Fabrication and Detailed Characterization of PLA/PEG Composite Nanofibers for the Co-Delivery and Synergistic Release of Quercetin and Rosmarinic Acid via Electrospinning

TL;DR

Researchers created a new drug delivery system using nanofibers to improve the effectiveness of two natural compounds for treating cancer and other conditions.

Contribution

A novel electrospun PLA/PEG nanofiber platform for co-delivering quercetin and rosmarinic acid with controlled release and enhanced stability.

Findings

The nanofibers achieved a biphasic release profile with an initial burst followed by sustained release.

The material showed high anticancer activity against melanoma cells with low toxicity to normal cells.

PLA/PEG nanofibers provided uniform morphology and high entrapment efficiency for both polyphenols.

Abstract

Natural polyphenols, particularly quercetin (QUE) and rosmarinic Acid (RA), possess significant synergistic therapeutic potential as potent antioxidants and anti-inflammatories. However, their poor stability, low water solubility, and resulting limited bioavailability severely hinder their effective clinical translation. This study addresses these fundamental limitations by designing a novel advanced drug delivery platform utilizing electrospinning. We have fabricated composite high-molecular-weight poly(L-Lactic Acid) (PLA)/polyethylene glycol (PEG) nanofibers for the simultaneous co-delivery of both QUE and RA, optimizing compound stability and release kinetics. PLA provided mechanical integrity and sustained release properties, while the incorporation of PEG strategically enhanced the mat’s wettability, enabling precise control over initial drug dissolution. Comprehensive characterization confirmed uniform, bead-free morphology and high entrapment efficiency for both polyphenols. Crucially, the PLA/PEG blend successfully achieved a biphasic release profile, featuring an initial burst release mediated by PEG followed by a sustained release phase governed by the PLA matrix. Furthermore, the performed in vitro investigations using SH-4 melanoma cells and HaCaT normal keratinocytes revealed that the prepared novel materials containing the polyphenols possessed high anticancer activity to the used cancer cell line. However, the toxicity to the normal cell line is much lower. Therefore, this novel electrospun composite scaffold offers an effective strategy to enhance the stability, control the delivery, and maximize the synergistic therapeutic benefits of quercetin and rosmarinic Acid for applications in areas such as advanced wound care, tissue regeneration, and antitumor therapies.

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1. Introduction

The exploration of phytochemicals has intensified globally, with natural polyphenolic compounds gaining prominence as powerful agents for promoting human health and as candidates for next-generation therapeutics [1,2]. These molecules are widely studied for their capability to scavenge reactive oxygen species, modulate cellular signaling pathways, and exhibit strong antioxidant, anti-inflammatory, and antimicrobial activities [3,4,5,6]. Among the most recognized and clinically significant polyphenols are quercetin (QUE), a ubiquitous flavonoid known for its pleiotropic biological effects, and rosmarinic Acid (RA), a high-value hydroxycinnamic acid derivative often noted for its potent radical-scavenging capabilities [7,8,9,10]. Critically, when combined, these two molecules have demonstrated synergistic anti-inflammatory and antioxidant properties, suggesting that co-delivery could dramatically enhance overall therapeutic efficacy [11]. Despite their undeniable potential, the successful clinical translation of both QUE and RA is severely constrained by shared physicochemical limitations. These compounds typically exhibit poor water solubility, which translates directly into low dissolution rates and subsequently poor oral and topical bioavailability [12,13,14]. Furthermore, their inherent chemical structure makes them highly susceptible to degradation upon exposure to light, elevated temperatures, and certain pH levels, rendering them unstable during processing, storage, and in vivo application [15]. Therefore, designing a robust, protective delivery platform capable of simultaneously stabilizing both agents while controlling their release kinetics remains a paramount pharmacokinetic challenge in natural product science.

To effectively circumvent the stability and solubility hurdles associated with QUE and RA, the development of advanced polymer-based functional materials is essential. Electrospinning has emerged as a leading technology in material science for fabricating continuous nanofibers with morphological features highly desirable for biomedical applications [16]. The technique’s scalability, combined with its ability to generate matrices characterized by an exceptionally high surface-area-to-volume ratio and high porosity, makes it ideal for achieving high drug loading efficiency and precise modulation of release profiles. Moreover, the resulting nanofibrous scaffolds structurally mimic the native extracellular matrix (ECM), which is crucial for applications in wound healing and tissue engineering, promoting cell adhesion and proliferation [17]. The selection of appropriate structural polymers is fundamental to tailoring the final properties of the electrospun material. Electrospun drug-loaded fibrous membranes are particularly suitable for localized antitumor therapy offering a remarkable versatility in terms of potential administration routes, incuding topical application for superficial tumors, implantation at the tumor site, and postoperative placement in the tumor resection area to prevent local recurrence [18]. Their unique structural and functional properties enable localized and sustained drug release, reduced systemic toxicity, and possibility for combination therapies involving multiple therapeutic agents. Therefore, the present study explores the potential of the developed electrospun mats as a promising platform for local anticancer drug delivery. In this study, we focus on evaluating the developed electrospun mats as a promising platform for local combination treatment of malignant skin melanomas.

Electrospun fibers incorporating bioactive polyphenols such as quercetin and rosmarinic acid have emerged as promising candidates for diverse biomedical applications [19]. Numerous studies have demonstrated that electrospun polymeric matrices can enable the controlled and sustained release of quercetin [20,21], maintaining therapeutically relevant concentrations over extended periods and thereby enhancing its overall biological efficacy. Similarly, rosmarinic acid—another naturally occurring phenolic compound with potent antioxidant, anti-inflammatory, and antimicrobial properties—has been successfully incorporated into electrospun systems to achieve prolonged and targeted delivery, improving its bioavailability and therapeutic potential [22,23]. The co-loading of quercetin and rosmarinic acid within electrospun fibers offers synergistic advantages, as both compounds exhibit complementary biological activities that can enhance cellular protection and tissue regeneration. Utilizing polymer blend systems composed of hydrophilic and hydrophobic components has been shown to facilitate dual-phase release behavior, allowing for both an initial burst and a sustained release profile suitable for different therapeutic needs [24,25].

Overall, the incorporation of quercetin and rosmarinic acid into electrospun fibrous matrices presents a versatile platform for biomedical use. The physicochemical and mechanical characteristics of PLA and PLA-PEG-based electrospun fibers can be precisely tuned to meet the structural and functional demands of specific tissues, expanding their potential applications in wound healing, regenerative medicine, and targeted cancer therapy. The combined antioxidant, anti-inflammatory, and anticancer properties of these polyphenols, together with the adaptable polymeric network, create a multifunctional system capable of addressing complex biological challenges [26,27,28,29]. Consequently, this innovative drug delivery approach holds significant promise for advancing therapeutic strategies across a wide range of biomedical fields [30,31,32,33].

Up to know the most of the studies have been addressed to encapsulate a single polyphenol into electrospun fibers. The novelty of the present work consists of creation of novel system for synergistic co-delivery of two distinct natural products within a tailored dual-polymer system fabricated by electrospinning. The primary objective of this research was the synthesis, characterization, and evaluation of the antioxidant and anticancer activity of the novel PLA/PEG composite nanofibers co-loaded with quercetin and rosmarinic Acid. The combination of the hydrophobic PLA structure with the hydrophilic PEG component would result in a robust, stable, and highly tunable delivery scaffold, simultaneously protecting both active compounds and enabling optimized synergistic release kinetics. Given the established biological activity of both QUE and RA, including their documented capacity to inhibit cell proliferation and induce apoptosis [34,35], this novel platform holds significant promise for applications in areas such as advanced wound care, tissue engineering, and localized antitumor therapy. This study presents a detailed analysis of the fiber morphology, behavior upon contact with water, antioxidant properties, in vitro release profiles and anticancer activity of the prepared in the present study electrospun mats.

2. Results and Discussion

In this work, electrospun fibrous matrices based on poly(lactic acid) (PLA) and a PLA/poly(ethylene glycol) (PLA/PEG) blend were produced from 10 wt.% polymer solutions. The optimized electrospinning parameters enabled the fabrication of continuous, uniform, and defect-free fibers exhibiting a homogeneous morphology. Due to their large specific surface area, high porosity, and excellent biocompatibility, these fibrous materials are well suited for the encapsulation and sustained release of biologically active compounds. In the present study, quercetin and rosmarinic acid—natural polyphenols with pronounced antioxidant and therapeutic properties—were incorporated into the PLA and PLA/PEG fibers both individually and in combination, in order to investigate potential synergistic effects.

Our previous investigations have revealed that electrospun PLA-based carriers are highly effective for the incorporation of natural extracts and phenolic compounds. For instance, PLA nanofibers loaded with quercetin demonstrated improved stability and a controlled release profile, which enhanced its anticancer efficacy against HeLa, 3T3, and SH4 cell lines [20,28]. Similarly, PLA matrices containing Melissa officinalis extract, rich in rosmarinic acid, exhibited strong antioxidant activity and favorable release characteristics, confirming the suitability of the electrospun PLA network for delivering hydrophilic phytochemicals [36]. Moreover, systems prepared with Portulaca oleracea extract revealed potent antimicrobial and antioxidant effects, further emphasizing the potential of PLA-based electrospun scaffolds as multifunctional carriers for natural bioactive substances [37,38]. Importantly, the inclusion of a hydrophilic polymer such as PEG within the PLA matrix has been reported to enhance the solubility, wettability, and diffusion-controlled release of entrapped molecules, thereby improving their overall biological performance.

Building on these findings, the current study focuses on a detailed evaluation of the antioxidant, antimicrobial, and anticancer activities of the newly developed PLA and PLA/PEG fibrous systems containing quercetin, rosmarinic acid, and their mixture, with particular emphasis on their cytotoxic effects against a melanoma cell line. In addition, cytocompatibility was assessed using HaCaT keratinocytes to determine the safety and potential biomedical applicability of these composite materials.

2.1. Preparation of Electrospun Fibrous Mats

Eight electrospinning formulations with distinct compositions were prepared and evaluated for their rheological properties prior to fiber fabrication. The dynamic viscosity of each spinning solution was measured to assess the effect of PEG, quercetin (QUE), and rosmarinic acid (RA) incorporation on the flow behavior. As reported previously, the viscosities of PLA, PLA/PEG, PLA/QUE, and PLA/PEG/QUE solutions were 1700, 1245, 1885, and 1450 cP, respectively [20]. The inclusion of RA resulted in viscosity values of 2107, 1562, 2135, and 1684 cP for PLA/RA, PLA/PEG/RA, PLA/QUE/RA, and PLA/PEG/QUE/RA formulations, respectively.

The results demonstrate that the viscosity of the spinning solutions is strongly composition-dependent. The addition of low-molecular-weight PEG reduced the viscosity of PLA-based systems, confirming its function as a plasticizing [39] and viscosity-lowering agent. Conversely, incorporation of individual bioactive compounds (QUE or RA) led to an increase in viscosity, likely due to enhanced intermolecular interactions and hydrogen bonding within the polymer matrix.

The correlation between the composition of the spinning solutions, their rheological properties, and the resulting fiber dimensions was further investigated. Among the parameters affecting electrospinning, viscosity plays a decisive role in determining fiber morphology by influencing jet uniformity and polymer chain entanglement. Generally, increasing the polymer concentration or solution viscosity leads to the formation of fibers with larger diameters, while lower viscosities favor the production of thinner fibers or bead-like structures. Therefore, the compositional variations introduced through PEG, quercetin, and rosmarinic acid are expected to modulate the fiber diameter by altering the overall viscosity of the spinning solutions.

The morphology of the novel obtained fibrous materials was observed by scanning electron microscopy (SEM). SEM micrographs reveal that all four types of fibers were defect-free and cylindrical. ImageJ software (ImageJ 1.54g) was used to analyze the SEM images in order to calculate the average fiber diameters. As previously was reported the production of PLA fibers with diameters of 768 ± 138 nm was achieved by electrospinning PLA solutions at a concentration of 10 wt.%. Both the resulting fiber diameters and the dynamic viscosity of the blend solution were decreased by adding the second polymer, PEG with a lower molecular weight, to the PLA solution. It was found that the PLA/PEG fibers’ diameter had an average value of 671 ± 137 nm. The average fiber diameter of PLA and PLA/PEG fibers upon quercetin loading was insignificantly increased to 807 ± 159 nm and 713 ± 143 nm, respectively [20]. Figure 1 reveals the morphology of novel fibrous materials based on PLA or PLA/PEG with RA or QUE and RA incorporated.

The mean fiber diameters were determined from representative micrographs. All samples exhibited continuous and uniform fibers without visible defects such as beads or irregular junctions, indicating stable electrospinning conditions. The average fiber diameters for the rosmarinic acid (RA)–containing systems were 846 ± 149 nm for PLA/RA and 720 ± 144 nm for PLA/PEG/RA (Figure 1c,d). For the polymer systems containing both flavonoids: 974 ± 148 nm for PLA/QUE/RA, and 826 ± 152 nm for PLA/PEG/QUE/RA (Figure 1e,f).

When compared with the previously reported diameters of PLA, PLA/PEG, PLA/QUE, and PLA/PEG/QUE fibers, a clear correlation between solution viscosity and fiber size is evident. The inclusion of PEG, a low-molecular-weight polymer, reduced the viscosity of the spinning solutions and consequently led to smaller fiber diameters. The combined incorporation of quercetin and rosmarinic acid into the polymers fibers resulted in slight increase in the fiber diameters. This is most probably due to the increase in the solution viscosities after the addition of the bioactive compounds. These findings confirm that modulation of the spinning solution composition effectively controls fiber morphology through viscosity-dependent mechanisms.

2.2. ATR-FTIR Analysis of Rosmarinic Acid-Containing Fibrous Materials

The chemical structure and molecular interactions of the electrospun fibers were examined using attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. The spectra of the PLA- and PLA/PEG-based fibrous mats containing rosmarinic acid (RA) are presented in Figure 2a,b, respectively. Both materials displayed the characteristic absorption bands of the polymeric matrix alongside additional signals associated with RA, confirming its successful incorporation into the fibers. The ATR-FTIR spectrum of rosmarinic acid (RA) powder (Figure 2a) displays the characteristic features of a polyphenolic ester. A broad O–H stretching region spanning 3600–3000 cm^−1^, with maxima near 3515, 3397, and 3165 cm^−1^, reflects extensive hydrogen bonding among phenolic and carboxylic groups. A strong carbonyl band at ~1705 cm^−1^ corresponds to the conjugated C=O group, while aromatic skeletal vibrations at ~1622 and ~1518 cm^−1^ confirm the presence of substituted benzene rings. Additional RA-related C–O and aryl–O stretching vibrations are observed at ~1282–1220 cm^−1^, with aromatic C–H out-of-plane bending modes appearing near ~961 and 842 cm^−1^, in agreement with published RA spectra [40,41].

The FTIR spectra presented in Figure 2a reveals that the PLA mat (green curve) exhibits the characteristic absorption bands of an aliphatic polyester. The intense peak at 1751 cm^−1^ corresponds to the ester carbonyl stretching vibration [42]. The bands at 1180–1080 cm^−1^ and ~1040 cm^−1^ are assigned to C–O–C stretching and C–O stretching of the ester backbone. A weaker band at 1452 cm^−1^ arises from CH_3_ bending, whereas the sharp features near 870–755 cm^−1^ correspond to crystalline-phase PLA. In the PLA/RA composite mat (red curve), the carbonyl stretching of PLA at ~1751 cm^−1^ remains evident, indicating preservation of the polymer backbone. The appearance of additional RA-derived peaks—particularly near 1609 cm^−1^ and 1516 cm^−1^, and the enhanced intensity in the fingerprint region between 1200–1000 cm^−1^—confirms the successful incorporation of RA into the PLA matrix. The slight broadening and partial overlap of peaks in the 1500–1650 cm^−1^ region suggest intermolecular interactions, likely hydrogen bonding between RA’s phenolic O–H groups and the carbonyl groups of PLA. The presence of RA-specific signals at 972, 878, 842, and 642 cm^−1^ further supports RA retention in the electrospun mat. Overall, the combined spectral changes indicate that RA is effectively embedded within the PLA fibers without disrupting the characteristic structural features of the polymer. For the PLA/RA system, the spectrum revealed the typical absorption bands of neat PLA, including a prominent ester carbonyl (C=O) stretching vibration at 1751 cm^−1^ and sharp bands at 2997 and 2945 cm^−1^ corresponding to the asymmetric and symmetric stretching of –CH_3_ groups, respectively. The band near 1452 cm^−1^ was attributed to –CH bending vibrations. A broad absorption region between 3600–3000 cm^−1^, with maxima at 3516, 3448, and 3344 cm^−1^, was associated with O–H stretching vibrations, likely originating from the hydroxyl groups of RA. The increased intensity and broadening of this region compared with pure PLA indicate the presence of hydrogen bonding between the hydroxyl groups of RA and the carbonyl groups of the polymer. Additional RA-related bands were observed at 1608, 1516, and 1452 cm^−1^, corresponding to aromatic C=C stretching vibrations, and at 1282 cm^−1^, linked to C–O stretching of aryl ether groups. Weak signals at 1356 and 1182 cm^−1^ were assigned to O–H bending and C–O stretching vibrations, while the fingerprint region (1045–758 cm^−1^) showed C–H out-of-plane bending characteristic of substituted aromatic rings [40,43]. These spectral features collectively confirm the successful encapsulation of RA within the PLA fibers and suggest molecular-level interactions, primarily through hydrogen bonding and van der Waals forces, between the phenolic compound and the polymer matrix.

The PLA/PEG mat (Figure 2b) exhibits the characteristic absorption profile of the polymer blend: a strong ester carbonyl band at ~1755 cm^−1^, aliphatic C–H stretching at 2995–2880 cm^−1^, and PEG-associated C–O–C vibrations at ~1283 and 961 cm^−1^ [40]. The absence of a broad O–H region confirms that no significant hydrogen-bonding species are present in the neat matrix. In contrast, the PLA/PEG/RA mat shows clear spectral evidence of RA incorporation. The broad O–H stretching region reappears and increases in intensity, indicating the presence of RA’s hydroxyl groups and their interaction with the polymer matrix. RA aromatic C=C stretching vibrations emerge at ~1608–1516 cm^−1^, a region not present in the neat PLA/PEG spectrum, confirming the presence of RA’s aromatic rings. Additional RA-specific bands in the 1285–1220 cm^−1^ region and weak aromatic C–H deformation peaks below 1000 cm^−1^ further support successful encapsulation. Minor broadening around the carbonyl region (~1755–1720 cm^−1^) suggests hydrogen bonding between RA hydroxyl groups and PLA ester carbonyls. Together, the appearance of RA-characteristic peaks, the re-emergence of the broad O–H stretching envelope, and shifts in the carbonyl region provide strong evidence that RA is successfully incorporated and interacts with the PLA/PEG matrix, primarily through hydrogen bonding, consistent with reported RA–polymer interactions [42].

The FTIR analysis verifies the successful incorporation of quercetin (QUE) and rosmarinic acid (RA) into the PLA-based electrospun mats [Figure S1a, Supplementary Materials]. A broadened and intensified O–H stretching band is evident within ~3500–3100 cm^−1^, which is not present in neat PLA and corresponds to the phenolic hydroxyl groups of both QUE and RA [23,25,44,45]. Furthermore, the FTIR analysis presented in Figure S1b confirms the successful incorporation of quercetin and rosmarinic acid into the PLA/PEG electrospun mats.

2.3. Contact Angle Measurements and Wettability

Static water contact-angle measurements were performed to evaluate the wettability of the electrospun PLA-based materials. Rectangular sample of each electrospun material were prepared for wettability assessment. A droplet of deionized water (7 μL) was gently dispensed onto the surface of the samples, and its profile was immediately recorded using a contact angle goniometer. The resulting droplet geometries and calculated contact-angle values are displayed in Figure 3. As previously demonstrated for PLA and PLA/QUE [36], both types of mats exhibited a hydrophobic surface, with water droplets maintaining a high and stable angle due to the limited number of hydrophilic functional groups exposed on the fiber surface. The presence of quercetin even induced a slight increase in hydrophobicity compared to neat PLA (contact angle around 100 degrees), which was attributed to its low aqueous solubility and restricted interaction with water.

A similar trend was observed for the PLA/RA mat, where a contact angle of 107.6° ± 2.6° confirmed a predominantly water-repelling character (Figure 3a). Although rosmarinic acid contains multiple phenolic moieties, its incorporation did not increase surface wettability, likely because RA remains mostly embedded within the PLA matrix rather than being oriented toward the fiber exterior. As can be observed at Figure 3c, the combined incorporation of RA and QUE into PLA fibers slightly increases the surface hydrophobicity of PLA/QUE/RA mat most probably due to the presence of low aqueous soluble quercetin.

In contrast, when PEG was introduced, a dramatic shift toward hydrophilic behavior was detected. The droplet immediately spread and penetrated the PLA/PEG/QUE/RA mat, resulting in a contact angle of 0° (Figure 3d). This behavior aligns with previously obtained PEG-containing systems [36], confirming that PEG effectively enhances water affinity through its hydrophilic chains and hydrogen-bonding capability. The superhydrophilic nature of PEG-based formulations is expected to facilitate faster hydration and improved release of the phenolic compounds.

2.4. In Vitro Release Study of Quercetin and Rosmarinic Acid from PLA-Based Electrospun Fibers: Effects of PEG and Co-Encapsulation

The design of efficient drug-delivery systems requires polymer matrices capable of providing predictable and sustained release of incorporated therapeutic agents. In this context, the present work examines the release profiles of two antioxidant polyphenols—quercetin and rosmarinic acid—from electrospun fibers based on poly(lactic acid) (PLA). The release behavior was evaluated under two distinct pH conditions selected to represent different biological environments: pH 7.4 (phosphate-buffered saline, PBS), which simulates the neutral physiological conditions of blood plasma and healthy tissues, and pH 5.5 (acetate-buffered saline), which mimics acidic tumor-associated and inflammatory environments, including intracellular compartments such as endosomes and lysosomes [46].

A series of formulations was produced to evaluate the effects of polymer composition and co-encapsulation on release behavior. These included single-loaded mats (PLA/QUE and PLA/RA), PEG-modified analogues (PLA/PEG/QUE and PLA/PEG/RA), and dual-loaded systems containing both polyphenols (PLA/QUE/RA and PLA/PEG/QUE/RA). Polyethylene glycol (PEG) was incorporated as a hydrophilic component to facilitate water uptake and potentially accelerate diffusion through the fibrous structure. In all cases, the total amount of incorporated bioactive compound(s) was fixed at 10 wt.% relative to the polymer, consisting of either 10 wt.% of a single polyphenol or 5 wt.% of each in the co-loaded systems.

2.4.1. Quercetin Release from PLA-Based Fibers

The cumulative release of QUE from the examined formulations is presented in Table 1. Overall, the data show clear differences among the systems depending on PEG incorporation and co-loading with RA.

As can be seen at Figure 4 a burst release up to ~60 min was observed in all studied fibers and actually it is very typical for the electrospun nanofiber systems. This is due to the fact that electrospun fibers possessed high surface-area-to-volume ratio and have interconnected pore spaces between fibers. At the 480 min at pH 5.5, the PLA/QUE fibers released 60.5% of their initial QUE content, which is consistent with the restricted water penetration and limited diffusivity characteristic of hydrophobic PLA matrices. Introducing PEG into the formulation (PLA/PEG/QUE) markedly accelerated release was detected, resulting in 87.3% cumulative release. This pronounced improvement can be attributed to the hydrophilic nature of PEG, which enhances water uptake and promotes matrix swelling, thereby facilitating QUE diffusion. Co-encapsulation with RA (PLA/QUE/RA) produced a moderate increase in QUE release, reaching 66.0% at pH 5.5. This behavior suggests that RA may induce subtle structural changes within the fiber network or slightly weaken polymer–drug interactions, both of which could support more efficient diffusion. The greatest release efficiency was observed for the PLA/PEG/QUE/RA system, which delivered almost complete release (96.8%), indicating a synergistic effect between PEG-induced hydrophilization and the presence of the second polyphenol. At pH 7.4, all materials showed slightly lower release, in line with reduced polymer swelling and slower hydrolytic relaxation under physiological conditions. PLA/QUE and PLA/QUE/RA reached 55.3% and 60.1% release, respectively. Incorporation of PEG again substantially enhanced the release profile, with PLA/PEG/QUE achieving 80.8%. The dual-loaded PLA/PEG/QUE/RA fibers maintained high efficiency at this pH as well, releasing 95.0% of their QUE content. The similarity of the release values for this formulation at both pH levels indicates that diffusion governs drug liberation from these PEG-containing systems. The obtained results were in accordance with previous studies revealing that addition of PEGs to the PLA matrix enhanced the release of hydrophobic drug–paclitaxel [47].

2.4.2. Rosmarinic Acid Release from PLA-Based Fibers

The release behavior of RA from the different fiber formulations (Figure 5, Table 2) showed trends comparable to those observed for QUE. At pH 5.5, the PLA/RA mats released 69.5% of their loaded RA, reflecting the restricted water uptake and slow diffusion typical of hydrophobic PLA matrices. Incorporation of PEG (PLA/PEG/RA) substantially improved the release performance, yielding 90.2% cumulative release. This enhancement can be attributed to the increased hydrophilicity and swelling capacity introduced by PEG, which promotes greater medium penetration and facilitates RA diffusion through the fiber structure. Table 2 summarizes the cumulative RA release under both acidic (pH 5.5) and physiological (pH 7.4) conditions for all prepared PLA-based electrospun systems.

Co-encapsulation of RA with QUE (PLA/QUE/RA) resulted in a moderate increase in RA release, reaching 76.5% at pH 5.5. This improvement suggests that the presence of QUE may influence the internal organization of the fibers or slightly weaken polymer–drug interactions, thereby enabling more efficient diffusion of RA. The highest release was observed for the PEG-containing dual-loaded system (PLA/PEG/QUE/RA), which achieved 96.2% cumulative release. This confirms a synergistic contribution of PEG-induced hydrophilicity and the secondary polyphenol, leading to a more permeable fiber structure.

At pH 7.4, RA release decreased across all formulations, consistent with the reduced swelling and slower hydrolytic relaxation of PLA under neutral conditions. The PLA/RA fibers released 51.1% of their content, while the PEG-modified PLA/PEG/RA mats showed a markedly higher release of 88.4%, only slightly lower than at pH 5.5. This minimal pH dependence highlights PEG as the primary factor governing RA diffusion from the fibers. The dual-loaded PLA/QUE/RA system reached 70.7% release, whereas the PEG-containing PLA/PEG/QUE/RA mats maintained high efficiency, delivering 86.8% of the RA payload. The similar release values for PEG-containing formulations at both pH conditions indicate that diffusion, rather than pH-triggered matrix degradation, predominates in regulating RA transport through the hydrophilized fibers.

The trends observed in this work are in agreement with previous reports on PLA-based delivery systems, which commonly exhibit accelerated release under mildly acidic conditions due to enhanced polymer relaxation, increased hydration, and pH-assisted hydrolysis of ester linkages [48,49]. Our results clearly demonstrate that both PEG incorporation and polyphenol co-encapsulation exert a significant influence on the release kinetics of QUE and RA. As anticipated, acidic pH promoted higher release from all formulations, likely reflecting greater water uptake and faster structural loosening of the PLA fibrous material. However, PEG-containing systems consistently outperformed their non-modified counterparts across both pH conditions, highlighting PEG as the primary factor governing water accessibility, polymer swelling, and overall diffusional transport through the fibrous matrix.

Co-loading QUE and RA further improved the release efficiency of each compound, suggesting that the presence of a second polyphenol modifies the microstructure of the fibers or disrupts drug–polymer interactions in a way that facilitates molecular mobility. Among all investigated systems, the PLA/PEG/QUE/RA formulation exhibited the most favorable release characteristics, achieving near-quantitative release of both polyphenols under physiological and acidic conditions. Such a highly efficient dual-delivery platform is particularly relevant for therapeutic contexts involving oxidative stress, chronic inflammation, or tumor-associated microenvironments, where sustained and substantial release of antioxidant and anti-inflammatory agents is desirable.

2.5. In Vitro Cytotoxicity Evaluation

The cytotoxic effects of the tested electrospun fibers and free compounds were evaluated in SH-4 melanoma cells and HaCaT normal keratinocytes following 24 h and 72 h of exposure (Figure 6).

In SH-4 cells at 24 h, PLA and PLA/PEG maintained high cell viability, comparable to control levels. Incorporation of quercetin or rosmarinic acid into PLA-based fibers (PLA/QUE and PLA/RA) significantly reduced cell viability. The decrease was even more pronounced for PEGylated formulations containing QUE and RA (PLA/PEG/QUE and PLA/PEG/RA). The strongest cytotoxic effect was observed for the combined PLA/PEG/QUE/RA mats. Overall, PEGylated drug-loaded mats exhibited higher cytotoxicity, as evidenced by lower cell viability values for PLA/PEG/QUE, PLA/PEG/RA and PLA/PEG/QUE/RA, than their non-PEGylated counterparts. Free QUE caused a marked reduction in SH-4 cell viability, whereas free RA showed comparatively lower cytotoxicity.

After 72 h of exposure, all treatments induced a more pronounced decline in SH-4 cell viability, indicating time-dependent cytotoxic effects. PLA and PLA/PEG remained well tolerated, with viability exceeding 90%. QUE- and RA-containing formulations, caused a substantial reduction in viability, with PEGylated variants again showing enhanced cytotoxicity. The strongest cytotoxic effects were associated with combined PLA/QUE/RA and PLA/PEG/QUE/RA mats. Free quercetin continued to exhibit high cytotoxicity, whereas free rosmarinic acid maintained relatively high cell survival.

In HaCaT cells at 24 h, all polymeric formulations demonstrated good compatibility, with only moderate reductions in viability observed for drug-loaded mats, including the combined PLA/QUE/RA and PLA/PEG/QUE/RA mats. At 72 h, HaCaT cells became more sensitive to quercetin and rosmarinic acid exposure, with PLA/PEG/RA, and in particular, PLA/PEG/QUE/RA significantly reducing cell viability. PEGylation intensified these effects. Free rosmarinic acid induced the strongest reduction in HaCaT cell viability at 72 h, while free quercetin was comparatively less toxic.

Overall, both cell lines exhibited similar response to the tested formulations; however, the magnitude of viability loss was significantly greater in melanoma cells, indicating cell-type-specific sensitivity and supporting the selective anticancer potential of the drug-loaded fibrous mats.

2.6. Fluorescent Microscopy of Cancer and Non-Cancerous Cells Exposed to Electrospun Fibrous Mats

Apoptosis, or programmed cell death, is a highly regulated biological process that plays a central role in normal tissue homeostasis and in the prevention and treatment of cancer. Fluorescence assays based on fluorochrome staining (acridine orange/ethidium bromide—AO/EtBr) are widely used to distinguish live from dead cells. Acridine orange stains viable cells with intact membranes green, whereas ethidium bromide penetrates cells with compromised membranes, staining late apoptotic or necrotic nuclei orange/red. In this study, the apoptotic morphology of human SH-4 melanoma cells after exposure to various treatments was assessed using AO/EtBr staining (Figure 7).

Control SH-4 cells had an elongated, spindle-like shape and intact cell membranes, and emitted predominantly green fluorescence, indicating high viability and lack of cytotoxicity (Figure 7a). PLA/PEG-treated cells demonstrated similar morphological features (Figure 7b). Cells treated with free RA or QUE alone showed changes characteristic of early apoptosis-rounding, chromatin condensation, and orange/red stained nuclei (Figure 7c,d). Treatment with the PLA/RA and PLA/PEG/RA fibrous mats further enhanced morphological alterations, as evidenced by brighter orange/red nuclear staining and reduced green cytoplasmic fluorescence (Figure 7e,f). Incubation with composite fibrous mats loaded with RA and QUE resulted in more pronounced cytotoxic responses, including increased numbers of late apoptotic/necrotic cells, nuclear fragmentation and loss of normal cellular architecture (Figure 7g,h). Notably, the cells treated with PLA/PEG/QUE/RA mats demonstrated the most significant effect, characterized by extensive orange/red fluorescence, a marked reduction in viable green-stained cells, and severe morphological abnormalities, indicating synergistic induction of apoptosis and loss of membrane integrity in SH-4 melanoma cells (Figure 7h).

DAPI staining revealed definite treatment-dependent changes in nuclear morphology of the melanoma cells (Figure 8).

Control and PLA/PEG-treated SH-4 cells displayed uniformly stained, round to oval nuclei with intact chromatin, indicative of healthy, viable cells (Figure 8a,b). Exposure to free RA or QUE led to noticeable nuclear alterations, including reduced cell density, irregular nuclear contours, and areas of chromatin condensation (Figure 8c,d). Composite fibrous mats containing RA (PLA/RA, PLA/PEG/RA) produced more pronounced effects, characterized by scattered nuclei, increased nuclear shrinkage, and intensified fluorescence, consistent with apoptotic condensation (Figure 8e,f). The most substantial nuclear disruption was observed in cells treated with the PLA/QUE/RA and PLA/PEG/QUE/RA fibrous mats, where nuclei appeared highly condensed, fragmented, or irregularly shaped, reflecting advanced stages of apoptosis (Figure 8g,h). Across treatments, the degree of nuclear changes correlated with the fibrous mat content, supporting their enhanced cytotoxic activity.

The effects of the electrospun mats on the cellular and nuclear morphology of normal human skin cells were also analyzed (Figure 9 and Figure 10).

Untreated HaCaT cells and the cells exposed to PLA/PEG mats exhibited dense monolayer growth, polygonal epithelial morphology, intact membranes, and displayed uniform green fluorescence (Figure 9a,b). Treatment with RA and QUE alone resulted in mild nuclear chromatin condensation, accompanied by a slight increase in fluorescence intensity (Figure 9c,d). Cells treated with PLA/RA and PLA/PEG/RA mats exhibited noticeable nuclear brightening and signs of shrinkage, indicating the onset of apoptosis (Figure 9e,f). The treatment with the PLA/QUE/RA composite mat resulted in more pronounced nuclear condensation and irregular cell shapes (Figure 9e). The most significant morphological alterations, characterized by intense nuclear fluorescence, rounded and shrunken cells, as well as a reduction in cell density, were observed following treatment with the PLA/PEG/QUE/RA mats (Figure 9h).

The nuclear morphology changes in non-cancerous HaCaT cells exposed to tested formulations and stained with DAPI are presented in Figure 10.

In the control and PLA/PEG-treated HaCaT cell cultures, nuclei appeared uniformly stained, round to oval in shape, and evenly distributed, reflecting intact nuclear morphology and normal chromatin organization (Figure 10a,b). Treatment with free RA or free QUE alone induces moderate nuclear alterations, including chromatin condensation and occasional nuclear shrinkage (Figure 10c,d). More pronounced nuclear damage was observed in cells treated with RA-loaded PLA formulations, characterized by increased nuclear condensation, fragmentation, and irregular nuclear contours—hallmarks of apoptotic cell death (Figure 10e,f). The combined treatment, particularly PLA/PEG/QUE/RA, exhibits the highest degree of nuclear alteration, with numerous condensed and fragmented nuclei and a marked reduction in normal nuclear morphology (Figure 10g,h).

Collectively, the findings of fluorescent microscopy analysis revealed clear differences in the morphological responses of SH-4 tumor cells and HaCaT keratinocytes to the tested fibrous formulations. While control and PLA/PEG-treated cells from both lines maintained normal morphology, single treatments (RA, QUE) induced noticeable apoptotic features in SH-4, including nuclear condensation, cell shrinkage, and increased membrane permeability, whereas HaCaT remained largely intact. Composite fibrous mats containing RA and/or QUE further intensified these effects, with SH-4 showing progressive membrane disruption, chromatin fragmentation, and loss of monolayer organization, particularly after PLA/PEG/QUE/RA treatment. In contrast, HaCaT cells exhibited only mild to moderate nuclear brightening and limited morphological alterations. These results demonstrate a pronounced and treatment-dependent cytotoxicity in SH-4, accompanied by substantially lower impact on HaCaT, indicating selective sensitivity of tumor cells to the composite fibrous mats.

These results demonstrate a pronounced and treatment-dependent cytotoxicity in SH-4, accompanied by substantially lower impact on HaCaT. Although the cytotoxicity and fluorescence microscopy results convincingly demonstrate selective anticancer activity of the developed drug-loaded nanofibers, the in vitro models cannot fully reproduce the complex tumor microenvironment, pharmacokinetics, and systemic interactions observed in living organisms. Therefore, further in vivo studies are required to validate the antitumor efficacy, biocompatibility, and safety profile of the electrospun mats prepared in this study and to assess their translational potential for clinical applications.

3. Materials and Methods

3.1. Materials

Polylactic acid (PLA; Ingeo™ Biopolymer 4032D, NatureWorks, Plymouth, MN, USA; Mw = 259,000 g·mol^−1^, PDI = 1.94) and polyethylene glycol (PEG; Serva, Heidelberg, Germany; Mw = 100,000 g·mol^−1^) were used as polymeric materials. quercetin (QUE ≥ 95%; Sigma–Aldrich, St. Louis, MO, USA), rosmarinic acid (RA) (Merck, Billerica, MA, USA) and Tween 80 (Acros Organics, Amsterdam, The Netherlands) were used. Analytical-grade dichloromethane (DCM), absolute ethanol (EtOH), and dimethyl sulfoxide (DMSO) served as solvents. Salts used for the preparation of the buffer solution of pH 7.4 (KH_2_PO_4_, Na_2_HPO_4_) were purchased from Merck Chemicals (Merck, Billerica, MA, USA). Glacial acetic acid (Merck, Billerica, MA, USA) and NaOH (Merck Chemicals) used for preparation of the buffer solution of pH 5.5 were of analytical grade of purity. Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were supplied by Gibco-Invitrogen (Leicestershire, UK). The penicillin–streptomycin antibiotic mixture was obtained from Lonza (Verviers, Belgium). Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and Trypsin–EDTA solution (2.5 g/L trypsin, 0.2 g/L EDTA) were provided by AppliChem (Darmstadt, Germany). MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,3-diphenyl tetrazolium bromide) was purchased from Sigma-Aldrich Chemie GmbH (Darmstadt, Germany), while acridine orange (AO) and ethidium bromide (EtBr) were supplied by Merck (Darmstadt, Germany). All sterile plastic laboratory consumables were sourced from Orange Scientific (Braine-l’Alleud, Belgium).

3.2. Cell Lines and Culture Conditions

In this study, the human skin melanoma cell line SH-4 (CRL-7724) was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). The non-tumorigenic human keratinocyte line HaCaT (CVCL_0038) was provided by CLS Cell Lines Service (Eppelheim, Germany). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) enriched with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (50 U/mL penicillin and 50 µg/mL streptomycin). The cell lines were maintained in 25 cm^2^ tissue culture flasks at 37 °C in a humidified atmosphere and 5% CO_2_, and were subcultured every 3–4 days to ensure continuous exponential growth. When cultures reached 60–80% confluency, the cells were detached using 0.25% trypsin-EDTA (pH 7.4), counted, and then resuspended in fresh medium at the concentrations required for the respective assays.

3.3. Fabrication of Electrospun Fibrous Mats

Electrospinning solutions were prepared using PLA, PLA/PEG, and their combinations with polyphenols. PLA and PLA/PEG were dissolved in a dichloromethane/ethanol mixture (80:20 v/v) under continuous stirring to obtain homogeneous 10 wt.% polymer solutions. For PLA/PEG mats, a weight ratio of PLA/PEG = 80:20 was used, based on preliminary optimization experiments [30]. Initially, PLA was dissolved in DCM and PEG was dissolved in ethanol at room temperature −21 °C for 2 h. After that the PEG solution was added to the PLA solution under continuous stirring in order to obtain homogeneous blend solution. Polyphenols were incorporated as follows: single-polyphenol mats (PLA/RA, PLA/PEG/RA, PLA/QUE, PLA/PEG/QUE) contained 10 wt.% of the respective compound relative to the polymer weight, while dual-polyphenol mats (PLA/QUE/RA and PLA/PEG/QUE/RA) were loaded with 5 wt.% of each polyphenol.

Electrospinning was performed using a syringe (12 mL) fitted with a 20 G needle connected to a high-voltage power supply. The applied voltage was set at 25 kV, with a needle-to-collector distance of 12 cm. Fibers were collected on a rotating drum at 1000 rpm, and the polymer solution was fed at a constant flow rate of 3 mL/h using an infusion pump (NE-300 Just Infusion™ Syringe Pump, New Era Pump Systems Inc., Farmingdale, NY, USA). After electrospinning, the mats were dried under vacuum at room temperature to remove residual solvents.

3.4. Characterization

3.4.1. Dynamic Viscosity Measurements

The dynamic viscosity of all solutions was determined using a Brookfield DV-II+ Pro viscometer (Middleboro, MA, USA) equipped with a CPE 52 cone spindle in the single-plate configuration. Measurements were performed at room temperature (25 °C) using a thermostatted sample cup.

3.4.2. Fiber Morphology Examination

The morphology of the electrospun fibers was examined using scanning electron microscopy (SEM, JEOL JSM-5510, JEOL Co., Ltd., Tokyo, Japan). Prior to imaging, samples were gold-coated under vacuum for 60 s using a JEOL JFC-1200 fine coater. Fiber diameters were measured from SEM micrographs using ImageJ software (version 1.54g), analyzing at least thirty fibers per sample to calculate the mean diameter and standard deviation.

3.4.3. ATR-FTIR Analysis

The chemical composition of the electrospun fibrous mats was analyzed by attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy. Spectra were recorded on an IRAffinity-1 spectrophotometer produced by Shimadzu Co., Kyoto, Japan, was used to capture the FTIR spectra. The spectra were recorded from 4000 to 600 cm^−1^.

3.4.4. Water Contact Angle Measurements

The hydrophilicity of the electrospun fibrous mats was evaluated using the static sessile drop method. Measurements were performed with an Easy Drop DSA20E (Krüss GmbH, Hamburg, Germany). A 10 μL droplet of distilled water was placed on the surface of each mat, and the contact angle was determined by image analysis over time. The reported values represent the mean of at least 20 measurements per sample.

3.4.5. Evaluation of In Vitro Release Profiles

The release behavior of QUE and RA from electrospun mats (PLA/QUE, PLA/RA, PLA/PEG/QUE, PLA/PEG/RA, PLA/QUE/RA, and PLA/PEG/QUE/RA) was investigated at 37 °C in both acetate buffer (0.1 M, CH_3_COONa/CH_3_COOH, pH 5.5) and phosphate-buffered saline (PBS, 0.1 M, pH 7.4). Samples (10 mg) were immersed in 100 mL of buffer and incubated in a JULABO SW23 shaking water bath (Allentown, PA, USA) under constant stirring. At predetermined time intervals, aliquots were withdrawn and replaced with fresh buffer to maintain sink conditions. The amount of released flavonoids was measured using a DU 800 UV–Vis spectrophotometer (Beckman Coulter, Brea, CA, USA) at 445 nm. Release quantities were calculated from calibration curves (R = 0.999). All experiments were performed in triplicate, and results are presented as mean values.

3.5. In Vitro Cytotoxicity Evaluation

The MTT colorimetric assay was performed to assess the cytotoxic effect of the tested fibrous mats in vitro. SH-4 melanoma and normal HaCaT cells were plated in 96-well plates at density of 10^4^ cells/well. After 24 incubation in 5% CO_2_ at 37 °C with 95 humidified air to form monolayer, the cells were treated with various fibrous mats for 72 h. The respective cells cultured only in medium or with QUE solution (100 µM) or RA solution (100 µM) were used as controls. Later, medium was discarded and the MTT solution (0.5 mg/mL) was added to all wells. Further, the plates were additionally incubated for 4 h at 37 °C. Finally, the MTT medium was removed and lysing solution (DMSO/ethanol 1:1) (100 µL) was added into each well in order to dissolve the formed formazan crystals. The absorbance was determined by using an ELISA microplate reader (TECAN, SunriseTM, Grodig/Salzburg, Austria) at 570 nm. Each variant was tested in five replicates. The results were expressed as mean ± standard deviation (SD) for three independent examinations. The cell viability was calculated by the following formula:

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3.6. Dual Fluorescent Staining with AO/EtBr for Cell Death Analysis

AO/EtBr dual staining was employed to assess morphological changes in cells after 24 h of incubation with the tested fibrous mats. AO stains both viable and non-viable cells, emitting green fluorescence, whereas EtBr is taken up only by non-viable cells, producing red fluorescence through DNA intercalation. Briefly, tumor and non-tumor cells were seeded onto sterile glass coverslips, which were placed in the bottoms of 24-well plates at a density of 2.0 × 10^5^ cells/well, and incubated for 24 h in a CO_2_ incubator to allow monolayer formation. The cells were then exposed to the fibrous samples, while cells cultured in medium alone served as controls. After 24 h of incubation, the coverslips were removed, washed with PBS, and stained with a fluorescent dye mixture containing AO (5 µg/mL) and EtBr (5 µg/mL). The stained cells were immediately examined under a fluorescence microscope (Leica DM 5000B, Wetzlar, Germany).

3.7. DAPI Fluorescent Staining

Nuclear morphology was assessed using the DNA-binding dye 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). DAPI effectively visualizes nuclear DNA in both live and fixed cells, as it can penetrate intact cytoplasmic membranes. Cells were cultured on glass coverslips and treated with various formulations as stated in the previous paragraph. After 24 h, the cells were fixed with methanol and incubated with DAPI solution (1 µg/mL in methanol) for 15 min in the dark. Stained cells were mounted on slides and examined using a fluorescence microscope (Leica DM 5000B, Wetzlar, Germany).

3.8. Statistical Methods

Data were expressed as mean ± standard deviation (SD) and analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Bonferroni post hoc tests, performed in GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA). A confidence level of 95% was applied, and significance was considered at p < 0.05, p < 0.01, and p < 0.001.

4. Conclusions

This study demonstrates for the first time that electrospun PLA/PEG composite nanofibers represent an effective and tunable platform for the co-delivery of quercetin and rosmarinic acid. By combining the structural robustness of hydrophobic PLA with the hydrophilizing properties of PEG, the fabricated mats exhibited uniform morphology, improved wettability, and high encapsulation efficiency for both polyphenols. Comprehensive physicochemical characterization confirmed successful incorporation of the active compounds in the prepared fibrous materials. Release studies conducted under physiological and mildly acidic conditions showed that PEG played a decisive role in accelerating hydration and diffusion, enabling near-complete release of both antioxidants from the hybrid matrices. Notably, the PLA/PEG/QUE/RA formulation achieved the most favorable release profile, combining rapid initial dissolution with sustained delivery. Additionally, the produced novel materials containing the polyphenols had strong anticancer activity against the used cancer cell line, but lower to the normal HaCaT cells, indicating selective sensitivity. Collectively, these results validate the potential of PLA/PEG nanofibers loaded with QUE and RA polyphenols as a versatile carrier system capable of delivering synergistic natural antioxidants in a controlled manner. The combination of structural stability, tailored release kinetics, and strong anticancer activity highlights their promise for biomedical applications such as advanced wound care, tissue regeneration, localized anti-inflammatory therapy, and cancer-related oxidative stress management.