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Karsten Baumgarten and Brian P. Tighe Viscous forces and bulk viscoelasticity near jamming

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DOI: 10.1039/D0SM01299H

Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x

Structural, Mechanical, and Biological Characterization of Hierarchical Nanofibrous Fmoc-Phenylalanine-Valine Hydrogels for 3D Culture of Differentiated and Mesenchymal Stem Cells Haniyeh Najafi a, Ali Mohammad Tamaddon Negar Azarpira d

a,b,*,

Samira Abolmaali

a,b,

Sedigheh Borandeh

a,b,c,

Fmoc-dipeptides are a class of short aromatic peptides featuring eminent supramolecular self-assembly, which is due to aromaticity of the Fmoc group that improves the association of peptide building blocks. This study aimed to introduce a new dipeptide hydrogel scaffold, Fmoc-phenylalanine-valine (Fmoc-FV), for 3D culture of various cells. Peptide hydrogel scaffolds were prepared by pH-titration method in various concentrations and temperatures, and characterized by spectroscopic methods, including circular dichroism, attenuated total reflection FT-IR and fluorimetry. Mechanical behaviors such as thixotropy and temperature-sensitivity were investigated by oscillatory rheology. Fmoc-FV hydrogels were then applied in 3D-culture of WJ-MSC (mesenchymal stem cell), HUVEC (normal endothelial cell), and MDA-MB231 (tumor cell line) by live-dead fluorescence microscopy and Alamar blue viability assay experiments. The results confirmed β-sheet structure is principally interlocked by π-π stacking of the Fmoc groups and entangled nanofibrous morphologies as revealed by FE-SEM. Fmoc-FV self-assembly in physiologic condition resulted in a thermo-sensitive and shear-thinning hydrogel. Notably, Fmoc-FV hydrogel exhibited cell type-dependent biological activity, so higher cell proliferation was attained in HUVEC or MDA-MB231 cells than WJ-MSCs, indicating possible need for incorporating cell-adhesion ligands in the Fmoc-FV hydrogel matrix. Therefore, the structural and biological properties of the Fmoc-dipeptide hydrogels are inter-related and can affect their applications in 3D cell culture and regenerative medicine. Keywords— Fmoc-dipeptide Hydrogel; Nanostructure; Self-assembly; 3D cell culture; Rheology

1. Introduction Molecular self-assembly is a phenomenon activated by noncovalent interactions such as π-π stacking, electrostatic interactions, hydrogen bonds, and hydrophobic interactions with basic building blocks, organized spontaneously into unique supramolecular nano-scale architectures 1-3. This bottom-up approach has been used in fabrication of numerous nanostructures with potential applications in 3D-cell culture 4, 5, regenerative medicine 6, 7, immune boosting 8, 9, sensing 10, 11, and drug delivery 12, 13. Over the past decades, supramolecular peptide hydrogels have gained much attention in biomedical

a. Pharmaceutical

Nanotechnology Department, Shiraz University of Medical Sciences, Shiraz, Iran b. Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz, Iran c. Polymer Technology Research Group, Department of Chemical and Metallurgical Engineering, Aalto University, 02152 Espoo, Finland d. Transplant Research Center, Shiraz University of Medical Sciences, Shiraz, Iran Corresponding Author: Prof. Ali Mohammad Tamaddon Office: +98-713-2424127 Fax: +98-713-2424126 Email: [email protected]

fields due to their numerous advantages, such as ease of synthesis, degradation over time into predictable metabolites without immunogenicity, easy integration to bioactive ligands, widely tuneable mechanical properties, cell signalling capacity, and most importantly high biocompatibility 2, 14-16. Owing to their interesting properties, short peptide hydrogels containing 2-5 amino acid residues, have attracted significant interest. Unlike many other nano-structured hydrogels, short peptide hydrogels form through self-assembly process in response to changes in physiological stimuli including pH 17, 18, ionic strength 19, 20, organic solvent 21, 22, or temperature 23. According to the recent reports 24-30, efficient gelation of oligopeptides generally requires a large aromatic ligand, such as benzyloxy carbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), naphthalene (Nap), and pyrene or phenothiazine (PTZ), facilitating self-assembly of peptides into supramolecular hydrogels. Apart from the aromatic ligand such as Fmoc, commonly used as a amine protecting group in peptide synthesis 31, 32, the amino acid sequence can alter physicochemical and biological properties of short peptide hydrogels. In general, the overall hydrophobicity determines gelation property of Fmoc-

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dipeptide. Ulijn et al. investigated the self-assembly behaviour of Fmoc-FG, Fmoc-GG, and Fmoc-GF in comparison to FmocFF. Their results indicated a shift in apparent pKa and thermosensitive gelation depending on Fmoc-dipeptide hydrophobicity. It was also found that Fmoc-dipeptides such as Fmoc-GF could form precipitates rather than hydrogel networks in pH lower than their apparent pKa, which was confirmed by micrometre scale sheet-like structures in TEM 33. Tang et al. formed hydrogels from leucine-based Fmocdipeptide homologues, including Fmoc-LL, Fmoc-LG, and FmocGL to study the impact of the aromatic side chains on the selfassembling behaviour of Fmoc-dipeptides. Their results indicated replacing the phenyl ring by isobutyl chain did not affect the propensity of the molecule to self-assemble into extended fibrillar structures. However, no hydrogel structure was formed in case of Fmoc-GL 24. Hence, aromatic amino acids such as L-phenylalanine have a synergistic effect with Fmoc on the self-assembly process through formation of π–π stacking interactions. All in all, the amido acid composition and sequence govern self-assembly and gelation properties of Fmoc-dipeptides. On the other hand, an ideal scaffold for cell delivery should possess an excellent mechanical properties and tailored structure with an interconnected pore network 34, 35. The scaffold moduli and shear-thinning/self-healing kinetics are important determinants of the scaffold suitability for biomedical applications. The shear-thinning systems are developed to form network structures under physiological conditions, flow under moderate pressure (during injection), and self-heal (after injection) 35. As addressed in literature, hydrogels with low storage and loss moduli, low yield strain, and low viscosity are generally well suited for injection 36, 37. Notably, the mechanical (i.e. stiffness, recovery rate, and injectability) and functional properties (i.e. permeability and aggregate modulus) of hydrogel scaffolds can play a crucial role in regulating the interactions between cells and extracellular matrix and directing the cells phenotype 38, 39. For example, mesenchymal stem cells (MSCs) proliferates on stiff hydrogels 40 whereas the neuronal stem cells prefer soft hydrogels for their growth 41. Also, the scaffold microstructure may direct differentiation of stem cells. Cheng et al showed porous scaffold derived from the articular cartilage can induce chondrogenic differentiation of adipose-derived adult stem cells without exogenous growth factors. In comparison with native cartilage, the designed scaffold exhibited suitable biomechanical properties such as the same aggregation modulus with 10-fold more permeability 42. Therefore, the present study aimed to introduce Fmoc-FV as a new dipeptide hydrogel scaffold for 3D-culture applications. To do so, we carried out synthesis of Fmoc-FV in liquid phase and formed hydrogels by the pH-titration method. The gelation property and morphology of Fmoc-FV were compared to Fmocphenylalanine-glycine (Fmoc-FG). Consequently, the structural and mechanical properties of Fmoc-FV were investigated by spectroscopic methods and oscillatory rheometry. Importantly, 3D-cell growth in the supported hydrogel was realized for Wharton’s jelly mesenchymal stem cells (WJ-MSC), human umbilical vein endothelial cells (HUVEC), and MDA-MB-231

breast cancer cell line. To the best of our knowledge, neither View Article Online DOI: 10.1039/D0SM01299H detailed mechanical nor biological studies were conducted on Fmoc-FV hydrogel, which will be discussed in this paper.

2. Result and discussion 2.1. Synthesis and charcterization of Fmoc-FV Fmoc-FV was synthesized with an acceptable recovery yield (70.6%). Fmoc-dipeptides were prepared in 2 consecutive steps: 1) coupling of the tert-butyl ester of valine or glycine to Fmoc-protected phenylalanine by carbodiimide chemistry, 2) elimination of the tert-butyl protecting group under acidic condition (Fig. 1). The products were characterized by FT-IR and 1H-NMR spectroscopy. Tert-butyl esters of Fmoc dipeptides like FmocFV-OtBu present peaks at 3302 cm-1 (N-H stretching), 3064, 3030, 2976, and 2932 cm-1 (C–H stretching), 1734 cm-1 (ester C=O stretching), 1656 cm-1 (amide C=O stretching), and 1542 cm-1 (N–H bending). After the elimination of tert-butyl protecting group by TFA, the ester C=O stretching bond disappeared and the characteristic peak of acidic C=O stretching bond appeared at 1709 cm-1, similarly for Fmoc-FG. Moreover, the FT-IR spectra of Fmoc-dipeptides showed a peak around 1660 cm-1, confirming the presence of amide bond between amino acids (ESI. 1) 43. 1H-NMR spectra of short peptides in DMSO-d6 (ESI. 2) showed multiple peaks at 2-4 ppm corresponding to the aliphatic carbon protons and the signals at 6.5-8 ppm, which refers to the phenyl protons. Also, the characteristic peak of hydroxyl proton of carboxylic group was observed at 12.5 ppm, confirming successful synthesis of dipeptides. 2.2. Fmoc-FV gelation and morphology Dispersion of Fmoc-FV in deionized water can lead to hierarchical assembly and gelation, which depends on the concentration, pH, and temperature 2, 23, 44. To prepare hydrogels, Fmoc-FV was dissolved by alkalizing the aqueous dispersion through adding concentrated NaOH solution 45. At high pH, the carboxylic terminal group of Fmoc-FV was expected to be ionized in the solution. Since the Fmoc moiety can be removed at pH>12, the filtered NaOH solution was added dropwise until pH=9.1±0.1 was reached 46. Then, Fmocdipeptide solutions were titrated by HCl until clear gels were formed. Vial inversion test confirmed the formation of selfsupporting gel without collapsing. When the pH was reduced (above the critical gelation concentration), antiparallel βsheets formed, the fibres entangled, and self-supporting hydrogel was obtained as it was indicated in sections 3.3 and 3.4 18, 47. The gelation occurred in concentrations above 20 mM in various pH values ranging from 3.2-7.5. Similar gelation behaviours were reported elsewhere for Fmoc-dipeptides containing leucine and alanine 18, 24, 27. Unlike Fmoc-FG which became viscous only in acidic pH (between 3-5) (data not shown), Fmoc-FV gelation readily occurred at physiologic pH (7.4). It has been shown that the gelation of Fmoc-dipeptides occurs in the pH below their apparent pKa. Moreover,

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depending on the hydrophobicity of Fmoc-dipeptides, apparent pKa shifts from the theoretical value due to their selfassembly in aqueous medium 33. Similarly, Fmoc-FF revealed two distinct pKa values (5.2-6.2 and 9.5-10.2), resulting in structural transitions 18. Apart from the gelation pH, the appearance of the Fmoc-dipeptides hydrogels varies with the amino acid composition. Unlike Fmoc-FG which became turbid by acid titration 45, Fmoc-FV hydrogel exhibited a transparent hydrogel. Similarly, Fmoc-LF unlike Fmoc-LA was reported to form transparent hydrogels 45. FE-SEM image of Fmoc-FV confirmed fibrous network with fibril thickness of 30±5 nm (Fig. 2A). In contrast, Fmoc-FG fibres were thin with thickness of 10±7 nm (ESI. 3), indicating that the fibre thickness depends on the amino acid composition. Interestingly, Fmoc-FV formed a “spaghetti-like” pile of thick fibres with favourable pores due to high hydrophobicity and aggregation number of Fmoc-FV precursor 48. As similarly shown elsewhere for Fmoc-FG or Fmoc-GF, glycine residues result in flexible fibres which do not favour βsheet formation 33. On the other hand, isopropyl side chain of valine can boost up the thickness and consequently overall rigidity of the peptide fibres. Also, Fmoc-GG and Fmoc-AA form nanofibers with average thickness of 33 and 68 nm, respectively 27. These results suggest that Fmoc moiety is playing the key role in self-assembly, and the peptide composition provides tunable physicochemical properties 24, 33, 49. Possible interplay of concentration and temperature on the gelation time of Fmoc-FV was investigated by the tube inversion method. As shown in Fig. 2B, by raising the temperature from 4°C to 37°C, the gelation time decreased. In addition, the gelation occurred in concentrations lower than 20 mM. Also, more transparent hydrogels of Fmoc-FV were attained upon heating. Therefore, the temperature can alter gelation property of Fmoc-FV that will be explained in the oscillatory rheology section. 2.3. Supramolcular assembly of Fmoc-FV To understand the mechanism by which the gelation occurs, the secondary structures of the Fmoc-FV was characterized by fluorescence pyrene assay, ATR-FTIR, fluorescence spectroscopy, and CD. Due to the availability of hydrophilic and hydrophobic moieties, the Fmoc-dipeptide can form assembly at concentrations above CAC. To investigate the critical concentration of fibre formation, the fluorescence intensities of samples were determined for increasing concentrations of Fmoc-dipeptide in the presence of fixed amount of pyrene. The critical concentration at intersection of the flat regions was observed at 0.5 mM (Fig. 3). Similarly, Fmoc-FF showed the CAC value of 0.337 mM with a lower propensity to self-assemble compared to the Fmoc-dipeptides comprising α-methyl-Lphenylalanine 50. ATR-FTIR spectra of Fmoc-FV in different pH values were collected in the wave number ranging from 1500-1800 cm1. After acid titration of Fmoc-FV alkaline solution, two dominant peaks appeared in the amide I region (around 1600 cm-1 and

1690 cm-1) (Fig. 4A), suggesting that the main Viewsecondary Article Online 10.1039/D0SM01299H structure was antiparallel β-sheets. DOI: However, the peaks around 1600 cm-1 were weak; hence, a lower proportion of βsheet structures was present. In contrast, a broad peak around 1655 cm-1 was observed, indicating abundance of random coil structure. π-π stacking interaction was proposed as a driving force triggering self-assembly of aromatic dipeptides 49, 51. For further characterization of Fmoc-dipeptide association, the arrangements of fluorenyl group in the supramolecular structures were observed by fluorescence spectroscopy (Fig. 4B). At pH around 9, the maximum emission wavelength was observed at 316 nm. Upon adding hydrochloric acid, the emission maximum wavelengths immediately shifted to 326 nm, and fluorescence quenching occurred, indicating antiparallel π–π stacking of fluorenyl rings 24. Complementary to the ATR-FTIR and fluorescence spectroscopy experiments, the CD spectrum of Fmoc-FV was collected in the wavelength ranging from 190-260 nm (Fig. 4C). A ‘v’-shaped spectrum with a negative peak around 218 nm and positive peaks around 190 nm and 240 nm was obtained, indicating anti-parallel structure with β-sheet arrangement. In addition, the negative peak between 210 and 230 nm was weak; hence, lower amount of β-sheet structures was present, as it was similarly shown in the ATR-FTIR investigation. It should be noted that this measurement was carried out at low concentration since the scattered light arising from the gel structure interferes with CD data collection 33, 51. Altogether, the spectroscopic data suggested that the Fmoc-FV peptide moiety should be in an anti-parallel β-sheet arrangement with interlocked chains through п-п stacking of the Fmoc moieties. 2.4. Oscillatory rheology Apart from the microscopic alteration in supramolecular structure of Fmoc-dipeptides, the peptide composition can macroscopically influence the visco-elasticity of the hydrogels. As shown in Fig. 6A, the elastic modulus (Gˊ) appeared 2-3 times higher than the viscous modulus (G˝) at pH 8.5 until a crossover of the moduli was observed at around 1 rad s−1. Both Gˊ and G˝ showed strong frequency dependence between 0.1 and 100 rad s−1, indicating the shear-thinning property and transition of gel to liquid-like materials by shear stress 33. By decreasing pH to 7.4 (Fig. 5A), Gˊ was mainly higher (4-6 times) than G˝ between 0.1 and 100 rad s−1 so both moduli displayed weak frequency dependence, confirming the stable gel-like structure with entangled fibrillar network as shown in the FESEM image. 27. Moreover, by comparing the modulus Gˊ at various pH values, Fmoc-FV exhibited stiffer hydrogel at pH 7.4 (Gˊ=12-26 kPa) than pH 8.5 (Gˊ= 0.0003-3.5 Pa). There are only few reports on mechanical properties of Fmocdipeptide hydrogels. Hence, dynamic frequency sweep was performed first at 4°C and 37°C. As shown in Fig. 5B, Gˊ values at 37°C were found to be 1.5-2 times higher than Gˊ values at 4°C, which showed hydrogel became stiffer upon heating. Then, dynamic temperature sweep was obtained at pH 8.5 and 7.4. Fig. 5C shows the temperature-dependency of mechanical moduli, so the Gˊ values appeared about 2 times higher than

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the G˝ values until a crossover of the moduli around 50°C (Tgel) occurred at pH 8.5. At pH 7.4, Gˊ was several times higher than G˝, indicating that the gelation occurred at a temperature lower than the studied range. Moreover, high stiffness of Fmoc-FV hydrogel was confirmed by the relatively high Gˊ values. The gel stiffness could be causally related to the intermolecular interactions associated with the amino acids (phenylalanine and valine). On the other hand, during the heating step, aggregated fibres were re-dissolved and locked in the self-assembled structures, resulting in an increase in effective fibre concentration and gel stiffness. These results clearly revealed that Fmoc-FV hydrogels became stiffer with raising the temperature, which is in line with our previous results (section 3.2) and visual observations 24, 33. As shown in Fig. 5D, by increasing the shear rate, a modest degeneration of the three-dimensional hydrogel network occurred, which in turn led to the decrement in viscosity of Fmoc-FV hydrogel 23. All in all, these results confirmed that Fmoc-FV formed a shearthinning hydrogel from the self-assembling amphiphiles at physiologic pH. 2.5. WJ-MSCs isolation and characterizations The plastic-adherent cells from Wharton’s jelly formed a monolayer with fibroblast-like morphology (Fig. 6A). After differentiation toward adipogenic and osteogenic lineage, the presence of lipid vacuoles was confirmed by Oil Red O, and calcium deposition revealed with Alizarin Red (Fig. 6B). The flow cytometry results showed that the WJ-MSCs expressed stromal markers (CD90, CD44), while they were negative for the hematopoietic markers (CD34 and CD45) (Fig. 6C). It has been reported that WJ-MSCs proliferated like plastic adherent cells under in vitro culture conditions. As similarly shown, they are positive for the expression of CD105, CD44, CD73, and CD90, and negative for the expression of hematopoietic cell surface markers such as CD34, CD19, CD45, CD11a, and HLA DR (human leukocyte antigen) 52-54. 2.6. 3D culture and Live/Dead assay Potential application of Fmoc-FV hydrogel as 3D scaffold was investigated for culturing WJ-MSC (mesenchymal stem cell), HUVEC (primary cell), and MDA-MB231 (tumour cell line). To encapsulate the cells, an equal volume of Fmoc-FV solution was mixed with cell suspensions prepared in serum-free DMEM. Interestingly, the self-supporting hydrogels were formed rapidly at 37°C; hence, homogeneity of the cell distribution within the hydrogel matrix was ensured. It seems that DMEM medium components such as the buffering agents and metal ions play a vital role in accelerating self-assembly and gelation of Fmoc-FV. As shown in Fig. 7, the gelation process did not change the cell viability according to the LiveDead assay performed 4 h post incubation. No dead cells (red) were detected within the gels and the green (live) cells were visualized with well-defined round contours. Within the first 24 h of culture, cell proliferation was noticed in Fmoc-FV scaffold for all cell cultures. In addition, the cells adopted a polyhedral shape with fine filopodia. After 72 h incubation, the cells

formed a 3D-network, which was more pronounced in MDAView Article Online DOI:showed 10.1039/D0SM01299H MB231. As expected, Fmoc-FV hydrogel cell typedependent growth, so only few dead cells appeared in the hydrogel containing HUVEC and MDA-MB231 cells. In contrast, the number of dead cells increased for WJ-MSC after 72 h post-incubation. Interestingly, WJ-MSCs almost maintained in spherical or ellipsoidal shape in the Fmoc-FV scaffold. This event can be attributed to low cell-matrix attachment of WJMSCs to the Fmoc-FV scaffold as similarly shown elsewhere 33. Zhou et al. reported that the Fmoc-FF hydrogel incorporated with Fmoc-RGD ligand promoted cell adhesion with subsequent spreading of human adult dermal fibroblast 55. 2.7. MTT and Alamar blue assays To investigate the cytocompatibility of the hydrogel construct and disentangled hydrogel nanofiber, the cell viability was determined by (A) MTT and (B) Alamar blue cytotoxicity assay, respectively. As shown in Fig. 8A, Fmoc-FV nanofibers showed no significant cytotoxicity at concentrations as low as 0.05 mM in HUVEC and MDA-MB231 cell lines for 24 h (P>0.05). However, their cytotoxicity increased significantly by the nanofiber concentration, so the cell viability reduced significantly at 0.001, 0.005, and 0.01 mM in both cell lines (P0.05); however, dissimilar letters indicate a significant difference (P0.05); however, dissimilar letters indicate a significant difference (P