Plasma treatment as an effective tool for crosslinking of electrospun fibers

Journal of Molecular Liquids 303 (2020) 112628

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Plasma treatment as an effective tool for crosslinking of electrospun fibers Kristof Molnar a,b, Benjamin Jozsa a, Dora Barczikai a, Eniko Krisch b, Judit E. Puskas b, Angela Jedlovszky-Hajdu a,⁎ a

Laboratory of Nanochemistry, Department of Biophysics and Radiation Biology, Semmelweis University, Nagyvarad ter 4, Budapest 1085, Hungary Department of Food, Agricultural and Biological Engineering, College of Food, Agricultural, and Environmental Sciences, The Sustainability Institute, The Ohio State University, 222 FABE, 1680 Madison Avenue, Wooster, OH 44691, United States of America

b

a r t i c l e

i n f o

Article history: Received 17 December 2019 Received in revised form 30 January 2020 Accepted 31 January 2020 Available online 03 February 2020 Keywords: Plasma treatment Electrospun fibers Crosslinking Polysuccinimide Allylamine

a b s t r a c t Electrospinning is a versatile method for the preparation of polymer fiber networks. From the medical application point of view, the stability of the fibrous structure in water is crucial. The aim of this research was to explore the possibility to induce crosslinking by treatment with non-equilibrium low pressure plasma to give structural stability to fibrous meshes. Low pressure plasma treatment was studied to induce crosslinking in allylaminemodified polysuccinimide nanofibers. Polysuccinimide was first modified with allylamine (PSI-AA) to attach the reactive allyl groups to the polymer chain. Allylamine-modification degrees varied between 10 and 100%. Then PSI-AA meshes were created by electrospinning followed by low pressure plasma treatment to allow the allylamine groups to establish crosslinks in the meshes. The crosslinked structure was confirmed by immersing both the plasma-treated, and untreated PSI-AA meshes in dimethyl sulfoxide, which is a good solvent of the non-crosslinked PSI-AA. Plasma-treated meshes kept their integrity, while the untreated samples dissolved almost immediately, which proved the formation of crosslinks due to plasma treatment. Structural changes in the meshes were studied with infrared spectroscopy (IR). X-ray photoelectron spectroscopy (XPS) investigates the structural changes on the surface of the samples and proved the presence of crosslinks. PSI-AA meshes were then hydrolyzed into poly(aspartic acid) (PASP) meshes and with the right choice of synthesis parameters (allylamine grafting degree, plasma treatment duration and power) PASP hydrogel meshes retained their fibrous structure, which was observed in SEM images of PSI-AA meshes. © 2020 Elsevier B.V. All rights reserved.

1. Introduction Electrospinning is a well-known technique for the preparation of fibrous polymer meshes from a large variety of polymers [1]. These fibrous structures already have promising applications in the field of energy-related devices such as solar cells, fuel cells or lithium-ion batteries [2] as well as in biomedical fields including drug delivery agents [3] or regenerative tissue engineering as they highly resemble the human extracellular matrix (ECM) [4,5]. In biomedical applications, the stability of these structures in aqueous solutions is a crucial factor that is mainly determined by the type of building polymer. No matter how great qualities a polymer exhibits, if it is unstable in water its biomedical applicability will be inevitably lost. A possible solution to this issue is the crosslinking of the polymer the electrospun fibers are built from, so the fibers can retain their integrity even in aqueous medium by forming polymer gel fibers.

⁎ Corresponding author. E-mail addresses: [email protected] (K. Molnar), [email protected] (A. Jedlovszky-Hajdu).

https://doi.org/10.1016/j.molliq.2020.112628 0167-7322/© 2020 Elsevier B.V. All rights reserved.

Crosslinking the polymer chains can be carried out using various methods. Polymer meshes can be crosslinked by treatment with liquids or vapors containing the crosslinking agents, but in this case, the number of crosslinks is difficult to control. In other cases, the polymer chain is first modified with various side groups and after fiber formation, the crosslinking reaction is initiated by UV light [6], gamma radiation [7], or heat [8]. The problem with these methods is that the modifying or initiating agents are often toxic, and even though the crosslinking can provide the structure with the required in vivo stability, the biocompatibility and biodegradability of the material are prone to be altered or even lost. Another, still quite unexplored method can be the post-electrospinning crosslinking of the meshes by plasma treatment. The process of exposing materials to a purposefully generated plasma medium is called plasma treatment. The plasma state of the treating gas can be initiated and maintained by continuously conveying energy to it. That can be achieved by gaseous, metallic, and laser plasma sources among others [9], which are available in various implementations resulting in a diverse array of plasma generating methods. The generated plasma can be equilibrium or nonequilibrium plasma, where the latter is also called “cold” or non-

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thermal plasma due to its relatively low temperature. Plasma treatment methods can also be differentiated by the pressure of the medium present during the treatment, low pressure and atmospheric variants are both used [10,11]. The plasma itself consists of an unregulated mixture of ions, free radicals, electrons, photons and unstable molecule fragments making it a very aggressive and reactive medium. Capitalizing on these properties' plasma treatment is used to accomplish various tasks where such traits are favorable. These are sterilization, surface modification, surface etching, crosslinking and plasma implantation just to mention a few of the most common applications [12]. However, only a few attempts are published in the literature about using plasma treatment for the crosslinking of polymers. The available articles are based on gelatin, which results are summarized in the recent review of Campiglio et al. [13]. Liguori et al. showed that plasma can effectively be used to induce crosslinking in fibrous gelatin membranes without the use of a crosslinking agent such as genipin [14]. In contrast to their work Sisson et al. showed that oxygen plasma could not adequately crosslink gelatin fibers as those dissolved after 12 h in cell culture medium regardless of plasma treatment optimization [15]. Dolci et al. also found that drug loaded gelatin films can be crosslinked using atmospheric air plasma [16]. Poly(aspartic acid) (PASP), a synthetic poly(amino acid) and consequently its anhydrous form, polysuccinimide (PSI), have a great potential in biomedical fields [17–19]. PSI can be synthesized by the thermal polycondensation of aspartic acid with well-defined molecular weight via a solvent-free method and due to its reactive succinimide units, it can be modified with a large variety of molecules. Water-soluble PASP and its derivatives can be generated from PSIs by hydrolysis in a mild alkaline medium. PASP and most of its derivatives are biodegradable and biocompatible, making them ideal for several biomedical applications, such as drug delivery systems or tissue engineering [17,19,20]. Our group has already published the synthesis of crosslinked electrospun PSI and PASP meshes. Crosslinking occurred either by using co-axial electrospinning, where the core of the needle contained the crosslinker, while the PSI was in the shell [21]. Crosslinked PSI meshes with fibrous structures were successfully synthesized, although the mesh had to be washed thoroughly to get rid of the excess crosslinkers and the number of crosslinks was undefined. Another approach was the crosslinking of thiol-modified PSI via air during the electrospinning process and thus disulfide crosslinked PSI and PASP fibers were synthesized [22]. These fibers were stable in water in vitro, although in the body disulfide linkages can disintegrate due to the presence of thiols [17]. Crosslinking of PSI fibers having reactive side groups by plasma treatment could be an easy and straightforward method to create biocompatible polymer fibers that are stable in vivo and thus provide several applications. Furthermore, plasma treatment has a sterilizing effect, thus sterile, crosslinked meshes can be produced without additional sterilizing steps. Hereby, we present the crosslinking of electrospun allylaminemodified PSI polymer nanofibers by low pressure plasma treatment and by their hydrolysis the preparation of PASP hydrogel fibers. The structure and morphology of the synthesized polymers and crosslinked polymer meshes are investigated by nuclear magnetic resonance, infrared spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. 2. Materials and methods 2.1. Materials Acetone (Sigma-Aldrich), agar agar (Thermo Fisher Scientific), allylamine (Sigma-Aldrich), L-aspartic acid (Sigma-Aldrich), dimethylformamide (DMF) (VWR International), dimethylsulfoxide (DMSO) (Sigma-Aldrich), nutrient broth (Fluka Analytical), ophosphoric acid (VWR), phosphate buffer saline (PBS) (Sigma-Aldrich), M9 phosphate buffer (1.3 g MgSO4, 10.0 g NH4Cl, 30.0 g KH2PO4, 155.5 g Na2HPO4*7H2O/10 L).

All the chemicals were of analytical grade and used as received. For the aqueous solutions, ultrapure water (Human Corporation ZeneerPower I Water Purification System) was used. 2.2. Synthesis of allylamine-modified polysuccinimide (PSI-AA) Polysuccinimide (PSI) was synthesized by thermal polycondensation of aspartic acid under vacuum, in the presence of phosphoric acid catalyst as reported [22]. Then PSI was reacted with allylamine (AA) to gain allylamine-modified PSI (PSI-AA) as follows (Fig. 1A): solutions of PSI (Solution B) and AA (Solution A) were prepared using DMF as solvent, and then Solution A was added to Solution B dropwise during intensive stirring. The mixture was stirred for five days (50 RPM, 60 °C), using an IKA RCT Basic stirrer and an IKA IKATRON ETS-D4 temperature controller with a Carousel 12 reactor (Radley). PSI-AAs with various grafting degrees (GD = 1; 2; 5; 10) were prepared, where GD shows the ratio of the number of succinimide repeat units to the number of AA molecules (hence, PSI-AA1, where GD = 1 has the most AA molecules, and PSI-AA10, where GD = 10 has the least). The amounts of the components used for the synthesis of PSI-AAx are shown in Table 1. After the synthesis, the reaction mixture was poured into a large excess of water to precipitate, then the precipitate was dialyzed against distilled water (cut off = 10 kDa) for 5 days while the distilled water was frequently replaced. After the DMF was completely washed out, the precipitated polymer with the supernatant water was freezedried. 2.3. Electrospinning of PSI and PSI-AA The electrospinning instrument consisted of a syringe pump (KD Scientific KDS100), a high voltage DC power supply (GENVOLT 73030P), and a collector made of aluminum foil, supported by a wood plate (Fig. S1). The solutions used for the preparation of electrospun PSI and PSI-AA meshes were obtained by dissolving PSI or the freezedried PSI-AA in DMF (Table 2). A solution was then poured into a 2.5 mL plastic syringe with a blunt end G18 metal needle. The syringe was fixed in the syringe pump and the pumping rate was set to 1 mL/h. The collector was installed in a distance of 15 cm from the end of the needle. Once the pumping started, high voltage was applied between the metal needle and the collector. The voltage was adjusted during the process (8–12 kV), to maintain a stationary flow of the polymer solution without excessive dripping or a temporary shortage of material. Once the solution run out the voltage was stopped and the aluminum foil collector with the electrospun polymer mesh on its surface was removed. Resulted polymer meshes were named according to Table 2. Electrospun samples are denoted M-PSI-AAx-y, where M stands for electrospun mesh, x stands for the grafting degree and y stands for the polymer concentration in the solution used for electrospinning. 2.4. Plasma treatment of electrospun meshes The electrospun M-PSI and M-PSI-AAx = − polymer meshes were subjected to a low pressure non-equilibrium air plasma treatment using a laboratory plasma treatment instrument (Diener Electronics – Zepto). Low pressure air plasma was generated with an LF plasma generator working at 40 kHz with adjustable power of 0–100 W surrounding closely a tubular plasma chamber with 105 mm diameter and 300 mm depth. Samples were placed into the center of the chamber on a Borosilicate plate (Samples under plasma treatment and the machine itself can be seen in Fig. S2). Low pressure was generated by a Pfeiffer Duo 1,6. The gas was laboratory air at 24 °C and approximately 30–50% relative humidity. Three spherical samples with diameters of 12 mm were cut from each PSI and PSI-AA mesh and placed in a Petri dish. The two adjustable parameters of the treatment were exposure time and generator power. The pressure of the plasma chamber was

K. Molnar et al. / Journal of Molecular Liquids 303 (2020) 112628

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Fig. 1. Synthesis of allylamine modified PSI (PSI-AA) (A) and hydrolysis of PSI-AA to allylamine-modified poly(aspartic acid) (PASP-AA) (B).

set to an initial value of 5 mbar at the beginning of each treatment and was not varied between treatments, although it decreased to approximately 0.3 mbar during the process. In the first series, the exposure time in each experiment was set to 13 min while the power was either 25, 50, 75 or 100 W. In the second series we have conducted additional experiments using only the MPSI-AA2 samples using four different plasma exposure times (13, 15.5, 17 and 30 min) with a fixed power of 25 W or 100 W.

2.8. X-ray photoelectron spectroscopy (XPS) For the surface analysis of PSI and PSI-AA meshes, a Kratos Axis Ultra XPS was used with a monochromated Al Kα source (1486.6 eV, 12 kV, 10 mA) and a charge neutralizer. Survey spectra were collected with 100 eV pass energy while high resolution spectra were collected with 20 eV pass energy. The CasaXPS software was used for data analysis. The corresponding reference signal was the C1s signal with a binding energy of about 285 eV. The fitting method was the Gaussian– Lorentzian curves with the deduction of the Shirley background.

2.5. Hydrolysis of PSI-AA meshes to PASP-AA meshes PASP-AA meshes were prepared by mild alkaline hydrolysis of the PSI-AA meshes in an imidazole-based buffer solution of pH = 8 (ionic strength: I = 250 mM). The chemical reaction of PSI turning into PASP is depicted in Fig. 1B. In order to ensure 100% conversion, samples were kept in the buffer for 24 h [17]. After the hydrolysis, samples were washed with ultrapure water to get rid of the side-product salts and were subsequently used either in their swollen state in further experiments or freeze-dried for SEM and ATR-FTIR.

2.6. Nuclear magnetic resonance (NMR) The chemical structure of PSI and PSI-AA polymers was studied by NMR. All NMR spectra were obtained using a Bruker AVANCE III spectrometer operating at 400 MHz for the 1H nucleus. Sample solutions were prepared by dissolving 40 mg of polymer powder in 0.6 mL of DMSO-d6 in 5 mm NMR tubes. All spectra were recorded at 27 ± 0.5 °C and tetramethylsilane was used as the internal standard. The pulse angle was set to 45o, 2 s delay was used with 8 spectral widths and 64 scans were recorded in every measurement. Spectrusprocessor by ACDLABS was used for spectrum analysis.

The chemical composition of the PSI and PSI-AA polymers and polymer meshes was investigated via Fourier Transform Infrared Spectroscopy (FTIR), using a JASCO FT/IR-4700 spectrophotometer fitted with an Attenuated Total Reflection (ATR) accessory (JASCO ATR Pro One). Spectra were collected in a range of (4000 cm−1–400 cm−1) with a resolution of 2 cm−1 and an accumulation number of 126.

Table 1 Compounds in the synthesis of allylamine-modified PSI (PSI-AA).

PSI-AA1 PSI-AA2 PSI-AA5 PSI-AA10

Grafting degree

1 2 5 10

Solution A

In order to verify the fibrous nature of the electrospun meshes and determine the diameter of the individual fibers, SEM studies were conducted. In the case of PSI and PSI-AA meshes a small part of the mesh was cut out and placed on conductive tape for coating and microscopy. In the case of PASP-AA meshes samples were washed thoroughly with ultrapure water and freeze-dried, then a small piece was placed on conductive tape for coating and microscopy. Afterward, all of the samples were sputter coated with gold (20–30 nm thickness) with a 2SPI Sputter Coating System. Micrographs were taken using a ZEISS EVO 40 XVP scanning electron microscope. The accelerating voltage was 20 kV. All presented images were captured using a magnification rate of 5000 unless stated otherwise. For calculating the average fiber diameter 50 fibers of each sample were measured using the ImageJ software. The fiber diameters were only measured in case of those meshes where the SEM images indicated a fully fibrous structure, with the fibers being separated. Measurement points were selected arbitrarily by a human operator and were measured by hand. 2.10. Sterility tests To evaluate the plasma treated PSI-AA meshes' sterility, samples were placed on the surface of hard agar under a sterile hood. Afterward a mixture of 1 mL of M9 phosphate buffer and 2 mL of semisolid (soft)

2.7. Fourier transform infrared spectroscopy (ATR-FTIR)

Sample name

2.9. Scanning electron microscope (SEM)

Solution B

Allylamine [μl]

DMF [g]

PSI [g]

DMF [g]

774.5 387.2 154.9 77.5

0.5

1

8.5

Table 2 Concentration of PSI-AA solutions used for electrospinning. Sample name of PSI and PSI-AA meshes

Polymer used for electrospinning

Concentration of polymer solution [w/w%]

M-PSI M-PSI-AA1-35 M- PSI-AA1-40 M- PSI-AA1-45 M- PSI-AA2-35 M- PSI-AA2-40 M- PSI-AA2-45 M- PSI-AA5-25 M- PSI-AA5-30 M- PSI-AA5-35 M- PSI-AA10-22.5 M- PSI-AA10-25 M- PSI-AA10-27.5

PSI PSI-AA1

25 35 40 45 35 40 45 25 30 35 22.5 25 27.5

PSI-AA2

PSI-AA5

PSI-AA10

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agar (kept at 54 °C) was gently poured on to the meshes and spread evenly on the surface of the hard agar. The Petri dishes were then placed into an incubation chamber at 37 °C. After 24 and 48 h, the Petri dishes were visually checked for colonies.

3. Results and discussion 3.1. Synthesis of electrospun PSI and PSI-AA meshes Polysuccinimde (PSI) was synthesized in a solvent-free process by thermal polycondensation of aspartic acid under vacuum, in the presence of phosphoric acid catalyst. The viscometric molecular mass average of PSI prepared under identical conditions was 28,500 ± 3000 Da based on viscosimetry and the Kuhn-Mark-Houwink equation [22]. PSI was reacted with allylamine, a primer amin, that is able to react with the succinimide rings in a nucleophile substitution reaction with a high conversion rate [23]. Allylamine-modified PSI with various grafting degrees (GD = 1; 2; 5; 10) were prepared (Fig. 1A), where GD shows the ratio of the number of succinimide repeat units to the number of AA molecules (hence, PSI-AA1, where GD = 1 has the most AA molecules, and PSI-AA10, where GD = 10 has the least). The physical appearance of the freeze-dried PSI-AAs showed a significant correlation with the GD (Fig. S3). The least modified polymer (PSI-AA10) looked like the unmodified dry PSI, while the most modified version (PSI-AA1) was constituted of crystal-like, yellowish flakes of low density. The modified polymers with a grafting degree in between (PSI-AA2, PSIAA5) were white, and powder-like. Success of the modification was confirmed by 1H NMR (see Supplementary information, Figs. S4 and S5) and FT-IR (discussed later in Section 3.3). For electrospinning, solutions of PSI and PSI-AAs were prepared in DMF. In general, with the increase of the grafting degree an increment in the applied concentration (22.5–45 w/w%) and voltage (8 → 12 kV) was necessary for successful electrospinning. The mesh obtained from the unmodified PSI at 25 w/w% was a dense and stable structure (Fig. S6). For the electrospinning of PSI-AAs, solutions of different concentrations were used to determine which concentration is the most suitable for each grafting degree to create good quality meshes. The viable concentrations are determined by the practical barriers experienced during the electrospinning process. Solutions of low concentration are often prone to forming isolated droplets when leaving the syringe instead of producing continuous flow. The upper limit of the applicable concentration is set by the viscosity of the polymer solution which increases with its concentration. The concentrations shown in Table 2 were tested, but not all yielded satisfactory meshes. The unsuitable concentrations are marked grey in Table 2. One of them is the PSI-AA1 solution with a concentration of 45%, which proved to be too viscous to be suitable for electrospinning. The other one is the attempt with PSI-AA10 using a concentration of 22.5%. In this case, the solution was vaporized when exiting the needle, instead of forming a continuous flow and as a result, the product obtained did not have the necessary structural integrity to be suitable for any further manipulation. All the other concentrations shown in Table 2 have resulted in satisfactory electrospun meshes with visibly good sample homogeneity and structural stability. The M-PSI-AA10 meshes with the least AA closely resembled the unmodified PSI mesh. On the other hand, the M-PSI-AA1 meshes with the highest modification rate, were thinner and more inhomogeneous in structure, resembling a close-knit spider web. Despite the higher concentration, the solutions of PSI-AA1 had lower viscosity than those of PSI-AA10, due to the difference in their molecular weights. During the electrospinning of PSI-AA1, more dripping occurred, and the M-PSIAA1 meshes appeared to be weaker leading to the conclusion that either the fibers prepared were thinner and weaker or instead of fibers, spheres were produced. Later these speculations were verified using SEM and are discussed in Section 3.5. In general, all grafting degrees

provided meshes of better quality at higher solution concentrations and PSI-AA2 provided the most stable and robust meshes. 3.2. Crosslinking of PSI-AA meshes by plasma treatment PSI and PSI-AA meshes were subjected to low pressure plasma treatment in order to establish covalent bonds between the polymer chains through the pending allylamine groups. No visible macroscopic change occurred during the plasma treatment of the samples. To examine the outcome of the crosslinking attempt, plasma treated PSI and PSI-AA meshes (marked as pM-PSI and pM-PSI-AAx) were immersed in dimethyl sulfoxide (DMSO), a good solvent of PSI. pM-PSI meshes, lacking any covalent bonds, immediately dissolved in DMSO. pM-PSI-AAx meshes, on the other hand, did not dissolve in DMSO (Fig. 2, Videos 1 and 2) that proves the presence of crosslinks that maintain the gel structure and protect the meshes from dissolution. All the meshes were tested for solubility in DMSO before the plasma treatment as well (MPSI-AAx), and all of them proved to be soluble in DMSO. That shows on one hand, that the allylamine modification, in itself, is insufficient to crosslink the polymer chains, however, combined with the plasma treatment it is already capable of doing that. On the other hand, the persisting solubility of the unmodified PSI meshes shows, that the crosslinking cannot be attributed solely to the plasma treatment either since it is ineffective without the presence of allylamine groups on the polymer chain. 3.3. Chemical structure of PSI and PSI-AA meshes (FT-IR spectroscopy) The chemical structure of PSI and PSI-AA meshes was studied by Fourier Transform Infrared Spectroscopy (FT-IR). Detail can be found in the Supplementary information and Fig. S7. In short FT-IR proved the modification of PSI with AA with results in line with the literature. The FTIR spectra of the plasma-treated samples were identical to those of the untreated samples. However, the solubility tests have clearly shown that the treatment is effective in establishing crosslinks, meaning that some chemical change had to occur. A reason for that could be, that the crosslinking only occurs on the surface of the fibers, where the plasma can reach them and not in the core of the fibers [24]. However, the infrared light in ATR-FTIR penetrates inside the fibers, and as a result, the bulk of the fibers takes considerably larger part in peak formation than the surface region, which can lead to the IR spectrum missing the peaks related to crosslinks, even though other experiments point to their presence. Hence, further measurements had to be executed to investigate the chemical structure of the surface of the fibers (see Section 3.4). The plasma treatment acting only on the surface of the fibers has the obvious advantage that in the case of drug loaded fibers the drug molecules concentrating in the middle of the fibers are protected from the damage that might occur due to plasma treatment. 3.4. Surface analysis of PSI and PSI-AA meshes (XPS) As opposed to FTIR, XPS collects information from only the top 7–12 nm of the samples [25]. Therefore, it is possible to see the surficial changes in the samples, which were not visible on the FTIR spectra. XPS analysis was carried out on M-PSI, M-PSI-AA2 and pM-PSI-AA2 meshes.

Fig. 2. Submerging plasma treated PSI and PSI-AA meshes in DMSO.

K. Molnar et al. / Journal of Molecular Liquids 303 (2020) 112628

First survey spectra were collected, to screen for all the elements present in the samples and only peaks related to carbon, nitrogen and oxygen were found in the spectra (Fig. S8). To make further analyses about the changes on the types and ratios of the surface functional groups, high resolution C1s and O1s spectra were analyzed. C1s spectra are composed of 4 main peaks: C\\C, C_C, and C\\H that overlap at 284.8 eV; C\\N at 286 eV; C\\O at 288 eV and C(=O)-NH at 290 eV [26–28]. Peak areas are presented in Table 3. If we compare the spectra of M-PSI and M-PSI-AA2 (Fig. 3A and B), it can be seen that due to the modification with allylamine there were considerable changes in the spectrum. The amount of C\\C, C_C and C\\N bonds increased on the surface proving that grafting was successful, and the allyl groups are present on the surface. In contrast to this after plasma treatment (Fig. 3C) the percentage of C\\C, C_C and C\\H bonds, as well as C\\N bonds, are reduced, while C\\O bonds rose to 45%. This indicates that even though according to FTIR there were no changes upon plasma treatment in the chemical structure of the polymer in the bulk, there were changes on the surface resulting in crosslinks. The O1s spectra further support this finding as to the C\\O peak in M-PSI (Fig. 3D) and M-PSI-AA2 (Fig. 3E) has the proportion of 23.01% and 26.15% respectively, whereas in the pM-PSI-AA2 sample it rose to 48.4% (Fig. 3F) [11].

3.5. Morphology of PSI and PSI-AA meshes (SEM) SEM was used to study the fibrous structure of the prepared PSI and PSI-AA meshes (Fig. 4). In the case of M-PSI-AA1, entirely fibrous meshes could not be obtained regardless of the chosen concentration. Images were taken from both the M-PSI-AA1-35 and M-PSI-AA1-40 meshes (Fig. 4B, C) clearly show the presence of larger globular, or spindle-like structures interrupting the continuous cylindrical geometry of the fibers. The ratio of these objects compared to the fibers appears to be higher in the lower concentration ranges. This tendency can be observed throughout all the grafting degrees, and it is most likely caused by the difference in the viscosity of the polymer solutions. This observation is in accordance with the results of Fong et al. who examined the formation of beads as a function of the solution viscosity [29]. In the case of the M-PSI-AA2 meshes (Fig. 4D-F), the concentration of 45 w/w% already yielded an entirely fibrous structure with an average fiber diameter of 955 ± 39 nm. At lower concentrations, the abovementioned globular structures start to appear, which is accompanied by a visible decrease in the average diameter of the fibers. The M-PSIAA5 meshes (Fig. 4G-I) are similar to the M-PSI-AA2 meshes, and the mesh with the highest concentration appears to be completely fibrous with an average fiber diameter of 464 ± 18 nm. As we go towards the lower concentrations the globular to fibrous ratio appears to be higher than in the case of the M-PSI-AA2 meshes. The M-PSI-AA10 meshes (Fig. 4J-K) have a very limited concentration range where their electrospinning was feasible. The two samples show the same tendencies as observed above, only in this case, the fibrous character of the meshes seems to be more sensitive to the concentration change. As these meshes are the least modified compared to the others, a high similarity can be observed to the unmodified PSI mesh shown in Fig. 4A

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Fig. 3. High resolution C1s and O1s XPS spectra of M-PSI (A, D), M-PSI-AA2 (B, E) and pMPSI-AA2 (C, F).

both in regard to appearance and average fiber diameter of 344 ± 17 nm. As it was mentioned earlier, meshes were examined using SEM both before and after plasma treatment in search of any visible difference. After the inspection of multiple images, it has been concluded that there is no visible characteristic difference between the SEM images of plasma treated and untreated meshes. To demonstrate the above conclusion, an example is provided in Fig. 5, where the M-PSI-AA10-27.5 and pM-PSI-AA10-27.5 meshes are shown, where the latter was plasma treated for 13 min at 25 W.

3.6. Conversion of PSI meshes into PASP-AA meshes PSI undergoes hydrolysis in alkaline medium (pH N 7.4) resulting in water soluble polyaspartic acid (PASP). The plasma treated PSI-AA meshes were proven to be insoluble in DMSO, one of the good solvents of PSI, which proved the development of crosslinking bonds, but the reaction of the meshes in aqueous medium was yet to be tested.

Table 3 Collection of C1s and O1s component peak data. Component

C1s

O1s

Bonds

C-C; C-H; C=C C-N C-O C(=O)-NH C(=O)-NH C-O

Binding energy (eV)

Area (%) M-PSI

M-PSI-AA2

pM-PSI-AA2

285 286 288 290 529 531

25.63 25.30 35.01 14.06 76.99 23.01

28.70 40.45 26.58 4.27 73.85 26.15

21.54 25.80 45.12 7.54 51.60 48.40

Fig. 4. SEM image(s) of M-PSI (A), M-PSI-AA1-35 (B) M-PSI-AA1-40 (C), M-PSI-AA2-35 (D), M-PSI-AA2-40 (E) M-PSI-AA2-45 (F), M-PSI-AA5-25 (G), M-PSI-AA5-30 (H) M-PSIAA5-35 (I) M-PSI-AA10-25 (J) and M-PSI-AA10-27.5 (K).

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K. Molnar et al. / Journal of Molecular Liquids 303 (2020) 112628

Fig. 5. SEM images of M-PSI-AA10-27.5 (A), and pM-PSI-AA10-27.5 (B).

After the plasma treatment, the meshes were submerged into a phosphate buffer solution (PBS, pH = 7.4) for 24 h. After this submerging period, the samples were still visible and kept their integrity. Their appearance became almost completely transparent as they hydrolyzed into PASP meshes as can be seen in Fig. 6A. The stability of the meshes in aqueous medium shows that even after converting into PASP, which is a water-soluble polymer, the samples kept their gel-like structure due to the establishment of crosslinks during plasma treatment. SEM images were taken of the dry PASP-AA meshes in order to verify whether the meshes retained their fibrous structure. As it is shown in Fig. 6B-C, the fibrous structure was almost entirely lost, the fibers merged to form an apparently continuous polymer layer. This is not unprecedented in the literature as it is often the case for fiber-based polymer networks [14,30–32]. The loss of the fibrous structure as a result of the hydrolysis is not a preferable outcome since it is this structure that grants special properties to the meshes such as the high surface to weight ratio and the structural resemblance to the extracellular matrix of the human body. The exact mechanism behind the fusion of the fibers is unknown. It can be speculated that it is caused by the free allylamine side-groups that do not crosslink between chains. As the plasma treatment only affects the surface of the fibers, the underlying domains may have free allylamine side-groups and are not crosslinked at the end of the treatment. That means that the inside of the fibers is enclosed in a crosslinked and thus insoluble shell, which may be quite thin. If this shell breaks or allows the inner water-soluble section to partly migrate outwards forming irregular structures upon freeze drying, ultimately leading to the fusion of the fibers. In order to increase the stability of the PSI-AA fibrous structure and thus avoid the merging of the fibers during hydrolysis, the effectiveness of the plasma treatment must be enhanced. Hence, additional experiments have been conducted, where the parameters of plasma treatments were varied as described under Section 2.4.

Fig. 6. pM-PSI-AA meshes before hydrolysis in PBS (left), right after submersion in PBS (middle) and after 24 h (right) (A). The red circle marks the sample in the rightmost image. SEM images of pM-PSI-AA (B) and pM-PASP-AA meshes (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

M-PSI-AA2 meshes were used for these experiments, as they had the best consistency and structural stability during sample handling. First, we have assessed the effect of plasma treatment duration with a fixed performance of 25 W. The SEM images of the hydrolyzed and freeze-dried meshes along with the treatment durations are summarized in Fig. 7. Apparently, the increased plasma treatment duration benefits the retention of the fibrous structure up until a certain point. The samples treated for 13, 15.5 and 17 min (Fig. 7A-C) provided progressively better results with 17 min being the best, but the ones treated for 30 min (Fig. 7D) were already worse compared to the previous one. The sample treated for 30 min also showed a macroscopically visible burnt area, therefore further increasing the treating time seemed unnecessary. After that, we have repeated the plasma treatment using only treatment durations of 13 and 17 min but with power increased to 100 W (Fig. 7E and F). 17 min treatment at 100 W was found to be the optimal treatment duration. The fibers do deform to a certain extent, but their retained fibrous structure is closely comparable to that of the PSI-AA mesh before hydrolysis. Since in most of the cases involving hydrogel fibers there is an unavoidable but minimal merging of fibers, we can consider the 100 W and 17 min treatment an optimized sample preparation protocol [30,33]. 3.7. Sterility of the membranes Freshly plasma treated PSI-AA meshes were placed onto hard agar, then covered with soft agar in a sterile hood. The reason why we used this setup is that the soft agar overlay provides easier counting of the colonies as microorganisms can't migrate into the hard agar and under sterile circumstances, only microorganisms from the surface or from the inside of the meshes could grow. The addition of 1 mL M9 phosphate buffer allows the meshes to slightly swell and therefore bacteria could grow in the surrounding soft agar. After 24 h, all membranes swelled visibly (Fig. S9A), which means that hydrolysis to PASP-AA occurred. Out of 6 samples, 1 had several colonies growing at the edge of the Petri dish, far away from the samples. 4 samples had 1–3 colonies farther away and 1 sample had none. After 48 h there was no change in

Fig. 7. SEM images of hydrolyzed and freeze dried pM-PASP-AA meshes. A-D) Plasma treatment power: 25 W. Treatment durations: 13 min (A), 15.5 min (B), 17 min (C), 30 min(D), E-F) plasma treatment power: 100 W, treatment durations: 13 min (E) 100 W, 17 min(F).

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the number of colonies (Fig. S9B). Therefore, the plasma treatment proved to be an effective method not just for crosslinking but also for the sterilization of the samples. 4. Conclusion Fibrous water-soluble polymers are inadequate for implantation or any application where they get in contact with water, as they dissolve rapidly due to their high surface to volume ratio. Crosslinking the polymers, thus preparing a polymer network to prevent dissolution and thus upon contact with water the fibers instead of dissolving, just uptake fluid and form hydrogels. In the present article, we explored the possibility to utilize low pressure plasma treatment to induce crosslinking and sterilization at the same time at the final stage of the synthesis of fibrous membranes. We showed that the base polymer, polysuccinimide, cannot be crosslinked by plasma as it dissolved immediately after immersion into its solvent. However, after grafting pendant allyl groups onto the polymer and electrospinning, plasma treatment induced crosslinking reaction in the fibers. According to FTIR and XPS, the bulk of the fibers are not crosslinked, but the surface layer is. This could be very useful in drug delivery when there is a sensitive material trapped inside the fibers which could be sensitive to plasma treatment. Upon hydrolysis in pH = 7.4 polysuccinimide turns into water-soluble poly (aspartic acid). Although the plasma induced crosslinks stopped the membranes from dissolution, the fibers fused together after hydrolysis, overall for the membrane losing its fibrous properties. However, with optimizing the processing parameters, the fibrous structure was retained proving low pressure plasma treatment to be a feasible and useful tool for simultaneous crosslinking and sterilization. Supplementary data to this article can be found online at https://doi. org/10.1016/j.molliq.2020.112628. CRediT authorship contribution statement Kristof Molnar: Conceptualization, Investigation, Methodology, Writing - original draft. Benjamin Jozsa: Formal analysis, Investigation, Writing - original draft. Dora Barczikai: Methodology, Formal analysis. Eniko Krisch: Formal analysis, Writing - original draft. Judit E. Puskas: Formal analysis. Angela Jedlovszky-Hajdu: Supervision, Writing - original draft. Declaration of competing interest Authors declare that there is no conflict of interest. Acknowledgments The authors are grateful for the help of Surface Analysis Lab at The Ohio State University and NSF-DMR grant #0114098 for the XPS measurements. This work was funded by the National Research, Development and Innovation Office – NKFIH FK 124147, the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (AJH) and by the ÚNKP-19-4-SE-04 new national excellence program of the Ministry for Innovation and Technology. Start-up fund of The Ohio State University #11232011000-11-PUSKAS. References [1] A. Greiner, J.H. Wendorff, Electrospinning: a fascinating method for the preparation of ultrathin fibers, Angew. Chem. Int. Ed. 46 (2007) 5670–5703, https://doi.org/10. 1002/anie.200604646. [2] Q. Liu, J. Zhu, L. Zhang, Y. Qiu, Recent advances in energy materials by electrospinning, Renew. Sust. Energ. Rev. 81 (2018) 1825–1858, https://doi.org/ 10.1016/J.RSER.2017.05.281. [3] X. Hu, S. Liu, G. Zhou, Y. Huang, Z. Xie, X. Jing, Electrospinning of polymeric nanofibers for drug delivery applications, J. Control. Release 185 (2014) 12–21, https://doi. org/10.1016/j.jconrel.2014.04.018.

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