Antibacterial Hydrogels Derived from Poly(γ-glutamic acid) Nanofibers

Abstract

Biocompatible hydrogels with antibacterial properties derived from γ-polyglutamic acid (γ-PGA) were prepared from bulk and electrospun nanofibers. The antibacterial drugs loaded in these hydrogels were triclosan (TCS), chlorhexidine (CHX) and polyhexamethylene biguanide (PHMB); furthermore, bacteriophages were loaded as an alternative antibacterial agent. Continuous and regular γ-PGA nanofibers were successfully obtained by the electrospinning of trifluoroacetic acid solutions in a narrow polymer concentration range and restricted parameter values of flow rate, voltage and needle-collector distance. Hydrogels were successfully obtained by using cystamine as a crosslinking agent following previous published procedures. A closed pore structure was characteristic of bulk hydrogels, whereas an open but structurally consistent structure was found in the electrospun hydrogels. In this case, the morphology of the electrospun nanofibers was drastically modified after the crosslinking reaction, increasing their diameter and surface roughness according to the amount of the added crosslinker. The release of TCS, CHX, PHMB and bacteriophages was evaluated for the different samples, being results dependent on the hydrophobicity of the selected medium and the percentage of the added cystamine. A high efficiency of hydrogels to load bacteriophages and preserve their bactericide activity was demonstrated too.

Keywords: antibacterial properties; bacteriophages; drug release; electrospinning; hydrogels; nanofibers.

Figure 1

Optical micrographs comparing the morphology of γ-PGA electrospun fibers obtained at concentrations of 3 (a), 8 (b) and 13 wt-% (c) using a voltage of 30 kV, a flow rate of 1 mL/h and a needle to collector distance of 25 cm.

Figure 2

SEM micrographs (low and high magnification) and fiber diameter distribution of γ-PGA electrospun fibers obtained at concentrations of 5 (a), 8 (b) and 11 wt-% (c) using a voltage of 30 kV, a flow rate of 0.5 mL/h (a,b) or 0.3 mL/h (c) and a needle collector distance of 25 cm.

Figure 3

Scheme showing hydrogel preparation from γ-PGA and representative images of the two types of prepared hydrogels.

Figure 4

SEM micrographs (low and high magnifications) comparing the morphology of 100% crosslinked hydrogels derived from electrospinning solutions with polymer concentrations of 5 wt-% (a) and 8 wt-% (b).

Figure 5

FTIR spectra of a theoretically 100% crosslinked hydrogel obtained from electrospun nanofibers. For comparison, the spectrum of electrospun γ-PGA nanofibers taken before the crosslinking process is also provided. Note that the 100% crosslinking reaction was not complete (see text for further details).

Figure 6

C 1s, O 1s, N 1s and S 2p high resolution XPS spectra for γ-PGA electrospun fibers (a) and hydrogel from electrospun fibers crosslinked with the theoretical stoichiometric amount of cystamine (b). Deconvolution of the spectra is also shown to indicate the presence of different types of atoms.

Figure 7

SEM micrographs (left) comparing the morphology of hydrogels prepared from the bulk (a) and from electrospun nanofibers (b) after reaction with the stochiometric ratio of cystamine 100%. In both cases pore diameter distributions are provided (right). For comparison purposes, the inset of (b) shows the electrospun fibers before performing the crosslinking reaction.

Figure 8

SEM micrographs (left) comparing the morphology of hydrogels from electrospun nanofibers and a crosslinking process involving 50% (a), 75% (b) and 100% (c) of the steochiometric amount of cystamine. Fiber diameter distributions are provided in the right.

Figure 9

Accelerated release of triclosan from hydrogels in a PBS-ethanol (70v:30v) medium. (a) TCS relative release percentages for electrospun and bulk hydrogels; both with a crosslinking degree of 100%. (b) TCS relative release percentages for electrospun hydrogels with different crosslinking percentages. In both graphics, methods A and B refer to the loading of TCS by absorption or during the crosslinking process, respectively. Insets show release determined at initial times.

Figure 10

Relative release percentages of CHX (a) and PHMB (b) from electrospun hydrogels in PBS medium. Two-step release of CHX (c) and PHMB (d) from electrospun hydrogels with a theoretical crosslinking degree of 50%. The first step was performed in PBS and the second one in the PBS-ethanol medium.

Figure 11

Inhibition of bacterial growth. Growth of the Staphylococcus aureus Gram-positive bacteria on a LB agar plate, control (a), bactericide effect (inner circle) and bacteriostatic effect (outer circle) of the ampicillin antibiotic (b), and bactericide activity of the hydrogels having a theoretical crosslinking degree of 50% and loaded with CHX and (c) and PHMB (d). For comparison, the outer circle is drawn the same size in (b–d). Inhibition of antimicrobial solution containing 6.2 w/v-% of CHX (e) and 1.5 w/v-% of PHMB (f). SEM images of the bacteria E. coli (g) and S. aureus (h) forming biofilms on the surface of the hydrogel.

Figure 12

Antibacterial activity of the Fersisi bacteriophages. Control of the Staphylococcus aureus bacterial growth (blue), relative growth of S. aureus in the commercial preparation of phages (green) and bacteriophage-loaded electrospun hydrogels (red) with crosslinking degrees of 100% (circles), 75% (squares) and 50% (triangles). TEM micrographs show the morphology of Fersisi bacteriophages (commercial preparation): (a) Myoviridae, (b) Siphoviridae, (c) Leviviridae, and (d,e) Podoviridae.

Figure 13

Cytotoxic effect of the γ-PGA hydrogels extract on different cell lines (COS-1, COS-7, SIAT and MDCK). pH (red circles) corresponds to dilution and was not adjusted. The bars are mean ± SD (n = 3). * p < 0.05, scaffold vs. control, ANOVA followed by Tukey test (a). Fluorescence images of MDCK cells cultured for 96 h on hydrogels with 50% (b), 75% (c), and 100% (d) crosslinking degrees. The discontinuity of the monolayer is due to the pores of the hydrogel.