and PDL was investigated using a Zeiss 1540 EsB dual beam focused ion beam/field emission SEM. IR spectra were carried out with Varian 3100 FT-IR Excalibur Series Spectrometer. Zeta potential measurements and DSL were done with Malvern Zetasizer Nano Series. The t-test was used as statistical analysis. The morphology of cells in hydrogels were determined using Keyence BZ-9000E fluorescence microscopy. Cary 100 (Varian) double beam spectrophotometer was used for the determination of the absorption spectrum of the different concentrations of PDL/PLL in water. S-3

5 2. Supplementary Results OH/PLL/PDL and OH/PLL/PDL were characterized by dynamic light scattering (DLS), scanning electron microscopy (SEM), infrared spectroscopy (IR) and zeta-potential measurements. The synthesized PMOs were spherical in shape (Figure S1A-S1C) and the diameters of OH and PLL/PDL were approximately 222 nm and 388/367 nm, respectively (Table S2). For the zeolite particles, SEM images of OH and PLL/PDL showed disc-shaped zeolite L particles (Figure S1D-S1F) having diameters of approximately 780 nm ( OH) and 940/913 nm ( PLL/ PDL) (Table S2). The IR bands of the ν(co) and ν(cn) vibrations for the amide bonds of PLL and PDL in PLL( PLL) and PDL( PDL), respectively, were observed at around 1632 cm -1 and 1550 cm -1. The OH( OH) particles showed no corresponding IR bands at that region, as expected (Figure S2). Additionally, the change in the zeta potential from -25.6/-36.0 mv ( OH/ OH) to 27.2/23.4 mv ( PLL/ PLL) and to 24.7/19.6 mv ( PDL/ PDL) confirmed the successful functionalization of OH/ OH with PLL and PDL (Table S2). The synthesized OH/PLL/PDL and OH/PLL/PDL particles were used to prepare the NC hydrogels. The prepared OH/PLL/PDL and OH/PLL/PDL particles were embedded into alginate hydrogels and then frozen and lyophilized to generate PLL/PDL- and PLL/PDL- scaffolds. Table S1. Quantitative amount of PLL and PDL (mg) on PMO/PLL and PMO/PDL (1 mg). PLL/PDL (mg) PLL ± PDL ± PLL ± PDL ± S-4

6 Table S2. Physicochemical properties of OH, PLL, PDL, OH, PLL, PDL. Diameter (DLS, nm) Water Zeta potential (mv) Water OH 222 ± ± 0.7 PLL 388 ± ± 1.6 PDL 367 ± ± 0.6 OH 780 ± ± 1.7 PLL 940 ± ± 0.9 PDL 913 ± ± 0.3 A B C 1 µm 1 µm 1 µm D E F 1 µm 1 µm 1 µm Figure S1. SEM images of OH (A), PLL (B), PDL (C), OH (D), PLL (E), PDL (F). S-5

7 OH PLL PDL OH PLL PDL Figure S2. IR spectra of OH, PLL, PDL, OH, PLL, and PDL. S-6

8 A B C D 2 µm 1 µm 1 µm 1 µm 50 µm 50 µm 20 µm 20 µm E F G 2 µm 2 µm 2 µm 20 µm 20 µm 20 µm Figure S3. SEM images of (A), OH- (B), PLL- (C), PDL- (D), OH- (E), PLL- (F), PDL- (G). Upper right zoomed-in images show the embedded PMOs and zeolites in. Table S3. Quantitative amount of OH, PLL, PDL in 0.2 ml NC hydrogels, [the number of repeated experiments (N) = 3]. (mg) w/v % OH ± ± 0.01 PLL ± ,029 ± 0.01 PDL ± ± 0.01 Table S4. OH, PLL, PDL distribution on 1 µm 2 area of, [the number of repeated experiments (N) = 3]. OH 13.8 ± 2.3 PLL 14.6 ± 3.3 PDL 13.9 ± 3.7 Number of PMOs on 1 µm 2 area of S-7

9 Table S5. Swelling Ratio of alginate and NC hydrogel scaffolds after 1 day and 4 days incubation at 37 C, [the number of repeated experiments (N) = 3]. OH- PLL- PDL- 1 day 10.9 ± ± ± ± days 10.7 ± ± ± ± 0.6 Table S6. EWC (%) of alginate and NC hydrogel scaffolds after 1 day and 4 days incubation at 37 C, [the number of repeated experiments (N) = 3]. OH- PLL- PDL- 1 day 90.9 ± ± ± ± days 90.6 ± ± ± ± 0.1 Table S7. Weight loss (%) of alginate and NC hydrogel scaffolds after 1 day and 4 days incubation in presence of cells at 37 C, [the number of repeated experiments (N) = 3]. OH- PLL- PDL- 1 day 0.4 ± ± ± ± days 1.2 ± ± ± ± 0.2 S-8

10 Table S8. Porosity (%) and pore size (µm) of alginate and NC alginate scaffolds,. OH- PLL- PDL- Porosity (%) Pore size (µm) 52 ± ± ± ± ± ± ± ± 42 Table S9. Quantitative numbers ( 10 3 ) of fibroblast cells and cell viability [a] after 1 day and 4 days incubation time (37 C) in the, OH-, PLL-, PDL-, OH-, PLL-, PDL- in serum-free media (N = 3). 1 day OH- PLL- PDL- OH- PLL- PDL- 1.7 ± 0.3 (79 %) 1.8 ± 0.3 (78 %) 5.5 ± 0.9 (78 %) 6.0 ± 0.1 (82 %) 3.0 ± 0.1 (56 %) 4.3 ± 0.6 (61 %) 5.3 ± 0.6 (61 %) 4 days OH- PLL- PDL- OH- PLL- PDL- 1.0 ± 0.6 (50 %) 1.3 ± 0.6 (56 %) 2.5 ± 0.7 (71 %) 2.6 ± 0.6 (70 %) 3.0 ± 0.6 (22 %) 4.0 ± 0.6 (42 %) 4.7 ± 0.6 (50 %) [a] in parentheses. Table S10. Quantitative amount of adsorbed proteins on, OH-, PLL-, PDL- after 1d incubation time, [the number of repeated experiments (N) = 3]. 1d Adsorbed proteins (µg) 1828 ± 115 OH ± 63 PLL ± 178 PDL ± 85 S-9

11 Table S11. Quantitative amount of adsorbed proteins on J-4: Janus-( PLL- OH- ); J-5: Janus-( PDL- OH-); J-6: Janus-( PLL- PDL- ), [the number of repeated experiments (N) = 3] after 1d incubation time. [a] in parentheses. 1d Adsorbed proteins (µg) PLL- / 2480 ± 62 / OH ± 50 PDL- / OH ± 80 / 2214 ± 43 PDL- / 2747 ± 57 / PLL ± 114 A B C D 50 µm 50 µm 50 µm 50 µm Figure S4. Fluorescence microscopy images of cells in (A), OH- (B), PLL- (C), PDL- (D). S-10