In Situ Cross-Linked Nanofibers by Aqueous Electrospinning of

Jul 17, 2018 - results. Regarding the thermal stability of the dynamic covalent diselenide bonds, the PEtOx-EI-Se2 membranes that were stored in the f...
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In Situ Cross-Linked Nanofibers by Aqueous Electrospinning of Selenol-Functionalized Poly(2-oxazoline)s Yin Li,† Maarten Vergaelen,‡ Xiangqiang Pan,*,§ Filip E. Du Prez,‡ Richard Hoogenboom,*,‡ and Karen De Clerck*,† †

Department of Materials, Textiles and Chemical Engineering, Ghent University, Technologiepark 907, 9052 Ghent, Belgium Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4, 9000 Ghent, Belgium § Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, Department of Polymer Science and Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China

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‡

S Supporting Information *

ABSTRACT: Poly(2-oxazoline)-based biomaterials have shown significant potential for various applications in the past decade. Herein, we present a methodology for the design of degradable diselenide-cross-linked nanofibers by aqueous electrospinning of selenol (SeH)-modified poly(2-ethyl-2oxazoline) (PEtOx). The selenol groups have been introduced into PEtOx by reacting partially hydrolyzed PEtOx (poly(2ethyl-2-oxazoline-)-co-ethylenimine (PEtOx-EI)) with γ-butyroselenolactone, whereby ring-opening upon reaction with the secondary amine groups in the polymer backbone yields selenol side chains. Subsequent aqueous electrospinning of the selenol-containing PEtOx was found to lead to in situ crosslinked water-stable nanofibers, ascribed to the formation of diselenide cross-links. The effects of changes in the experimental parameters and the influence of the selenium content on the electrospinning process were investigated in detail. Dynamic exchange between the remaining free SeH groups and diselenide bonds formed upon cross-linking enabled a tunable dissolution of the PEtOx-based nanofibers, which could be controlled by changing both the temperature and cross-linking density. Furthermore, the dissolution of the diselenide cross-linked nanofibers could also be induced by the exchange of diselenide groups under ultraviolet (UV) irradiation.

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called “stealth” behavior, and demonstrated the reproducibility as well as transferability of the electrospinning parameters.18 Poly(2-alkyl/aryl-2-oxazoline)s (PAOx) have a wide range of potential applications in many areas,19−22 especially in the biomedical field.23−25 The chemical tunability and potential of PAOx as an alternative to poly(ethylene oxide) (PEO) were already demonstrated in an earlier report by Hoogenboom et al.26 Additionally, functionalization of PEtOx, a crucial process to this study, is further achieved through acidic (or basic) hydrolysis of PEtOx revealing poly(2-ethyl-2-oxazoline)-co(ethylenimine) (PEtOx-EI) statistical copolymers (Scheme 1a), which may serve as a broader functionalization platform toward side-chain modification.22,27 Although our group and others have successfully prepared electrospun PEtOx nanofibers,18,28,29 the control over the dissolution of these nanofibrous materials is nonexisting since the obtained fibers consist of highly water-soluble PEtOx polymers. Despite the existence of well-known methods to achieve cross-linking of

INTRODUCTION

Electrospinning is an inexpensive and reliable technique to create nanometer- to micrometer-sized fibers for various applications. Nonwoven fabrics or membranes comprising electrospun fibers have good mechanical properties, ease of functionalization for various purposes, and a large specific surface area.1−4 In recent years, electrospinning applications could be found in preparation of materials for filtration,5,6 protective textiles,7,8 sensors,9,10 and nanofiber-reinforced composites.11−13 Electrospinning has also attracted a strong and rapidly growing interest from researchers in biotechnology and medicine for the development of e.g. nanofibrous materials for medical implants, drug delivery, wound dressings, dental materials, and enzyme immobilization scaffolds.14−17 However, many electrospinning processes are conducted in organic solvents, which are mostly toxic or carcinogenic. In a biomedical context, polymer processing avoiding harmful organic solvents and sophisticated operations is highly desirable. In previous work, we reported the electrospinning of aqueous solutions of poly(2-ethyl-2-oxazoline) (PEtOx), a water-soluble polymer with good biocompatibility and so© XXXX American Chemical Society

Received: May 27, 2018 Revised: July 17, 2018

A

DOI: 10.1021/acs.macromol.8b01113 Macromolecules XXXX, XXX, XXX−XXX

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Scheme 1. (a) Partial Hydrolysis of PEtOx To Form PEtOx-EI; (b) Reaction of PEtOx-EI with γ-Butyroselenolactone To Form Poly(2-ethyl-2-oxazoline)-co-ethylenimine (PEtOx-EI-SeH) and Its Subsequent Cross-Linking in Air into PEtOx-EI-Se2

water-stable nanofibers,30−32 up to date nobody reported the formation of cross-linked PEtOx nanofibers based on aqueous electrospinning. Recently, we developed water-stable PEtOx nanofibers via in situ photoinitiated radical thiol−ene crosslinking during electrospinning from DMF/THF solution.33 However, this cross-linking method was found to be noncompatible with an aqueous electrospinning process. Therefore, in this study, we attempt to produce cross-linkable PEtOx nanofibers that are electrospinnable from water and that feature postelectrospinning tunable redissolution. For this purpose, we investigated the use of selenol chemistry, which has similar chemical properties to thiol− disulfide-based chemistry, but with higher reactivity, to create cross-linkable electrospun fibers using selenol-containing polymers that are cross-linked with diselenide bonds (Scheme 1b). The advantages of diselenide bonds have been showcased in many studies. First, diselenide bonds are formed very easily, and the cross-linking process occurs under much milder conditions than disulfide cross-linking methods. Xu et al. found that diselenide bonds are dynamic covalent bonds and can undergo a dynamic exchange reaction under mild conditions.34 In addition, compared to disulfide bonds, diselenide bonds have lower bond energy (diselenide bonds: 172 kJ mol−1; disulfide bonds: 240 kJ mol−1).35 Moreover, because of their rapid response to external triggers, such as light or redox environment, reversible diselenide metathesis can be easily controlled, although rearrangements can alter the originally obtained cross-linked network. Very recently, γ-butyroselenolactone was used to produce diselenide-containing polymer networks in which reversible diselenide metathesis could be controlled by UV light.36 Thus, introducing diselenide bonds onto nanofibers is expected not only to improve the properties of PEtOx electrospun fibers but also to endow those nanomaterials with new functions, such as tunable water solubility. The ultimate aim of this study is the design of water-stable, cross-linked electrospun nanofibers from PEtOx-EI copolymers by using only aqueous solutions and to explore the potential of selenol-containing copolymers for preparing cross-linked

nanofibers. Because the diselenide bonds can be easily generated in air, the cross-linking process can occur during the electrospinning process. Thus, our research is expected to produce uniform and smoothly cross-linked electrospun nanofibers. Moreover, because of the reversible of diselenide bonds, the cross-linked nanofibers will exhibit tunable water solubility that will be useful for biomedical applications such as controlled drug delivery and release, which is, however, beyond the scope of this report.

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EXPERIMENTAL SECTION

Materials. γ-Butyroselenolactone was synthesized according to protocols described in the literature.37 Poly(2-ethyl-2-oxazoline) (PEtOx, i.e., Aquazol) (average Mw = ∼200000, PDI = 3−4) was supplied by Polymer Chemistry Innovations, Inc. (Tucson, AZ). Deionized water (Laborpure, Behr Labor Technik) was used as solvent for the electrospinning solutions and for fiber solubility tests. Dichloromethane (DCM) (99.8%), hydrochloric acid (37%), sodium hydroxide (≥90%), toluene (99.9%), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (99%), and ethylene glycol methyl ether acrylate (98%) were obtained from Sigma-Aldrich and used as received. Synthesis of Poly(2-ethyl-2-oxazoline)-co-ethylenimine (PEtOx-EI). PEtOx (50 g) was dissolved in 500 mL of deionized water, to which 50 mL of HCl (37%) was then added. Under a nitrogen atmosphere, the solution was stirred for 2 h at 75 °C. Subsequently, the pH value of the solution was adjusted to be higher than 9 with NaOH solution (6 mol L−1) to ensure full deprotonation of the released propionic acid. The neutralized solution was dialyzed in deionized water for 3 days and freeze-dried to give pure PEtOx-EI. The degree of the hydrolysis of PEtOx was determined by 1H nuclear magnetic resonance (NMR) spectroscopy to be 10 mol %, calculated by the integral ratio between the peaks at 2.6−2.8 ppm (PEI backbone) and 3.2−3.5 ppm (PEtOx backbone).20 Synthesis of Selenol-Functionalized Poly(2-ethyl-2-oxazoline)-co-ethylenimine (PEtOx-EI-SeH). PEtOx-EI (1.5 g) was dissolved in 3.5 mL of deionized water, and then 45.2 mg of γbutyroselenolactone was added. The solution was stirred at 50 °C under an argon atmosphere for 4 days. The polymer (PEtOx-EI-SeH) solution was used directly for electrospinning without further purification. Conversion of selenolactone was determined with 1H NMR analysis by comparing the integrated areas of characteristic signals of selenolactone (CH2, 1.91 ppm) and PEtOx-EI (CH3, 0.97 ppm). The extent of γ-butyroselenolactone functionalization of the B

DOI: 10.1021/acs.macromol.8b01113 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules polyoxazoline was 2, 4, 5, and 6 mol % when 45.2, 90.4, 113, and 135.6 mg of γ-butyroselenolactone were used, respectively. Electrospinning. The PEtOx and PEtOx-EI solutions for electrospinning were prepared by dissolving the polymers in deionized water and stirring at room temperature for 24 h. The PEtOx-EI-SeH polymer solutions are transferred into the syringe pump for electrospinning directly after preparation. To prevent cross-linking of the solution before electrospinning, the solutions were prepared under an argon atmosphere. The electrospinning experiments were conducted on a mononozzle setup. The separation distance between the needle tip and the collector was set at 15 cm, and the flow rate was set at 0.5 mL h−1. To obtain a stable Taylor cone, the voltage was adjusted between 15 and 20 kV. The electrospinning process was performed in a climate chamber to maintain a relative humidity of 30% and a constant temperature of 23 °C. The nanofibers resulting from spinning the 2, 4, 5, and 6 mol % PEtOx-EI-SeH solutions are termed 2, 4, 5, and 6 mol % PEtOx-EI-Se2 fibers, respectively. Characterization. All 1H NMR spectra were recorded in D2O on a Bruker Avance 400 at 400 MHz. Chemical shifts (δ) are presented in parts per million (ppm) relative to D2O (4.70 ppm in 1H NMR) as an internal standard. The viscosity of PEtOx, PEtOx-EI, and PEtOxEI-SeH solutions was characterized by an MCR302 rheometer (Anton Paar), equipped with a Paar Physica Viscotherm VT2. All the measurements used a lower measuring plate (area diameter 25 mm) and a parallel plate (length 100 mm). The tests were performed with a shear rate ranging from 1 to 10 s−1, and the temperature was set at 25 °C. The morphology and diameters of the nanofibers were analyzed by a scanning electron microscope (SEM, FEI Quanta 200F). Prior to SEM analysis, a gold coating was applied onto the samples using a sputter coater (Balzers Union SKD030). The nanofiber diameters were determined via ImageJ software, and the average fiber diameter and standard deviations were calculated from the SEM images by analyzing at least 50 fibers. The PEtOx-EI-Se2 nanofibrous membranes were cut into 1 × 3 cm2 pieces and immersed in 3 mL of Milli-Q water at 23 and 37 °C. After different time intervals 0.5 mL samples were taken from the solution and analyzed by dynamic light scattering (DLS), which was performed on a Zeta Sizer Nano device (Malvern) using 1.1 mL polystyrene cuvettes. To maintain a constant volume, the same amount of Milli-Q water was added back to the solution. The temperature was equilibrated at 25 °C, and the results were reported based on the average of at least five measurements. UVirradiations occurred in a Metalight Classic from Primotec, with 12 double 365 nm UV lamps of 9 W each (intensity measured in the middle, 5 mW cm−2). The visible light irradiations were performed in the lab without any dedicated device and can be regarded as standard tungsten lamp irradiation. Fiber Solubility Test. The PEtOx-EI-Se2 nanofibrous membranes were cut into samples with a size of 1 × 3 cm2 and immersed in 3 mL of deionized water at 4, 23, 37, or 50 °C for different periods of time to test their solubility.

Figure 1. Representative SEM images of PEtOx fibers: (a) PEtOx, (b) PEtOx-EI, (c) PEtOx-EI-Se2 with 2 mol % SeH, (d) PEtOx-EI-Se2 with 4 mol % SeH, (e) PEtOx-EI-Se2 with 5 mol % SeH, and (f) and PEtOx-EI-Se2 with 6 mol % SeH. All polymer solutions were electrospun from 30 wt % solutions in deionized water.

butyroselenolactone compared to that of thiolactone could result in a successful reaction. As shown in Scheme 1, the selenol groups were introduced onto the backbone of PEtOx-EI copolymers through aminolysis of γ-butyroselenolactone with the secondary amines of the PEI units in the backbone. The reaction occurs in deionized water, and the prepared PEtOx-EI-SeH polymers show good water solubility as long as they were shielded from oxygen by preparation under an argon atmosphere. Exposure of aqueous PEtOx-EI-SeH solutions to the air was found to lead to formation of insoluble cross-linked polymer networks, which is ascribed to deselenide formation, and the resulting diselenide cross-linked polymers are termed PEtOx-EI-Se2. When shielded from the air, the PEtOx-EI-SeH solutions are readily applicable to the aqueous electrospinning process while exposure of the PEtOx-EI-SeH copolymer chains to air during electrospinning process leads to in situ cross-linking; that is, the obtained nanofibrous are stable against water directly after the electrospinning process. The large specific surface area of the electrospun fibers is hypothesized to lead to fast and efficient diselenide formation. In contrast to other cross-linking methods,33 the conditions for PEtOx-EI-SeH cross-linking are very straightforward as no additives are needed nor are pretreatment and follow-up treatments, which is favorable for the large-scale industrial production of such nanofiber networks. To study the influence of the γ-butyroselenolactone functionalization on the electrospinning process, the degree of partial hydrolysis of PEtOx was kept constant at approximately 10 mol % according to the repeat units; i.e., on average one out of 10 repeat units is hydrolyzed, as confirmed by 1H NMR spectroscopy (Figure S1). Subsequently, PEtOx-EI-SeH polymers were prepared containing 2, 4, 5, and 6 mol % γ-butyroselenolactone content that still have 8, 6, 5, or 4 mol % remaining EI units, respectively. The viscosity range of PEtOx, PEtOx-EI, and PEtOx-EI-SeH solutions was determined to vary between 2000 and 5000 mPa·s (Figure S2). These measurements show that the viscosity decreases after hydrolysis of PEtOx to PEtOx-EI copolymers due to increasing chain flexibility resulting from removal of some rigid amide units, whereas an increase in the viscosity is noticed when the PEtOx-EI copolymers were functionalized with γ-butyroselenolactone that reintroduces the amide-containing 2-oxazoline repeat units. As a result, a

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RESULTS AND DISCUSSION Preparation of PEtOx-EI-Se2 Nanofibers with Tunable Solubility in Water. In line with the results of our previous study,18 uniform PEtOx fibers (Figure 1a) were successfully prepared by electrospinning an aqueous 30 wt % polymer solution. In addition, the hydrolyzed PEtOx-EI copolymers were also well electrospinnable (Figure 1b). However, as expected, the resulting PEtOx and PEtOx-EI nanofibers immediately dissolved when submersed in water. To develop water-stable PEtOx nanofibers with tunable solubility, γbutyroselenolactone was added to the PEtOx-EI copolymers to introduce selenol side chains aiming for PEtOx-EI-Se2 nanofibers. It is important to note that initial preliminary attempts to react thiolactone with PEtOx-EI in water did not result in ring-opening of the thiolactones, most likely due to the steric bulk around the secondary amines in the PEtOx-EI. It was hypothesized that the higher reactivity of γC

DOI: 10.1021/acs.macromol.8b01113 Macromolecules XXXX, XXX, XXX−XXX

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that the water-resistance test occurred at room temperature and shielded from any incoming light. Thus, the rearrangement of diselenide bonds can be ignored within the short time frame. The different cross-linking density is considered to be the principal reason for the different swelling behavior of the crosslinked fibers. Influence of Temperature and Cross-Linking Density on PEtOx-EI-Se2 Solubility. Because of the dynamic behavior of diselenide bonds,38,39 reversible metathesis may occur between polymer chains when the fiber is soaked in water, ultimately resulting in dissolution of the cross-linked structure. Furthermore, the presence of residual SeH groups may induce reshuffling of the diselenide bonds inducing dissolution of the fibers. A nanometer-sized gel is anticipated to be formed and released in solution based on these dynamic exchange reactions, eventually resulting in complete dissolution of the PEtOx-EI-Se2 fibers (Scheme 2). To evaluate the fiber solubility behavior, 2, 4, and 5 mol % PEtOx-EI-Se2 fibers were immersed in deionized water for 1 h to 28 days (Table 1). All the samples for these dissolution studies were covered with aluminum foil to eliminate the impact of UV light on the results. Regarding the thermal stability of the dynamic covalent diselenide bonds, the PEtOx-EI-Se2 membranes that were stored in the fridge (4 °C) were found to be more stable than those kept at room temperature (23 °C). As shown in Table 1 and Figure 3, the PEtOx-EI-Se2 nanofibrous membranes slowly dissolved over time, even at low temperature. In addition, it is observed that higher temperatures accelerate the dissolution process. These findings demonstrate that the dynamic exchange reaction of the diselenide bonds in the electrospun fibers can occur under mild conditions. The 5 mol % PEtOxEI-Se2 membrane was found to maintain its shape after 2 weeks in the fridge, whereas the 2 mol % PEtOx-EI-Se2 membrane dissolved within 7 days in the fridge. It is evident that nanofibers with a higher diselenide cross-linking density will be more stable as more diselenide exchange reactions need to occur to fully release nanogel structures from the fibers. Furthermore, an increase in solution temperature will accelerate the diselenide exchange reactions leading to faster fiber dissolution. The results listed in Table 1 clearly demonstrate that the dissolution time of the PEtOx-EI-Se2 fibers can be controlled by changing the temperature and cross-linking density. Blocking of the Free Selenol Groups in PEtOx-EI-Se2 Nanofibrous Networks To Prepare Water-Stable PEtOx Nanofibers. As indicated in the previous paragraph, the dissolution of the diselenide cross-linked PEtOx-EI-Se2 membranes can be ascribed to either diselenide metathesis or SeH inducing diselenide reshuffling reactions. Because of the fast electrospinning process, we assume that not all the SeH groups can be transformed into diselenide bonds, leaving some remaining groups inside the nanofiber. These free SeH groups are expected to be responsible for the dissolution of the nanofibrous networks, when in contact with water. To verify this assumption, the presence of free SeH groups within the PEtOx-EI-SeH polymers was investigated with Ellman’s reagent, a well-known detection reagent for free thiols and selenols.40 Therefore, Ellman’s reagent was added into a solution of PEtOx-EI-SeH, under an argon atmosphere, and after 30 min the color of the polymer solution changed from light yellow to dark yellow, which showed that free SeH groups were indeed still present as expected. However, upon addition

gradual increase in viscosity was noticed with increasing SeH amount in the polymer solution. Meanwhile, the SeH content drastically influences the optimal conditions for the electrospinning process of the PEtOx-EI-SeH copolymers, revealing it as one of the important parameters for achieving stable and reproducible nanofiber production. Uniform nanofibers were found to be accessible (Figure 1c−e) by electrospinning of 30 wt % aqueous solutions of PEtOx-EI-SeH with up to 5 mol % SeH contents while a SeH content of 6 mol % or higher resulted in too elevated viscosity, which made the electrospinning process unstable, at least at 30 wt % polymer concentration. The resulting PEtOx-EI-SeH 6% fibers were difficult to collect, and a 5-fold increase in the diameters was observed (Figure 1f). It is expected that this can be remediated by lowering the solution concentration of the copolymer. Figure 1 shows SEM images of the electrospun PEtOx fibers, PEtOx-EI fibers, and PEtOx-EI-Se2 fibers based on PEtOx-EISeH with different SeH contents. The PEtOx-EI-Se2 fibers exhibit a cylindrical shape, a smooth surface, and a uniform size, which are similar to the morphology of the PEtOx fibers. Partial hydrolysis of the PEtOx reduces the resulting nanofiber diameter significantly due to the decreased solution viscosity at 30 wt %. After modification of the PEtOx-EI copolymers with γ-butyroselenolactone, increasing the SeH group content was found to result in a larger diameter of the PEtOx-EI-Se2 fibers, again correlated to the solution viscosity. The results obtained from the SEM images (Figure 1c−e) showed that the diameters of the 2, 4, and 5 mol % PEtOx-EI-Se2 fibers are 865 ± 139, 995 ± 99, and 1289 ± 190 nm, respectively, which are in line with the observed viscosity increases of the corresponding polymer solutions. For water-resistance tests, the PEtOx, PEtOx-EI, and PEtOxEI-Se2 nanofibrous membranes were immersed in deionized water for 10 min and then dried at room temperature. The results show that within a few seconds both the PEtOx and PEtOx-EI nanofibers fully dissolved in water. In comparison, complete PEtOx-EI-Se2 membranes could be observed after the same treatment, which confirmed that the PEtOx-EI-Se2 fibers were successfully cross-linked during the electrospinning process. Figure 2 presents the fiber morphologies of the

Figure 2. Cross-linked electrospun PEtOx-EI-Se2 fibers after a waterresistance test: (a) 2 mol % PEtOx-EI-Se2, (b) 4 mol % PEtOx-EI-Se2, and (c) 5 mol % PEtOx-EI-Se2.

samples after the 10 min water-resistance tests. Water uptake resulted in swelling of the cross-linked PEtOx-EI-Se2 fibers compared to the original fibers (Figure 1c). After the waterresistance test, a significant variation was noticed in the morphology of the 2 mol % PEtOx-EI-Se2 fiber (Figure 2a). On the other hand, the 4 mol % PEtOx-EI-Se2 fibers (Figure 2b) and 5 mol % PEtOx-EI-Se2 fibers (Figure 2c) did not significantly change their structure after water exposure. Therefore, based on the SEM results, the cross-linking density of 2 mol % PEtOx-EI-Se2 nanofiber is considered to be insufficient for obtaining water-stable fibers. It should be noted D

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Scheme 2. Proposed Formation and Release of Nanometer-Sized Gel Particles from the PEtOx-EI-Se2 Fibers Resulting from Dynamic Exchange of the Covalent Diselenide Bonds

Table 1. Solubility Test of 2, 4, and 5 mol % SeH Groups of PEtOx-EI-Se2 Membranes and 4 mol % Blocked PEtOx-EI-Se2 Membrane at Different Temperaturesa 2 mol % PEtOx-EI-Se2 1h 3h 6h 12 h 1 day 2 days 3 days 4 days 7 days 9 days 14 days 28 days

4 mol % PEtOx-EI-Se2

5 mol % PEtOx-EI-Se2

4 mol % blocked PEtOx-EI-Se2

4 °C

23 °C

37 °C

50 °C

4 °C

23 °C

37 °C

50 °C

4 °C

23 °C

37 °C

50 °C

23 °C

+ + + + + + + + − − − −

+ + + + + − − − − − − −

+ + + − − − − − − − − −

+ − − − − − − − − − −

+ + + + + + + + + + − −

+ + + + + + − − − − − −

+ + + + − − − − − − − −

+ + − − − − − − − − − −

+ + + + + + + + + + + −

+ + + + + + + + + − − −

+ + + + + + − − − − − −

+ + + − − − − − − − − −

+ + + + + + + + + + + +

All samples were shielded from incoming light. +: membrane samples remained after the period of soaking. −: membrane samples totally dissolved in water. a

stability tests in water revealed that the resulting cross-linked and blocked PEtOx-EI-Se2 nanofibers did not dissolve in water, even after 4 weeks at 23 °C (Table 1 and Figure S4), confirming that indeed the free SeH groups are responsible for the dynamic diselenide exchange reactions and the accompanied dissolution of the PEtOx-EI-Se2 fibers. DLS measurements were performed to further investigate the dissolution of the nonblocked (as obtained) and blocked PEtOx-EI-Se2 fibers at different time scales (Table 2, Figures S5 and S6). The nonblocked PEtOx-EI-Se2 nanofibers, which showed a slow dissolution process, resulted in the formation of nanosized polymer aggregates in solution as observed by DLS. The size of the polymer residue leaching out of the cross-

Figure 3. Slow dissolution of 4 mol % PEtOx-EI- Se2 membrane at 23 °C with increasing immersing times: (a) 0, (b) 24, and (c) 48 h as illustrative examples.

Table 2. Size (nm) and Polydispersity Index (PDI) as Function of Time Obtained by DLS Measurements of the Solutions in Which 4 mol % PEtOx-EI-Se2 Fiber and 4 mol % Blocked PEtOx-EI-Se2 Fiber Were Dispersed at Room Temperature

of Ellman’s reagent to a solution containing dispersed PEtOxEI-Se2 nanofibers, the white cross-linked nanofibers also turned yellow (Figure S3) in 30 min, indicating that the fibers indeed contain free SeH groups (Scheme S1). To verify whether these free SeH groups in the nanofibers are responsible for the dissolution and to potentially further stabilize the nanofibers, we attempted to block the free SeH groups inside the nanofibers. Therefore, the free selenol groups in the PEtOx-EISe2 fibers were successfully modified (blocked) via Michael addition with ethylene glycol methyl ether acrylate.41 The

samples

60 min

4 mol % PEtOx-EI-Se2 125 nm (PDI 0.296) 4 mol % blocked 0.66 nm PEtOx-EI-Se2 (PDI 0.419) E

1 day

3 days

177 nm (PDI 0.275) 0.82 nm (PDI 0.652)

330 nm (PDI 0.460) 0.92 nm (PDI 0.619)

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Macromolecules linked nanofibers was determined to be approximately 125 nm after 60 min and increased to 177 nm after 1 day and to 330 nm after 3 days when the tested membrane was visually fully dissolved. Thus, the DLS observations confirm the dissolution of the polymer from the PEtOx-EI-Se2 fibers, which may be anticipated to be nanogels resulting from SeH-diselenide exchange reactions as the diselenide bonds do not readily hydrolyze under these conditions. All samples show rather high polydispersity values, indicating that there is a broad distribution of particle sizes in solution. On the contrary, the blocked nanofibers did not show the presence of polymer aggregates in the DLS measurements, as no accumulation of dissolved polymer chains can occur due to a rearrangement of cross-links since all selenol groups were blocked (Table 2). The SEM results are consistent with the results from the solubility test and DLS analysis. Figure 4 shows the fiber

Table 3. Solubility Test of the 2, 4, and 5 mol % PEtOx-EISe2 Membranes and 4% Blocked PEtOx-EI-Se2 Membrane under Visible Light and UV Light Irradiation at 23 °Ca 2 mol % PEtOx-EI-Se2

4 mol % PEtOx-EI-Se2

5 mol % PEtOx-EI-Se2

4 mol % blocked PEtOx-EI-Se2

visible light

UV

visible light

UV

visible light

UV

visible light

UV

+ + + + + − − − − −

+ − − − − − − − − −

+ + + + + + − − − −

+ + − − − − − − − −

+ + + + + + + + + −

+ + + − − − − − − −

+ + + + + + + + + +

+ + − − − − − − − −

5 min 10 min 15 min 30 min 1 day 2 days 3 days 4 days 7 days 14 days

a +: membrane samples remain after the period of soaking; −: membrane samples totally dissolved in water.

days to even a few minutes. The UV light can be absorbed by the diselenide groups and offers sufficient energy to speed up the reversible diselenide metathesis process. This is true for both the PEtOx-EI-Se2 nanofibers still containing free SeH groups as well as for the nanofibers for which the SeH groups were blocked. The results are further analyzed by DLS measurements of the aqueous solution in which the fibers were dispersed (Table 4, Figures S7 and S8). After UV irradiation for 5 min, both the Table 4. Size (nm) and Polydispersity Index (PDI) as a Function of Time Obtained by DLS Measurements of the Solutions in Which 4 mol % PEtOx-EI-Se2 Fiber and 4 mol % Blocked PEtOx-EI-Se2 Fiber Were Dispersed under UV Light Irradiation samples

Figure 4. SEM images of 4 mol % blocked PEtOx-EI-Se2 fiber soaked water for (a) 30 min, (b) 3 days, (c) 7 days, and (d) 28 days.

5 min

4 mol % PEtOx-EI-Se2 69 nm (PDI 0.493) 4 mol % blocked 78 nm PEtOx-EI-Se2 (PDI 0.244)

morphologies of the 4 mol % blocked PEtOx-EI-Se 2 membranes before and after soaking in deionized water. After immersion in water for 30 min, as shown in Figure 4a, the morphology of the 4 mol % PEtOx-EI-Se2 nanofibers was well retained. When the blocked membrane was immersed in deionized water for 3 days (Figure 4b), the fiber continued to swell and the porosity reduced significantly but the fibrous nature remains. Finally, when the immersion time was prolonged to 28 days, the fibrous structure is still observed. It is clear that after blockage of the SeH groups in the fibers the dynamic exchange between the free SeH groups and diselenide bonds was prevented. UV Treatment To Control the Solubility of Nanofibers. Diselenide bonds are known to be photoresponsive,38 suggesting light as another stimulus to control the dissolution of the PEtOx-EI-Se2 nanofibers. Visible light and UV light are chosen to evaluate the nanofiber’s photoresponsiveness, and the results are illustrated in Table 3. The effect of visible light on the dissolution process is negligible as expected because the diselenide bonds do not absorb visible light. On the other hand, UV light noticeably accelerated the dissolution process, reducing the timespan to fully dissolve the membrane from

15 min

30 min

340 nm (PDI 0.518) 271 nm (PDI 0.316)

182 nm (PDI 0.314) 256 nm (PDI 0.288)

4 mol % PEtOx-EI-Se2 nanofibers and the blocked nanofibers showed an average diameter of 69 and 78 nm, respectively, indicating that UV irradiation caused the nanofibers to partially dissolve in a shorter time. Moreover, during the UV-induced dissolution process, larger aggregates were found in the deionized water after 15 min, which may be attributed to the faster UV-induced diselenide exchange reactions leading to the release of larger kinetically formed nanogels. Finally, the nanofibers totally dissolved in water and the average diameter of the nanogels in solution was found to be 182 and 256 nm for the PEtOx-EI-Se2 nanofibers and blocked nanofibers, respectively, indicating that the larger kinetically formed nanogels further exchanged and fragmented in solution to the smaller nanogels.

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CONCLUSIONS In summary, PEtOx-EI copolymers modified with reactive selenol groups were developed and used for waterborne electrospinning. Stable and reproducible cross-linked nanofibers were obtained using PEtOx-EI-SeH aqueous solution F

DOI: 10.1021/acs.macromol.8b01113 Macromolecules XXXX, XXX, XXX−XXX

Macromolecules

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without any other required processing step. The morphology and diameter of the PEtOx-EI-Se2 nanofibers could be controlled by adjusting the selenol group content when keeping the polymer solution concentration constant. With a higher content of selenol groups, the cross-linking density increased, and the PEtOx-EI-Se2 nanofibers were found to be more stable in water for a longer time. Because of the exchange reaction between SeH groups and diselenide bonds, the (slow) dissolution of the PEtOx-EI-Se2 nanofibers could be controlled by temperature and cross-linking density. Furthermore, UV treatment could be used for faster controlled dissolution of the nanofibers. Through blockage of the remaining SeH groups inside the cross-linked fiber, a photoresponsive nanofiber could be obtained without any diselenide exchange originating from the reaction of any free selenol groups. These dissolution results could be confirmed by DLS and SEM measurements. Finally, we believe that the developed cross-linked PEtOx-EISe2 nanofibers have potential for a variety of applications, such as for medical sutures, pharmaceutical therapy, and controlled release techniques in medicine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b01113. 1

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Article

H NMR spectra of PEtOx-EI and PEtOx-EI-SeH; viscosity versus shear rate graphs of PEtOx, PEtOx-EISeH, and PEtOx-EI; PEtOx-EI-Se2 fiber color change in DTNB solution; 4 mol % blocked PEtOx-EI-Se 2 membrane immersed in water for prolonged times; determination of the free selenols in PEtOx-EI-Se2 fibers using DTNB; effect of time on DLS measurements of the 4 mol % PEtOx-EI-Se2 fiber, blocked PEtOx-EI-Se2 fiber, and these fibers with UV irradiation (DOCX)

AUTHOR INFORMATION

Corresponding Authors

*(K.D.C.) E-mail [email protected], Tel +3292645740, Fax +3292645846. *(R.H.) E-mail [email protected], Tel +3292644482, Fax +3292644998. *(X.Q.P.) E-mail [email protected], Tel +8651265883343, Fax +8651265882787. ORCID

Filip E. Du Prez: 0000-0001-7727-4155 Richard Hoogenboom: 0000-0001-7398-2058 Karen De Clerck: 0000-0002-5650-3075 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Y.L. acknowledges the financial support of the China Scholarship Council (201308410056). X.Q.P. thanks the National Science Foundation of China (21774080 and 21302132), the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Suzhou Key Lab of Macromolecular Design and Precision Synthesis. F.D.P. and R.H. thank BOF-UGent (GOA-funding). K.D.C. thanks BOF-UGent (BAS-funding). G

DOI: 10.1021/acs.macromol.8b01113 Macromolecules XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.macromol.8b01113 Macromolecules XXXX, XXX, XXX−XXX