Blend Electrospinning of Poly(Ɛ-Caprolactone) and Poly(Ethylene Glycol-400) Nanofibers Loaded with Ibuprofen as a Potential Drug Delivery System for Wound Dressings

PCL (Mw = 80,000 g/mol) and PEG (Mw = 400 g/mol) were purchased from Sigma-Aldrich and were used as received. Chloroform and THF were purchased from Carlo Erba with purity >99.0%. Ethanol and Acetone by Honeywell with purity >99.5% and o-xylene from Sigma-Aldrich with purity 98.0% were procured. IBU (>98% (Mw (= 206.28 g/mol) was sourced by from Sigma Sigma-Aldrich.

A homemade setup of single-needle ES was used for this research work with a syringe pump to maintain a uniform flow rate of solution. A high voltage source (0–30 kV) was connected to an enclosed poly (methyl methacrylate) chamber to supply a controlled electric field between needle tip and the grounded collector that was made of 20 × 20 cm Teflon plate. All nanofibrous samples were collected on this grounded plate that was covered with aluminum foil, and the samples have been collected using random location on the aluminum foil to be representative of the nano-filaments’ population. PCL solution was filled in a 20 mL plastic syringe attached to a needle with an inner diameter of 0.4 mm gauge. All experiments were carried out at room temperature (25 ± 2 °C) and relative humidity was kept constant at 35 ± 4%. A schematic diagram of single-needle ES setup is shown in Figure 1 [23].

Figure 1

A homogeneous solution of 10 wt% PCL was prepared by using a mix of solvents, composed of chloroform and ethanol in 88:12 wt/wt ratio, respectively. To obtain a clear solution of PCL, the solution was magnetically stirred overnight at room temperature. 10 wt% PEG-400, with respect to weight of PCL, was added to this clear solution and it was again stirred for 15 min with magnetic stirrer. Finally, the solution was left in an ultrasonic bath for 5 min to remove air bubbles. Similarly, 5 wt% and 7 wt% IBU with respect to the weight of PCL, were directly added to the clear PCL and PCL/PEG-10% solutions respectively and were magnetically stirred for 10 min each. Later on, these solutions were left in an ultrasonic bath for 5 min to remove air bubbles.

Viscosity measurements were performed for PCL solutions in different solvents such as chloroform, THF, acetone, o-xylene, and a binary solvent system of chloroform:ethanol (88:12 wt/wt). For this purpose, an Anton Paar MCR-302 plate–plate rheometer was employed. All measurements were taken at a shear rate of 100 /s and at room temperature (25°C). For each sample, 20 measuring points were taken and for each point the time-setting was 12 s. All solutions were clear and homogeneous and viscosity was measured as a function of time.

A PCL film was used as a reference to measure the water contact angle of PCL and PCL/PEG-10% nanofibers. To prepare the film, the PCL solution was prepared by using a solvent mix of chloroform, namely ethanol (88:12 wt/wt), and was magnetically stirred overnight. This solution was utilized to cast a smooth film on a Teflon sheet by using a manual bar coater of 100 μm thickness. After coating a thin film of PCL solution on the Teflon sheet, the sheet was placed in a drying oven for 3 h at 40°C to evaporate all the solvents. After complete drying, it was used to measure the water-contact angle.

SEM-JSM model IT100, was used to take high resolution images of nanofibers. Prior to SEM, all samples were sputter-coated with gold. Three images for each type of samples were taken at 10 μm scale. Diameters of nanofibers were measured by using ImageJ software, which was used to take bar measurements. Fifty measurements were made per sample to ensure accuracy. Mean diameters, standard deviations, and coefficients of variation (CV%) for all samples were calculated to determine the overall morphology and homogeneity of nanofiber.

Water contact angle measurements were carried out by Drop Shape Analyzer (DSA 100 KRUSS GmbH, Germany) apparatus by Sessile drop method. A water droplet of 2 μL was dispensed from the needle and was dropped on the samples placed underneath on a glass plate. The PCL film prepared on Teflon sheet was peeled off and then used for taking measurements. Distilled water was used as reference liquid and was allowed to drop automatically on the electrospun nanofibers. Contact angle measuring time was 10 s after the water droplet was dropped. Measurements were recorded by CCD video camera installed inside the instrument. Five droplets for each sample were deposited and analyzed.

Thermal properties of pure PCL, pure PEG, and all sets of PCL and PCL/PEG nanofibers were determined by using DSC model TA instrument Q200. Specimens of approximately 9 mg in weight were sealed in non-hermetic aluminum capsules. Experiments were performed under nitrogen atmosphere with a single cycle of heating from −80°C to 100°C at a rate of 10°C/min. The glass transition and melting temperatures of each sample were determined and the crystallinity ratios (Xc) of PCL and PCL/PEG nanofibers were calculated. The values of X_c% of all nanofibers were calculated with reference to the content of PCL used for PCL/PEG blends. X_c% was determined by using the following Equation 1: (1)Xc(%)=ΔHm/ΔHm.PCL×100{\rm{Xc}}\left( \% \right) = \Delta {{\rm{H}}_{\rm{m}}}/\Delta {{\rm{H}}_{{\rm{m}}{\rm{.PCL}}}} \times 100 where ΔH_m is the melting enthalpy of PCL nanofibers and Δ_Hm.PCL is the melting enthalpy of 100% crystalline PCL, that is 142 J/g [24].

To determine the thermal stability of nanofibers, TGA was performed by using TA instrument model Q500. All samples were analyzed under nitrogen with 10°C/min rate of heating ramp till 800°C.

Rectangular strips of nanofibrous webs were cut from different areas of sample with a 20 mm gauge length and 5 mm width using a roller cutter. The values for thickness of each sample, at five different points, were measured using Schut's digital micrometer with a precision value up to 0.001 mm. All the specimens were weighed on a micro balance. Different areas were chosen to cover all possible thicknesses of nanofibers. The specimens were conditioned in atmospheric conditions at 20 ± 2°C and 65 ± 2% relative humidity for 48 h before testing. Double-edge duct tape was placed on both edges of the sample to facilitate its grip between the jaws of tensile tester. Cut samples were sandwiched between two cardboard layers, which functioned as templates and made it possible to easily handle and grip the specimens in jaws for mechanical testing, as shown in Figure 2. The edges of the cardboard along with the specimen ends were placed between the grips of a tensile testing machine, thereby ensuring that the area of nanofibrous webs between the two jaws remained at 10 mm. Tensile test was performed on MTS M/20 tensile testing machine using a load cell with a capacity of 10 N and an elongation rate of 10 mm/min. The cardboard templates were cut after fixing specimens between pneumatic clamps of the tensile machine and tests were performed at room temperature. Four samples for each specimen were tested to analyze the tensile strength.

Figure 2

The in-vitro drug-release profile of IBU-loaded nanofibers was studied by using dialysis method. Each sample was introduced into a dialysis membrane and was suspended into 10 mL of buffer solution (phosphate buffer solution with pH 7.4) at 37 ± 0.5°C under constant stirring. Certain volumes of solution (approximately 2 μL) were taken out at pre-established time intervals and the concentration was determined by UV-vis spectroscopy according to a calibration curve, which is shown in Figure 3. The percentage release efficiency of IBU (RE%) was calculated using the following Equation 2: (2)RE%=mr/ml×100{\rm{RE}}\% = {\rm{mr}}/{\rm{ml}} \times 100 where mr is the amount of released IBU (mg) and mL is the amount of loaded IBU (mg) in nanofibrous webs.

Figure 3

10 wt% concentration of PCL was taken to prepare the solutions with each solvent i.e., chloroform, chloroform:ethanol (88:12) wt/wt, THF, acetone, and o-xylene. The viscosity values of PCL in chloroform and chloroform:ethanol (88:12 wt/wt) solvents were the highest in a range of 3,200–3,600 MPa/s. On the other hand, the PCL solutions in THF, acetone, and o-xylene exhibited a different behavior with very low viscosity values of 600, 500, and 300 MPa/s respectively. It is established from the literature that viscosities that are excessively high or low are not favorable for the production of continuous and bead-free nanofibers [24, 25]. This vast difference in viscosities indicated the interaction among PCL molecular chains with different solvents. Higher viscosity value indicates the better solubility of PCL in the respective solvent, which translates into stronger intermolecular interactions between polymer chains and solvent molecules. For problem-free ES and bead-free nanofibers, the choice of solvent and its solution viscosity plays a vital role. Therefore, a homogenous polymer solution with optimum viscosity is needed to perform successful ES processes. Higher solution viscosity and reduced surface tension contribute to the bead-free formation of nanofibers. The concentration of polymer controls the final solution-viscosity, and the coefficient of surface tension depends upon the interaction between polymer and solvent. For example, the addition of ethanol to the solvent system can reduce the surface tension coefficient, and this will avoid the bead formation and will ensure smooth ES of nanofibers under electric field [26]. Therefore, after analyzing these viscosity values, the binary solvent system of chloroform:ethanol (88:12 wt/wt) was selected as a suitable solvent for preparing PCL solutions. All the experiments in this study were performed using this binary solvent system.

On the basis of preliminary studies of ES parameters and their impact on nanofibers’ characteristics, the optimum ES parameters were selected. These optimum ES parameters, for all the samples, are mentioned in Table 2. These best parameters were selected on the basis of homogeneity of these nanofibers with a small coefficient of variation of their mean diameters. The temperature of ES chamber was 25 ± 2°C and the relative humidity was 35 ± 4% for all the experiments.

3.2.1

Impact of PEG-400 on morphology of PCL nanofibers

The morphology of nanofibrous webs was studied by SEM analysis and their diameters were measured by using ImageJ software, which was used to take 50 measurements per specimen. The SEM images of PCL, PCL/PEG, and their IBU-loaded nanofibers showed predominantly round morphology with varying thicknesses along their length, as shown in Figure 4. No bead formation was observed in any sample. All samples were heterogeneous in morphology including nano and micro fibers and showed variations in their mean diameters. The diameters of PCL nanofibers were ranging from 500 nm to 3.5 μm whereas, in case of PCL/PEG-10%, nanofibers’ diameters ranged from 400 nm to 1.5 μm [27]. It was obvious that the addition of PEG-400 noticeably reduced the diameters of nanofibers. Moreover, the morphology of PCL/PEG nanofibers was also different because numerous pores were seen on their surface that could be due to the phase separation of both polymers; such separation was considered to be an expected result according to previously reported studies [19].

Figure 4

Despite its good mechanical properties, slow degradation and biocompatibility, PCL lacks hydrophilicity, and this lacuna limits its applicability for wound healing. Therefore, a hypothesis was made that blending of PCL with a hydrophilic polymer such as PEG-400 would solve this problem. This theory was verified by taking water contact angle measurements for pristine PCL film, PCL, and PCL/PEG-10% nanofibrous webs, as given in Table 3.

It was observed that PCL nanofibers showed a larger water contact angle as compared to pure PCL film, as illustrated in Figure 5. A larger angle in case of PCL nanofibers could be due to the random arrangement of PCL chains in nanofibrous web structure, which resulted in a more compact surface and depicted enhanced hydrophobic behavior. The topology of these nanofibers plays an important role in determining their wettability. Surface roughness is known to amplify the hydrophobicity by mimicking the lotus effect [28]. Another reason for this phenomenon could be the stretching of PCL chains during ES, under the influence of electric fields that cause a rearrangement of polymer chains to form these nanofibers [29]. Contrastingly, in PCL/PEG-10% nanofibers, it was not possible to measure the contact angle, since the water droplet was quickly absorbed on the surface of nanofibrous webs. This rapid absorption of the water droplet could be due to the presence of hydro soluble PEG-400 on the surface of nanofibers. Therefore, it was proved that the addition of PEG-400 improved the wettability of PCL nanofibers, and this was actually a desired feature for its potential application as a wound dressing material to absorb wound exudate [18, 30].

Figure 5

The results of TGA analysis enabled comparison of the thermal properties of PCL, PCL/PEG-10%, and their IBU-loaded nanofibers with their pure states, as shown in Table 5. No residue was found at the end of the heating cycle for any sample. No significant difference among the degradation temperatures of PCL, of PCL/PEG-10% nanofibers, and of pure PCL was observed, which implicates that addition of PEG-400 did not affect the thermal stability of PCL nanofibers. The IBU-loaded nanofibers showed a decline in their degradation temperatures, and this degradation is in agreement with their decreased crystallinity ratios determined by DSC analysis. Despite the decrease in their degradation temperatures, IBU-loaded nanofibers still exhibit adequate thermal stability for their potential application as wound-dressing material.

3.5.1

Presence of IBU determined by TGA

Thermal analysis of IBU-loaded nanofibers also confirmed the presence of IBU in the nanofibers’ structure. The degradation curves of pure IBU, 5%IBU + PCL, and 7%IBU+PCL-PEG-10% nanofibers are shown in Figure 6 (a, b and c) respectively.

Figure 6

The degradation curve of pure IBU showed that a complete degradation of drug takes place at 226°C, as shown in Figure 6(a). Therefore, similar intermediate peaks at 228°C and 230°C were noted in degradation curves of 5%IBU + PCL and 7%IBU + PCL/PEG-10% nanofibers, respectively. Presence of these corresponding peaks of IBU during thermal degradation of these electrospun nanofibers provided strong evidence that the drug was successfully incorporated into the network of nanofibers during their ES.

Fundamental studies on mechanical properties of electrospun nanofibers typically focus on uniaxial tensile-strength testing of randomly aligned nanofibers. In this study, tensile-strength testing of PCL/PEG-10% and 7%IBU + PCL/PEG-10% nanofibrous webs (electrospun with chloroform:ethanol (88:12 wt/wt) as solvent) were performed. These nanofibrous webs consisted of randomly oriented nanofibers having heterogeneous morphology with their mean diameters ranging from nano to micro scale. The stress-strain curves of these randomly oriented PCL/PEG-10% and 7%IBU + PCL/PEG-10% nanofibrous webs are shown in Figure 7, which highlights their mechanical behavior. Five samples of each type of nanofibers were tested.

Figure 7

These curves can be divided into three sections i.e., elastic, yielding, and strain-hardening regions. Specifically, the initial linear elastic region occurs up to 2% strain, where stretching and alignment of nanofibrous mats occurred along the direction of applied load. The values of applied tensile stress corresponding to their percentage strain for each sample are given in Table 6.

It was observed that in PCL/PEG-10% nanofibers, when the applied stress increased up to 5 MPa, the percentage strain in material was 710%, with a total elongation in material up to 66 mm. On the other hand, in IBU-loaded nanofibers, the value for maximum applied stress only reached up to 1.8 MPa and the resulting strain was up to 380% with elongation at a break of 32 mm in the material. The values for the tensile strength of IBU-incorporated nanofiber webs were lower than that of PCL/PEG-10% nanofiber webs [18, 19].

The main objective of drug incorporation into PCL and PCL/PEG-10% nanofibers was to devise a drug delivery system in these non-woven electrospun nanofibrous webs for their wound dressing applications. The performance of these IBU-loaded nanofibrous webs was tested through in-vitro drug-release kinetic study. Samples of these nanofibrous webs were immersed in phosphate-buffered saline (PBS) solution (pH = 7) at 37°C. Drug-release efficiency of IBU was monitored for a 24 h-duration. Percentage release efficiencies (RE%) of both samples, 5%IBU + PCL and 7 wt%IBU + PCL/PEG-10%, are given in Table 7.

The graphical release profile of IBU in both samples is presented in Figure 8. The drug release analysis was performed for a 24 h-duration.

Figure 8

An initial burst release of IBU was observed during initial hours of analysis, followed by a gradual release until completion of 24 h. This rapid release of IBU during the first 2 h could be attributed to the presence of IBU agglomerates on the surface of nanofibers. However, in case of 7 wt%IBU + PCL/PEG-10%, this effect of burst release was rather slow as compared to the 5%IBU + PCL nanofibers [31,32,33]. This steady release could be due to the fact that IBU was embedded into the nanofibers’ network and was not dispersed on the surface of nanofibrous webs. Furthermore, the amount of IBU released from these nanofibrous webs was not 100%, which indicated that IBU was not homogenously disseminated into the nanofibrous network. Another possibility could be that some amount of IBU was lost during the ES process.