Electrospun Zein/PCL Fibrous Matrices Release Tetracycline in a Controlled Manner, Killing Staphylococcus aureus Both in Biofilms and Ex Vivo on Pig Skin, and are Compatible with Human Skin Cells

Abstract

Purpose: To investigate the destruction of clinically-relevant bacteria within biofilms via the sustained release of the antibiotic tetracycline from zein-based electrospun polymeric fibrous matrices and to demonstrate the compatibility of such wound dressing matrices with human skin cells.

Methods: Zein/PCL triple layered fibrous dressings with entrapped tetracycline were electrospun. The successful entrapment of tetracycline in these dressings was validated. The successful release of bioactive tetracycline, the destruction of preformed biofilms, and the viability of fibroblast (FEK4) cells were investigated.

Results: The sustained release of tetracycline from these matrices led to the efficient destruction of preformed biofilms from Staphylococcus aureus MRSA252 in vitro, and of MRSA252 and ATCC 25923 bacteria in an ex vivo pig skin model using 1 × 1 cm square matrices containing tetracycline (30 μg). Human FEK4 cells grew normally in the presence of these matrices.

Conclusions: The ability of the zein-based matrices to destroy bacteria within increasingly complex in vitro biofilm models was clearly established. An ex vivo pig skin assay showed that these matrices, with entrapped tetracycline, efficiently kill bacteria and this, combined with their compatibility with a human skin cell line suggest these matrices are well suited for applications in wound healing and infection control.

Keywords: biofilm; controlled drug delivery; electrospinning; nanofibre; pig skin.

Fig. 1

The activity of clinically used antibiotics (200 μL containing 50 μg/mL, for 24 h) against preformed (24 h) MRSA252 biofilms (normalised relative to crystal violet assayed biofilm formation in the absence of any antibiotic) tested using the MTP model showing the efficiency of biofilm removal. The error bars represent the standard deviation (n = 3, in triplicate).

Fig. 2

¹H NMR spectra (CDCl₃) of (in ascending order): Tet, Tet-free electrospun zein, electrospun zein matrix containing Tet, and electrospun zein containing Tet but with the zein spectral data subtracted.

Fig. 3

The Tet release profile from the electrospun triple-layered electrospun matrices zein 3L and zein/PCL 3L with the Tet payload (5%) encapsulated in the middle layer only. The error bars represent the standard deviation (n = 3, in triplicate). The insets on the right show representative SEM images of electrospun zein 3L (upper) and electrospun zein/PCL (20:10) 3L (lower) matrices after immersion in PBS at 37°C (12). Below are other representative images showing, from the left: electrospun zein 3L, electrospun zein 3L after immersion in PBS at 37°C, electrospun zein/PCL (20:10) after immersion in PBS at 37°C, and a fluorescence microscopy image of Tet HCl loaded zein.

Fig. 4

The activity of the electrospun matrices against preformed MRSA252 biofilms (normalised absorbance) tested using the MTP model showing the percentage of biofilm remaining after treatment, where the untreated biofilm acts as a control for normalisation. The error bars represent the standard deviation (n = 3, in triplicate).

Fig. 5

Effects of the Tet containing electrospun matrices on MRSA252 biofilms grown on polycarbonate discs tested using the CBM model expressed as normalised live bacteria/disc. The error bars represent the standard deviation (n = 3).

Fig. 6

The antibacterial effects of Tet loaded formulations after 5 days of incubating MRSA252 on pig skin, 37°C. The formulations were added on the fifth day and left for 24 h (n = 3). The error bars represent the standard deviation.

Fig. 7

The MTS cell metabolism assay (normalised absorbance) demonstrates the biocompatibility of zein/PCL+Tet, 72 h after seeding. The error bars represent the standard deviation (n = 3, in triplicate).