Enabling low molecular weight electrospinning through binary solutions of polymer blends

Research article Enabling low molecular weight electrospinning through binary solutions of polymer blends Victor M. Perez-Puyana a , Alberto Romero a , Antonio Guerrero a , Lorenzo Moroni b , Paul A. Wieringa b , * a Departamento de Ingeniería Química, Universidad de Sevilla, Facultad de Química, Sevilla 41012, Spain b Department of Complex Tissue Regeneration, MERLN Institute for Technology-Inspired Regenerative Medicine, Maastricht University, Maastricht 6200 MD, the Netherlands ARTICLE INFO Keywords: PCL Gelatin Electrospinning Binary solution, Nanofibers ABSTRACT The formation of nanofibrous membranes via electrospinning is typically restricted to high molecular weight polymers in an appropriate solvent, correlated with the necessary formation of polymer chain entanglements that are needed to achieve successful production of electrospun fibers. The present work extends the electrospinning of low molecular weight polymers by investigating the electrospinning of a binary solution system consisting of two different low molecular weight polymers, using as a model system polycaprolactone (PCL) and gelatin in different ratios. The viscosities of the polymer solutions were characterized as a proxy for polymer chain entanglement and the resulting fibers were morphologically characterized by SEM imaging and further assessed water contact angle and molecular composition to determine the impact and homogeneity of the binary mixtures. We found that unitary solutions of either PCL or gelatin failed to generate proper fibers despite indications of chain entanglement. In contrast, binary solutions of low molecular weight PCL and gelatin generated different fiber quality and size distributions, depending on the ratio used, with direct correlations between fiber properties and the PCL:Gelatin ratio. It was discovered that the ratio of PCL to gelation was most predictive for successful fiber generation, with effective electrospinning occurring only for a define intermediate range of high blend ratios while both low and high blended binary solutions resulted in poor fiber production. Our study confirmed that this behavior was independent from absolute polymer concentration, indicating a unique interaction between these binary species which exists only under specific ratio concentrations and indicates promising new avenues to process low molecular weight polymers solutions. 1. Introduction The electrospinning technique is a process that can produce nonwoven fibrous membranes comprised of fibers with diameters ranging from nanometers to micrometers [1]. Thus, these membranes present interesting properties, such as moderate porosity and high specific area. During electrospinning, a polymeric solution is pumped through a metallic spinneret and a high voltage is applied between the spinneret and a surface, called collector, that is held a fixed distance way. The resulting electric field electrostatically extrudes the polymer solution from the spinneret, forming a jet of polymer solution that travels from the spinneret to the collector. Under optimum conditions, the jet undergoes high speed chaotic whipping which increases evaporation rate of the solvent, forming solid fibers that are eventually deposited onto the collector to create a fibrous membrane [2]. The fiber formation and the properties of the membranes depend on the correct evolution of the polymer jet during the electrospinning process. Fiber formation is an interplay between processing parameters, environmental conditions and polymer solution properties. Processing parameters can be adjusted to ensure stable jet formation and to tune the fiber diameter [3]. In this sense, several authors have evaluated the influence of the different processing parameters (voltage, humidity, working distance, etc.) on the electrospinning process [4–7]. Environmental conditions have been studied in terms of their influence on the evaporation rate of the solvent and the subsequent effects on fiber formation [8,9]. But polymer solution is a critical aspect in terms of surface tension, conductivity or viscosity. The latter is used as an indication of the degree of chain entanglement. If chain entanglement is insufficient, * Corresponding author. E-mail address: [email protected] (P.A. Wieringa). Contents lists available at ScienceDirect Next Materials journal homepage: www.sciencedirect.com/journal/next-materials https://doi.org/10.1016/j.nxmate.2024.100306 Received 1 May 2024; Received in revised form 18 June 2024; Accepted 4 July 2024 Next Materials 6 (2025) 100306 Available online 30 July 2024 2949-8228/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC license ( http://creativecommons.org/licenses/bync/4.0/ ). the jet cannot be continuously maintained and the process transitions from electrospinning to electrospraying where the jet breaks up into droplets that quickly disperse as a result of columbic forces and deposit onto the collector as polymer microspheres [10]. As described by Colby et al. [11], a polymer solution falls into “four different concentration regimes: dilute, semidilute unentangled, semidilute entangled, and concentrated”. For electrospinning, it is the boundary between the semidilute unentangled and semidilute entangled regimes, commonly known as the entanglement concentration (C e ), which is of most interest; this is the point that the number of polymer chains in solution is sufficiently high to constrain each other, thus producing entanglement couplings [12] that resist the breakup of the evolving polymer jet and promote consistent fiber formation [13]. Although the entanglement concentration differs depending on the working system, McKee et al. found that a concentration between 2 and 2.5 times the entanglement concentration is necessary to achieve defect-free nanofibrous mats [14]. This has been a generally accepted guideline and is followed by the electrospinning-research community. The C e of a polymer/solvent system is often determined by measuring the solution viscosity, since this depends on the intermolecular interactions in these polymer solutions and can serve as a proxy for the degree of chain entanglement [15]. The solvent system used has a great influence on viscosity, due to their effect on the dissolution and homogenization of the polymer in solution [16]. Similarly, the polymer molecular weight and polymer concentration in solution have a significant effect on chain entanglement within the solution, with an increase in molecular weight or concentration causing an increase in viscosity [17]. Therefore, the limiting concentration from which homogeneous fibers are obtained may differ depending on the polymer or the solvent used. In the current study, we investigated a binary polymer system in a single solvent, under the premise that intermolecular interactions of two distinct polymer types in solution can be optimized and correlated to an improved electrospinning process compared to respective unitary polymer solutions. From a wide variety of polymers currently available for electrospinning, we selected a binary system of PCL and Gelatin as two distinct polymer types of synthetic and natural origin, respectively, with good solubility in similar solvents and with reported applications in tissue engineering applications. Gelatin, in particular, has been used extensively in the electrospinning of tissue engineered fibrous constructs as a natural protein polymer that has high biocompatibility compared to synthetic polymers [18–20]. However, the electrospinning of gelatin alone is difficult to carry out properly and it often requires the use of organic solvents to achieve appropriate solution viscosity and the use of a blended solution of synthetic and natural polymers to achieve homogeneous fibers [21–23]. Polycaprolactone (PCL) is similarly a common synthetic polymer for electrospun meshes due to its suitable mechanical properties and easy processability [24], though molecular weight and polymer concentration remain critical for nanofiber formation. Consistent electrospinning of PCL has been reported when using an average molecular weight of 45,000 or 80,000 and concentrations ranging from 5 to 20 wt% [18,25–28] while no successful reports could be found of electrospinning low molecular weight polycaprolactone (LW-PCL, M w =14,000 g/mol). Poor electrospinning performance is typically indicated by the formation of beads, which is attributed to the low surface tension of the polymer solution or low molecular weight of the dissolved polymer, which prevents a continuous polymer jet during the electrospinning process. For low molecular weights, in particular, it is suggested that small polymers chains make it difficult to achieve sufficient chain entanglement for fiber formation even at high concentrations in solution. Instead of fibers, polymer solutions of this type often result in the consistent production of electrosprayed microspheres, where LW-PCL are reported to produce microspheres over a range of concentrations up to 30 % w/v (21.4 mM) [29]. Our main hypothesis is that gelatin and LW-PCL, two polymers which cannot be successfully electrospun alone, can be combined to create a binary polymer solution with optimized chain entanglement and, thus, successful electrospinning. This would lead to nanofiber production using lower cost polymers of lower molecular weight. The viscosity of polymer solutions with varying concentrations of LW-PCL and gelatin was measured and the electrospinning outcome of the same polymer solutions were evaluated using HFIP as solvent, with special focus on how the addition of gelatin altered fibrous membrane production. To this end, PCL and PCL:Gelatin nanofibrous scaffolds were fabricated and a physicochemical (water contact angle and energy dispersive analysis) and microstructural characterization (scanning electron microscopy) were performed. The main novelty of this work is the identification of an optimal regime of concentrations within a binary system, as defined by the viscosity measurements and relative polymer ratios. This study was focused on the role of change the solution properties, so all other parameters, such as the processing conditions, were maintained constant. This study provides a proof-of-principle for further exploration to produce fibres with lower cost processing conditions. 2. Materials and methods 2.1. Materials Gelatin protein (gelatin type B, 80–120 g Bloom) was supplied by Henan Boom Gelatin Co. Ltd (China). Gelatin is mainly composed of protein (ca 98 wt%) and minor components, such as ash, lipids and moisture. Very low molecular weight poly( ε -caprolactone) (LW PCL, M w =14,000 g/mol) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from Sigma Aldrich (Germany). 2.2. Electrospinning process PCL and gelatin in different concentrations were mixed together directly in HFIP at room temperature by stirring for ca 24 h on a magnetic stirrer following the different ratios evaluated. The selection of the solvent was based on the optimization of the electrospinning process since HFIP dissolve both PCL and gelatin protein. Once the solutions were prepared, the electrospinning process was carried out using a Fluidnatek LE-100 equipment (Bioinicia). Membranes with PCL and gelatin in different ratios were processed following the same processing conditions used in previous studies [30]: 14 kV of voltage, 0.4 mL/h of flow rate and 14 cm as the needle-collector distance. The systems processed with different proportions of PCL and gelatin are shown in Table 1. The mats obtained were characterized by analysing the mean fiber size and the fiber size distribution measured from SEM imaging. In addition, the contact angle and the N content (with EDAX) were also evaluated. The previous polymeric solutions were characterised by means of their viscosity. Table 1 Systems processed with different PCL:Gelatin ratios modifying the PCL or gelatin concentration. Ratio PCL Concentration Gelatin Concentration Total polymer Concentration Nomenclature 3:1 12 w/v% 4 w/v% 16 w/v% 12/4 4:1 16 w/v% 20 w/v% 16/4 5:1 20 w/v% 24 w/v% 20/4 6:1 24 w/v% 28 w/v% 24/4 8:1 32 w/v% 36 w/v% 32/4 6:1* 6 w/v% 38 w/v% 32/6 5:1* 6.4 w/v% 38.4 w/v% 32/6.4 4:1* 8 w/v% 40 w/v% 32/8 3:1* 10.7 w/v% 42.7 w/v% 32/10.7 V.M. Perez-Puyana et al. Next Materials 6 (2025) 100306 2 2.3. Characterization studies 2.3.1. Fiber morphology characterization 2.3.1.1. Scanning electron microscopy (SEM). Microscopy examination of scaffolds was assessed with a XL 30 (Philips XL Series) at an acceleration voltage of 10 kV, a spot size of 3 and a working distance of 10 mm. The samples were covered with an Au film in a high-resolution sputter coater. ImageJ (a digital processing software) was used to analyze the mean fiber size and the porosity. The fiber size distribution was also obtained for the different systems produced. Furthermore, the ratio of their standard deviation to their mean was calculated to determine the uniformity of the systems (Eq. 1): Uniformity(%) = (1−standard deviation mean fiber diameter )⋅100 (1) The parameter “uniformity”has been defined to evaluate the homogeneity of the membranes since it allows comparing the standard deviation and mean fiber diameter of the fiber size distribution obtained. In this sense, an increase in uniformity implies a decrease in the standard deviation, which represents a lower variability of fiber size obtained. 2.3.1.2. Energy dispersive x-rays spectroscopy (EDAX). The composition of the electrospun membranes was evaluated using the energy dispersive spectroscopy capability of the SEM equipment (EDAX Si(Li) detector). An acceleration voltage of 20 kV, a spot size of 3 and a working distance of 10 mm were the parameters selected. 2.3.2. Water contact angle (WCA) The hydrophobicity and wettability of the scaffolds were analyzed by measuring the water contact angle (WCA) with the sessile drop method (droplets with an approximate volume of 5 μ L). WCA values were obtained as the average between the right/left sides values of the deionized water droplets. The equipment used was a Drop Shape Analyzer (Krüss). 2.3.3. Viscosity A glass capillary Ubbelohde viscosimeter (VWR, The Netherlands) was used to measure the viscosity of the PCL:Gelatin solutions. The temperature was maintained at 20±2ºC for all the measurements. The specific viscosity was obtained using the following equation [31] (Eq. 2): Specific Viscosity = η − η 0 η 0 (2) Where η is the viscosity of the solution (Pa⋅s) and η 0is the viscosity of the solvent used (1.65 mPa⋅s at 20 ◦C for HFIP). 2.4. Statistical analysis Each measurement was performed, at least, in triplicate. Statistical analyses were carried out with t-tests and one way analysis of variance (p <0.05) using PASW Statistics for Windows (Version 18: SPSS, Chicago, IL). Standard deviations were obtained for selected parameters. 3. Results and discussion 3.1. Unitary PCL and gelatin solutions: electrospinning and viscosity As mentioned above, previous reports have correlated the outcomes of the electrospinning process to the polymer solution properties, specifically solution viscosity and polymer concentration. These two parameters are interconnected, obtaining a correlation with the minimum concentration needed to achieve a suitable solution viscosity to carry out the electrospinning process properly. This is achieved thanks to the entanglement between the polymer chains, which favours the electrospinning process. It has been confirmed that fibers cannot be consistently generated from unitary solutions produced with PCL (supplementary material) or gelatin [30] by electrospinning. To correlate this to viscosity, unitary solutions of PCL and gelatin were first studied in a specific solvent (HFIP in this case). Fig. 1A shows the evolution of the viscosity for PCL solutions by increasing its concentration. Two different regions can be highlighted by a change in the slope (from 4.95 to 10.35). The first region ends when the total polymer concentration is 20 w/v%, starting a new region in which the influence of the concentration over the viscosity exhibits a higher slope. Considering the study conducted by Colby et al. [11], these two regions observed correspond to the semidilute unentangled and entangled regimes, with the crossover point at 20 w/v% as the entanglement concentration (Ce). As expected, the viscosity of unitary solutions of gelatin was also observed to increase with increasing protein concentrations (Fig. 1B). Similarly, the results show an initial linear region up to a concentration of 8 % w/v (with a slope of 0.061), with a sharp increase in viscosity at 10.7 % w/v (with a slope of 1.39). This sudden increase suggests gelatin molecules start to increase their interaction with each other, indicative of increased entanglement. Despite these indications of an increase in entanglement, the electrospinning outcome of both solutions remains poor. The electrospinning of LW-PCL results in microspheres at all concentrations, both above and below the 20 %w/v transition (Figure S1). Similarly, our previous finding of gelatin-only studies found that the electrospinning process could not be performed properly [30]. This behavior can be explained by the low entanglement of the polymer chains despite the increasing gelatin concentration and subsequent increase in viscosity (Fig. 1B). This low entanglement is a possible consequence of a low molecular weight, which is known to contribute to bead formation since the electrospinning process cannot be carried out continuously. However, in contrast to both unitary solutions, which failed to generate fibers, we observed that combinations of LW-PCL and gelatin in different ratios were successfully electrospun under certain conditions. 3.2. Influence of the PCL: gelatin ratio with increasing PCL concentration 3.2.1. Viscosity measurements To better understand this phenomenon, we further studied this binary system whereby the gelatin concentration was held constant and the amount of LW-PCL was systematically varied. Therefore, different PCL concentrations (12, 16, 20, 24 and 32 w/v%) were combined with a constant amount of gelatin (4 w/v%) and the measured viscosity of these mixtures measured, in order to correlate and predict possible electrospinning outcomes of these solutions (labelled as ratio of PCL/ Gelatin, specifically 12/4, 16/4, 20/4, 24/4 and 32/4). Fig. 2 shows the evolution of the specific viscosity for the systems produced with a 4 % w/v of gelatin and increasing concentrations of PCL (from 12 % to 32 % w/v), with an initial slope of 2.49 from 12 % to 16 % w/v that increases to 13.57 from 16 % to 32 % w/v. This defines two well-differentiated regions, where the first region (from 12 % to 16 %) corresponds to the semidilute unentangled regime and the second region correlates to a semidilute entangled regime; above the concentration of 16 %w/v PCL, chains start to entangle and, as a consequence, the viscosity of the solutions markedly increases. Therefore, the combination of 16 % of PCL and 4 % of gelatin (12/4 PCL:Gelatin ratio) can be considered the entanglement concentration (Ce, with a 20 % of total polymer concentration). According to Kong and Ziegler [32], the spinning of good fibers requires that the solution concentration be 1.2–2.0 times the entanglement concentration. Of the different values studied, a PCL:Gelatin ratio between 20/4 and 24/4, for a total polymer concentration of 24 % and 28 % w/v, respectively, should represent the start of good fiber formation since the polymer concentration is 1.25 and 1.50 times the entanglement concentration. V.M. Perez-Puyana et al. Next Materials 6 (2025) 100306 3 3.2.2. Microstructural evaluation SEM imaging of the evolution of the PCL-based membranes generated with a constant amount of gelatin (4 %w/v) and increasing concentrations of LW PCL (12, 16, 20, 24 and 32 %w/v) is shown in Fig. 3. These starting solutions have relative ratios of PCL to gelatin of 3:1, 4:1, 5:1, 6:1, and 8:1, labelled respectively as 12/4, 16/4, 20/4, 24/4 and 32/4 to clarify the polymer content per condition. The lowest ratio (12/ 4) showed a combination of fibers and beads on its structure. However, the increase in the ratio lead to the homogenization of the system with the disappearance of the beads and the formation of consistent fibers. This improvement is mainly observed for the 16/4 and 20/4 ratio, which correspond with the optimal range found in the viscosity measurements shown in the previous section. Interestingly, when the ratio is higher than 20/4 (24/4 and 32/4), the SEM images showed again the formation of spheroids in the structure. From the SEM images, the distribution of sizes of the elements (either fibers or beads) present in the structure were analysed and plotted in Fig. 3. A shift in the profile can be seen, from a wide distribution observed for the system with a 12/4 (due to the presence of both fibers and beads) to a narrow distribution towards a central value (around 0.5 µm) for the 20/4 system and a return to a wider distribution when the ratio is higher at 24/4 and 32/4, which gives rise to a system with more heterogeneous fiber sizes and the re-emergence of spheroid formation. To better assess the effect of the PCL:Gelatin ratio, we quantified fiber uniformity (Fig. 3) as function of fiber and bead size (Table 2) for each of the mixed PCL:Gelatin systems. This clearly shows an optimal region, where uniformity increases to a maximum between PCL:Gelatin ratios of 16/4 and 24/4, reflecting an increase in the system homogeneity and a reduction in bead formation. However, moving away from that optimal region encourages the appearance of beads results in a decrease in the mean fiber size. The prolife follows a trend of an inverted second polynomial, reaching the maximum value at a ratio of 20/4 that coincides with the disappearance of the beads in its structure and a narrowed distribution of fiber diameters. Overall, with the transition from a semidilute unentangled to a semidilute entangled polymer solution we observed the expected shift from poor quality electrospun fiber generation, denoted by bead formation and a broad range of fiber diameters, to the production of consistent, homogeneous fibers. Interestingly, as the polymer concentration continued to increase, we observed a return to poor fiber formation. This finding is inconsistent with current theory which apply to electrospinning of unitary polymer solutions and suggests other underlying mechanisms are involved when considering a semidilute entangled binary polymer system. 3.3. Influence of the PCL: gelatin ratio with increasing gelatin concentration We wanted to explore the importance of maintaining a specific PCL: Gelatin ratio, irrespective of the absolute concentrations or viscosity. We started from an initial PCL and gelatin concentration of 32 % and 4 %, Fig. 1. Evolution of the specific viscosity of unitary systems as a function of the (A) PCL concentration and (B) Gelatin concentration. Fig. 2. (A) Evolution of the specific viscosity of binary systems PCL:Gelatin as a function of the PCL concentration. (B) Evolution of the specific viscosity of binary systems PCL:Gelatin as a function of the PCL concentration with a dashed region in which the spinning process cannot be carried out properly. SEM images of the different binary systems have also been included (scale bar: 10 µm). V.M. Perez-Puyana et al. Next Materials 6 (2025) 100306 4 respectively, for a PCL:Gelatin ratio of 32/4 in an HFIP solvent; in relative terms, this has a ratio of 8:1. This constituted semidilute entangled polymer solution that, despite sufficiently high viscosity, was already shown to have poor fiber formation and extensive microsphere production. To better understand the transition from beaded fibers to consistent fibers, we held the PCL concentration constant this higher value of 32 % w/v and systematically increased the quantity of gelatin with concentration of 4.0, 6.0, 6.4, 8.0 and 10.7 w/v%, thereby achieving the same relative ratios of 8:1, 6:1, 5:1, 4:1, and 3:1 (in reverse order); to distinguish these solutions from the previous conditions, the relative ratios include an asterisk ‘*’when mentioned and the conditions are labelled with the absolute ratio of PCL to gelatin, specifically 32/ 10.7, 32/8, 32/6.4, 32/6 and 32/4. In addition, a polymer solution of 32 % PCL with no gelatin was also included to assess the impact of adding gelatin. SEM images and fiber/bead size distributions of these systems are shown in Fig. 4. The systems composed of only PCL presented a structure mainly formed by beads. Interestingly, the addition of gelatin to a constant concentration of PCL (32 %) followed the same trend as was previously observed. The morphology of electrospun deposition evolved from heterogeneous fibers with bead formation (system 32/4) to homogeneous fibers for intermediate ratios (32/6, 32/6.4 and 32/8), followed again by beads and small fibers (system 32/10.7). Bead formation was largest for the 32/4 ratio, while both highest and lowest ratios also displayed a decrease in the size of the fibers generated. An analysis of fiber uniformity shows the same inverted second order polynomial trend for this 32 % PCL-based binary system (Fig. 5), where the 32/10.7 and 32/4 PCL:Gelatin ratios led to less than 40 % uniformity that reflects the emergence of spheroids. Conversely, uniformity and fiber diameter increase for the intermediate ratios (shown in Fig. 5). Fig. 3. SEM images (scale bar: 10 µm) and fiber/bead size distributions of the membranes generated with mixed PCL:Gelatin systems with different ratios: 12 %+4 % (12/4, ratio 3:1), 16 %+4 % (16/4, ratio 4:1), 20 %+4 % (20/4, ratio 5:1), 24 %+4 % (24/4, ratio 6:1) and 32 %+4 % (32/4, ratio 8:1). Table 2 Mean fiber and beads diameters of membranes processed with different PCL and gelatin percentages and PCL:Gelatin ratios: 12 %+4 % (12/4), 16 %+4 % (16/ 4), 20 %+4 % (20/4), 24 %+4 % (24/4), 32 %+4 % (32/4), 32 %+6 % (32/6), 32 %+6.4 % (32/6.4), 32 %+8 % (32/8) and 32 %+10.7 % (32/10.7). SYSTEM Nomenclature Ratio Fiber Size (nm) Beads Size (nm) PCL (12 %) +Gelatin (4 %) 12/4 3:1 288 ±86 807 ±223 PCL (16 %) +Gelatin (4 %) 16/4 4:1 485 ±170 - PCL (20 %) +Gelatin (4 %) 20/4 5:1 415 ±91 - PCL (24 %) +Gelatin (4 %) 24/4 6:1 443 ±95 874 ±176 PCL (32 %) +Gelatin (4 %) 32/4 8:1 308 ±100 1.160 ±450 PCL (32 %) +Gelatin (6 %) 32/6 6:1* 590 ±218 - PCL (32 %) +Gelatin (6.4 %) 32/6.4 5:1* 357 ±93 - PCL (32 %) +Gelatin (8 %) 32/8 4:1* 724 ±376 - PCL (32 %) +Gelatin (10.7 %) 32/10.7 3:1* 290 ±107 1.299 ±328 V.M. Perez-Puyana et al. Next Materials 6 (2025) 100306 5 Fig. 4. SEM images (scale bar: 10 µm) and fiber/bead size distributions of the membranes generated with mixed PCL:Gelatin systems with different ratios: 32 %+4 % (32/4, ratio 8:1), 32 %+6 % (32/6, ratio 6:1*), 32 %+6.4 % (32/6.4, ratio 5:1*), 32 %+8 % (32/8, ratio 4:1*) and 32 %+10.7 % (32/10.7, ratio 3:1*). Fig. 5. SEM images and fiber/bead size distributions of the membranes generated with mixed PCL:Gelatin systems with different PCL:Gelatin ratio: (A) 12 %+4 % (12/4), 16 %+4 % (16/4), 20 %+4 % (20/4), 24 %+4 % (24/4), 32 %+4 % (32/4); (B) 32 %+10.7 % (32/10.7), 32 %+8 % (32/8), 32 %+6.4 % (32/6.4), 32 %+ 6 % (32/6) and 32 %+4 % (32/4). V.M. Perez-Puyana et al. Next Materials 6 (2025) 100306 6 Similar to the PCL:Gelatin (4 %) systems, these results of the PCL (32 %):Gelatin systems are partly consistent with the proposed explanation regarding polymer concentration and chain entanglement as it pertains to the electrospinning process. As the polymer solution transitions from a lower concentration (e.g. 4 % gelatin) to higher concentration (with gelatin concentrations between 6 % and 8 %), heterogeneous fibers give way to the production of homogeneous fibers with no beads. However, from this concentration on, homogeneity is lost again, and beaded membranes are obtained, with the subsequent decrease in uniformity. One possibility is that the overall high viscosity of the solution, when both PCL and gelatin concentrations are high, prevents a consistent electrospinning jet and, therefore, increasing the heterogeneity of the resulting membranes. Alternatively, another possible reason is a change in miscibility of the two polymers as the relative concentrations increase; this has been previously reported for other natural-synthetic polymer solution blends [33,34]. With such a decrease in polymer miscibility and subsequent polymer phase separation, it would be expected that less inter-species polymer chain interaction would occur, consequently resulting in poor electrospinning outcomes, and that there would be a consistent inhomogeneous polymer species distribution within the deposited fibers. 3.4. Physicochemical evaluation Directly measuring the behaviour of a binary polymer solution during electrospinning is a challenge given the dynamic nature of the process, including the continual evaporation of solvent, subsequent rapid changes in polymer concentration, and high-speed evolution of fluidic jet during processing. To look for evidence of polymer phase separation in the resulting fiber, water contact angle (WCA) and EDAX measurements were used to assess fiber properties as indicators of inhomogeneous fiber composition. These two techniques provide an indication of the surface composition of the fibers, wherein we expected homogeneous fibers to have a change in WCA and EDAX readout that is directly proportional to the changes in polymer concentration. In contrast, we anticipated any inhomogeneity in the fiber surface composition, resulting from phase separation and a subsequent underor overrepresentation of one polymer species versus the other on the fiber surface, would present as a nonlinear relationship with respect to polymer composition. As can be observed in Fig. 6A and 6B, the value of the contact angle increased when the ratio between the polymers was higher, due to the presence of the hydrophobic polymer (PCL) in a higher concentration compared to the concentration of gelatin, which is the hydrophilic polymer of the mixture. For that reason, the contact angle varied (almost doubled) comparing the values obtained for the systems with the lowest (around 40º, ratio 12/4) and the highest (around 75º, ratio 32/4) ratio between PCL and gelatin. Moreover, an evolution towards a more hydrophilic system took place when the concentration of gelatin was increased, switching from ca 70º(32/4 system) to ca 40º(32/10.7 system). Furthermore, similar contact angle values were obtained for the systems processed with a similar PCL:Gelatin ratio. Performing a linear regression of the contact angle measurements with respect to the PCL:Gelatin ratios from 12/4–32/4 and from 32/ 4–32/10.7, it was possible to determine a clear linear trend (Fig. 5A and 5B) that resulted in the following equation (Eqs. 3 and 4, respectively): WCA =6.68⋅[PCL :Gelatin Ratio] + 20.28 (3) WCA =6.91⋅[PCL :Gelatin Ratio] + 36.03 (4) This confirms expectations that an increase in the ratio between PCL and gelatin produces an increase in the contact angle, since the system obtained is more hydrophobic due to the greater presence of PCL. Further, according to fit parameters of different correlations shown in Table 3, a linear trend was observed, which achieved an improved fit (an increase in R 2 value) when the outermost ratios (12/4 and 32/4) were excluded. Since the overall distribution of material deposition is the same for all samples, this improved fit can reflect the change in fiber morphology (beads to fibers) but can also indicate an improved homogeneity in fiber composition for intermediate PCL:Gelatin ratios. To further investigate the correlation between the homogeneity of fiber composition and initial polymer solution ratio, the relation between PCL and gelatin was assessed via EDAX analysis of the electrospun fibers, where the % amount of nitrogen per unit area was used as a metric for protein distribution within the fiber structure (Table S1). The nitrogen present in the surface obtained from the EDAX profiles, shown in Fig. 5A and 5B, shows an obvious correlation between N content and the changing ratios between the two polymers. The results shown Fig. 6. (A) Correlation between the WCA and N content with the PCL:Gelatin ratio and (B) relation between the uniformity and mean fiber size of the membranes processed with different PCL:Gelatin ratio: 12 %+4 % (12/4), 16 %+4 % (16/4), 20 %+4 % (20/4), 24 %+4 % (24/4), 32 %+4 % (32/4), 32 %+6 % (32/6), 32 %+ 6.4 % (32/6.4), 32 %+8 % (32/8) and 32 %+10.7 % (32/10.7). Table 3 R 2 values for the different correlations for Contact Angle and Nitrogen content of membranes processed with different PCL and gelatin percentages and PCL: Gelatin ratios: 12 %+4 % (12/4), 16 %+4 % (16/4), 20 %+4 % (20/4), 24 %+ 4 % (24/4), 32 %+4 % (32/4), 32 %+6 % (32/6), 32 %+6.4 % (32/6.4), 32 %+8 % (32/8) and 32 %+10.7 % (32/10.7). CORRELATIONS Systems R 2 R 2 (without 12/4 system) R 2 (without 12/4 and 32/4 systems) Contact Angle 12/4–32/4 0.9607 0.9768 0.9839 Nitrogen content 12/4–32/4 0.8908 0.9809 0.9998 Contact Angle 32/ 10.7–32/4 0.9511 0.9082 0.7992 Nitrogen content 32/ 10.7–32/4 0.9774 0.9871 0.9886 V.M. Perez-Puyana et al. Next Materials 6 (2025) 100306 7 conclude that there is a similar decreasing linear trend (Eqs. 5 and 6): Ncontent = − 0.63⋅[PCL :Gelatin Ratio] + 5.26 (5) Ncontent = − 0.70⋅[PCL :Gelatin Ratio] + 5.94 (6) This confirms that increasing the PCL:Gelatin ratio results in a lower amount of protein-associated nitrogen. Furthermore, the analysis of the different correlations performed (Table 3) showed the similar behaviour exhibited by the contact angle measurements, where an increase in the R 2 fitting value is achieved when excluding the outermost ratios of 12/4 and 32/4. While is it currently not possible to directly confirm the response of the two polymer species as they are subjected to a rapid increase in concentration and simultaneous mechanical forces during the evaporation and whipping phases of the electrospun polymer jet, the observed changes in fiber morphology and both the contact angle and evaluation of nitrogen distribution provide indirect support with the hypothesis of inhomogeneity in the distribution of gelatin and, thus, phase separation within the resulting fibers [35]. 4. Conclusions Suitable PCL/Gelatin nanofiber membranes can be obtained using low molecular weight (LW) PCL. The typical formation of beads during the electrospinning process using LW PCL can be avoided with the addition of gelatin, which is homogeneously distributed in the membrane. The greater hydrophilicity, the lower surface tension and the increased viscosity of the polymer systems leads to improve the electrospinning process. However, a further increase in gelatin protein concentration leads to an increase in viscosity, which could cause greater difficulties in the electrospinning process, or could trigger phase separation within the binary solution. Consequently, it would be convenient to characterize the solution evolution during the electrospinning process in terms of viscosity, surface tension, and composition, although this currently presents a challenge due to the dynamic nature of this manufacturing process. Different correlations between the PCL:Gelatin ratio and the different properties measured (N content and contact angle) were established. Linear correlations were obtained, although with opposite slopes, since the hydrophobicity of the systems increases with the PCL: Gelatin ratio whereas the N content decreases as the ratio increases. On the other hand, the homogeneity of the systems was analyzed calculating the uniformity of the nanofibrous membranes from the SEM imaging. A maximum was found for the ratio with the lowest mean fiber size (ratio 20/4). In fact, higher mean sizes lead to an increase in the heterogeneity of the systems. The present work demonstrates that not only the total polymer concentration affects the fiber formation, but also the ratio between the two polymers when using a binary system; initial indications suggest that phase separation may occur for some polymer systems as they become more concentrated during the electrospinning process, resulting in poor entanglement and fiber uniformity. For PCL and gelatin, the most suitable ratios were found to be 16/4, 20/4 and 24/4. The use of binary solutions presents a new avenue to process low molecular weight polymers via electrospinning, where low molecular weight polymers are also typically more economic to produce. While a caveat is the use of HFIP as a necessary universal solvent for both species of this binary system, thereby not in line with emerging obligations for green manufacturing processes, it provides a case study for the development of low cost, low molecular weight polymer species that are compatible with suitable solvent systems. In any case, membranes with controllable morphology, structure and properties could be achieved by adjusting the PCL:Gelatin ratio. Funding sources This work is part of a research project sponsored by the “Ministerio de Economía y Competitividad”(MCI/AEI/FEDER, EU) from the Spanish Government (Ref. RTI2018-097100-B-C21). The authors gratefully acknowledge their financial support. The authors also acknowledge the University of Seville for the VPPI-US predoctoral grant and Junta de Andalucía (European Social Fund, PAIDI DOCTOR –Convocatoria 2019–2020, DOC_00586) for the postdoctoral contract of Victor M. Perez-Puyana. Part of this work was carried out at the Department of Complex Tissue Regeneration (Maastricht University, MERLN-Institute for Technology-Inspired Regenerative Medicine) and by financial support from the program “Estancias breves en Espa˜ na y en el extranjero para beneficiarios de Becas predoctorales o PIF de la US y de Becas de la Fundaci´ on C´ amara”from the University of Seville. This research project was made also possible thanks to the Dutch province of Limburg. Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.nxmate.2024.100306. 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