Thermally-controlled spherical peptide gel architectures prepared using the pH switch method

Self-assembling nanostructured peptide gels are promising materials for sensing, drug delivery, and energy harvesting. Of particular interest are short diphenylalanine (FF) peptides modified with 9-fluorenylmethyloxycarbonyl (Fmoc), which promotes the association of the peptide building blocks. Fmoc-FF gels generally form fibrous networks and while other structures have been demonstrated, further control of the gelation and resulting ordered three-dimensional structures potentially offers new possibilities in tissue engineering, sensing, and drug release applications. Herein, we report that the structure tunability of Fmoc-FF gels can be achieved by controlling the water content and the temperature. We further explore the incorporation of metal nanoparticles in the formation of the gel to enable optical sensing applications based on hybrid Fmoc-FF-nanoparticle microspheres. Finally, fluorescence lifetime imaging microscopy reveals a correlation between lifetime and reduced bandgap, in support of a semiconductor-induced charge transfer mechanism that might also increase the stability of an excited state of a probe molecule. The observations potentially further widen the use of these peptide materials in bioimaging and sensing applications.

The formation of spherical micro vesicles has been previously demonstrated and used for drug delivery with other peptides. [15,16] Boc-Phe-Phe-OH dissolved in hexa-fluoro-2-propanol (HFIP) and diluted with ethanol led to the formation of spheres with sizes ranging from 30 nm to 2 μm. [15] In addition, microspheres with diameters ranging from 2 to 3 μm were prepared using Boc-Trp-Leu-Trp-Leu-OMe in ethanol and methanol. [16] In a separate study, Arnon et al. [17] reported that fluorenylmethoxycarbonyl-β,β-diphenyl-Ala-OH (Fmoc-Dip) in ethanol formed needle-like crystals. When the assembly conditions were altered using a "solvent switch" method (i.e., the addition of water to peptide solution prepared in ethanol), spherical particles formed. These spherical particles scattered different wavelengths of light depending on their size (≤2 μm). [17] In these examples, the size of the microspheres did not exceed 3 μm. There is a reported need for larger microspheres that could serve as better carriers for drug delivery. [18] This has motivated us to investigate the possibility of making larger particles from Fmoc-FF by tailoring the sample preparation through solvent and temperature control.
Beyond investigating the role of solvent on the formation of such peptide gels and resultant structures, the effect of temperature is important for several scientific and practical purposes. Some of the applications include fluorescence and Raman based biosensing. [19,20] Despite the fact that peptide-based hydrogel materials have been studied extensively to achieve diverse architectures, including fibrils and ribbons, the role of heating and the interplay with solvent has not been fully explored, [21][22][23] especially in the presence of metal NPs. Understanding the key factors that control gelation, structure formation, and the functional properties of the resulting gels could potentially open up the use of those materials in new applications. [24][25][26] Fmoc-FF gels generally form fibrous networks, [4,27] yet porous microspheres have been demonstrated, [28] and both temperature and solvent have been used to tailor the structure of other peptide-based materials. [28] We have previously reported that bioinspired semiconducting diphenylalanine peptide nanotubes (FFPNTs) can result in structural transition when annealed. Such structures can support Raman detection of 10 À7 M concentrations for a range of molecules, including mononucleotides. The enhancement is attributed to the introduction of electronic states below the conduction band that facilitate charge transfer to the analyte molecule. [29] Thus, in this study, we have investigated the influence of both temperature and water content on Fmoc-FF gel formation. We have found that microsphere formation can be tuned by increasing the water content and by heating the solution. We further explored the incorporation of metal NPs during gelation to enable optical-based applications, demonstrating the generation of hybrid Fmoc-FF-NP microspheres. Raman, UV-vis, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) imaging were used to characterize the Fmoc-FF structure. We have also performed computational simulations to study the effect of heat on Fmoc-FF structure.

| Theoretical calculations
The corresponding molecular structure was fully optimized using Density Function Theory (DFT) as implemented in the QuantumATK software [30] using a local combination of the atomic orbitals (LCAO) approach, the Perdew, Burke, Ernzerhof (PBE) functionals, [31] the norm-conserving PseudoDojo [32] pseudopotential with medium basis set, and a mesh cut-off energy of 10 5 Ha. The calculation of the self-consistent field (SCF) considered a tolerance limit of 10 À6 Ha for energy convergence. The geometry structure and ion relaxations were performed using the limited-memory Broyden-Fletcher-Goldfarb-Shanno (LBFGS) algorithm, including the force on each atom less than 0.05 eV/Å. In addition, a molecular dynamics simulation, based on heating runs through a simulated annealing protocol, is conducted using the reactive force field (ReaxFF) at temperatures of 100 C. NVT Nose Hoover algorithm [30] was used in the calculations, which has a 1 fs calculation time step along with 100 fs of thermostat timescale. The reservoir temperature, which specifies the temperature of the heat bath, is kept at 100 C. At the study temperature, the starting velocities of the atoms follow the Maxwell-Boltzmann distribution. Water molecules were not included in the calculation.

| RESULTS AND DISCUSSION
In this work, we have investigated the influence of increasing ddH 2 O content and temperature on Fmoc-FF hydrogel formation using widely used [4,33] "pH switch" (Figures 1 and 2 and Figure S1) and "solvent switch" ( Figure S2) methods, initially without and subsequently with Ag NPs. The "solvent switch" method relies on the addition of ddH2O water to a stock peptide solution (100 mg/mL) prepared in an organic solvent (DMSO). [5] The ddH 2 O water has lower solubility, triggering spontaneous self-assembly into an organized structure. The peptide backbones form hydrogen bonds necessary to create β-sheets, while the aromatic nature of the fluorenyl group and the amino acid side groups allow for π-π stacking interactions, which are highly anisotropic and short-range relative to the size of the Fmoc-FF molecule. [3][4][5]24] In the "pH switch" method, gel formation is achieved by lowering the initial pH of the peptide solution (pH = 10.5) with the progressive addition of HCl. [4] Studies have demonstrated that Fmoc-FF can assemble in a β-sheet structure when prepared via "pH switch." [4,24] For the titration experiment with ddH 2 O, Fmoc-FF was mixed with ddH 2 O at different ratios from 1:1 to 1:6.

| Role of water content
An SEM image of a Fmoc-FF gel prepared using "pH switch" is shown in Figure 1a. Typically, Fmoc-FF self-assembles into a nanofibrillar hydrogel in ddH 2 O via hydrogen bonding and π-π interactions, leading to the formation of β-sheet structures. [1][2][3][4]34] The addition of water to Fmoc-FF leads to the formation of larger β-sheet structures and therefore larger fibrils, in agreement with previous reports. [1][2][3][4][5]34] Ordered fibrils were produced when the Fmoc-FF was prepared via "solvent switch"  Table S1.
To  [19] previously associated a red shift of N H  both associated with Fmoc-FF fibril formation, were present. [1][2][3]34] Bands associated with parallel β-sheets [15,16] such as 1536 cm À1 (amide II N H bending) and 1690 cm À1 (amide I carbonyl C O stretching) were observed (Figure 2d and Figure S2e). Other studies have shown that parallel β-sheet structures in FF-based (i.e., Boc-Phe-Phe-OH) microspheres form through the creation of a network of hydrogen bonds and π-π interactions, whereby the carbonyl groups at $1693 cm À1 result in the formation of a spherical structure. [35,36] FTIR spectra remained largely unchanged when Fmoc-FF was prepared via "solvent switch." All band assignments were in agreement with literature reports for Fmoc-FF in fibrillar form ( Figure S2e). [4,5,24] UV-vis spectra for Fmoc-FF prepared using "pH switch" and "solvent switch" methods as a function of ddH 2 O content are shown in Figure 1f and Figure S2f. Fmoc-FF prepared using both methods exhibited a peak at the long-wavelength edge (314 nm), in line with literature reports. [1][2][3] With the addition of water to Fmoc-FF prepared using "pH switch," the absorption increased and with high water content (1:4 and 1:6). The UV-vis spectra lose their structure and become flat. The bandgap was determined from UV-vis data following previous reports [37,38] to decrease from 4.8 ± 0.1 (control samples) to 3.9 ± 0.1 eV at 1:6 ratio. Studies have shown that the presence or addition of ddH 2 O leads to changes in the electronic properties of peptide-based materials that reduce the bandgap and lead to increased conductivity. [39] The increased conductivity could be associated with the alignment of the ddH 2 O molecules' dipole moments that results in a larger dipole moment of the overall structure. [39] Fmoc-FF prepared using "solvent switch" experienced only slight changes in absorption and a smaller decrease in the calculated bandgap from 4.6 ± 0.1 to 4.1 ± 0.1 eV ( Figure S2f). This observation suggests that Fmoc-FF prepared using "pH switch" is more sensitive to changes in the ddH 2 O content.
Raman spectra for Fmoc-FF prepared using both methods as a function of ddH 2 O content are shown in Figure 1g and Figure S2g.
Raman bands for Fmoc-FF prepared using "pH switch" were more apparent (Figure 1g) in comparison to Fmoc-FF prepared using "solvent switch" where DMSO bands (682 and 713 cm À1 ) dominated the spectra ( Figure S2g). Raman of Fmoc-FF prepared using "pH switch" presents the typical breathing modes at 998 and 1020 cm À1 and other characteristic bands (719, 736, 1265, 1350, 1412, 1452, and 1507 cm À1 ) all in agreement with the literature reports. [3] As the water content increased, bands associated with Fmoc-FF, such as the aforementioned breathing modes, decreased in intensity, as expected.
A new sharp band formed at around 1160 cm À1 (C H stretching) that could be an indication of microsphere formation (Figure 1g).  temperature. [40] Given the role of hydrophobic interactions and hydrogen bonding on the self-assembly of peptides, and as evidenced above for Fmoc-FF in particular, we next investigated the effect of heating on Fmoc-FF prepared using "pH switch" (with no changes in water content) at 20-100 C for 5 min in closed containers. During self-assembly, Fmoc-FF molecules form cylindrical nanofibrils by interlocking four twisted antiparallel β-sheets through lateral antiparallel π-π interactions, [9] as shown in Figure 2a. Scanning electron microscope (SEM) images of Fmoc-FF prepared via "pH switch" as a function of temperature are shown in Figure 2a-c. At 20 C, Fmoc-FF exhibited a dense network of fibrous structures with diameters of 102 ± 43 nm (n = 30 fibrils), in line with literature reports. [3] As the temperature increased, the microspheres formed and increased in diameter, as shown in Figure S4. The diameter was 1.0 ± 0.3 μm at 40 C, 1.1 ± 0.2 μm at 60 C, and 1.2 ± 0.3 μm at 100 C. Not only do microspheres form after 5 min of heating at 100 C, but they also form when heating at 50 C for 10 min when using Fmoc-FF prepared by "pH switch" ( Figure S5). When using "solvent switch," no microspheres were observed at any temperature; only slightly increased fibril diameters were observed (up to 120 ± 65 nm [n = 30 fibrils]) at 100 C ( Figure S6).

| Role of temperature
Fmoc-FF samples prepared at different temperatures were further analyzed by FTIR, UV-vis, and Raman spectroscopy, as illustrated in Figure 2d-f. The FTIR spectrum of Fmoc-FF prepared using "pH switch" (Figure 2d and Figure S7) at 20 C ( Figure S4a) has weak amide I bands in the range 1500-1600 cm À1 , similar to the band assignments reported in Figure 1f. However, more intense bands started to appear with increasing temperature, such as the band at 1539 cm À1 associated with N H bending (amide II), other amide II bands at 1693 and 1746 cm À1 , and the band at 1746 cm À1 attributed to C O stretching. [5] The red shift and split of the N H stretching band at 3223 and 3354 cm À1 after heating to 100 С might indicate a rearrangement of hydrogen bonds. [19] All bands between 3000 and 3500 cm À1 are attributed to O H, N H, and C H, as described previously. [19] No significant changes were observed in FTIR measurements when Fmoc-FF was prepared via "solvent switch" at different temperatures.
UV-vis spectroscopic analysis of Fmoc-FF prepared using "pH switch" and "solvent switch" methods as a function of temperature is shown in Figure 2e and Figure S6f. From both methods, there was a slight increase in the absorption intensity with temperature compared to Fmoc-FF (Figure 1g). When using "pH switch" at 100 C, the UVvis spectrum becomes flat in around 200-220 nm. There was an increase in the bands at 260 nm (as shaded in purple in Figure 2e) and a blue shift in bands located at 320 nm. These changes were not present when Fmoc-FF was prepared via "solvent switch," suggesting that "pH switch" is more sensitive to temperature. The bandgap was determined from UV-vis data to have decreased from 4.8 ± 0.1 eV at 20 C to 4.6 ± 0.1 eV at 60 C, and to 4.1 ± 0.2 eV at 100 C for "pH switch." From this observation, we have concluded that Fmoc-FF prepared by the pH switch method is more sensitive to temperature changes; however, further studies are needed to fully understand the underlying mechanism.

| Computational simulations of the effect of heat
To gain an in-depth understanding of the effect of heating on the sheets. Figure 3b shows the optimized hydrogen-bonding network structure, while the bond lengths and angles of the hydrogen bonds are presented in Table S2. It is observed from the plots that the molecular packing is described by intra-and intermolecular hydrogen bonds characterized by different functional groups present in the structure ( CO, COOH, O , NH). HN(1) and HN(5) form intermolecular hydrogen bonds to O(9) and O(8) atoms in a carbonyl group of an adjacent molecule, respectively, that stabilize the molecular sheet structure along the y-axis (see Figure 3b).
A molecular dynamics simulation, based on heating runs through a simulated annealing protocol, is carried out at temperatures of 100 C, lasting for 5 ps with a time step of 1 fs. As illustrated in Figure S9, the potential energy of the Fmoc-FF system varies around a constant value of À1204 eV with a considerably small fluctuation magnitude, showing the thermal stability of the newly obtained structure. The fully relaxed structure after 5 ps at 100 C is plotted in Figure 3c, where geometry reconstruction occurs, demonstrating a deformation of the stacked arrangement of Fmoc-FF sheets during the temporal evolution. Moreover, aromatic π-π stackings were reduced for two Fmoc-FF sheets with centroid distances of 3.309 Å. The molecular sheet structure after heating shows pronounced deformations via rotations of the different hydrophobic side groups (e.g., C19-O5-C20-C21 torsion angle has a larger deviation than that of the molecular structure before heating, see Table   S3). These results are consistent with our experimental measurements.
Water molecules were not included in the simulation.

| Influence of the presence of Ag NPs
In order to understand the influence of the presence of Ag NPs, which would normally widen the use of Fmoc-FF in optical sensing applications, [4,8] Ag NPs were incorporated within the structures.
Adding NPs to Fmoc-FF gels prepared using "solvent switch" (Fig-ure S10) yields results in line with literature reports. [3] SEM images of Fmoc-FF prepared by "pH switch" in the presence of Ag NPs (Figure 4a) revealed fibrils with a typical diameter of 83 ± 15 nm; however, when heated at 100 C, the fibrils formed a microsphere-like structure that encapsulated the fibers (Figure 4b).
The small fibers assembled into thick fiber bundles and formed more densely packed and entangled three-dimensional networks. However, upon cooling after heating the solution, the encapsulated fibers start to spread again over the surface upon drying, highlighting the reversibility of the process ( Figure S11).
To understand the influence of NPs on gel formation and optical properties, FTIR, UV-vis, and Raman investigations were undertaken.
FTIR data ( Figure S12) are comparable to those in Figure 2, revealing the appearance of strong amide I bands located at $1690 and bands.
From UV-vis data, it was determined that the bandgap decreased from 4.6 ± 0.1 eV at 20 C to 3.8 ± 0.2 eV at 100 C, slightly lower than in the absence of the Ag NPs. In addition, a broadening and red shift ($20 nm) of the surface plasmon resonance (SPR) of Ag NPs (at 420 nm) was observed (Figure 4f). [5] Tuning the SPR of NPs, as demonstrated here, is of interest for optical and bio sensing applications.
Heat plays a significant role in the gel formation, as Raman data show changes including increased Raman intensity with increasing temperature in gels prepared using both methods (Figure 4g and   3.5 | Effect of increasing water content at a fixed temperature of 100 C Given that hydrophobic interactions and hydrogen bonds are key to the formation and stability of secondary structure of peptides, they can be affected by temperature. [40] We have investigated the effect of both heat and water content on Fmoc-FF prepared only by "pH switch" as "solvent switch" did not produce spherical structures. Interestingly, combining heating at 100 C and titration experiments (e.g., 1:1 ratio) without and with NPs ( Figures 5 and 6) resulted in the transformation of most of the fibrils to spherical structures with a range of nanometer to micrometer sized diameters, as illustrated in the histograms shown in Figures S13 and S14. At 100 C, the diameter was 1.6 ± 0.4 μm at 1:1 ratio with ddH 2 O, 2.6 ± 0.7 μm at 1:2, 3.1 ± 1.0 μm at 1:4, and 4.2 ± 1.8 μm at 1:6.
As before, the FTIR spectrum of the heated Fmoc-FF (Figure 5e Table S4. and 1744 cm À1 . The peak at 1693 cm À1 has been assigned in the literature to peptides adopting a sphere shape. [21,22] The findings suggest that Fmoc-FF loses its antiparallel β-sheet organization to form microspheres with a predominant parallel β-sheet structure. The exact nature of the induced structure, however, has not been yet but charge transfer and changes in band structure likely contribute. [41] The results highlight the potential for using the microspheres in SERS sensing applications and a potential avenue to explore charge transfer via hydrogen bond-tuning. [41] Not only was an enhancement in SERS intensity observed but also an increase in PL signal was seen. Figure   S19 shows emission spectra of CV and MB on pristine and annealed Fmoc-FF at 100 C with and without water content and with and without Ag NPs. It can be seen from both molecules used that the highest PL intensity was recorded when using Fmoc-FF spheres at 1:1 water ratio at 100 C with Ag NPs. Both the addition of water and heat result in increased PL intensity in comparison to control samples when using CV or MB on cover slips, for instance, or when using Fmoc-FF only without further treatment, resulting in a 16-fold increase with NP and a 7-fold increase without NPs in PL for MB on Fmoc-FF spheres at 1:1 ratio with water and 100 C ( Figure S19c,d). The increase in PL intensity could be attributed to the fact that both heat and water change the hydrophobic interactions and hydrogen bonding, affecting intermolecular interaction and hence enhancing the PL intensity. [38,42] Similar results were observed when using CV ( Figure S19).
To better understand the Raman enhancement mechanism, fluorescence lifetime imaging microscopy (FLIM) by multidimensional time-correlated single-photon counting was performed (Figure 8)  lifetime for the excited singlet state between Fmoc-FF and TMPyP. [43] Our findings are in agreement with previous studies [38] reporting that charge transfer from peptide-based materials can stabilize the excited state of the photosensitizer or dye molecules.
The formation of FF fibrils, nanotubes, and nanospheres by aromatic dipeptides is facilitated by the closure of two-dimensional peptide layers. [44] Previous reports have shown that solvent com-  type and valence state, ionic strength, pH) and external conditions (temperature, homogenization method). [9,45]