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. 2025 Aug 31;10(36):41104–41112. doi: 10.1021/acsomega.5c03670

Influence of Solvent Choice on Mitomycin C Loading and Stability in Electrospun Polyvinylidene Fluoride Nanofibers

Jana Horakova †,*, Radek Jirkovec †, Stanislava Vrchovecka ‡, Andrea Sidova †
PMCID: PMC12444496  PMID: 40978389

Abstract

This study investigates the influence of solvent systems on incorporating the hydrophilic drug mitomycin C (MMC) into electrospun polyvinylidene fluoride (PVDF) nanofibers, with a focus on the often-overlooked issue of active substance degradation during storage. MMC was dissolved in either water or acetone and added to the electrospinning solution, followed by quantification of drug loading using liquid chromatography. Dissolution of MMC in water resulted in poor drug loading efficiency. In contrast, acetone led to significantly higher incorporation, with 50.66% loading for PVDF containing 0.01% MMC and 26.63% for PVDF with 0.1% MMC. However, storage at 4 °C over 21 days resulted in a substantial decline in MMC content, indicating challenges in long-term stability. In vitro testing using mouse fibroblasts revealed no cytotoxic effects of the nanofibrous layers and demonstrated a reduction in fibroblast proliferation in MMC-loaded samples compared to pure PVDF controls. These findings highlight the importance of solvent selection for effective drug incorporation and point to the potential of PVDF-based nanofibers for antifibrotic applications.

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1. Introduction

Over the past few decades, the field of drug delivery has been investigated, driven by the need for targeted, controlled, and efficient therapeutic strategies. Nanofiber-based drug delivery systems have gained significant attention among emerging platforms due to their unique structural and functional properties. These ultrafine fibers, typically produced through techniques such as electrospinning, offer a high surface-area-to-volume ratio, tunable porosity, and the ability to encapsulate a wide range of bioactive agents. ,

Nanofibrous materials have emerged as ideal candidates for glaucoma drainage devices, primarily owing to their distinctive structural characteristics, excellent mechanical properties including flexibility and conformability, and their inherent capability to serve as effective drug delivery systems. , In this study, we investigated the incorporation of the active agent mitomycin C (MMC) into polyvinylidene fluoride (PVDF) nanofibers. Our previous work has demonstrated the potential of electrospun PVDF as a material for nanofibrous glaucoma drainage devices. However, such devices face a significant challenge of fibroblast proliferation, which can alter device function and ultimately lead to failure over time. Given the need for antifibrotic properties in such applications, we previously explored PVDF blended with poly­(ethylene oxide) (PEO) to enhance material performance. Building on these findings, the present research aims to develop nanofibrous materials capable of inhibiting fibroblast proliferation by incorporating MMC directly into the nanofiber layers. Similar efforts to incorporate MMC into glaucoma drainage devices have been reported by other authors. Specifically, Dong et al. and Sahiner et al. demonstrated promising results by coating the surface of existing glaucoma drainage device with MMC, which mitigated scar tissue formationa critical factor leading to device failure.

Polyvinylidene fluoride is a semicrystalline fluoropolymer that has shown versatility for a wide range of biomedical and pharmaceutical applications, such as wound dressings, cardiovascular and nerve regeneration, etc. , Mitomycin C is a chemotherapeutic agent whose incorporation has been extensively investigated using a variety of delivery carriers. , Besides its chemotherapeutic properties, mitomycin C also functions as an antimetabolite and is commonly used during glaucoma surgeries such as trabeculectomy to inhibit fibroblast proliferation and prevent excessive postoperative scarring. , Mitomycin C is soluble in water; however, its stability in water is limited up to 4 days. Only a few studies have explored the incorporation of MMC into electrospun nanofibers. Shi et al. developed nanofibrous membranes composed of polycaprolactone and chitosan, incorporating MMC and meloxicam, to prevent epidural adhesions. In another study, Zhao et al. encapsulated MMC-loaded hyaluronan hydrosol within polylactic acid nanofibers to promote balanced tendon healing. These examples highlight the potential of MMC-functionalized nanofibers for localized drug delivery in tissue repair and fibrosis prevention.

When evaluating drug delivery systems, drug loading represents a critical parameter that must be thoroughly analyzed. Most studies primarily focus on drug loading efficiency and release kinetics under conditions that simulate the in vivo environment. − However, a factor often overlooked is the stability of the incorporated drug over storage time, which may significantly influence therapeutic performance. In this study, we investigated the incorporation of MMC into nanofibrous layers following its dissolution in two different solventswater and acetone. Drug loading was assessed at three time points: 1 day, 7 days, and 21 days postfabrication, to evaluate the potential impact of storage on MMC content. In addition, we examined the cytotoxicity of MMC and the nanofibrous scaffolds and their effect on fibroblast proliferation to assess their antifibrotic potential and cytocompatibility.

2. Materials and Methods

2.1. Preparation of Electrospinning Solutions

Polyvinylidene fluoride (PVDF, M w 180,000, Merck, Germany) was dissolved in a solvent system composed of dimethylacetamide (DMAc) and acetone (both from Penta Chemicals, Czech Republic) in a concentration of 26 wt %. PVDF was first dissolved in DMAc at 80 °C for 24 h. Afterward, the heating was stopped, and once the solution had cooled, acetone was added to the PVDF solution (final weight ratio of DMAc/acetone 4:1). The solution was stirred for 1 h at room temperature. After dissolution, the mitomycin C (MMC, Roche Diagnostics, Germany) solution was added to the PVDF solution. MMC was dissolved in water (1 mg MMC per 0.25 g of water) in the first round of experiments and added to the electrospinning solution. Two concentrations of MMC in the PVDF solution were selected: 0.1 mg per 1 g of PVDF (referred to as PVDF + 0.01% MMC) and 1 mg per 1 g of PVDF (referred to PVDF + 0.1% MMC). After the aqueous mitomycin solution was added to the PVDF solution, precipitation occurred, and the mixture was then stirred at room temperature for another hour until the precipitate dissolved. In the work’s second part, MMC in the same concentrations was dissolved in acetone. After adding MMC in acetone, no precipitation of the solution was seen.

2.2. Fabrication of Nanofibrous Materials

A Nanospider NS 1WS500U (Elmarco, Czech Republic) device with a string electrode was used to prepare nanofibrous layers. The schematic of the device is shown in Figure . Electrospinning parameters are listed in Table . After the fabrication of the layers, materials were kept at 4 °C before further analysis.

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Schematic of the needleless electrospinning device used for material fabrication.

1. Parameters of the Electrospinning Process Used for the Fabrication of the Layers.

the distance between the electrode and the collector 180 mm
cartridge movement speed 130 mm/s
electrode voltage 50 kV
collector voltage –10 kV
substrate towing speed 10 mm/min
temperature 22 °C
relative air humidity 10%
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2.3. Characterization of Produced Materials

Nanofibrous materials were characterized by their thickness, area weight, and morphology. The thickness of materials was measured by a digital thickness gauge (2871-101, INSIZE, Czech Republic, n = 10). Area weight was measured by cutting a circle with an area of 100 cm2 from produced materials and subsequent weighing (n = 1). The morphology of the materials was assessed by scanning electron microscopy (SEM, Vega 3SB Easy Probe, Czech Republic). The fiber diameter was analyzed using NIS Elements software (LIM s.r.o., Czech Republic). For fiber diameter analysis, three SEM photos with a magnification of 5000× were measured to obtain 150 measurements of fiber diameter (50 measurements per image).

2.4. Measurement of MMC Loading

A Sciex X500R mass spectrometer coupled with an Exion LC liquid chromatograph (AB Sciex, Framingham, USA) was used to determine MMC in nanofibrous samples. The spectrometric conditions for MMC were optimizedspecifically, the detection mode, collision energy, and declustering potential were adjusted to obtain specific m/z transitions with the highest possible response intensity.

The analysis was performed in multiple reaction monitoring (MRM) mode, which utilizes specific mass transitions from precursor ions to fragment ions under precisely defined conditions. A YMC Triart C18 column (100 mm length; 2.1 mm internal diameter; YMC Triart) was used for separation. The mobile phase consisted of a mixture of 0.1% formic acid (phase A) and pure methanol (phase B), with a flow rate of 0.42 mL/min. A sample volume of 50 μL was injected. The instrument operated in positive ESI ionization mode with an ion source temperature of 500 °C, using the following gas parameters: GAS1 at 40 PSI, GAS2 at 50 PSI, curtain gas at 35 PSI, and CAD gas at 7 PSI. The spray voltage was set to 5500 V for the positive mode. Quantification was performed using MRM HR in positive mode, applying one quantifier and one qualifier MRM transition.

The real samples were prepared by dissolving the nanofibrous material in DMAc. To ensure complete dissolution, the samples were vortexed and subsequently diluted with demineralized water to the desired volume. During this step, PVDF reprecipitation was removed by filtration using PTFE filters. The filtered sample was then transferred into a 2 mL vial and immediately subjected to analysis. To ensure reproducibility and reliability, each sample was analyzed in triplicate.

2.5. Cytotoxicity Evaluation of Mitomycin C

Cytotoxicity of MMC was evaluated according to ISO 10993:5:2009 utilizing a cell line of 3T3 mouse fibroblasts. The cells were cultured in a complete medium composed of Dulbecco’s modified Eagle medium (DMEM, Capricorn Scientific, Germany) with the addition of fetal bovine serum (FBS, 10%, Capricorn Scientific, Germany) and penicillin, streptomycin, and amphotericin B (1%, Capricorn Scientific, Germany). On the first day of testing, 3T3 mouse fibroblasts (ATCC, USA) were seeded into the 96-well plate (104 cells/well). On the second day, the cells were checked by optical microscopy (Nikon, Czech Republic); the medium was removed from the wells and replaced by controls: negative control (NC) composed of complete DMEM, positive control (PC) consisting of complete DMEM + 0.1% cytotoxic Triton X-100, and mitomycin C dissolved in complete medium in concentrations of 0.01; 0.1; 0.3; 0.5; 0.7; 0.9; 1.1; 1.3 and 1.5 μg/mL (n = 8). Such prepared well plate was incubated at 37 °C (5% CO2) for 24 h. On the third day, the cells were microscopically checked, and their viability was measured by colorimetric cck-8 assay (Dojindo, Japan). Medium containing 10% cck-8 was prepared and incubated with the cells for 2 h. Afterward, absorbance was detected. The measured absorbance of negative control was considered 100% viability of cells. The absorbance values of positive control and MMC concentrations were recalculated to the % viability compared to cells in the presence of complete media (negative control). Data were presented in a column graph showing the mean and standard deviation.

2.6. In Vitro Testing of PVDF Nanofibrous Layers with Mitomycin C

Testing of produced layers (PVDF, PVDF + 0.01% MMC, PVDF + 0.1% MMC in acetone) was conducted 3 weeks following material production. After electrospinning, the materials were cut for cytotoxicity and proliferation tests and sterilized by ethylene oxide (Anprolene). Following ethylene oxide sterilization, the materials must be aerated for at least 2 weeks.

Cytotoxicity testing was also performed according to ISO 10993:5:2009 using an extract test. On the first day of testing, cells were seeded in a 96-well plate (104 cells/well). Extracts of tested materials were prepared by soaking sterile materials in complete media at 10 mg/mL. Such prepared test tubes containing tested material and an appropriate amount of media were placed in a shaker (37 °C, 60 rpm). On the second day, the cells were checked by optical microscopy, and the medium was replaced by controls: negative control (NC) composed of complete DMEM, positive control (PC) composed of DMEM + 0.1% Triton X-100, and tested extracts. After 24 h of incubation, the absorbance was measured and recalculated as in the previous experiment.

To assess cell proliferation, materials were prepared in the 24-well plates and weighted with glass rings to prevent material floating during testing. Fibroblast cells were seeded on top of the tested materials at 104 cells/well concentrations. After 7 days of culturing, the materials were assessed by measurement of metabolic activity using cck-8 (n ≥ 3), fluorescence staining of cells by phalloidin-FITC and DAPI (n = 1), and scanning electron microscopy (n = 1).

3. Results

3.1. Electrospinning of PVDF with MMC Dissolved in Water

Electrospinning of the control PVDF solution and the MMC-modified PVDF solutions was carried out on a needleless Nanospider device without any obstacles. The addition of active substances did not affect the productivity of electrospinning.

Nanofibers made from pure PVDF possessed homogeneous nanofibers with occasional beaded structures (Figures A and A). When MMC dissolved in water was added to the electrospinning solution, the number of beads increased significantly, as seen in Figure B, C. Nanofibrous PVDF material possesses a mean fiber diameter of 195 ± 77 nm. Adding water containing MMC slightly reduces fiber diameter to 159 ± 60 nm (PVDF + 0.01% MMC) and 175 ± 81 nm (PVDF + 0.1% MMC).

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Scanning electron microscopy photos of electrospun PVDF (A), PVDF + 0.01% MMC in water (B), PVDF + 0.1% MMC in water (C), scale bar 20 μm.

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Scanning electron microscopy photos of electrospun PVDF (A), PVDF + 0.01% MMC in acetone (B), PVDF + 0.1% MMC in acetone (C), scale bar 20 μm.

3.2. Electrospinning of PVDF with MMC Dissolved in Acetone

With the addition of acetone containing MMC, the electrospinning process also did not show any differences compared to the electrospinning of the control PVDF layer. When MMC was dissolved in acetone, the nanofibrous layer was composed of fibers having a mean fiber diameter similar to the pure PVDF layer (187 ± 49 nm for PVDF + 0.01% MMC, 192 ± 53 nm for PVDF + 0.1% MMC). The presence of beads was higher than in the pure PVDF layer but lower than after the dissolution of MMC in water, as depicted in Figure A–C.

Characterization of the layers in terms of their thickness, area weight, and fiber diameter is reported in Table . The electrospinning process of PVDF solution under the conditions listed in Table led to the fabrication of a layer with a thickness of 104 ± 6 μm and an area weight of 26.7 g/m2. The layers containing MMC reached similar values of thickness (±10 μm) and area weight (±4 g/m2) except for the materials PVDF + 0.1MMC_W with a lower area weight of 19.2 g/m2.

2. Structural Features of Produced Materials (PVDF, PVDF + MMC_W Refers to Mitomycin Dissolved in Water, PVDF + MMC_A Relates to the Materials Fabricated after Dissolution of MMC in Acetone).

  PVDF PVDF + 0.01MMC_W PVDF + 0.1MMC_W PVDF + 0.01MMC_A PVDF + 0.1MMC_A
the thickness of the layer [μm] mean ± SD 104 ± 6 107 ± 5 98 ± 7 109 ± 3 92 ± 3
area weight [g/m2] 26.7 25.8 19.2 30.0 24.7
fiber diameter [nm] mean ± SD 195 ± 77 159 ± 60 175 ± 81 187 ± 49 192 ± 53
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3.3. Quality Control

The LC/HRMS (liquid chromatography/high resolution mass spectrometry) method was partially validated to verify suitability. The linearity, limit of detection (LOD), and limit of quantification (LOQ) were determined for MMC. The LOD and LOQ were based on signal-to-noise ratios of 1:3 and 1:10, respectively. The specific values of the analytical parameters monitored are given in Table . Emphasis was also placed on monitoring the stability of MMC in aqueous solution. Repeated analysis of the standard MMC solution found that during storage at 4 °C, there was a 37.50% decrease in MMC concentration within 3 days. The key was to verify the method’s accuracy by using the recovery. The recovery was carried out at three concentration levels and in triplicate. Pure PVDF fibers were spiked with standard MMC solution and processed as a real sample. After the measurements, the recovery of the whole process was calculated, which was 80.08 ± 1.31%, within the desired 80–120% range. Blank samples consisting of pure solvent were continuously measured at the beginning of the measurement to avoid interferences caused by the chromatographic system.

3. Analytical Parameters for the Quantification of MMC (Limit of Detection LOD, Limit of Quantification LOQ).

compound linear range (ng/L) correlation coefficient LOD (ng/L) LOQ (ng/L)
mitomycin C 50–10,000 0.9998 15 50
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3.4. Loading Efficiency and Stability of MMC in PVDF Nanofibers

Mitomycin C was dissolved using two solvent systems to evaluate its incorporation into electrospun PVDF nanofibers. In the first approach, MMC was dissolved in water and added to the electrospinning solution at concentrations of 0.01% and 0.1%. However, this method resulted in very low drug-loading efficiency. As shown in Figure A, the MMC content measured 1 day after layer fabrication reached only 0.34% and 0.35% for PVDF + 0.01% MMC and PVDF + 0.1% MMC, respectively, relative to the initial amount added. Additionally, MMC proved unstable within the fibers, with further reductions in concentration observed after 7 days of storage.

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Concentration of mitomycin C within the fabricated materials PVDF + MMC dissolved in water (A), PVDF + MMC dissolved in acetone (B) measured by liquid chromatography/high resolution mass spectrometry after 1, 7, and 21 days of storage, n = 3. Individual measurements with marked medians are depicted.

MMC was subsequently dissolved in acetone, a more suitable solvent for compatibility with the PVDF electrospinning solution to improve incorporation. The same concentrations of 0.01% and 0.1% MMC were used. As shown in Figure B, this approach resulted in significantly higher drug-loading efficiencies. One day after fabrication, the PVDF + 0.01% MMC layer reached a loading efficiency of 50.66%, while PVDF + 0.1% MMC achieved 28.63%. Nevertheless, a progressive decline in MMC content was observed during storage at 4 °C. For PVDF + 0.01% MMC, the MMC content decreased by 42% after 7 days and by 74% after 21 days, relative to the initial amount. Similarly, PVDF + 0.1% MMC exhibited a 26% reduction after 7 days and a 42% reduction after 21 days. These results highlight both the importance of solvent selection and the challenges of maintaining long-term stability of MMC within PVDF nanofibrous matrices.

3.5. Cytotoxicity of MMC

The concentration range between 0.01 and 1.5 μg/mL MMC in a complete medium was tested to quantify the impact of MMC concentrations on fibroblast cell lines. The cytotoxicity test results are depicted in Figure , where the red line symbolizes cytotoxic values (70% viability) given by the norm. Concentrations up to 0.3 μg/mL were nontoxic; cell viability was above 70%. Concentrations of 0.5 μg/mL and higher were cytotoxic for the fibroblast cell line, and cell viability decreased with an increase in MMC concentration.

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Cytotoxicity test of MMC in complete medium measured by cck-8 assay (n = 8).

3.6. In Vitro Testing: Cytotoxicity and Proliferation of Fibroblast Cell Line on Produced Materials

Prepared nanofibrous layers, PVDF + MMC dissolved in acetone, were tested for their cytotoxicity and fibroblast proliferation upon 7 days of culturing. Due to the ethylene oxide sterilization, the tests were conducted 3 weeks after production. Therefore, the relevant amount of MMC is according to the data in Figure B after 21 days. Extracts of the materials were tested with fibroblast cells after 24 h incubation. The results shown in Figure revealed no cytotoxic behavior of the materials.

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Cytotoxicity assessment of material extracts (concentration of 10 mg/mL of complete media) after 24 h of incubation measured by cck-8 assay (n = 8).

Since the materials did not exhibit cytotoxic behavior, the proliferation of the cells on the material surface was conducted. After 7 days of culturing, metabolic activity was measured by metabolic cck-8 assay. Moreover, cells were captured using fluorescence microscopy and scanning electron microscopy. Fibroblast metabolic activity is depicted in the graph in Figure . Cells cultured on pure PVDF reached the highest absorbance values (3.25 ± 0.85). Incorporation of MMC into the layers led to decreased cell viability; the measured absorbance of cells on PVDF + 0.01% MMC was 1.41 ± 0.57 and on PVDF + 0.1% MMC, 1.27 ± 0.31.

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Metabolic activity of fibroblasts cultured for 7 days on the surface of nanofibrous materials measured by cck-8, n ≥ 3.

Fluorescence microscopical pictures following the staining of actin filaments of the cells in green and cell nuclei in blue are depicted in Figure . An almost confluent layer of fibroblast cells was found on nanofibrous PVDF. Occasional cell clusters were found on MMC layers in both concentrations (0.01% and 0.1%).

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Fluorescence microscopy pictures of phalloidin-FITC and DAPI stained fibroblasts on PVDF (A), PVDF + 0.01% MMC (B), and PVDF + 0.1% MMC (C), scale bar 50 μm.

Similar findings were confirmed by scanning electron microscopy. Cell clusters were found on MMC-enriched materials, while spread cells covering the material’s surface were found in the pure PVDF layer, as seen in Figure .

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Scanning electron microscopy pictures of fibroblasts after 7 days of culturing on nanofibrous PVDF (A D), PVDF + 0.01% MMC (B,E), PVDF + 0.1% MMC (C,F). The first row shows photos with smaller magnifications (1000×, scale bar 50 μm), the second row shows pictures with higher magnifications of 5000×, scale bar 10 μm.

4. Discussion

Due to its favorable physicochemical and mechanical properties, polyvinylidene fluoride is gaining increasing attention across various fields, including biomedical applications. − In this study, we present the fabrication of a material intended for use in glaucoma drainage devices based on electrospun PVDF with mitomycin C incorporation to suppress fibroblast proliferation. PVDF can be electrospun from a range of solvents such as dimethylformamide or dimethylsulfoxide, usually with the addition of a volatile component such as acetone. The choice of solvent system significantly affects the resulting fiber morphology. , In our approach, PVDF was dissolved in a solvent mixture of dimethylacetamide and acetone in a ratio of 4:1, enabling a uniform fibrous mat formation with a mean fiber diameter of 195 ± 77 nm.

The addition of MMC did not significantly alter the electrospinning process, as indicated by the structural features of the resulting nanofibrous layers shown in Table . However, two adverse effects were observed when MMC was dissolved in water. First, the fiber morphology was affected, with the appearance of beaded structures within the layers, as illustrated in Figure B,C. Bead formation during electrospinning generally depends on several factors, including the composition of the electrospinning solution, processing parameters, and ambient conditions. , In the present context, the composition of the electrospinning solution is considered as the principal determinant of bead formation. While occasional bead formation during pure PVDF electrospinning is a recognized occurrence, extensively studied by Trupp et al., the introduction of water-dissolved MMC into the PVDF solution (comprising DMAc and acetone as solvents) can induce immiscibility issues. These issues lead to localized alterations in solution properties, notably viscosity and surface tension. During the electrospinning process, such inhomogeneities generate fluctuations within the polymeric jets, which subsequently manifest as bead formation. This observation is corroborated by Sarma et al., who theoretically predicted and experimentally established that the electrospinning of pure PVDF from various solvent systems results in similar material characteristics. Their comprehensive research concluded that heightened inhomogeneity within the electrospinning solution instigates instabilities in jet formation, culminating in bead formation within the final material.

Second, and more importantly, the drug loading was markedly low, with values of only 0.34% and 0.35% of the initial MMC content in the electrospinning solution. This outcome can be attributed to thermodynamic incompatibility between the components of the electrospinning solution. It is possible to quantify the Flory–Huggins interaction parameter to predict system compatibility, an approach frequently employed in the literature for investigating nanofibrous drug delivery systems. , This theory can also be useful for predicting phase separation. The solvents typically used for PVDF electrospinning are hydrophobic, whereas water, used to dissolve MMC, is hydrophilic. This significant disparity in polarity results in a pronounced tendency toward phase separation, which was visually observed during solution preparation. Even though the precipitate appeared to dissolve before electrospinning, the incorporation of MMC into the fibers remained minimal. Furthermore, the difference in solvent volatility also plays a roleacetone evaporates much more rapidly than water, which influences fiber formation and can contribute further to the low drug loading. However, thermodynamic incompatibility leading to phase separation is considered the leading cause of the low MMC loading.

As a second approach, MMC was dissolved in acetone, a polar aprotic solvent less polar than water but capable of dissolving small organic molecules. Upon dissolution of MMC in acetone and subsequent addition to the electrospinning solution, no precipitation was observed, indicating improved compatibility with the PVDF solvent system. From a thermodynamic perspective, the homogeneity and compatibility of the electrospinning solution’s components were significantly improved. This enhancement is attributed to acetone’s ability to dissolve MMC and its complete miscibility with DMAc, thereby promoting a more uniform and stable solution. Following electrospinning, the morphology of the resulting nanofibers remained consistent with control samples, showing no significant structural alterations. Importantly, drug loading efficiency was markedly improved compared to the water-based approach. In the case of PVDF with 0.01% MMC, the drug loading reached 50.66% of the initial MMC content 1 day after fabrication. MMC instability and loss of MMC due to the fast evaporation rate of acetone during electrospinning are considered the causes of the achieved drug loading efficiency. When a higher concentration of MMC (0.1%) was used, the drug loading decreased to 28.63%. This suggests that drug retention does not scale linearly with initial concentration and may be affected by the limited solubility of MMC in acetone, which is about ≤1000 ppm. Similar findings were found in a study by Böncü et al., who electrospun hydrophobic polymer polylactic acid with hydrophilic active substance ampicillin trihydrate. A higher amount of incorporated drug leads to decreased drug loading efficiency.

The stability of MMC within the electrospun layers was another key aspect investigated in this study. To assess the preservation of MMC content over time, PVDF-based nanofibrous layers containing MMC dissolved in acetone (PVDF + MMC_A) were analyzed 1, 7, and 21 days after fabrication, with samples stored at 4 °C. A significant decline in MMC content was observed over the storage period, as shown in Figure B. These findings indicate that although incorporating MMC into PVDF fibers using acetone as a solvent enhances initial drug loading, long-term stability remains challenging. Assessing drug loading is essential for evaluating a drug delivery system; however, drug stability during storage is rarely investigated. Our results highlight the importance of including such analyses in future studies.

Although a decrease in MMC content was observed during storage, the electrospun layers were tested after 21 days to evaluate the impact of MMC on fibroblast proliferation. Initial tests confirmed that MMC concentrations above 0.5 μg/mL are cytotoxic to fibroblast cell lines. Chen et al. investigated the cytotoxic effect of MMC on nondermal fibroblasts, demonstrating a time- and concentration-dependent effect. They tested MMC at 0.4, 4, and 40 mg/mL over 7 days, observing a visible decrease in viability with all concentrations over time. Notably, the lowest concentration of 0.4 mg/mL showed results similar to our study: fibroblast viability after 1 day was comparable to controls, with no apparent cytotoxic effect. However, Chen et al.’s study reported a complete loss of viability (0%) after longer exposure of 7 days.

The material extracts did not exhibit any cytotoxic effects, as shown in Figure . Furthermore, fibroblast proliferation on the materials was assessed. The results in Figures – demonstrated delayed fibroblast proliferation on MMC-enriched nanofibrous layers compared to electrospun PVDF alone. These findings confirm the desired antiproliferative properties of the developed materials. Similar outcomes have been reported following the incorporation of MMC into hydrogels.

Future work will optimize the solvent system to enhance MMC solubility and uniform dispersion within the polymer solution. In addition, blending PVDF with poly­(ethylene oxide) (PEO) will be explored to further improve drug retention and leverage the synergistic antifibrotic effects of PEO and MMC. This approach may offer improved biocompatibility, enhanced drug incorporation, and more significant inhibition of fibroblast proliferation, making it a promising direction for developing next-generation antifibrotic materials.

While this study demonstrates the feasibility of incorporating MMC into electrospun PVDF nanofibers and its antifibrotic potential, several limitations should be acknowledged. First, the stability of MMC within the nanofibrous matrix was evaluated only under one storage condition (4 °C) and for a limited duration of 21 days; longer-term studies and evaluation under different environmental conditions are needed to fully understand the degradation kinetics. Second, the drug release profile was not assessed, which is critical for predicting in vivo performance and therapeutic efficacy. Moreover, the biological testing was limited to in vitro fibroblast proliferation assays using a mouse cell line; thus, further validation using other cell lines is necessary to confirm the clinical relevance. Lastly, the study focused solely on PVDF as the polymer matrix and did not explore whether alternative or blended polymers could enhance MMC stability or release characteristics.

5. Conclusion

This study focused on developing nanofibrous materials incorporating MMC as an active antifibrotic agent for potential biomedical applications. The use of acetone as a solvent for MMC proved to be more effective than water, resulting in significantly higher drug loading and improved compatibility with the PVDF electrospinning solution. Despite this advantage, the stability of MMC over time remained a challenge, with a notable decrease in drug content observed after 21 days of storage at 4 °C. The biological performance of the MMC-loaded materials was evaluated using mouse fibroblasts, where a reduction in proliferation rate was observed compared to nanofibrous layers composed of pure PVDF. These results demonstrate the potential of PVDF/MMC nanofibers for antifibrotic applications, highlighting the need for further optimization to improve long-term drug stability.

Acknowledgments

The study was supported by the project of Ministry of Health of the Czech Republic (NU23-08-00586: Antifibrotic fibrous material for reducing of intraocular pressure in glaucoma disease). The authors acknowledge the assistance the Research Infrastructure NanoEnviCz provided, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2023066.

Glossary

Abbreviations

PVDF

polyvinylidene fluoride

MMC

mitomycin C

PEO

poly­(ethylene oxide)

DMAc

dimethylacetamide

SEM

scanning electron microscopy

MRM

multiple reaction monitoring

DMEM

Dulbecco’s modified Eagle medium

FBS

fetal bovine serum

LOD

limit of detection

LOQ

limit of quantification

LC/HRMS

liquid chromatography/high resolution mass spectrometry.

The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.

The study was supported by the project of Ministry of Health of the Czech Republic (NU23-08-00586: Antifibrotic fibrous material for reducing of intraocular pressure in glaucoma disease). The authors acknowledge the assistance the Research Infrastructure NanoEnviCz provided, supported by the Ministry of Education, Youth and Sports of the Czech Republic under Project No. LM2023066.

The authors declare no competing financial interest.

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