Multilayer Alginate–Polycaprolactone Electrospun Membranes as Skin Wound Patches with Drug Delivery Abilities

A multilayer nanofibrous membrane consisting of a layer of polycaprolactone and one of physically cross-linked alginate-embedding ZnO nanoparticles is prepared via electrospinning technique as potential wound healing patches with drug delivery capabilities. A washing–cross-linking protocol is developed to obtain stable materials at the same time removing poly(ethylene oxide), which was used here as a cospinning agent for alginate, without interfering with the membrane’s peculiar nanofibrous structure. The mechanical behavior of the samples is assessed via a uniaxial tensile test showing appropriate resistance and manageability together with a good thermal stability as proved via thermogravimetric analysis. The polycaprolactone external layer enriches the samples with good liquid-repellent properties, whereas the alginate layer is able to promote tissue regeneration owing to its capability to promote cell viability and allow exudate removal and gas exchanges. Moreover, using methylene blue and methyl orange as model molecules, promising drug delivery abilities are observed for the mats. Indeed, depending on the nature and on the dye-loading concentration, the release kinetic can be easily tuned to obtain a slow controlled or a fast burst release. Consequently, the proposed alginate–polycaprolactone membrane represents a promising class of innovative, simple, and cost-effective wound healing patches appropriate for large-scale production.


S1.
Assessment of the biological and anti-bacterial properties of alginate-based membranes embedding ZnO nanoparticles S2.
Mechanical properties of the prepared multilayer membranes S5. Evaluation of the different MB uptake mechanism for PCL and SA-based layers S1.

Assessment of the biological and anti-bacterial properties of alginate-based membranes embedding ZnO nanoparticles
The biological response and the anti-bacterial properties of alginate/ZnO electrospun membranes were assessed as thoroughly described in reference 22. Here, Figure S1-a reports the results of the cytotoxicity test over a time period of 48h undoubtedly proving the safety of the proposed mats. The adhesion of mouse fibroblasts (L929) on the alginatebased mats is reported in Figure S1-b (gray bars) together with the cell proliferation (orange bars). Similarly, the results attained by using human keratinocytes (HaCaT) are shown in Additionally, the anti-bacterial properties of the mats were assessed against E. Coli bacteria as described in detail in reference 22. Table S1 summarizes the obtained results pointing out the strong effect of ZnO nanoparticles and confirming the possibility to use such membranes as infection control systems.

S2. Evaluation of PCL solution viscosity decrease
Owing to the fact that PCL can easily undergo to hydrolysis phenomena, as broadly described in the literature, the viscosity of PCL 30% w/v glacial acetic acid/acetone solution was evaluated over a time period of 48h after its preparation. The experimental data were fitted with a straight line at low shear rate values in order to calculate the zero-shear viscosity (  ). Figure S2 reports the obtained curved, whereas Table S2

S3. ZnO morphological investigation
The morphology of the synthetized ZnO nanoparticles is shown in Figure S3-a. ZnO-NPs were found to form irregular clusters with various dimensions, besides the single nanoparticles could still be recognised showing a dimension in the range 20-30 nm.
The distribution of ZnO nanoparticles within the electrospun alginate-based layer was evaluated via EDS. Figure S3-b indicates ZnO-NPs as red points homogenously distributed both on the surface and in the inner part of nanofibers. Green points are instead related to the presence of strontium, which was here used as crosslinking agents.  Figure S4 reports the stress-elongation curves obtained by the mechanical tensile tests performed on both the as-is (black squares) and crosslinked (red circles) multilayer membranes. Figure S4. Stress-elongation curves obtained from the mechanical tensile test of the prepared multilayer membranes.

S5. Evaluation of the different MB uptake mechanism for PCL and SA -based layers
The color difference between PCL layer (on the left) and SA-based layer (on the right) of the multilayer membrane after the uptake of MB is shown in Figure S5. As clearly observed and as suggested by the fitting of the experimental data with several mathematical models, a much higher amount of dye was adsorbed by the alginate layer owing to the attractive electrostatic interactions occurring between them (i.e. MB is positive charged and SA is negative charged), besides diffusive phenomena could not be completely neglected. Figure S5. Colour of the multilayer membrane on both sides after the adsorption of MB.