Composite of Peptide-Supramolecular Polymer and Covalent Polymer Comprises a New Multifunctional, Bio-Inspired Soft Material

Abstract

Peptide-based supramolecular hydrogels are utilized as functional materials in tissue engineering, axonal regeneration, and controlled drug delivery. The Arg-Gly-Asp (RGD) ligand based supramolecular gels have immense potential in this respect, as this tripeptide is known to promote cell adhesion. Although several RGD-based supramolecular hydrogels have been reported, most of them are devoid of adequate resilience and long-range stability for in vitro cell culture. In a quest to improve the mechanical properties of these tripeptide-based gels and their durability in cell culture media, the Fmoc-RGD hydrogelator is non-covalently functionalized with a biocompatible and biodegradable polymer, chitosan, resulting in a composite hydrogel with enhanced gelation rate, mechanical properties and cell media durability. Interestingly, both Fmoc-RGD and Fmoc-RGD/chitosan composite hydrogels exhibit thixotropic properties. The utilization of the Fmoc-RGD/chitosan composite hydrogel as a scaffold for 2D and 3D cell cultures is demonstrated. The composite hydrogel is found to have notable antibacterial activity, which stems from the inherent antibacterial properties of chitosan. Furthermore, the composite hydrogels are able to produce ultra-small, mono-dispersed, silver nanoparticles (AgNPs) arranged on the fiber axis. Therefore, the authors’ approach harnesses the attributes of both the supramolecular-polymer (Fmoc-RGD) and the covalent-polymer (chitosan) component, resulting in a composite hydrogel with excellent potential.

Engineering of extracellular matrix (ECM) mimics, with most of the necessary features of a natural ECM, is a crucial requirement for the design of biomaterials. The natural ECM encompasses a 3D network of intertwined protein nanofibers which contains complex biomolecules for communication between cells.^[1] Short peptide-based 3D nanostructured supra-molecular hydrogels are excellent candidates for ECM mimicry as they provide networks of fibers which resemble the ECM structure.^[2–21] Modifications of certain oligopeptide termini with bio-active ligands, aimed to introduce bio-active components into the system, have also been reported.^[22–25] The tripeptide sequence Arg-Gly-Asp (RGD), which was first recognized in fibronectin as an independent cell attachment site, is the most commonly used bio-active ligand.^[26] This tri-peptide sequence is recognized by the α_v β₃ and α₅β₁ integrins located in cell membranes, thus facilitating the coupling of the ECM with the cytoskeleton.^[27] Owing to these attributes, the RGD ligand has been introduced into supramolecular or polymeric substrates to facilitate cell attachment.^[28–31]

Fmoc-RGD [Fmoc = N-(fluorenyl-9-methoxycarbonyl)] is one of the simplest RGD derivatives capable of forming hydrogels with an inter-connected fibrous network.^[15,27] Ulijn’s group reported Fmoc-RGD based supramolecular hydrogels in neutral aqueous solution by appropriate mixing of two short peptide-based building blocks, Fmoc-FF (F = phenylalanine) and Fmoc-RGD and utilized them as scaffolds for cell culture.^[15] They proposed that Fmoc-RGD alone was not able to form β-sheet fibrils, although in the composite gels of Fmoc-FF and Fmoc-RGD (10-30 wt% Fmoc-RGD), signatures of β-sheet structures were found. We later reported flanking of the RGD sequence by aromatic moieties, demonstrating the hydrogelation of RGD derivatives, including Fmoc-FRGD and Fmoc-RGDF.^[32] Hamley and co-workers showed that the simple Fmoc-RGD moiety, without any modification or mixing with other hydro-gelator peptides, could from nano-fibrous hydrogels.^[27] Castel-letto and co-workers prepared monoliths of Fmoc-RGD gels at a high peptide concentration of 10 wt% which showed excellent stability in water.^[33] Hydrogelation of the RGD-based tetrapeptides Fmoc-RGDS and Fmoc-GRDS at a peptide concentration of 2 wt% was also reported by Hamley’s research group.^[34] They observed that the Fmoc-RGDS peptide exhibited significant syneresis behavior upon application of very low stress or strain.^[34]

One major problem of most of the above mentioned hydrogels is their low mechanical properties and lack of stability. This results in scaffolds devoid of the appropriate durability necessary for culturing cells for longer periods of time. Moreover, it was observed that most of these hydrogels failed to endure either, the contact with cell culture media, or, the physiological temperature (37 °C).^[35] One simple way to improve the mechanical properties of these systems is to combine them with covalent polymers. Covalent polymers are robust, and their non-covalent functionalization with supramolecular polymers results in composite hydrogels with much improved mechanical and other physical properties.^[36–50]

Here, aiming to engineer a bio-active hydrogel capable of culturing cells for sufficient period (7 days), we adapted the “supramolecular polymer-covalent polymer” combination approach. The bio-active hydrogelator, Fmoc-RGD (Figure 1A) was chosen as the supramolecular polymer component. As the covalent polymer component, we chose chitosan (Figure 1A), a biocompatible, biodegradable, antibacterial biopolymer, which is widely used for tissue engineering.^[51,52] Furthermore, this biopolymer has the propensity to form H-bonding with the C terminal free carboxylic acid groups^[36] of RGD, resulting in non-covalent functionalization of the peptide. The resulting composite Fmoc-RGD/chitosan hydrogels exhibited enhanced mechanical properties and gelation rate compared to the native Fmoc-RGD gels. Moreover, these gels exhibited much improved durability when in contact with cell culture media. The composite hydrogels were utilized as 2D and 3D cell culture scaffolds, and they also exhibited impressive antibacterial properties due to the presence of chitosan. The physical appearance of the hydrogels (Figure 1A) indicated the composite Fmoc-RGD/chitosan hydrogel to be more homogenous in nature, although with decreased transparency.

Figure 1. Preparation and morphology of the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels.

A) Chemical structures of Fmoc-RGD and chitosan, and digital images of the hydrogels. TEM micrographs of B) Fmoc-RGD and C) Fmoc-RGD/chitosan hydrogels (Scale bars are 1 μm).

Scanning electron microscopy (SEM) was first performed to investigate the ultrastructure of the hydrogels (Figure S1, Supporting Information). While Fmoc-RGD hydrogels exhibited fibrous aggregates on flake like structures, Fmoc-RGD/ chitosan exhibited bundled fibers. Chitosan alone exhibited irregular aggregates. This type of fibrous aggregates, flakes, or bundled fibers could be artefacts during the drying process of the hydrogels.^[53] Therefore, TEM was necessary to visualize the individual fibers.

TEM micrographs (Figure 1B,C) revealed that although both the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels were comprised of nano-fibrous networks, the fiber diameters varied significantly. A histogram of the fiber diameters (Figure S2, Supporting Information) clearly showed them to be smaller in the Fmoc-RGD/chitosan hydrogels (25 ± 4 nm) compared to that of the Fmoc-RGD hydrogels (119 ± 32 nm). This observation is in agreement with previous reports, where a decrease in fiber diameter was observed after addition of a polymeric additive to a supramolecular gelator.^[36,39,53] This signifies that chitosan has an influence during the growth process of the fibers. Expectedly, chitosan solution did not exhibit fibrous structures and some irregular aggregates were observed in their TEM micrographs (Figure S3, Supporting Information).

FTIR spectra (selected portion, for full spectra see Figure S4, Supporting Information) of the Fmoc-RGD hydrogel (Figure 2A) exhibited a peak at 1623 cm^-1 for the amide vibration^[54] possibly indicating the presence of ß-sheet structure. ^27] In the Fmoc-RGD/chitosan hydrogel (Figure 2A), the position of this peak remained unchanged, indicating that chitosan did not affect the secondary structure of the peptide. Another strong peak at 1660 cm^-1 in both the hydrogels may arise from the TFA counter-ions.^[27] The small hump at 1710 cm^-1 observed in the Fmoc-RGD hydrogel spectra may result from C=O stretching vibrations of the carboxylic acid groups.^[55] Notably, this hump was absent in the Fmoc-RGD/chitosan hydrogel, suggesting H-bonding interactions between the carboxylic acid group of Fmoc-RGD and —NH₂/–OH groups of chitosan.^[55] This type of H-bonding interaction in chitosan-based supramolecular gels was also recognized earlier in literature.^[36] Some additional peaks in the 1200-1000 cm^-1 region were also observed in the composite gels due to the presence of chitosan (Figure S4, Supporting Information).^[56] Therefore, from the FTIR spectra it can be surmised that chitosan might have been adsorbed on the Fmoc-RGD core assembly, rather than getting inserted into the fibers.^[57] Apart from H-bonding, electrostatic interactions may also play a crucial role in non-covalent attachment of Fmoc-RGD and chitosan. In fact, the electrostatic interaction between bovine serum albumin (BSA) and positively charged chitosan derivatives has been well documented in literature.^[58–60] To explore this in our system, we have calculated the pH values of chitosan solution, Fmoc-RGD, and Fmoc-RGD/chitosan composite hydrogels, which were found to be 4.6, 2.3, and 3.2, respectively. Therefore, it is apparent that the chitosan chains are positively charged in the solution as well as in the composite hydrogels. The pK_a1 (i.e., pK_a of the α-carboxyl group) of aspartic acid is 1.88. Consequently, it can be assumed that, both in the solution and in the composite hydrogel, the α-carboxyl group of Fmoc-RGD is ionized. Therefore, the α-carboxyl group of Fmoc-RGD should electrostatically interact with the positively charged chitosan chains.

Figure 2. Characterization of the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels.

A) FTIR spectra of dried hydrogels and chitosan film (Selected portions). B) WAXS patterns of the dried hydrogels and chitosan film. C) UV–vis spectra of the hydrogels. D) Fluorescence spectra of the hydrogels. E) CD spectra of the hydrogels.

Figure 2B presents the XRD patterns of chitosan alone and the dried Fmoc-RGD and Fmoc-RGD/chitosan hydrogels. Fmoc-RGD neither exhibited any crystalline peak nor any broad hump. Chitosan alone exhibited an amorphous halo which was also conferred to the Fmoc-RGD/chitosan composite hydrogels. The absence of crystalline peaks in Fmoc-RGD and Fmoc-RGD/chitosan hydrogels suggests the lack of long range order in both of their structures.

UV–vis spectra of the Fmoc-RGD hydrogels exhibit the characteristic Fmoc absorbance at 266, 289, 279, and 300 nm (Figure 2C).^[61] These peak positions remained almost similar in the Fmoc-RGD/chitosan hydrogels (Figure 2C). Fluorescence spectra (Figure 2D) showed an emission peak at 373 nm for the Fmoc-RGD hydrogel, which could be attributed to overlapping of the fluorenyl groups in a parallel fashion.^[62] In the composite Fmoc-RGD/chitosan hydrogel, the peak position was very similar (369 nm). Therefore, the UV-vis and fluorescence spectra both indicate that chitosan did not effectively hamper the aggregation pattern of Fmoc-RGD.

Finally, we performed the CD spectra of Fmoc-RGD and Fmoc-RGD/chitosan hydrogels (Figure 2E) to assess their secondary structures. Fmoc-RGD hydrogel exhibited a minimum at 220 nm which indicates the signature of β-sheet structures.^[27] The red shift of this β-sheet minimum to 232 nm in the Fmoc-RGD/chitosan hydrogel (Figure 2E) may represent a more twisted arrangement which increases the entropy of the β-sheet. HV values of both gels (Figure S5, Supporting Information) were in the acceptable region of 550-750 V, which are lower than the limited value of the instrument (<1000 V).

It is evident that chitosan does not penetrate the β-sheet structure of Fmoc-RGD, and, the H-bonding between carboxylic acid groups of RGD with –NH₂/–OH groups of chitosan along with the electrostatic interactions are responsible for the attachment between the components. TEM micrographs further reveal the decreased fiber diameter in the composite hydrogel. Therefore, chitosan is assumed to be adsorbed on the long axis of the fibers through H-bonding and electrostatic interactions and consequently blocking the access of additional Fmoc-RGD molecules to the growing fibers (Scheme 1).^[53] As a consequence, the composite fibers become thinner. Moreover, no irregular aggregates of chitosan (Figure S3, Supporting Information) were observed in the composite hydrogels. Therefore, clearly, the composite hydrogel material is a homogeneous co-assembly rather than a heterogeneous array.

Scheme 1.

Schematic model explaining the formation of Fmoc-RGD and Fmoc-RGD/chitosan composite fibers. The randomly oriented monomers of Fmoc-RGD arrange into ordered fibrils. These fibrils further aggregate laterally to form fibers, which subsequently entangle to yield the Fmoc-RGD hydrogels. When chitosan is present in the system, it adsorbs on the long axis of the Fmoc-RGD fibrils, thus inhibiting the lateral growth and producing thinner fibers.

Appropriate mechanical properties of a hydrogel material are very important for biological applications. A particular mechanical rigidity of the hydrogels (≈1 kPa) is suitable for the culture of soft tissues.^[63] Therefore, optimization of the mechanical properties is indeed necessary. To evaluate the mechanical rigidity of the hydrogels, we studied their rheological properties. First, the gelation was monitored by time sweep experiments (Figure 3, which revealed that the composite Fmoc-RGD/ chitosan system had gelled much faster compared to Fmoc-RGD gels. The gelation times, defined as the time required for the storage modulus (G′) value to reach a plateau, were ≈239 min and ≈46 min for the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels, respectively. Therefore, chitosan enhances the gelation rate of Fmoc-RGD. The improvement in gelation rate after chitosan incorporation may be attributed to the increment in supramolecular interactions within the system. Chitosan could also serve as an additional heterogeneous nucleation center thereby enhancing nucleation of Fmoc-RGD and gelation rate.^[53]

Figure 3. Mechanical properties of the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels at peptide and polymer concentrations of 1% and 0.5%, respectively.

A) Time sweep experiment carried out on the hydrogels. Frequency-dependent oscillatory rheology of B) Fmoc-RGD and C) Fmoc-RGD/chitosan hydrogels. D) Moduli values as a function of chitosan concentration in Fmoc-RGD/chitosan hydrogels. Continuous step strain measurement at alternate 0.1% and 200% strain over time carried out on E) Fmoc-RGD and F) Fmoc-RGD/chitosan hydrogels.

Strain sweep experiments were carried out on the hydrogels to measure the linear viscoelastic region (LVR) (Figure S6, Supporting Information) at a constant frequency of 1 Hz. Fmoc-RGD and Fmoc-RGD/chitosan hydrogels exhibited breakage at 158% and 42% respectively. Therefore, the time sweep and frequency sweep experiments were carried out at 0.1% strain (within the LVR).

Dynamic frequency sweep experiments carried out on the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels (Figure 3B,C), depicted a wide LVR, and the storage modulus (G′) and loss modulus (G″) showed linear response with variation of frequency. The storage modulus (G′) values were also higher than the loss modulus G″ values in each case. Both these behaviors confirm the gel nature of Fmoc-RGD and Fmoc-RGD/ chitosan samples.^[64,65] Interestingly, a significant increase in modulus values was observed in the composite Fmoc-RGD/ chitosan hydrogels. To quantitatively establish this observation, we compared the G′ values of both the gels at an angular frequency of 10 rad s⁻¹, which were found to be 529 and 3436 Pa for the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels, respectively. A probable reason may be the decreased fiber diameter in the composite gels, which, in turn, increased the aspect ratio of the fibers. These higher aspect ratio fibers entrapped the solvent more tightly, thereby improving the mechanical properties.^[36] Another reason for the improvement of mechanical properties of the Fmoc-RGD/chitosan hydrogels may be attributed to the increased network density of the Fmoc-RGD/chitosan hydrogels as observed in the SEM images of the hydrogels (Figure S1, Supporting Information). Chitosan plays the crucial role of additional nucleation sites enhancing the number of fibers and consequently, the fiber network density, which in turn improves the mechanical properties.^[53]

Next, we examined the effect of chitosan concentration on the storage modulus values of the resultant hydrogels (Figure 3D) keeping Fmoc-RGD concentration constant at 1% w/v. It was observed that at a chitosan concentration below 0.5% w/v, the modulus values increased with increasing chitosan concentrations. However, at a higher concentration (1% w/v), the modulus values decreased. Interestingly, both the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels exhibited thixotropic properties. Thixotropy is typically a viscoelastic property, in which a gel material is isothermally converted into sol state by mechanical stimuli and returns to the gel state on standing. This behavior is very important for hydrogels used in bio-medical applications.^[66] To illustrate the thixotropic properties of the hydrogels, we performed step strain measurements, in which, a time sweep experiment was carried out on the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels with alternating strain values (0.1% and 200%) in each step (Figure 3E,F). Both the hydrogels showed thixotropic properties, as the gels transferred into a sol state at 200% strain (G′ < G″) and recovered again when the strain was lowered to 0.1% (G′ > G″).^[39] We further calculated the recovery ratios of both the hydrogels. For the Fmoc-RGD hydrogel, the percentage of recovery was found to be 85% after two cycles of breakage. However, for the Fmoc-RGD/chitosan hydrogel, the recovery was 209%, indicating that the mechanical strength of the gel increased following its mechanical breakage. This behavior is very interesting and has rarely been observed.

Shinkai and co-workers have demonstrated that the thixo-tropic/self-healing property results from re-entanglement of the fibers after mechanical force detaches their junction points.^[67] Here, the Fmoc-RGD fibers re-entangle immediately after decreasing the strain due to the strong supramolecular interactions between them, causing ≈85% recovery of the original mechanical property. Interestingly, for the Fmoc-RGD/chitosan hydrogel, the recovery was 209%, indicating that the mechanical strength of the gel increased following its mechanical breakage. This behavior suggests formation of tougher network (i.e., increase in the number of junction points between the fibers) after mechanical breakage in the Fmoc-RGD/chitosan hydrogels.

In order to check the cellular viability, MTT analysis was performed on dried Fmoc-RGD/chitosan hydrogel, Fmoc-RGD hydrogel, and dried chitosan films at three different time points of 48 h, 72 h, and 7 days (Figure S7, Supporting Information). More than ≈90–95% cells were viable for both Fmoc-RGD/chitosan hydrogel and Fmoc-RGD hydrogel up to 72 h which was reduced to ≈70–75% after 7 days. However chitosan film alone showed 97% cell viability at 48 h which was successively reduced to ≈87% at 72 h and further decreased to ≈65% at 7 days. Next, we sought to assess the potential of the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels as 2D/3D cell culture scaffolds. First, we tested the stability of the hydrogels in contact with cell culture medium. After 30 min, the Fmoc-RGD gel completely dissolved in the cell culture medium, whereas the Fmoc-RGD/chitosan hydrogel remained stable (Figure 4A). Notably, the Fmoc-RGD/chitosan hydrogel was observed to be stable for months in cell culture medium (Figure 4A). We, therefore, performed the cell culture experiments using only the composite Fmoc-RGD/chitosan hydrogels. The composite hydrogels were first prepared on 24-well cell culture plates and washed with cell culture medium for 2 days to remove the monomers and acetic acid used during the preparation of chitosan solution. The medium was changed twice a day. Chinese hamster ovarian (CHO) cells were seeded on the top of the gels following UV-light sterilization. The viability of the cells after 2, 3, and 7 days was analyzed using Live/Dead staining (Figure 4B–D). High cell viability and cell adherence were indicated by the presence of a large population of green cells showing a typical morphology, with no stained red cells, even after 7 days. To rule out the possibility of the cells adhering to the plate rather than the gels, we scratched the gels with a micro-pipette tip and imaged them (Figure S8, Supporting Information). It was clearly observed that the scratched portion (the surface of the cell culture plate) did not contain any cells, demonstrating that the cells were adhered to the surface of the gels. As a control, we examined cell viability in the presence of chitosan solution of the same concentration as used for preparing the gels (0.5%). The Live/Dead staining (Figure S9, Supporting Information) after 2 days of incubation showed a large population of red cells, indicating very low cell viability. This emphasizes the significance of integrating the polymer with 3D networks of supramolecular hydrogels, so that the cytotoxic components used during preparation of the polymer solution could be adequately washed out. Furthermore, we seeded Mouse Swiss Albino Embryo Fibroblast cells (3T3) on the gels in order to check cell adhesion using another cell line. A large population of green cells with spindle or polyhedral shape was observed (Figure S10, Supporting Information) indicating good cell viability and adherence. 3D images obtained from Z-stacks of laser microscopy scans after 3 days revealed that the cells did not penetrate inside the 3D environment of the hydrogel (Figure 4E). Interestingly, after 6 days, the cells were observed inside the hydrogels (Scheme 2), with a migration depth of 300 μm (Figure 4F), higher than previously reported amino-acid or peptide based supramolecular hydro-gels.^[68,69] This cell migration, an effective method for 3D cell culture, can be attributed to the excellent cell attracting property of the RGD ligands.^[70] Thus, the Fmoc-RGD/chitosan cell-loaded hydrogels have immense potential as materials for 3D bio-printing, and as injectable hydrogel scaffolds owing to its thixotropic property.

Figure 4. Biological studies on the hydrogels.

A) Digital images showing much enhanced stability ofthe Fmoc-RGD/chitosan gels compared to the Fmoc-RGD hydrogels with cell medium. Live-dead staining of Chinese hamster ovary (CHO) cells cultured on the Fmoc-RGD/chitosan hydrogels surface (2D cell culture) for B) 2, C) 3, and D) 7 days. Green staining indicates live cells; red staining indicates dead cells. Scale bars are 500 μm. 3D volume rendering images obtained by Z-stack of confocal microscopy showing 3T3 fibroblast cells in Fmoc-RGD/chitosan hydrogels after E) 3 days (thickness 135 μm) and F) 6 days (thickness 300 μm).

Scheme 2.

Schematic model of the simplistic strategy for 3D cell culture. The cells were first seeded on the top of the gels. Over time, the cells migrated from the surface to the bulk.

The composition of hydrogel has an overall effect on the growth and proliferation of cells. Fmoc-RGD is a well reported tri-peptide motif which is recognized by α_vβ₃ and α₅β₁ inte-grins located in cell membranes which makes this sequence particularly important for attachment of cells to the ECM mimicking matrix.^[27] Fmoc-RGD itself forms a weak gel which is not rigid enough to support the culture of cells. Alternatively, chitosan solution alone at this concentration does not form gel and is toxic to the cells (Figure S9, Supporting Information). The optimum integration of Fmoc-RGD with chitosan hereby produces a hydrogel with superior biocompatibility throughout the cell culture span of 7 days. Cells showed a higher preference for adhesion and spreading on the hydrogel surface up to 3 days of culture. After 6 days, the cells migrated inside the hydrogels, and the migration depth was found to be 300 μm which further supports the cell integrating property of the designed composite hydrogel.

It is also important to discuss the effect of mechanical stiffness of the hydrogel on cellular properties. It has been previously reported that surface stiffness and mechanical rigidity of the materials play a crucial role in cellular adhesion and morphology.^[71–74] Soft, lightly cross-linked gels having rigidity nearly 1 kPa show diffuse and dynamic adhesion of cells.^[63] After washing with cell culture medium, Fmoc-RGD/chitosan hydrogels exhibited a G′ value close to 1 kPa (Figure S11, Supporting Information), which was ideal for the adherence of the cells, which were further observed to be in good morphology. Moreover, with increase in time the hydrogel supported the migration of cells from surface to the bulk. These observations indicate that an optimal concentration and structure of the building blocks lead to adequate physico-chemical property for cell biocompatibility and attachment, which could be utilized for cell proliferation in tissue engineering applications.

We next set out to evaluate the antibacterial properties of the composite hydrogels. While the inherent antibacterial activity of chitosan is well documented,^[52] its ability to both retain and confer this activity within the context of the composite hydrogels needs to be verified. The antibacterial activity of the Fmoc-RGD hydrogels was also evaluated and standard LB/Agar was used as a control. Escherichia coli bacteria, at early log phase, were grown on each of the hydrogels overnight, and bacterial viability was assessed via Live/Dead analysis (Figure 5). As expected, the chitosan solution, used as a control, exhibited complete inhibition of bacterial growth (Figure 5 and Table S1, Supporting Information). Similarly, the composite Fmoc-RGD/ chitosan hydrogels exhibited significant, near complete, inhibition of bacterial growth (Table S1, Supporting Information). Interestingly, the Fmoc-RGD hydrogels were able to inhibit bacterial growth to about 47% of the control (Table S1, Supporting Information). These results demonstrate the inherent antibacterial capabilities of the composite hydrogels mainly stems from their chitosan component, but may also be supported by the Fmoc-RGD component as well.

Figure 5.

Antibacterial activity of the hydrogels. The effect on bacterial viability was evaluated using the live/dead backlight bacterial viability kit. Green fluorescence of the Syto9 probe indicates bacterial cells with an intact membrane while red fluorescence of propidium iodide (PI) indicates dead bacterial cells. Scale bars are 50 μm.

AgNPs have been demonstrated to be utilized as plasmonic substrates for sensing and imaging, catalysts, or antimicrobial systems.^[75–77] The properties of AgNPs vary according to their shape, size, composition, as well as spatial arrange-ment,^[78] emphasizing the significance of their synthesis with controlled size and spatial structural design. Stupp’s research group reported the synthesis of mono-dispersed AgNPs grown along the length of nano-fibers produced from supramolecular assemblies of peptide-amphiphiles.^[79] This hybrid metal-organic nanostructures combine the advantages of AgNPs with organic fibers, generating a 1D nanoparticle array. Short peptide or even amino acid based supramolecular hydrogels have also been used as templates to produce AgNPs.^[80–82] Here, the ability of Fmoc-RGD and Fmoc-RGD/chitosan composite hydrogels to form AgNPs was assessed. Ag⁺ ion incorporated hydrogels were kept under diffused sunlight for the production of AgNPs. A pinkish coloration was observed in both cases, indicating the formation of nanoparticles inside the hydrogels (Figure 6A and Figure S12A, Supporting Information). However, the Fmoc-RGD hydrogel was observed to become unstable after the formation of AgNPs (Figure S12A, Supporting Information). Next, time dependent UV-vis spectra were used to monitor the formation of AgNPs in the Fmoc-RGD and Fmoc-RGD/chitosan composite hydrogels (Figure 6B and Figure S12A, Supporting Information). The appearance of a peak at ≈521 nm was observed in the Fmoc-RGD/chitosan composite hydrogel after 30 min of irradiation (Figure 6B). After further irradiation, the absorbance of this peak increased, and after 40 min, this peak red shifted to longer wavelengths (590 nm). The appearance of the 521 nm peak may indicate the formation of Ag_n nanoclusters with n = 2–8 atoms.^[83] The red shift signifies the complexation of suitable functional groups present in the Fmoc-RGD/chitosan complex with AgNPs.^[83] In the Fmoc-RGD hydrogel, a peak at ≈484 was observed after 50 min and its absorbance increased with further irradiation with no significant red shift. This may indicate the formation of larger AgNPs inside the Fmoc-RGD hydrogel.

Figure 6. Reduction of ionic silver within the Fmoc-RGD/chitosan hydrogels triggered by diffused solar radiation.

A) Digital images depicting the formation of AgNPs over time. B) Time-dependent UV–vis spectroscopic analysis depicting the formation of AgNPs within the Fmoc-RGD/chitosan hydrogels. C) TEM micrographs of the AgNPs-embedded Fmoc-RGD/chitosan hydrogels (Scale bars are 200 nm). D) Distribution of AgNP diameters (n = 50).

To confirm the predictions regarding the dimensions of the AgNPs based on the UV–vis results, we studied their morphology by TEM after completion of formation of AgNPs (70 min and 90 min for Fmoc-RGD and Fmoc-RGD/chitosan hydrogels, respectively). The AgNPs produced in the Fmoc-RGD/chitosan hydrogels were ultra-small and were arranged on the long axis of the fibers (Figure 6C and Figure S13, Supporting Information). A distribution of the AgNPs diameter (calculated from the TEM micrographs, Figure 6D) revealed the formation of monodispersed AgNPs with an average diameter of 3.5 ± 0.6 nm. Although the average diameter of the AgNPs was larger than typical nanoclusters (<2 nm), the UV–vis spectra indicated that Ag nanoclusters had probably formed in the Fmoc-RGD/chitosan hydrogels. On the other hand, AgNPs formed inside the Fmoc-RGD hydrogels had higher dimensions (25 ± 15.1 nm) and they were poly-disperse in nature (Figure S12B, Supporting Information). Interestingly, the fibers of Fmoc-RGD were not visible in the TEM micrograph, possibly indicating the destruction of the network structure of the gels, resulting in their breakage.

Based on these characterization details, we can suggest a possible formation mechanism of AgNPs of different dimensions in the Fmoc-RGD and Fmoc-RGD/chitosan hydrogels. It is known that carboxylic acid residues co-ordinate with Ag⁺ ions, resulting in the reduction of the standard electrode potential of Ag⁺/Ag couple (0.8 V for the bulk system).^[81] Stupp and co-workers proposed the attachment of Ag⁺ ions on the fiber surface by coordination with glutamic acid residues, which create a negative charge on the fiber surface.^[79] Therefore, we envision that the free carboxylic acid groups of the aspartic acid residue generate a negative charge on the fiber surface, thereby coordinating the Ag⁺ ions. These Ag⁺ ions are then reduced in the presence of ambient solar radiation to produce AgNPs which are stabilized inside the 3D networks of the Fmoc-RGD/ chitosan hydrogels. However, the Fmoc-RGD fibers disappeared, leading to the breakage of the gel. The pH of the AgNP embedded Fmoc-RGD hydrogel was found to be ≈2, similar to that of only Fmoc-RGD hydrogels. Therefore, the disappearance of the fibers might be related to the solar exposure, or nanoparticle formation. Banerjee and co-workers also observed the disappearance of fibers after incorporation of Ag⁺ ions into the hydrogels.^[81] The formation of ultra-small AgNPs in the Fmoc-RGD/chitosan hydrogels may arise from the synergetic effect of both Fmoc-RGD and chitosan. To explore this possibility, we studied the ability of chitosan to produce AgNPs under identical conditions by TEM microscopy which suggested the formation of AgNPs (Figure S14, Supporting Information). Therefore, it can be envisioned that chitosan reduced Ag⁺ ions and stabilized the AgNPs, probably through coordination with its -OH and -NH₂ groups. Furthermore, we propose that in the Fmoc-RGD/chitosan hydrogels, AgNPs can bind to the composite fibers by interacting with 1) the −OH and −NH₂ of chitosan and 2) the free carboxylic acid groups (not H-bonded with chitosan) of Fmoc-RGD. This improved interaction consequently minimizes the electrostatic repulsion of the system, resulting in smaller size and mono-dispersity of the AgNPs.^[79]

In summary, we demonstrate the fabrication of a composite hydrogel composed of Fmoc-RGD and chitosan by a simple, non-covalent functionalization approach. The composite Fmoc-RGD/chitosan hydrogel is comprised of nano-fibrous networks, similar to Fmoc-RGD hydrogels, although with a reduced fiber diameter. FTIR spectra implies the signature of β-sheet structures in the Fmoc-RGD hydrogels, which remained unaffected after non-covalently binding with chitosan. Improvement of the gelation rate and mechanical rigidity of the hydrogels were observed in the Fmoc-RGD/chitosan hydrogel compared to Fmoc-RGD hydrogels. Both hydrogels were also capable of exhibiting thixotropic properties. Interestingly, the Fmoc-RGD hydrogel showed poor stability in cell culture medium, whereas the Fmoc-RGD/chitosan hydrogels showed excellent stability. Therefore, Fmoc-RGD/chitosan hydrogels were utilized as candidates for 2D and 3D cell culture. Interestingly, the inherent antibacterial properties of chitosan were conferred on the composite hydrogels, and consequently, the Fmoc-RGD/ chitosan hydrogels exhibited significant, near complete, inhibition of bacterial growth. The combination of excellent cell adherence and cell migration along with antibacterial properties make this composite hydrogel a potential candidate for wound healing applications. In addition, mono-dispersed, ultrasmall (3.5 ± 0.6 nm) AgNPs were grown on the long axis of the Fmoc-RGD/chitosan composite fibers. These metallo-organic fibers have significant promise in future applications, such as sensing, imaging, and catalysis. The composite Fmoc-RGD/ chitosan hydrogel thus assimilates the attributes of Fmoc-RGD and chitosan providing a symbiotic, self-sustaining, and mechanically stable hydrogel for multiple applications.