Improvement of the Stability of Functionalized PVA Nanofibers for Sensitive Compound Incorporation by Gentle Freeze–Thaw Treatment

Abstract Recent advances in tissue engineering focus on enhancing the biological performance of nanofibrous materials that mimic the extracellular matrix. These materials provide mechanical stability and form nanofibrous membranes that support cell adhesion, migration, and growth. Their functionality can be further improved by incorporating bioactive compounds, especially in water-soluble polymer-based nanofibers, enabling controlled release. However, a major limitation is the “burst release” effect, where more than 90% of the embedded substances are released within the first 24 h. The second challenge is the stabilization of these water-soluble systems. The most commonly used chemical crosslinking methods require the addition of chemical agents (crosslinkers) that are potentially toxic and may lead to structural and chemical changes in the nanofibrous materials, while also causing denaturation of the incorporated bioactive compounds. This study presents the development of functionalized nanofibers based on water-soluble poly(vinyl alcohol) (PVA), produced by an advanced alternating current (AC) electrospinning method, and their subsequent stabilization using a non-toxic Freeze-Thaw (FT) method. This physical stabilization technique avoids chemical crosslinkers and instead induces crystallinity through polymer chain reorganization, reducing solubility while preserving structural integrity. The impact of FT stabilization on the physicochemical properties and solubility of PVA nanofibers was evaluated, along with its effect on the release profile of human platelet lysate (PL), a growth factor-rich additive. The results show that FT treatment effectively stabilizes PVA nanofibers and significantly slows down the release of incorporated PL. Specifically, the solubility of neat PVA nanofibers after 10 FT cycles under thawing at +4 °C decreased by 19.5%, whereas in PVA + PL nanofibers the reduction in PVA solubility under the same conditions reached 12.2%. The release of PL itself slowed by 18.2% after 10 FT cycles. Changes in crystallinity were also observed in the FT-treated materials. This approach offers a promising platform for developing safe, functional nanofibrous materials for chronic wound treatment and skin regeneration.