Polymer mixtures contain at least one component besides the polymer, such as nanoparticles, inorganic salts, or other polymers. These types of polymer mixtures are used for medical purposes; for example, metronidazole/poly(ε-caprolactone) (PCL)/alginate for dental implants or poly(lactic acid)/hydroxyapatite in orthopedics . Biocompatible polymers are widely used in biomedical fields, such as stents, drug delivery systems in cancer therapy, bone repair, dentistry, joint prostheses, and tissue engineering . Polymers have several advantageous properties for these applications as they are flexible, variable in weight, have excellent biocompatibility, are resistant to biochemical degradation, and have suitable physical and mechanical characteristics. Moreover, they can be formed to the desired shape . It is also worth mentioning that different drugs or other additives can be easily linked to the polymer or distributed to them in the same solvent; thus, a multicomponent composite system can be generated. Based on the parameters mentioned above, wound-dressing application is one of the most relevant research areas for these systems .

An effective wound dressing should fulfill multiple needs: shielding the wound against bacterial infection, facilitating proper gas exchange, providing an environment that promotes healing, and controlling biofluid production . Furthermore, it should be nontoxic and hypoallergenic .

By using electrospinning, nanofibers can be created and used in numerous biomedical applications, such as tissue engineering, wound dressing, and drug delivery . Electrospinning has many advantages: it is a simple technique, cost-effective, reproducible, scalable, and reliable. In addition, various polymers can be used as starting material, and fibers can be produced with uniform diameters in a controlled way . Electrospun fibers are similar to macromolecule networks (e.g., collagen, fibrinogen, elastin) around the cells, which are called extracellular matrix (ECM). The ECM has fiber diameters in the size range of 50–500 nm and has several functions in the human body, including wound healing . Nanofibers produced by electrospinning have beneficial structural attributes, such as elevated porosity, high specific surface area, and nanoscale fiber dimensions; thus, adequately mimicking the ECM and promoting cellular adhesion, growth, proliferation, and differentiation. The electrospinning technique offers the possibility of using the formed scaffold as a wound dressing with fibers of proper size and morphology. The porous nature of the scaffold enables the drainage of wound fluids and facilitates the entry of oxygen from the atmosphere .

To achieve an antibacterial effect, one option is the introduction of salts to the polymer solution. The addition of salts to the electrospinning solution leads to changes in the conductivity, viscosity, shearing strength and morphology, and fiber diameter of the prepared scaffolds . The antibacterial effectiveness of the fibrous structure is significantly influenced by incorporating salts or nanoparticles. When electrospun fibers are combined with inorganic nanoparticles , they can become resistant to bacteria. However, their ability to enhance antibacterial properties is limited due to the encapsulation of certain nanoparticles within the fibers . One of the most common approaches is the introduction of antibiotics. However, with the misuse and/or overuse of these types of drugs, there is the risk of antibiotic resistance (AMR), which is one of the top-ten global public health threats, according to the World Health Organization . A 2022 cross-sectional study in Hungary revealed that multidrug-resistant (MDR) bacterial infections caused extended hospital stays and increased patient mortality . One option to solve this problem could be to apply silver ions, which are frequently used in clinical practice. However, they can cause a large environmental burden and, in some cases, allergies . Since urgent actions are needed to stop AMR and avoid the negative environmental impact, developing other agents and treatment strategies to fight bacterial pathogens with similar effects to antibiotics and silver is essential. In our study, inorganic salts, namely Zn(O2CCH3)2 and Sr(NO3)2, were added to the polymer fibers. These salts possess antibacterial properties and stimulate cell proliferation as well as tissue regeneration .

Based on the special requirements to fulfill as a wound dressing material, such as biocompatibility, biodegradability, good gas permeability, and water retention capacity, polysuccinimide (PSI) was used for the preparation of electrostatic fibers. Polysuccinimide is a nontoxic , biocompatible, and biodegradable polymer. Based on the literature, it is extensively researched for biomedical applications . It is also known that PSI turns into polyaspartic acid (PASP) at physiological conditions, which is a degradable biomaterial .

This study aimed to create a two-component polymer-composite scaffold as a potential wound dressing material by electrospinning, using antibacterial salts (Zn(O2CCH3)2 or Sr(NO3)2) in addition to PSI. We performed the following experiments: physical and chemical characterization of the fibers by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), mechanical tests, investigation of salt dissolution from the scaffolds, examination of their antibacterial activity against four different bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis), and finally cytotoxicity assays on two human cell types (i.e., MG-63 tumor cell line and 155BR fibroblasts).

N,N-Dimethylformamide (DMF ≥99.5%, Alfa Aesar, USA); dimethylsulfoxide (DMSO ≥99.9%, Fisher Chemical, USA); zinc acetate (99,99%, Sigma-Aldrich, USA); strontium nitrate (Reanal, Hungary); ʟ-aspartic acid (99%, Amresco, USA); phosphoric acid (≥99.0%, Sigma-Aldrich, USA); E. coli (ATCC 25922); P. aeruginosa (ATCC 27853); B. subtilis (ATCC 6633); S. epidermidis (ATCC 14990); Mueller–Hinton agar (Biolab Zrt., Hungary); minimum essential medium (MEM, Gibco, USA); Dulbecco's modified Eagle medium (DMEM, Gibco, USA); MEM, no glutamine, no phenol red (Gibco, USA); phosphate-buffered saline (Gibco, USA); fetal bovine serum (FBS, Gibco, USA); penicillin/streptomycin mix (Gibco, USA); ʟ-glutamine (Gibco, USA); non-essential amino acid solution (NEAA, Gibco, USA); trypsin/EDTA solution (Sigma-Aldrich, USA); cell proliferation reagent WST-1 (Roche, Switzerland); and ultra-purified water (Zineer Power I Water Purification System). All reagents were used without any further purification.

The PSI was produced by thermal polycondensation in the presence of a phosphoric acid catalyst . Phosphoric acid (20 g) and ʟ-aspartic acid (20 g) were mixed in a round-bottom flask and placed into a rotatory vacuum evaporator (RV10, digital rotary evaporator, IKA, Germany) with a rotation speed of 130 rpm. The temperature was gradually changed from room temperature to 180 °C, and the pressure was gradually reduced to 3 mbar. The synthesis lasted 8 h. The DMF was added to the synthesized polymer, stirred at 80 rpm, and left to dissolve for 60 min. The solution was dripped into distilled water, precipitating the PSI as a pellet. The pellet was filtered and washed using a vacuum filter to remove the phosphoric acid catalyst and the unreacted ʟ-aspartic acid monomeric molecules. Then, the pellet was mixed in distilled water for 10 min. This step was repeated until the pH of the filtrate changed from acidic to neutral. Finally, the PSI powder was dried at 40 °C and stored at room temperature. Based on our previous publications, the molecular weight of the polymer is 28 500 ± 3000 Da .

Table 1: The components of the polymer/salt solutions and the applied voltages and flow rates at the electrospinning in the case of the PSI and different PSI + salt samples. The numbers before the polymer and the salts mean % (w/w). All experiments were performed at room temperature.

The presence of inorganic salts in the scaffolds was investigated by using infrared spectroscopy. For this purpose, a JASCO FT/IR-4700 spectroscope with a diamond ATR head was applied. In all cases, the measurements were taken between 400 and 4000 cm−1 wave numbers with a 2 cm−1 resolution. A deuterated triglycine sulfate (DTGS) detector was used, and 256 scans were accumulated for each sample. The background spectra (H2O, CO2, ATR head exclusion) were obtained on a diamond crystal and were subtracted from the sample spectra. The peaks of the spectra were determined with the SpectraGryph software, and the graph was made using the Origin 2018 program (OriginLab, USA).

To prove the dissolution of inorganic salts from the scaffolds, the conductivity of the dissolution medium was measured. Stock solutions (50 mL, 5% w/v) were prepared from Sr(NO3)2 and Zn(O2CCH3)2 salts with distilled water. Then, by dilution, a concentration series was made for the calibration: 2.5, 1, 0.5, 0.2, 0.1, 0.05, and 0.01% (w/v). The conductivity of these solutions with different salt contents was determined using a conductivity meter and the belonging sensor (SevenCompact Duo S213 Benchtop pH/mV/Conductivity Meter; Cond Sensor InLab® 710, Mettler Toledo, USA). The salt-containing scaffolds were wrapped into silk paper and dropped into 20 mL of distilled water to investigate how the salts can be dissolved from them. The solutions were stirred at 100 rpm such that the salt content could be properly distributed in distilled water. The samples for the measurements were taken from the media outside the wrapped scaffolds. The experiment was performed in 8 h: in the first hour conductivity values were measured every 10 min, then the measurements were taken every hour after that.

Scanning electron microscopy images were taken to determine the fiber diameters of salt-containing polymer scaffolds. They were made at the Budapest University of Technology and Economics, Department of Polymer Technology, applying a high-performance scanning electron microscope (JSM-6380LV, JEOL, Japan), with a resolution of 3.0 nm. Before the measurements, all the samples were fixed on a tape and coated with gold using a JFC-1200 Sputter Coating System (JEOL, Japan). Images of the samples were taken at 1000×, 5000×, and 10000× magnification, applying a 10 kV accelerating voltage. The fiber diameters of the scaffolds were determined by the Fiji ImageJ software (open-source software). In each case, 50 fiber diameters were measured. The results were statistically analyzed by the software GraphPad Prism 8.0.1 (GraphPad Inc., USA), including the normality tests regarding the distribution of fiber diameters (Shapiro–Wilk normality test) and the significant difference between different experimental groups (Kruskal–Wallis test). The fiber diameter distributions were plotted in the Origin 2018 program (OriginLab, USA). To complete the FTIR analysis, we used an energy-dispersive X-ray spectroscopy (EDX) detector during the SEM measurements to show the presence of salts in the fibers. The EDX spectra were taken at a voltage of 15 kV.

From the elongation values and the length of the polymer scaffold captured at the beginning of the experiments, the percentage of elongation was determined using the following formula:

The cross-sectional area of the scaffolds was calculated from the product of the thickness and the width. The thickness of the scaffolds was measured by a digital micrometer (IP65, Mitutoyo, Japan). The Young’s modulus was calculated from the linear part of the stress–strain curve . The slope of the linear function gives the Young’s modulus.

An osteosarcoma cell line (MG-63, ECACC 86051601) and skin fibroblasts (155BR, ECACC 90011809), both of human origin, were cultured as monolayers at 37 °C and 5% CO2 in a humidified atmosphere, and subcultivated at a subconfluent state by using trypsin/EDTA (0.05%). The medium used for the MG-63 cell line was composed of MEM supplemented with 10% of fetal bovine serum, 1% of non-essential amino acids, 2 mM of ʟ-glutamine, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin. The medium for the 155BR cells contained 15% of fetal bovine serum, 1% of non-essential amino acids, 2 mM of ʟ-glutamine, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin in DMEM.

Indirect cytotoxicity tests were based on a previous publications of our research group . The tests were performed first on tumor cells to optimize the experimental conditions and then on fibroblasts to investigate if the scaffolds have a cytotoxic effect. The discs with diameters of 16 mm (m = 0.5 ± 0.1 mg) were sterilized under UV light for 1 h. After that, they were incubated in cell culture medium for 24 h (37 °C and 5% CO2). The mass of the discs varied, so the applied volume of culture medium was proportionally adjusted (0.1 mg of scaffold in 1 mL of medium). The control medium containing the same salt concentration as the scaffolds were sterile filtered by 0.2 µm syringe filters (Sarstedt AG & Co. KG, Germany).

Table 2: Names of the samples used in the cytotoxicity test and their treatments.

After one day of incubation, the cells were investigated under a phase-contrast microscope (Nikon Eclipse TS100, Nikon, Japan), and photomicrographs were taken using a CCD camera (COHU, USA) to track any changes in cell morphology. Then, 100 µL of old medium was replaced in each well with 200 µL of media: positive or negative control, extracts from the scaffolds, or media with the same salt concentrations.

After 24 and 72 h treatments, phase-contrast microscopy images were taken. The relative cell viability was determined using the WST-1 reagent (diluted in medium without phenol red (colorless) at 1:20). A volume of 200 µL of old medium was removed from each well and 200 µL of phosphate-buffered saline was added to eliminate the residual media as well as cells that were either loosely attached or did not attach to the plate. Then, 100 µL of diluted WST-1 reagent was added to the cells and incubated for 4 h. The absorbance values of the samples were measured in a microplate reader (model 3550, Bio-Rad Laboratories, Japan) at 450 nm. A reference wavelength of 655 nm was also used. The statistical analysis of the relative cell viability values was done using one-way ANOVA in the GraphPad Prism 8.0.1 software (GraphPad Inc., USA).

To create a proper wound dressing material containing inorganic salt for potential antibacterial purposes, the first step was the synthesis of the polymer, the characterization of the solution, and the final scaffold with different techniques.

Figure 1: A) The synthesized PSI powder, 25 PSI solution, 20 PSI + salt solutions, and fibrous scaffolds were made using the electrospinning technique. B) FTIR spectra of the Sr(NO3)2 salt, scaffolds containing this salt, and 25 PSI scaffold. C) FTIR spectra of the Zn(O2CCH3)2 salt, scaffolds containing this salt, and 25 PSI scaffold.

Table 3: The ratio of dissolution (in percentage) regarding Sr(NO3)2 and Zn(O2CCH3)2 salts from salt-containing PSI scaffolds after 1, 3, 5, and 8 h.

Figure 2: A) Fiber diameter distribution of PSI and PSI + salt scaffolds and B) SEM images at a lower (1000×) and a higher (5000×) magnification. The Gaussian lines on the histograms are guides to the eye.

Table 4: Mechanical parameters of the PSI and PSI + salt scaffolds.