Stimuli-responsive electrospun nanofibers based on PNVCL-PVAc copolymer in biomedical applications

Graphical abstract

Poly(N-vinylcaprolactam) (PNVCL) is a suitable alternative for biomedical applications due to its biocompatibility, biodegradability, non-toxicity, and showing phase transition at the human body temperature range. The purpose of this study was to synthesize a high molecular weight PNVCL-PVAc thermo-responsive copolymer with broad mass distribution suitable for electrospun nanofiber fabrication. The chemical structure of the synthesized materials was detected by FTIR and ¹HNMR spectroscopies. N-Vinyl caprolactam/vinyl acetate copolymers (159,680 molecular weight (g/mol) and 2.51 PDI) were synthesized by radical polymerization. The phase transition temperature of N-vinyl caprolactam/vinyl acetate copolymer was determined by conducting a contact angle test at various temperatures (25, 26, 28, and 30 ${}^{\circ }\mathrm{C}$). The biocompatibility of the nanofibers was also evaluated, and both qualitative and quantitative results showed that the growth and proliferation of 929L mouse fibroblast cells increased to 80% within 48 h. These results revealed that the synthesized nanofibers were biocompatible and not cytotoxic. The results confirmed that the synthesized copolymers have good characteristics for biomedical applications.

Introduction

Smart polymeric materials have been found intriguing for researchers over the last two decades owing to the fact that some of their chemical and physical properties change by external stimuli, which is generally controlled by the ratio of hydrophilic to hydrophobic groups on the polymer chains. Smart polymers are divided into two main groups as natural and synthetic polymers. These polymers respond to external and internal stimuli like magnetic field, light, temperature, electric field, pH, ion strength and etc. Smart polymers have been used in medical fields, textile, solar cell, aerospace, and other industries (Hu et al. 2012; Shimada and Maruyama 2013; Puoci 2015; Teotia et al. 2015; Wang et al. 2016a; Lubben et al. 2018; Palza et al. 2019; Heggannavar et al. 2019; Sponchioni et al. 2019).

Lower critical solution temperature (LCST) or upper critical solution temperature (UCST) of polymers may change by copolymerization, application of co-solvents, surfactants and salts with great impact on the interaction between polymer chains and water molecules(Reis et al. 2006; Lue et al. 2011; Gandhi et al. 2015; Zhu et al. 2016; de Oliveira et al. 2017; Zhang et al. 2018; Käfer et al. 2018). This phenomenon turns the hydrophilic interactions into hydrophobic interactions above the LCST, leading to phase separation and precipitation of polymer in water (Zuo et al. 2020). Smart polymers show an abrupt change in phase transition temperature, which depends on the system entropy and free energy (Patterson 1972; Ma and Yes 2001; Teotia et al. 2015; Bruschi et al. 2017; Sponchioni et al. 2019).

Poly (N-vinyl caprolactam) (PNVCL) is a synthetic, amphiphilic, biocompatible, and non-toxic polymer with LCST around 30–50 ${}^{\circ }\mathrm{C}$, which is fully reversible and depends on the molecular weight and concentration of the polymer. This polymer is also soluble in both polar and non-polar solvents such as dimethylformamide, alcohol, tetrahydrofuran, and water (below LCST) (Ponce-Vargas et al. 2013; Sugawara and Nikaido 2014; Gandhi et al. 2015; Mohammed et al. 2018).

Through copolymerization, introducing hydrophobic polymeric segments into the polymer chains would reduce LCST and increase of UCST in the final synthesized copolymer, whereas using a hydrophilic polymeric segment increases the LCST and reduces the UCST (Ponce-Vargas et al. 2013; Moghanizadeh-Ashkezari et al. 2019).

Poly(N-vinyl caprolactam) shows the solution phase transition at 30–50 ${}^{\circ }\mathrm{C}$, so the copolymerization of NVCL with VAc would change the solution phase transition temperature. The synthesized PVAc-b-PNVCL copolymer by Marie Hurtgen and co-workers with cobalt-mediated radical copolymerization showed phase transition temperature of 26.5–29.5 ${}^{\circ }\mathrm{C}$ and maximum molecular weight of 86,000 g/mol with a low polydispersity (M_w/M_n = 1.1) (Hurtgen et al. 2012). High molecular mass and wide molecular mass distribution are two important factors in the choice of polymer type in electrospinning method. As molecular mass and PDI grow, lower polymer concentration is needed to fabricate nanofibers which could be economical (Palangetic et al. 2014). In this research, high molecular weight PVAc-b-PNVCL copolymer (Mw159,000 g/mol) was synthesized with a radical copolymerization method which is proper for the electrospinning process.

Nanofibers have been used in a diversity of fields such as drug delivery, wound healing, and tissue engineering applications because of high surface to volume ratio, porosity, and controlled morphology (Irani et al. 2014; Teotia et al. 2015; Ghanian et al. 2015; Jung et al. 2019; Al CET 2020).

Thermoresponsive polymers have been considered in the field of biomaterials recently. Although a vast number of materials are being used in biomaterial, only a small number is suitable to make nanofibers by electrospinning (Hsieh et al. 2020; Schoolaert et al. 2020). One of the factors which affects the fabrication of fibers is molecular weight. Polymers with low molecular weight are not suitable for fabrication of fibers. Hurtgen et al. (2012) synthesized a low molecular weight of a biocompatible PVAc-b-PNVCL copolymer (M_w3100 g/mol) that can be used in biomedical applications (Achilleos and Krasia-Christoforou 2018; Jung et al. 2019; Young et al. 2019). In this paper, we intended to synthesize high molecular weight PVAc-b-PNVCL copolymers to create fibers by electrospinning method.

Experimental section

Materials

N-Vinylcaprolactam (NVCL 98%), (2,2,6,6-tetramethylpiperidin-1-yl) oxyl (TEMPO), vinyl acetate (VAc), N,N-dimethylformamide (DMF), acetone and azobisisobutyronitrile (AIBN) were purchased from AkzoNobel, United Arab Emirates. The aqueous 50% glutaraldehyde solution, 99% ethanol and salts for making PBS phosphate buffer, including NaCl, KCl, Na₂HPO₄, KH₂PO_4, were obtained from Merck, Germany. The fetal bovine serum (FBS), trypsin, and mammalian cells medium (RPMI 1640) were purchased from Gibco, Canada.

Synthesis of PNVCL-PVAc copolymer

A 6-mL (0.069 mol) dried vinyl acetate was injected into a round-bottomed flask by a syringe, and 150 mg (0.9 mmol) of recrystallized AIBN was added into the flask and purged under three vacuum-argon cycles. The mixture was heated to 40 ${}^{\circ }\mathrm{C}$ with a slow stirring rate for 4 h. Next, 10 g (0.071 mol) vinylcaprolactam was dissolved in 10-mL (0.136 mol) dried DMF and the solution was injected into the flask by a syringe, and the reaction mixture was heated to 40 ${}^{\circ }\mathrm{C}$ with a slow stirring rate of about 40 h to acquire high molecular weight copolymer of (PVAc-co-PNVCL). By addition of 150 mg (0.9 mmol) TEMPO into the solution the reaction was stopped. After the complete synthetic procedure, the mixture was poured into a flat plate and placed under a vacuum to remove the solvent, and at the end the copolymer film was obtained.

Characterization of PNVCL-PVAc copolymer

Fourier transform infrared (FTIR)

FTIR was carried out with Bruker- Equinox55 spectrometer (Germany) to determine the structure and identification of chemical groups to characterize the synthesized copolymers in the 4000–400 cm⁻¹ range.

¹H nuclear magnetic resonance (¹H NMR)

¹H NMR was performed by Bruker spectrometer (Germany) with a frequency of 500 MHz to characterize copolymer structure. Deuterated dimethylsulfoxide was used as a solvent.

Gel permeation chromatography (GPC)

GPC measurement was performed to measure copolymer molecular weight and the molecular weight distribution with an instrument model Agilent-1100. Tetrahydrofuran (THF) was used as an eluent with a flow rate of 1 mL/min at 29 ${}^{\circ }\mathrm{C}$, and the concentration of polymer solution was 1 g/L. The measurement was carried out with PL gel-MIXD C column and was also calibrated with polystyrene standard.

Differential scanning calorimetry (DSC)

The thermal behavior of copolymers was investigated by METTLER TOLEDO (Switzerland) calorimeter under nitrogen atmosphere with a heating rate of 10 ${}^{\circ }\mathrm{C}$/min from 50 to 260 ${}^{\circ }\mathrm{C}$ for PNVCL-b-PVAc.

Thermogravimetric analysis (TGA)

The thermal behavior of the copolymers was carried out with a thermogravimetric analyzer, PL-1500 model from Polymer Laboratory (England) under the nitrogen atmosphere from room temperature to 600 ${}^{\circ }\mathrm{C}$ with 10 ${}^{\circ }\mathrm{C}$/min heating rate.

Contact angle measurements (CAM)

Contact angle measurements were carried out using the SESSILE DROP technique with a contact angle instrument (KRUSS G10 (Germany)). The experiment was repeated three times for each sample.

Water uptake of PNVCL-PVAc film

This test has been used to determine the water absorption of bulk copolymers. The polymeric films were prepared, weighed, placed in distilled water and kept in an incubator for 48 h. After 48 h, the specimens were taken out of water and after drying with a paper filter, weighed again. The process was repeated for 2 days with a 24-h interval. The absorption percentage of the samples was calculated from the following formula (Eq. 1), where W1 is the weight of dry film and W2 is the weight of wet film.

$$\mathrm{Water}\mathrm{uptake}\left(\%\right)=(\left(W_2,-,{\mathrm{W}}_1\right)/{\mathrm{W}}_1)\times 100$$ 1

Preparation of PNVCL-PVAc nanofibers

The most suitable solvent would be acetone to dissolve and formulate uniform solutions of PNVCL/PVAc copolymer, though due to the low boiling point of the acetone, it is not possible to produce high thickness nanofibrous mats (Augustine et al. 2016). To solve this problem, due to the high boiling point of dimethylformamide, a mixture of dimethylformamide/acetone was used as a solvent system in this study. To prepare the appropriate PNVCL/PVAc copolymer solution for the electrospinning process, a 12% (w/v) solution of the copolymer in dimethylformamide/acetone solution system with a volume ratio of 30:70 was selected. The PNVCL/PVAc copolymer was dissolved in dimethylformamide/acetone by stirring with a magnetic stirrer at room temperature for 30 min to obtain a uniform solution. The solutions were placed in a 10-mL plastic syringe after complete dissolution and subjected to a horizontal electrospinning apparatus with a cylinder collector (Co 881007 NYI, ANSTCO, Iran). In all experiments, the distance between the needle tip and the collector was adjusted to 125 mm, and the chosen rotational speed of the collector was 250 rpm. The applying voltage was varied from 21 to 32 kV with flow rates of 0.1 and 0.5 ml h⁻¹. Finally, the nanofibers were dried through a vacuum pump several times to remove residual DMF in the nanofiber samples.

Characterization of PNVCL-PVAc nanofibers

Scanning electron microscopy (SEM)

To observe the morphology of PNVCL/PVA nanofibers, SEM (Tscan© Vega, Czech Republic) was used. All the nanofibrous samples were coated by a gold-sputter under nitrogen to be electron conductive for to be imaged by applying an accelerating voltage of 15–20 kV.

Porosimetry of nanofibrous scaffolds

The Archimedean principle was used to measure the porosity of nanofibrous scaffolds (Pati et al. 2012; Gürbüz et al. 2014). The samples were immersed in deionized water for 15 min. The sample weight in the water was measured using a four decimal 4-digit laboratory electronic weighing balance. To measure the weight of the contained water sample, the absorbed water at the sample surface was removed by paper filter and quickly weighed to prevent its evaporation. The porosity of the sample was calculated using Eq. (2), where W_w is the weight of the sample containing water; W_d is the weight of dry sample and W₁ is the weight of the sample in the water.

$$\mathrm{Porosity}\left(\%\right)=\left(\frac{\left(W_{\mathrm{w}},-,W_{\mathrm{d}}\right)}{\left(W_{\mathrm{w}},-,W_1\right)}\right)\times 100$$ 2

Mechanical properties

The tensile strength test was used to test the tensile strength of the nanofiber samples. In this test, nanofibers were examined under a load of 10 N and at a stretch rate of 0.5 mm/min. The experiment was executed for a dry sample and a wet sample immersed in PBS for 1 h.

Cell viability and cytotoxicity

To investigate cytotoxicity, a flask of mouse fibroblast cell (L 929) was placed under a fume hood in completely sterile conditions. The surface of the cells was washed with PBS, and then trypsin was added to detach the cells and change the cells from stretched to spherical state. Then, 10% FBS medium (Bovine serum) was added to neutralize trypsin. The cells were centrifuged in a falcon tube at 1200 rpm for 5 min, the cells were precipitated at the bottom of the falcon tube.

The falcon tube was removed from the centrifuge, and supernatant was discharged, and then 10% FBS medium was poured on the cells, and cell-count was carried out using a Lam Neobar. According to ISO 5-10993 standard, the experiment was performed, and the toxicity of nanofibers was evaluated using L929 fibroblast cells. Nanofiber samples with 1 cm² area were transferred to 24 well-plate, and for each well of the plate, there were 50,000 cell/cm² cells in 1640 RPMI medium with 10% FBS. The nanofiber samples were sterilized by UV irradiation and washed with PBS, and then placed in the center of each well. The cell suspension was added to the wells as well, and the well plate was left in a 37 ${}^{\circ }\mathrm{C}$ incubator with 5% CO₂ for 24 h. According to the standard, after 24 and 48 h, the supernatant was removed, and the cellular layers were washed with PBS, and then the MTT solution (1 mg/ml) was sufficiently charged on the cellular layer. The culture plate was placed in the incubator for 3–5 h in the dark. Then, the cells were washed with PBS to remove the unreacted MTT. The formazan product was dissolved by the isopropanol solution, and the absorption of each well was read at 540 nm. The amount of cellular viability resulted from the division of absorbance of each sample by the absorbance of the control sample (polystyrene container) (Akbarzadeh et al. 2019).To evaluate the quality control of nanofibers' toxicity, after 24 and 48 h, microscopic images were prepared by an optical microscope to observe the structure of fibroblasts cultured on the scaffold.

Cellular attachment

The SEM was used to observe the morphology of the cells cultured on nanofibrous samples. After cultivating L929 cells on nanofibrous samples for 24 and 48 h, the samples were rinsed twice with PBS to wash the unattached cells away and then were immersed in 2.5% solution of glutaraldehyde for 1 h, the cells were stabilized, and then the dehydration was performed using graded alcohol series (30%, 50%, 60%, 70%, 80%, 90%, and 100%). Samples were left in a vacuum oven to dry for 24 h, after stabilization in 100% ethanol solution. The dried samples were coated with gold and examined by electron microscopy.

Degradation of nanofibrous scaffolds in PBS

To simulate the degradation of fabricated nanofibrous scaffolds in the body environment, degradation was performed in a PBS buffer with pH  7.4. First, nanofibers were weighed with four decimal 4-digit laboratory electronical weighing balance and then placed in PBS and left in an incubator at 37 ${}^{\circ }\mathrm{C}$. The degradation test was carried out at periods of 1, 7, 14, and 21 days. After passing the mentioned times, the nanofibrous scaffolds were washed with deionized water and dried in a vacuum oven. To observe the morphology of the nanofibers at specified times, SEM tests were also performed on samples.

PBS/water uptake

The nanofibrous scaffolds were placed in a PBS buffer/water after weighing in the dry state. The nanofibers were drawn out of the aqueous medium at specific time intervals and then re-weighed after the removal of extra water using the filter paper. The period after which the sample weight did not change notably (15 min) was considered as the reference time, and, according to Eq. (3), the PBS/water absorption was calculated where W₁ was the weight of dry nanofiber sample, and W₂ was the weight of nanofiber sample after PBS/water uptake and removal of extra water by filter paper.

$$\mathrm{PBS}/\mathrm{water}\mathrm{uptake}\left(\%\right)=(\left(W_2,-,W_1\right)/W_1)\times 100$$ 3

Results and discussion

FTIR

As it is shown in Fig. 1a, carbon–carbon double bonds of =CH, C=C, and =CH₂ appear at 3105, 1666, and 994 cm⁻¹ which attributed to NVCL and C=C at 1658 cm⁻¹ in VAc spectrum disappeared in PNVCL-b-PVAc copolymer spectrum. Also C=O (stretching) at 1739 cm⁻¹ and C=O(stretching) at 1650 cm⁻¹ prove the presence of PVAc and PNVCL in the copolymer chain. Minor water absorbance peak in the PNVCL-PVAc spectrum is because of the presence of PNVCL, which is an amphiphilic polymer (Yelil Arasi et al. 2009).

Fig. 1.

a FTIR spectra of VAc, NVCL, PNVCL-PVAc and b 1H NMR spectra of PNVCL-PVAc

¹H NMR

The ¹H NMR spectrum of PNVCL-PVAc in Fig. 1b, indicates the copolymer structure existence. Proton peaks of CH₂(g), (CHO)f, and (CH₃CO₂)h reveal the existence of PVAc while e(CH₂), d(CNH), c(CNH₂), b(CH₂), and a(CH₂CO) proton peaks prove the existence of PNVCL (Pal et al. 2018).

Monomer fractions of VAc and NVCL in copolymer can be acquired from integral data of h and c proton peaks:

n = number of VAc monomers, m = number of NVCL monomers

1. n = h/3, h = 6, n = 2

2. m = c/2, c = 2, m = 1

3. VAc% $=n/(n+m)$ = 66%

4. NVCL% $=m/(n+m)$ = 34%

GPC results of synthesized PNVCL-PVAc

In accord with GPC chromatogram, Fig. 2, the molecular weight of copolymer has been increased up to 159,680 g/mol with high molar mass distribution (PDI = 2.45). High polydispersity causes the extensibility of polymer chains because of different polymer chain lengths, improving the electrospinning process (Palangetic et al. 2014).

Fig. 2.

Molecular mass distribution of PNVCL-PVAc copolymer

DSC

Since PNVCL-PVAc is an amorphous copolymer, the thermogram corresponding to the mentioned copolymer exhibited only glass transition temperatures (Tg) because of PNVCL, PVAc which are both amorphous polymers (Karlsson et al. 2002; Chen et al. 2002; From et al. 2004). As Fig. 3a points out, PNVCL-PVAc copolymer discloses two glass transition temperatures. Tg₁ = 42.39 ${}^{\circ }\mathrm{C}$ is ascribed to the midpoint glass temperature of PVAc, and Tg₂ = 174.86 ${}^{\circ }\mathrm{C}$ refers to the midpoint glass temperature of PNVCL segments (Meeussen et al. 2000; Jelinska et al. 2010). The synthesized copolymer, proportional to the size of two incompatible polymer blocks, showed two glass transition temperatures, which were slightly displaced in comparison with the Tg of their homopolymers (Fig. 3b, c). It would be the reason of the incomplete phase separation of the blocks (Halligan et al. 2017).

Fig. 3.

a DSC thermogram of PNVCL-PVAc, b DSC thermogram of PVAc, c DSC thermogram of PNVCL

TGA/DTG

The thermal degradation of PNVCL-PVAc is studied through TGA/DTG thermogram, Fig. 4a. According to the thermogram, the copolymer consists of 6% moisture because PNVCL is an amphiphilic polymer, so the first weight loss happened at 110 ${}^{\circ }\mathrm{C}$, which is related to water evaporation. PVAc segments in the copolymer started to degrade at 280 ${}^{\circ }\mathrm{C}$, and the maximum weight loss happened at 330 ${}^{\circ }\mathrm{C}$, Fig. 4b. PNVCL degradation started at 380 ${}^{\circ }\mathrm{C}$, and the maximum degradation took place at 441 ${}^{\circ }\mathrm{C}$ Fig. 4c (Rimez et al. 2008; Kozanoǧlu et al. 2011).

Fig. 4.

a TGA thermogram of PNVCL-PVAc, b TGA thermogram of PVAc, c TGA thermogram of PNVCL

CAM results of PNVCL-PVAc

CAM was carried out on synthesized PNVCL-PVAc film at different temperatures to follow the surface tension behavior with increasing temperature. As results indicate in Fig. 5, by increasing temperature, polymer chains tend to form more hydrophobic structures(Lin et al. 2013; Zhuang et al. 2016).

Fig. 5.

Water drop contact angle with PNVCL-PVAc film surface pictures at 25, 26, 28 and 30 °C

At 25 ${}^{\circ }\mathrm{C}$, the average contact angle of a water drop with the surface was 28 degrees which shifted to 83 degrees at 26 ${}^{\circ }\mathrm{C}$. As a matter of fact, polymer chains turned into a hydrophobic structure by increasing temperature from 25 ${}^{\circ }\mathrm{C}$ to 26 ${}^{\circ }\mathrm{C}$. At 25 ${}^{\circ }\mathrm{C}$, the copolymer conformation started to change into a hydrophobic structure, so it would be considered as LCST.

Water uptake characterization of PNVCL-PVAc film

According to Fig. 6, the highest amount of water absorption is 1.3%, which is the low water absorption due to the copolymer hydrophobicity. Most part of the copolymer consists of hydrophobic PVAc, and the other part is the amphiphilic PNVCL chain, which causes a slight absorption of water. After 24 days, the copolymer film was left in a vacuum oven to dry and weighed again. 2.84% mass loss of copolymer film revealed the partial dissolution of PNVCL chains in water (Ali et al. 2015).

Fig. 6.

Water uptake of PNVCL-PVAc copolymer film

Fabrication of PNVCL-PVAc nanofibers by electrospinning process

Since the purpose of this study is to fabricate nanofibers based on PNVCL-PVAc copolymer, a suitable solvent should be selected that would be able to dissolve the copolymer. Different solvents have been investigated in which acetone and dimethyl had the ability to dissolve copolymer, as well as suitable conditions for electrospinning. To prepare the appropriate electrospinning solution, the specified amount of copolymer was chosen to form a uniform solution with a constant concentration. To dissolve copolymer in acetone/dimethylformamide solution (70/30), the solution was stirred by a magnetic stirrer for 1 h. After 1 h, the solution was poured into a syringe of 10 ml and the syringe was connected to the connector and placed in the device, and then the electrospinning process was performed under appropriate conditions.

After this stage, SEM was used to observe morphology and fiber diameter determination. Images with the magnification of 2000 and 5000 were taken from each sample, and Image J software was used to determine the diameter of the fibers. Figure 7 represents the fiber morphologies and fiber diameter-frequency plots for each sample.

Fig. 7.

SEM images of nanofibrous scaffolds with a magnification of a 2000 nm, b 5000 nm, and nanofiber diameter distribution for samples 1 (21, 0.5), 2 (30, 0.5), 3 (32, 0.5), and 4 (32, 0.1)* (in constant rotational speed of 250 rpm, the distance of 125 mm). Asterisk: The first number is voltage, and the second number is related to flow rate.

In Fig. 7, the experiment was executed in 21, 30, and 32 kV voltages for samples 1, 2, and 3, respectively. The fiber diameter distribution histograms showed that the uniformity of nanofibers was improved by increasing the voltage of samples (Fig. 7). These results were expectable since Buchko 2001 and lee 2004 showed that more uniformed nanofiber with more elasticity could be fabricated by higher voltage.

Studies have shown that the diameter of the nanofibers initially increases with increasing voltage, but beyond the certain voltage, the diameter of the nanofiber begins to decrease. While the tension of the solution droplet may increase with increasing voltage, the high voltage may also accelerate the solution towards the collector due to a greater potential difference, resulting in less jet flight time for stretching the fiber before sitting on the collector (Mazoochi et al. 2012). As it is obvious, sample 3 is more uniform than samples 1 and 2 (Fig. 7). Therefore, by decreasing the flow rate of sample 3 from 0.5 to 0.1 ml/h with similar other factors, sample 4 was fabricated. This specimen was selected as the optimal situation due to higher uniformity and less diameter of fibers compared to other nanofibers.

Porosity measurement of PNVCL-PVAc nanofibrous scaffold

Porosity is one of the important features that influence the performance of polymer scaffolds. The porous structure of the scaffold will be very useful for penetrating the cells as well as replicating them. In the electrospinning method, pores are created by spinning the nanofibers on each other. Although it is not possible to produce specifically sized pores in the electrospinning method due to the fact that the fibers are placed randomly, the overall network structure is the best simulation of the normal ECM, and this can be very useful in tissue engineering (Jiankang et al. 2007; Milašius and Malašauskienė 2014). To check the repeatability and accuracy of the results, each measurement was repeated three times. According to the result of calculations, the mean porosity percentage of the samples was 90.75 ± 1.14%.

Mechanical strength of PNVCL-PVAc nanofibers

To investigate the mechanical properties of electrospun PNVCL-PVAc nanofibers, its stress–strain behavior in wet and dry conditions was examined using tensile tests. In wet conditions, the nanofibers were placed in PBS for 1 h. As Fig. 8a, b indicate, the tensile strength of the scaffold in the dry state is more than the wet state, and the failure strain in the wet state is much higher than in the dry state. In general, with increasing vinyl acetate fraction in copolymers based on vinyl acetate, the higher the relative humidity gets, the tensile strength decreases, and the failure strain increases. The polymeric chain density changes with water and affects the movement of acetate groups, which changes intermolecular forces and that would lead tensile properties to change. Also, the change in tensile properties can be due to a change in the conformation or polymer degradation in water (Covt et al. 1979).

Fig. 8.

a Elongation at break of PNVCL-PVAc nanofibers in a dry and wet state, b tensile strength of PNVCL-PVAc nanofibers in a dry and wet state

MTT assay for determination of cell viability

The cell viability can be evaluated using the MTT assay. In this method, the number of cells on the surface is measured at 24 and 48 h. The cell viability is shown in Fig. 9a, b in quantitative and qualitative terms, and the results for 24 and 48 h are 83% and 85%, respectively. There is no significant difference in the cellular behavior of nanofibrous scaffolds in 24 and 48 h. The results of the MTT test indicate the high compatibility of the PNVCL-PVAc scaffold. Optical microscopy images also show the lack of toxicity of the sample and the proliferation of cells over time.

Fig. 9.

a MTT assay of 929L fibroblast cells for a PNVCL-PVAc nanofibrous scaffold at two different times. b Optical microscopic images of 929L fibroblast cells cultured in the presence of PNVCL-PVAc scaffold after 24 and 48 h

Cellular attachment of L929 mouse cells on PNVCL-PVAc scaffold

To evaluate the biocompatibility of PNVCL-PVAc scaffolds, the behavior of living cells is evaluated by the cell culture method. In this method, the biocompatibility of the material was studied by observing the adhesion of the cells on the surface, morphological changes, and their growth. Growth and reproduction of cells on the surface require clinging and stacking cells on the surface, which is a sign of biocompatibility. In a cell culture assay, nanofibrous scaffolds were tested by 929L fibroblast cells from the mouse. The morphology of the cells on the scaffold surface was investigated by SEM imaging, and the corresponding images of this test are shown in Fig. 10. SEM images of 929L fibroblast cells indicate that the cellular adhesion and spreading have been enhanced by increasing time, indicating that the PNVCL-PVAc nanofibrous scaffolds show good biocompatibility. The PNVCL-PVAc copolymer was dissolved in alcohol. To maintain and observe cell structure and cell growth, graded ethanol was used to remove the water, which caused the dissolution of nanofibers.

Fig. 10.

SEM images of mouse fibroblast cells on PNVCL-PVAc scaffold after 24 and 48 h

PNVCL-PVAc nanofibers degradation testing

The degradation test of PNVCL-PVAc electrospun nanofibers was carried out in PBS buffer and 37 ${}^{\circ }\mathrm{C}$ temperature. The morphology of the nanofibers was investigated by SEM after 1, 3, 7, 14, and 21 days as shown in Fig. 11. As shown in Fig. 11, the morphology of the hydrophobic nanofibers significantly changed over a period of 21 days. Nanofibers conformation changed at the human body temperature (37 ${}^{\circ }\mathrm{C}$), which is a temperature above the critical temperature (LCST) of the PNVCL-PVAc nanofibers. The nanofibers were stacked together due to the dominance of the polymer–polymer interactions on the polymer-water interactions. The morphology of the nanofibers did not change significantly between 1 and 3 days. After 7 days, the nanofibrous structure had slightly changed, and the surface of the nanofibers had become rougher, but the nanofiber structure was still recognizable. After about 14 to 21 days, the nanofibers penetrated into each other and formed a rough surface, and the nanofiber structure was not recognizable anymore. Regarding calculations, after a 21-day period, 4.71% degradation for PNVCL-PVAc nanofibers was achieved. The aqueous environment hardly penetrated into the nanofibers, so the weight reduction and degradation rate were too low (Loh et al. 2010).

Fig. 11.

SEM images of PNVCL-PVAc nanofibers after weight reduction (degradation) in PBS at 37 °C during 1, 3, 7, 14 and 21 days

Water and PBS uptake of PNVCL-PVAc nanofibrous scaffold

The values of PBS and water uptake of PNVCL-PVAc nanofibrous scaffold were measured after 48 h (Fig. 12). As can be seen in the graphs, the figures for PBS and water uptake are 183.14 ± 15.15 and 198.33 ± 1.52, respectively. To check the repeatability and accuracy of the results, each measurement was repeated three times, and the error rate was specified on the graph.

Fig. 12.

PBS and water uptake of PNVCL-PVAc nanofibrous scaffolds after 48 h

The porosity and chemical structure (hydrophilicity) of scaffolds affect the PBS and water absorption.

In general, the PBS and water uptake of scaffolds in the experiment was due to the porosity of the scaffolds and the chemical structure (hydrophilicity) of the materials used in the construction of nanofibrous scaffolds which were in good agreement with the results. As can be seen, the results of water and PBS uptake were approximately the same.

Contact angle of PNVCL-PVAc nanofibers

The average contact angle of water droplet on PNVCL-PVAc nanofibers at room temperature (25 ${}^{\circ }\mathrm{C}$) was 49.2° (Fig. 13)which was much more than the contact angle of water droplet on copolymer film (28°). The nanofibrous surface is more hydrophobic than the film surface due to its porous surface; as porous structure entrapped air pockets could be one of the main factors of polymer surface hydrophobicity (Schoolaert et al. 2020).

Fig. 13.

Water drop contact angle with PNVCL-PVAc nanofibers surface pictures at 25 °C

Discussion

The purpose of this study was to synthesize and investigate the properties of high molecular weight PNVCL-PVAc thermo-responsive copolymer with broad mass distribution for preparation of nanofibers. Synthesis of PNVCL-PVAc copolymer was performed by free radical copolymerization by Hurtgen and co-workers. Due to low molecular weight of previous synthesized copolymer, the process of electrospinning was not possible (Wang et al. 2016a, b). Because PNVCL-PVAc copolymer is biocompatible and has a low phase transition temperature, fabrication of nanofibers would be very practical (Shi et al. 2017). Copolymerization of PNVCL-PVAc was confirmed by HNMR, FTIR, and GPC. Two glass transition temperatures were observed at 42.39 and 174.86 ${}^{\circ }\mathrm{C}$, which were attributed with respect to PVAc and PNVCL segments, through DSC thermal analysis of PNVCL-PVAc copolymer. The molecular mass and molecular mass distribution of N-vinyl caprolactam/vinyl acetate copolymer were measured at 159,680 g/mol and 2.45 by GPC test. The phase transition temperature of N-vinyl caprolactam/vinyl acetate copolymer was determined at 25 ${}^{\circ }\mathrm{C}$ by conducting a contact angle test. The tensile strength and strain at their failure point were also measured at 0.109 MPa, 19.45% in dry and 0.045 MPa, 73.11% under wet conditions. The PNVCL-PVAc nanofibers were fabricated through the process of electrospinning where results are shown in SEM pictures (Fig. 7).The biocompatibility of nanofibers was investigated using MTT test which was reported 83% to 85%, as ideal. The results showed that the growth and proliferation of L929 mouse fibroblast cells increased with an increase in time, indicating the biocompatibility of the nanofibers and the absence of toxicity in the specimen.

Conclusion

The chemical structures of the synthesized materials were detected and identified by FTIR and ¹HNMR spectroscopy methods. Molecular mass and molecular mass distribution of n-vinyl caprolactam/vinyl acetate copolymer were measured by GPC test. The phase transition temperature of n-vinyl caprolactam/vinyl acetate copolymer was determined by conducting a contact angle test at different temperatures. The results showed that the phase transition of the copolymer started at 25 ${}^{\circ }\mathrm{C}$. The compatibility of nanofibers was also evaluated. The results showed that the growth and proliferation of L929 mouse fibroblast cells increased with an increase in time, indicating the compatibility of the nanofibers and the absence of toxicity in the specimens. The results show that the synthesized copolymers have phase transition temperature at 25 ${}^{\circ }\mathrm{C}$ with good characteristics for biomedical applications.

Declarations

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.