2.1. Protonation and Complex Formation Thermodynamics
A preliminary potentiometric titration study for understanding the interaction strength of the different ionic groups present on the polymer was carried out mainly on the monomer precursors. The latter, being more soluble than the corresponding polymers, can give more information about the complexing ability towards silver ions in a wide pH range. As was to be expected, the N
-phenylalanine (PHE) monomer exhibits no significant deviation of the titration curve in the presence and absence of silver ion (Figure 1
) in the low pH range involving the ionization of the carboxylic group.
The superimposed titration curves (pH vs. mL titrant) show deviations at high pH values attributable to the formation of stoichiometric silver hydroxide. On the other hand, the monomer N
-histidine (MHist) shows titration curves that vary significantly with a regular increase in the amount of silver ion (Figure 2
In fact, any increase in the Ag(I)/L molar ratio (L is the monomer ligand) is reflected in a decrease in the pH range of the buffer zone involving the ionization of the imidazole nitrogen. An approximate graph of pH = pKa
in relation to the Ag(I)/L molar ratio shows a good linearity (Figure 2
On the other hand, a copolymer compound, the poly(MHist-co
-Nip), shows a complexing ability towards the Ag+
ions considerably higher than the ligand monomer. As shown in Figure 3
, the copolymer protonation behaves exactly like the precursor monomer, whereas in the presence of silver ions a considerable flattening of the titration curve occurs at low pH values and in the pH range that involves the complexing equilibria.
The presence of Ag+
ions produces a lowering of the pH as a consequence of its complexation to the ligand; this causes a release of free H+
ions in solution from the ligand. The protons released are stoichiometric with respect to the Ag+
ions present, which suggests a possible stoichiometry whereby an Ag+
ion is complexed to a ligand and two ligands are involved in two complex species with a single Ag+
ion. The complexation behavior with the silver ion of the copolymer seems to be similar to that reported in the literature for simple imidazole and histidine, but different to that reported above for the MHist monomer [31
]. The titration curves of the latter highlight the formation of complexes in the presence of silver ions but are not as discriminated and synergistic as in the case of copolymers. It is reasonable to hypothesize a synergy also for hydrogels and that, therefore, the presence of silver ions favors the interaction of several ligand units towards the metal ion [33
]. This is what actually happens and is confirmed by the behavior of the swelling of hydrogels in the presence of the metal ion, as described below.
2.2. Syntheses and Characterization
Among the different methods of formation of silver nanoparticles in aqueous solution, we used the reduction of Ag+
ion, complexed/absorbed in suitable polyelectrolyte hydrogels, by means of the reducing sodium borohydride NaBH4
]. Usually, this reaction is carried out in the presence of colloidal stabilizers to avoid the aggregation of the nanoparticles. This is a conventional method for the synthesis of nanoparticles that makes use of materials with potential hazards, especially for biomedical applications. There is therefore a general interest in developing environmentally friendly processes to obtain metal nanoparticles through the use of appropriate polyelectrolytes. Recently some polysaccharide hydrogels [35
] and some based on chitosan modification [36
] have shown reducing properties towards metal ions and in particular Ag+
ions; at the same time, they have been shown to stabilize the nanoparticles in colloidal solution. However, the reduction reaction must take place at temperatures far above the ambient temperature. In the case of our hydrogels, their thermosensitive properties limit the temperature rise above their LCST (Lower Critical Solution Temperature). The presence of the Nip units in some hydrogels involves an LCST of just over 32 °C. So a reduction of Ag+
ion would see the hydrogel always in a collapsed state. Acrylic hydrogels with Nip units make use of NaBH4
ion reduction [37
]. However, a green process is being developed to synthesize metal nanoparticles from our proposed hydrogels.
In this paper we plan to use the functional groups present on the hydrogel to mitigate the high surface energy of the nanoparticles during the reduction. The functional groups of α-amino acid residues help to stabilize the particles. Furthermore, the silver ion has a higher affinity towards the amine group than the carboxylic group; this involves the formation of relatively stable silver-binding complexes, as suggested by the values of the stability constants [30
]. As shown during the synthesis procedure, the interaction between the Ag+
ion and the ligand, in the ionized form, leads to a sudden collapse of the swollen hydrogel, as a consequence of a strong electrostatic interaction between opposite charges (Scheme 2
All four hydrogels considered, despite their different degree of cross-linking, suffer a considerable collapse in the presence of Ag+
ions. While the measured value of the equilibrium degree of swelling (EDS) is practically negligible for the zwitterionic hydrogel H5-EBA, the degree of swellability for the other carboxyl hydrogels can be related to the ionic unit content present on the polymer. A lower content of carboxyl groups determines a higher EDS value due to the presence of the N
-isopropylacrylamide (Nip) non-ion units. This is shown by the comparison of the hydrogels reported in Figure 4
A greater EDS value for PHE-Nip-PEG and AVA-Nip-PEG hydrogels in the presence of Ag+
ions is to be attributed to the decreased amount of PHE and AVA ion units, despite their different hydrophobic properties. As a consequence, the collapse of the hydrogel is mainly due to charge neutralization following the Ag+
ion complexation with the imidazole nitrogen and the carboxylate ion. On the other hand, the subsequent Ag+
ion reduction step with metallic silver, by NaBH4
, restores the swollen hydrogel state less quickly. This means that the whole Ag+
ion is reduced and that the Ag+
ion bound to the ligand probably ensures the nucleation of the neighboring silver atoms for the formation of stable silver nanoparticles. The first macroscopic observation of hydrogels, in the presence of metallic Ag, was the significant color difference of the obtained product: black in the case of the H5-EBA hydrogel, while the others are more clearly brown. In any case, even if less evident for Ag-H5-EBA, the external surface of all the dry nanocomposite hydrogels is typically of metallic silver. A topographic evaluation shows the SEM reported in Figure 5
and Figure 6
, along with the UV-visible spectra of the hydrogels in the native state, complexed with Ag+
ions and silver nanoparticles.
In any case, the surface of the nanocomposite hydrogel sample is different from that of the corresponding native hydrogel. While the H5-EBA hydrogel shows greater flattening in the presence of metallic silver, the more homogeneous hydrogel PHE-Nip-EBA shows residual patches probably of externally agglomerated silver. A particular behavior is observed for the PHE-Nip-PEG hydrogel (Figure 6
); the homogeneity of the native hydrogel is transformed into crystal-like in the presence of Ag. This point, which may raise a particular interest, requires further investigation. Furthermore, due to the reduction of Ag+
ion, the white hydrogel immediately became dark on the surface. To confirm the formation of silver nanoparticles, a UV-visible analysis clearly showed an absorption peak around 400 nm, due to the surface plasmon resonance effect (Figure 5
and Figure 6
); this peak is not present in the native and in the Ag(I)-complexed hydrogel [38
]. A great deal of information about the physical state of the nanoparticles can be obtained by analyzing the spectral properties of silver nanoparticles incorporate into the hydrogel. The initial UV-visible broad spectrum contracted after 24 h and remained unchanged over time. This time interval allowed us to monitor the formation of the nanoparticles inside the hydrogel that did not undergo any aggregation so that their size remained unaltered. This optical response shows how active hydrogel groups exert a kind of stabilization towards the formed nanoparticles. The spectral response is compatible with a fairly low nanoparticle size. In fact, a TEM analysis (Figure 7
) shows that the nanoparticles have a spherical shape of about 15 nm in size and are well distributed within the hydrogel.
It is worth mentioning that in the case of the zwitterionic hydrogel (H5-EBA) the silver nanoparticles are markedly spherical and well-defined, uniformly distributed, and considerably spaced from each other throughout the polymer network. On the other hand, albeit to a lesser extent, carboxyl hydrogels show some irregularities, with sporadic aggregations of different shapes.
Some dynamic mechanical analysis of nanocomposite hydrogels provided quantitative information on the viscoelastic and rheological properties. Storage elastic modulus G’ and loss elastic modulus G” of the hydrogels were measured, and the viscoelastic characteristics of the anionic hydrogels are reported in Figure 8
As shown in the figure, all the composites are characterized by a well-developed network, since the storage modulus G’, which exhibits a plateau in the 0.1–100 rad/s frequency range, is always greater than the loss modulus G”. In contrast to the high elastic modulus of PHE-Nip-PEG and AVA-Nip-PEG, the hydrogel PHE-Nip-EBA showed a low modulus. The weaker behavior of the hydrogel PHE-Nip-EBA may be due to the lower crosslinking density. It is known that G’ correlates with the rigidity of hydrogels, so the cross-linked hydrogels PHE-Nip-PEG and AVA-Nip-PEG are strong gels [39
]. Moreover, for all the nanocomposites the loss factor (tanδ) was 0.1, which was almost the same as that of a general chemically cross-linked gel. Since the strain characteristics and the angular frequency characteristics showed the same tendency, it was found that similar viscoelastic properties are exhibited except for sample hardness.
2.3. In Vitro Antimicrobial Activity
The in vitro antibacterial screening of native hydrogels and their silver analogues was carried out against Gram-positive (B. subtilis
) and Gram-negative (E. coli
) bacteria, and a fungus (S. cerevisiae
). The results reported in Figure 9
suggest that the silver nanocomposite hydrogel (1–4, indicated with arrow heads), compared to the corresponding native one (1B–4B), shows inhibitory effects, since a clear inhibition zone was observed under similar conditions. The possible mechanism for this inhibition zone may be only attributed to the silver ion released by the nanocomposite hydrogel containing the silver nanoparticles; the latter could be toxic because they release silver ions, which are well known for their antibacterial and other destructive behaviors [40
]. The tests for the release of silver nanoparticles by hydrogels, both in pure water and in phosphate buffer at pH 7.4, have shown that only the PHE-Nip-EBA nanocomposite hydrogel is able to release free silver nanoparticles in solution over time, as evidenced by the increasing spectrophotometric peak at about 400 nm and due to the surface plasmonic effect. Unlike other hydrogels, the low cross-linking content of the PHE-Nip-EBA hydrogel allows the release of nanoparticles, despite their similar size. However, in both cases, hydrogels with silver nanoparticles could interact with the lipid layer of the cell membrane. From the results, we revealed that the silver nanocomposite hydrogel showed activity towards both Gram-positive B. subtilis
and Gram-negative E. coli
, A1 and B1, or Figure 9
, A3 and B3). Compounds including antibiotics are generally hard to permeate through periplasm, but no significant difference between Gram-positive and Gram-negative bacteria in this study.
Moreover, it was noticeable that the antifungal activity of the silver nanocomposite hydrogels was significantly enhanced. The diameter of the inhibition zone against S. cerevisiae
was larger than the antibacterial one (Figure 9
C (1–4)). This can be a good result for the treatment of contact lenses [25
]. Fungal keratitis is a severe ocular disease that could cause blindness and is very often observed in developing countries. It is known that the variety of the forms and characteristics of various silver nanoparticles are also responsible for differences in their antibacterial mode of action and probably bacterial mechanism of resistance [17
]. The size and shape of the silver nanoparticles from the present hydrogels might be useful for their antifungal effects.