The approach is based on the ELR5/PA co-assembling system (Inostroza-Brito et al., 2015, 2017) and takes advantage of its reaction-diffusion mechanism to recruit, localize, and organize GO flakes at multiple length scales (Figure 1). We used 0.04% (v/v) GO (Wick et al., 2014) (pH = 2) with monolayer content >95% and oxygen content >36% given its water dispersability and low cost (Figure 1E). We used a cationic PAK3 (C₁₅H₃₁CONH-VVVAAAKKK-CONH₂) to co-assemble with the oppositely charged ELR5 (MESLLP-[(VPGVG VPGVG VPGEG VPGVG VPGVG)₁₀-(VGIPG)₆₀]₂-[(VPGIG)₁₀-AVTGRGDSPASS(VPGIG)₁₀]₂-V) molecule to evaluate the capacity of the assembling process to recruit and organize GO flakes within the distinctive ELR5/PA multilayers (Figures 1A–C). Furthermore, given our previous findings that distinct PA molecules can generate different ELR5/PA membrane structures (Inostroza-Brito et al., 2015), PAs with different charge densities including PAK2 (C₁₅H₃₁CONH-VVVAAAKK-CONH₂) and PAK4 (C₁₅H₃₁CONH-VVVAAAKKKK-CONH₂) were also used to co-assemble with ELR5 (Figure 1D). In addition, the resulting materials were characterized according to their mechanical properties and used as cell culture substrates to investigate their effect on cell adhesion, proliferation, metabolic activity, and morphology.

PA molecules were synthesized following standard solid-phase peptide synthesis methods as previously reported (Mata et al., 2012). PA purity and structure were characterized by reverse phase HPLC and electrospray ionization mass spectrometry (Figure S1). ELR5 molecules were obtained from Technical Proteins Nanobiotechnology S. L., Spain and GO aqueous dispersions were obtained from Sigma Aldrich, UK.

The ELR5/PA system relies on electrostatic, hydrophobic, and H-bond interactions (Inostroza-Brito et al., 2015). Consequently, we first used circular dichroism (CD) to investigate the secondary structure in aqueous solution of both PAs and ELR5 with and without GO. CD revealed that PAK3 exists in a random coil conformation when dissolved in MilliQ™ water at pH 4.5 and room temperature (RT), and does not undergo conformational change when mixed with GO under the same conditions (Figure 2A). However, a slight red-shift at the 195 nm minimum was observed, which might indicate an interaction between PAK3 and GO as previously reported (Pashuck et al., 2010). CD on ELR5 samples dissolved in MilliQ™ water at pH 5 and RT exhibited both random coil and β-sheet conformations (Figure 2A) that did not change upon addition of GO. However, lower intensity of the negative 195 nm signal might indicate that GO reduces the content of random coil structures in ELR5 while increasing the content of type I β-turns (Perczel and Fasman, 1992).

We further investigated the ELR5/PA/GO interactions by conducting zeta potential and dynamic light scattering (DLS) measurements. Zeta potential of PAK3 decreased after addition of GO (Figure 2B). This is likely a consequence of a drop in the PAK3's surface charge as a result of its electrostatic interactions with carboxyl, hydroxyl, or carbonyl groups present in GO (negatively charged), partially screening the positive charges of PAK3. On the other hand, the zeta potential of ELR5 molecules increased slightly after addition of GO, suggesting absence of electrostatic interactions between these components, which is in agreement with the CD results (Figure 2A). DLS measurements revealed a dramatic decrease in GO size after mixing with both PAK3 (Figure 2C) and ELR5, which suggests disruption of GO aggregates, known to form in aqueous solutions (Tang et al., 2015), due to electrostatic interactions. It is important to mention that DLS is a well-suited technique for estimating the size of spherical particles. However, this technique has been also used to provide relative changes in size of non-spherical components including GO (Stankovich et al., 2006), PAs (Raymond and Nilsson, 2018), and ELRs (Navon and Bitton, 2016).

Hydrophobic interactions also play a key role in the ELR5/PA system above the ELR5's transition temperature (Tt) (Inostroza-Brito et al., 2015). To investigate whether addition of GO into the ELR5/PA system influences hydrophobic interactions, a turbidity assay was performed. The results confirmed that significant levels of aggregation between ELR5, PAK3, and GO are only present when the temperature is above the Tt (19C) of ELR5 (Figure 2D), which indicates that presence of GO in the solution does not influence the hydrophobic interactions between PAK3 and ELR5, necessary for membrane formation (Inostroza-Brito et al., 2015). Analysis of interactions between ELR5, GO and both PAK2 and PAK2-K4 is presented in Figures S2 and S3.

These results indicate that molecular interaction between GO and both molecular building blocks (PAK3 and ELR5) relies mostly on electrostatic forces. However, both components retain most of their secondary structures in presence of GO and, consequently, hydrophobic interactions from ELR5 seem to also play a key role above the ELR5 transition temperature.

Upon ELR5/PAK3 co-assembly, a diffusion barrier is formed across which the PAK3 diffuses (Figure 3A) (Inostroza-Brito et al., 2015). Electron microscopy was used to investigate the assembly of GO within the ELR5/PAK3 membrane when added into the system. We first mixed GO with the PAK3 solution and subsequently inoculated it into the ELR5 solution. Scanning electron microscopy (SEM) revealed poor incorporation of GO (Figure 3B), likely as a result of the inability of the relatively large GO flakes to diffuse through the ELR5/PAK3 diffusion barrier. We then reasoned that combining GO with ELR5 solution might surpass this obstacle. We prepared an ELR5/GO solution followed by inoculation of PAK3 into the mixture. In this case, we observed much higher incorporation of GO in the final membrane (Figure 3C). Given these findings, we then mixed GO with both ELR5 and PAK3 solutions, followed by inoculation of PAK3/GO in the ELR5/GO solution. In this setup, membranes qualitatively exhibited the highest incorporation of GO (Figure 3D) while maintaining their multi-layered structure and capacity to adhere to interfaces and open controllably, transforming sacs into tubular structures (Figures 4C–E). Transmission electron microscopy (TEM) revealed presence of GO flakes (visible as black lines) throughout the thickness of the ELR5/PAK3/GO membrane, positioned within and parallel to the membrane layers (Figure 4B). SEM performed on the membranes confirmed the multi-layered microarchitecture with embedded GO flakes (Figure 3F-row) seen on TEM. These results indicate that GO can be successfully incorporated within the ELR5/PAK3 system by taking advantage of the diffusion-reaction mechanism of assembly to recruit, localize, and organize GO flakes within the multi-layered architecture and as part of a more complex geometrical structure.

To investigate the possibility to use supramolecular self-assembling processes to guide the organization of GO more broadly, we repeated the experiments using variations of PAK3, PAK2 (C15H31-VVVAAAKK–CONH2) and PAK4 (C15H31-VVVAAAKKKK-CONH2) (Figure 1D). We have previously demonstrated that these PA molecules with a different number of lysine residues were also able to form stable ELR5/PA membranes, but exhibiting different cross-sectional architectures (Inostroza-Brito et al., 2015). Membranes formed using PAK4 exhibited a multi-layered architecture with increased thickness and a looser structure compared to ELR5/PAK3 membranes (Inostroza-Brito et al., 2015) while those formed using PAK2 exhibited a different three-level structure with orthogonal fibers (Figure 3E-row) (Inostroza-Brito et al., 2015). To study the interaction between GO and the ELR5/PAK2-K4 system, we also attempted to generate ELR5/PA membranes combining both PAK2 and PAK4 (1:1 mixture). We hypothesized that the different diffusion mechanisms arising from the co-assembly of these different components would guide and organize GO flakes differently. To investigate this hypothesis, experiments were conducted using ELR5/PAK2 and ELR5/PAK2-K4 combinations (Figures 3E,G-rows). As before, GO was mixed with (i) PAs alone, (ii) ELR5 alone, and (iii) both components (Figures 3B–D, columns). ELR5/PAK2/GO and ELR5/PAK2-K4/GO membranes were stable and robust (Figures 3E,G insets) and exhibited darker color suggesting GO incorporation in these conditions, which was assessed by observations under an optical microscope. SEM investigations on membranes revealed GO flakes were incorporated and organized within and throughout the multi-layered architecture of the ELR5/PAK2-K4/GO membranes (Figure 3G-row). As with the ELR5/PAK3/GO system, highest incorporation of GO was achieved when the GO was mixed with both PA and ELR5 solutions (Figures 3D,F). In contrast, ELR5/PAK2/GO membranes exhibited large quantities of GO on the outer and inner side of the membrane but only marginal incorporation within the membrane.

These results suggest that GO can be incorporated within the ELR5/PA system independently of the PA molecule used. However, the level of incorporation of GO within the membrane depends on the supramolecular mechanism of assembly, with both ELR5/PAK3/GO and ELR5/PAK2-K4/GO presenting more GO in the multilayers than ELR5/PAK2/GO membranes, which present a different mechanism of co-assembly (Inostroza-Brito et al., 2015). Initial formation of a strong diffussion barrier prevents large flakes of GO from diffusing through the barrier, forming membranes with less GO flakes. Addition of GO flakes in the ELR5 solution helps to overcome this obstacle, forming membranes with higher content of GO. Highest incorporation, however, can be achieved only when GO is combined with both PA and ELR5 solutions, particularly in the case of the ELR5/PAK3 and ELR5/PAK2-K4 systems.

To study the effect of introducing GO into the ELR5/PA system, possible changes in membrane thickness were investigated by SEM (Figure 5B). SEM revealed that the thickness of ELR5/PAK3/GO membranes increased when GO was added in either PA or ELR5 solutions prior to assembly, forming the thickest membrane when GO was mixed with both ELR5 and PAK3 (Figure 5D). Similar results were observed in ELR5/PAK2-K4/GO membranes (Figure 5D). In contrast, the thickness of ELR5/PAK2/GO membranes gradually decreased with addition of GO to either PAK2, ELR5, or both PAK2 and ELR5 solutions. This result suggests that the supramolecular mechanism of assembly of ELR5/PAK2 membranes, which differs from ELR5/PAK3 and ELR5/PAK4 systems (Inostroza-Brito et al., 2015), prevents incorporation of GO. These experiments are in alignment with the SEM observations (Figure 3) and confirm that, while supramolecular processes may be able to organize GO flakes at multiple scales (ELR5/PAK3 and ELR5/PAK2-K4 systems), the size of GO flakes may limit its incorporation in some co-assembling systems (ELR5/PAK2).

To further characterize GO incorporation, the amount of GO within each of the ELR5/PA systems was quantified. After co-assembly, membranes were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and GO absorbance was monitored spectrophotometrically in the UV-Vis region. The technique enabled quantification of mass of GO per membrane (Figure 5C) and the results revealed the highest incorporation of GO occurred in ELR5/PAK2-K4 membranes, followed by ELR5/PAK3 membranes, and, as expected, ELR5/PAK2 membranes with the lowest incorporation of GO. These results correlate with the previously discussed SEM observations (Figure 3) and membrane thickness experiments (Figure 5B).

SEM and TEM demonstrated that GO flakes are incorporated and organized within the ELR5/PAK3/GO and ELR5/PAK2-K4/GO membranes. To further characterize the level of incorporation throughout the thickness of the membrane, 3D z-stacked bright field images of the membrane were generated (Figure 5A). Using ImageJ software (Schneider et al., 2012), we quantified the number of GO flakes in each layer of the different membranes by generating z-stack images and producing distribution profiles depicting number of GO flakes vs. layer number, which was normalized as % of the thickness of the membrane (Figure 5B). These profiles revealed that in ELR5/PAK3/GO membranes, GO is distributed throughout the thickness of the membrane with higher amounts present in the middle. In addition, ELR5/PAK2-K4/GO membranes exhibited similar level of GO incorporation but the flakes were more evenly distributed throughout the membrane (Figure 5B). Interestingly, ELR5/PAK2/GO membranes exhibited uniform distribution throughout the membrane but with much lower levels of incorporation (Figure 5B). These results confirm that GO flakes were in fact distributed throughout the membranes of all the systems studied but with different levels of incorporation. Highest incorporation and distribution was observed in ELR5/PAK2-K4/GO and ELR5/PAK3/GO membranes, on which we focused to assess applicability.

Given the unique mechanical properties of GO (Zhu et al., 2010) as well as its incorporation and organization within the ELR5/PA systems, we hypothesized that the mechanical properties of the resulting ELR5/PA/GO membranes would improve compared to the control ELR5/PA membranes. Atomic force microscopy (AFM) measurements were conducted on both luminal (inner) and abluminal (outer) sides of ELR5/PAK3/GO and ELR5/PAK2-K4/GO membranes assembled with GO added to both PA and ELR5 solutions (Figure 6). The results revealed significant increase in Young's Moduli of the ELR5/PAK2-K4/GO membrane on both luminal and abluminal sides compared to ELR5/PAK2-K4 membranes (Figure 6B). This increase was also evident in ELR5/PAK3/GO membranes compared to ELR5/PAK3 ones (Figure 6A). These results suggest that stiffness of the ELR5/PA membrane increases after incorporation of GO on both sides of the membrane, which correlates with the SEM observations (Figure 3), membrane thickness measurements (Figure 5D), and GO distribution and incorporation (Figure 5B) within the membrane's microstructure when GO is added to both ELR5 and PAs solutions.

ELR5 and PA molecules were dissolved separately in MilliQ water (10 and 15 mg/mL, respectively). pH was adjusted to pH = 5 (ELR5) and pH = 4.5 (PAs). 190 μl of ELR5 solution was placed in a well in a 48 well plate. 10 μl of PA solution was added by immersing the pipette tip into the ELR5 solution and slowly releasing the liquid. The membrane was left to develop for 48 h at 30°C. For GO membranes: GO 4 mg/mL (pH = 2) was diluted to the required concentration with pH adjusted to pH = 4.5 and mixed with either ELR5 solution or PA solution before membrane formation. Ten microliter of PA-GO solution was added by immersing the pipette tip into the ELR5-GO solution and slowly releasing the liquid. The membrane was left to develop for 48 h at RT.

ELR5 and PA molecules were dissolved separately in MilliQ water (10 and 15, mg/mL respectively). GO 4 mg/mL was diluted to the required concentration and mixed with either ELR5 solution or PA solution before membrane formation. 100 μl of ELR5/GO solution was placed in a glass vial. 5 μl of PA-GO solution was added by immersing the pipette tip into the ELR5 solution and slowly releasing the liquid. Additional 20 μl of ELR5/GO solution was carefully added to the vial every 10 min to observe growth of the membrane.

Zeta potential was measured to investigate the changes in surface charge density of ELR5, PAs and GO molecules when mixed together. ELR5 and PAs were dissolved in MilliQ water [0.1 and 0.15% (w/v), respectively]. GO was diluted to the final concentration of 0.001% (w/v). pH was adjusted to pH = 5 (ELR5), pH = 4.5 (PAs), and pH = 4.5 (GO). All samples were sonicated for 30 min prior taking the measurement. Zeta potential was measured at 25°C on a Zetasizer (Nano-ZS ZEN 3600, Malvern Instruments, UK).

ELR5 and PAs were dissolved in MilliQ water [0.025 and 0.01% (w/v), respectively]. In order to carry out the measurement at the same conditions as membrane formation, pH was adjusted to pH = 5 (ELR5) and pH = 4.5 (PAs). GO was diluted to the final concentration of 0.001% (w/v) and pH was adjusted to pH = 4.5. CD spectra were obtained using 1 mm path length and 300 μl volume quartz cuvette (Chirascan, Applied Photophysics, UK) between 195 and 270 nm with a 0.5 nm interval at 25°C. CD measurement was conducted using Chirascan™ CD spectrometer (Chirascan, Applied Photophysics Ltd, UK) equipped with a Peltier temperature controller, under a constant nitrogen purging at a constant pressure of 0.7 MPa. Each represented spectrum is the average of three consecutive spectra.

ELR5 and PA were dissolved in MilliQ water [0.025 and 0.01% (w/v), respectively]. GO was diluted to the final concentration of 0.001% (w/v). Absorbance of the solutions was measured at 300 nm using a microplate reader (Spetrostarnano, BMG Labtech, UK) at 4 and 40°C.

Membranes were formed as described. After washing with MilliQ water membranes were crosslinked with TEM grade glutaraldehyde followed by dehydration with a gradient 30–100% of ethanol. Membranes were then embedded on LRWhite resin and ethanol 50–50% for 1 h and 100% LRW 1 h and again 100% LRW overnight. The following day they were encased in capsules filled with resin and left in the oven at 60°C for 5 h to harden. The block was sectioned with a Reicht microtome to a thickness of 70 nm. The sections were loaded onto a copper grid and stained with 2% uranyl acetate for 4 min and rinsed in MilliQ water. The samples were visualized on a JEOL JEM 1230 electron microscope operating at 80 kV.

ELR5/PA/GO membranes were left to develop for 48 h and then washed in MilliQ water and fixed with 2.5% glutaraldehyde in MilliQ water for 2 h at room temperature. Then, the samples were washed in MilliQ water followed by dehydration by immersion in an increasing concentration of ethanol (20, 50, 70, 90, 96, and 100%). The samples were then subjected to a process of critical point drying (K850, Quorum Technologies, UK) followed by sputter-coating with gold for 90 s. SEM imaging was carried out using an Inspect F50 (FEI Comp, The Netherlands).

Membranes were formed as previously described. After washing in MilliQ water membranes were cut open, put flat on a microscope slide and then covered with a slide cover. 3D images were obtained with Zeiss LSM710 confocal microscope.

Fully formed and washed membranes were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) followed by sonication for 30 min. GO absorbance was measured spectrophotometrically in the UV-Vis region. Standard curve was prepared using a solution of GO flakes in HFIP at a known concentration.

Atomic force microscopy was used to measure Young's Modulus of the investigated system. Membranes were attached to a Petri dish using a drop of cyanoacrylate adhesive and left for a minute for the adhesive to dry followed by immersion in ultrapure MilliQ water. Young's Modulus measurements were taken with JPK Nanowizard-1 (JPK Instruments, Germany) in force spectroscopy mode, which was mounted on an inverted optical microscope (IX-81, Olympus, Japan). Indentation was carried out using quadratic pyramidal cantilevers (MLCT, Bruker, MA, USA) with spring constant of 0.07 N/m and half-angle to face of 17.5°. Measurements were taken in multiple areas per sample and multiple times per area.

Fully developed membranes were washed with MilliQ water and crosslinked with genipin at a concentration of 25 μl/ml at 37°C overnight. Tubes were then washed in MilliQ water and sterilized under UV light for 20 min. After sterilization, tubes were washed three times in Hank's balanced salt solution. 50,000 mADSCs re-suspended in DMEM (10% FBS, 1% P/S) were seeded on each ELR5/PA/GO tube. Media was changed every 2 to 3 days.

Cells were seeded as previously described in a serum free DMEM media and incubated for 4 h followed by additional 4 h in full media (DMEM with 20% FBS). Cells were fixed with 4% paraform aldehyde for 1 h and stained with blue dye 4'-6-diamino-2-phenylindole (DAPI), followed by imaging under an epifluorescent microscope (Leica DMi8).

Cell morphology was assessed by using epifluorescent microscopy (Leica DMi8). After the cell culture membranes were fixed with 4% paraform aldehyde for 1 h and stained with DAPI and Phalloidin CruzFluor™ 647.

Cell metabolic activity was assessed on days 2, 7, and 14 with AlamarBlue™ cell metabolic assay. Membranes were incubated for 2 h at 37°C in a 10% (v/v) solution of AlamarBlue™ in DMEM. Fluorescence of the solution was then read at 570 and 595 nm using a microplate reader (Spetrostarnano, BMG Labtech, UK).

Cell proliferation was assessed by quantifying the number of adherent cells to membranes with Quant-iT™ PicoGreen^TM assay on days 2, 7, and 14. Briefly, cells were lysed and the supernatant solution was diluted in assay buffer followed by addition of Quant-iT™ PicoGreen^TM reagent and incubation for 5 min at RT. Fluorescence of the samples was measured at 480 nm (excitation) and 520 nm (emission) using a microplate reader (Spetrostarnano, BMG Labtech, UK). The DNA concentration for each sample was calculated by using a standard curve.