Hybrid nanofibers from biodegradable polylactide and polythiophene for scaffolds

Abstract

Hybrid scaffolds constituted of polylactide (PLA) as a biodegradable polymer and poly(3-thiophene methyl acetate) (P3TMA) as an electroactive polymer were prepared and studied. Both polymers had a similar solubility and consequently could be easily electrospun using a common solvent. Electrospinning operational parameters were optimized to get continuous micro/nanofibers with a homogeneous diameter that ranged between 600 and 900 nm depending on the PLA–P3TMA ratio. Electrospinning was only effective when the P3TMA content was at maximum 50 wt%. The incorporation of P3TMA slightly decreased the fibre diameter, led to smoother fibre surfaces and gave rise to some heterogeneous clusters inside the fibers. PLA was highly oriented inside the electrospun fibers and able to easily cold crystallize by heating. Thermal degradation was not highly influenced by the presence of P3TMA, although the onset temperature slightly increased since the first decomposition step of PLA was prevented. New scaffolds had promising electrochemical properties and even provided a good substrate for cell adhesion and cell proliferation. Therefore, these hybrid materials are suitable to improve the cellular response towards physiological processes.

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Introduction

Polymeric materials with both biodegradable and electrically conducting properties have growing interest in biomedical applications because of both the lack of long-term health risk and their good behavior as the supportive matrix for tissue regeneration. The electrochemical response of conducting polymers makes feasible the local stimulation of desired tissue and the enhancement of either the proliferation or differentiation of various cell types.^1–4 Nevertheless, it remains a considerable challenge to synthesize an ideal electroactive polymer that meets the biocompatibility and biodegradability requirements to minimize the inflammatory reaction in the host tissue that could be raised by the use of non-degradable materials. An alternative strategy is the use of conducting polymer/biopolymer blends since unique properties that justify their potential technological applications in biomedical devices can be achieved.

The requirements for materials used in tissue engineering applications are biocompatibility and biodegradability since they should degrade with time and should be replaced with newly regenerated tissues. The architecture of the biomaterial is also very important and, specifically, scaffolds constituted by electrospun nanofibers have promising features, such as big surface area for absorbing proteins and abundance of binding sites for cell membrane receptors.

Ultrathin fibers from a wide range of polymer materials can be easily prepared by electrospinning.^5–11 This electrostatic technique involves the use of a high voltage field to charge the surface of a polymer solution droplet, held at the end of a capillary tube, and induce the ejection of a liquid jet towards a grounded target (collector).

Two different approaches have been applied to develop scaffolds constituted by conducting and biodegradable polymers: (a) by coating an electrospun mat of a well known biocompatible and reabsorbable biomaterial with the conducting polymer; and (b) by direct electrospinning of a conducting/biodegradable polymer mixture. The second option is easier to perform but requires a good solubility of the conducting polymer in the electrospinning solution and gives rise to scaffolds with lower conductivity.

First works providing novel conductive materials well suited as biocompatible scaffolds for tissue engineering involved polyaniline–gelatin blend nanofibers.¹² Picciani et al.¹³ considered the use of poly(L-lactide) as the support polymeric matrix for the preparation of polyaniline-based conducting nanofibers and evaluated the influence of operational parameters on the morphology of electrospun fibers. Several polyaniline and poly(D,L-lactide) mixtures at different weight percents were also successfully electrospun from 1,1,1,3,3,3-hexafluoroisopropanol solutions and their conductivity and biocompatibility evaluated.¹⁴ Nanofibrous blends of HCl-doped poly(aniline-co-3-aminobenzoic acid) copolymer and poly(lactic acid) (PLA) were fabricated by electrospinning solutions of the polymers in a dimethyl sulfoxide–tetrahydrofuran mixture.¹⁵ Scarce works concern the electrospinning of mixtures based on polypyrrole and basically deal with scaffolds constituted by polypyrrole/polycaprolactone (PCL)/gelatin nanofibers.¹⁶ Specifically, conductive nanofibers containing 15% polypyrrole exhibited the most suitable balance of electrical conductivity, mechanical properties and biodegradability, matching the requirements for the regeneration of cardiac tissue. Furthermore, such scaffold promoted cell attachment and proliferation as well as the interaction and expression of cardiac-specific proteins.

Polythiophenes constitute a group of conducting polymers with high technological potential due to their optical, electroluminescents, electronic and, specially, electrochemical properties.^17,18 Different derivatives can be considered and, specifically, several works were focused on the preparation of nanofibers from mixtures of poly(3-hexylthiophene) (P3HT) and PCL or poly(lactic-co-glycolic acid) (PLGA) as biodegradable polymers.^19,20 Thus, P3HT domains in concentrated PCL solution were highly stretched from the electrospinning electrode and formed fibrils with very small diameters (i.e. ∼30 nm) embedded inside PCL composite fibers. Interestingly, fibrils became connected one to another during the volume shrinkage of the solution by solvent evaporation, generating PCL composite fibers with continuous P3HT fibrils embedded inside. On the other hand, it was found that PLGA–P3HT nanofibers have a significant influence on cell adhesion and proliferation. These new electrically conducting axially aligned nanofibers provided both electrical and structural cues and could be potentially used as scaffolds for neural regeneration.

Poly(3-thiophene methyl acetate) (P3TMA) is another polythiophene derivative that is characterized by bearing carboxylate substituents in the 3-position of the heterocyclic ring. The polymer can be easily prepared via oxidative chemical²¹ and photochemical²² reactions and appears a suitable candidate for being processed by electrospinning since has a good solubility in organic solvents like chloroform. Furthermore, it has currently been demonstrated that very stable free-standing nanomembranes with electroactive and biodegradable properties can be prepared by combining P3TMA and polyesters, such as poly(tetramethylene succinate), and even thermoplastic polyurethanes and poly(vinylidene fluoride).^21–24

The main goal of the present work is the establishment of the electrospun conditions required to get continuous micro/nanofibers from mixtures of P3TMA with PLA as well as to perform a basic characterization of morphology and properties (e.g. ability to store charge and biocompatibility) of the derived scaffolds. PLA has been just selected as the biodegradable component due to its excellent properties and its wide use in the biomedical field.

Experimental section

Materials

3-Thiophene acetic acid (3TAA) (98.0%) was purchased from Fluka (Sigma-Aldrich). Iron chloride anhydrous (97.0%), dry methanol (99.5%), chloroform (99.9%) were purchased from Panreac Quimica S.A.U. (Spain) and used as received without further purification.

PLA, a product of Natureworks (polymer 2002D), was kindly supplied by Nupik International (Polinyà, Spain). According to the manufacturer, this PLA has a D content of 4.25%, a residual monomer content of 0.3%, a density of 1.24 g cc⁻¹, a glass transition temperature (T_g) of 58 °C and a melting point of 153 °C.

Kidney epithelial cells derived from African green monkey (VERO) were purchased from ATCC (USA).

Synthesis of poly(3-thiophene methyl acetate)

The 3-thiophene methyl acetate (3TMA) monomer was obtained with a 74% yield by refluxing 3TAA in dry methanol for 24 hours at a temperature of 90 °C.²¹ The polythiophene derivative, P3TMA, was subsequently prepared by a chemical oxidative coupling in dry chloroform following the procedure described by Kim et al.²⁵ Anhydrous ferric chloride (FeCl₃) was used as oxidant and dopant. The polymerization yield was ca. 61% after removing the residual oxidant and oligomers.

Molecular weights and polydispersity index (PDI) were estimated by size exclusion chromatography (SEC) using a liquid chromatograph (Shimadzu, model LC-8A) equipped with an Empower computer program (Waters). A PL HFIP gel column (Polymer Lab) and a refractive index detector (Shimadzu RID-10A) were employed. Polymers were dissolved and eluted in 1,1,1,3,3,3-hexafluoroisopropanol at a flow rate of 0.5 mL min⁻¹ (injected volume 100 μL, sample concentration 1.5 mg mL⁻¹). The number and weight average molecular weights were calculated using polymethyl methacrylate standards. The resulting number and weight average molecular weights were [M_n = 59 300 g mol⁻¹ and M_w = 117 500 g mol⁻¹] and [M_n = 10 700 g mol⁻¹ and M_w = 22 500 g mol⁻¹] for PLA and P3TMA, respectively.

Electrospinning

Mixtures of PLA and P3TMA were electrospun from different solvents such as chloroform, acetone and chloroform–acetone mixtures at polymer concentrations of 5 w/v% and 1–5 w/v% for PLA and P3TMA, respectively. Samples will be named indicating only the PLA weight percentage (e.g. PLA–P3TMA-100, and PLA–P3TMA-67 corresponds to PLA alone, and a mixture with 67% PLA and 33% P3TMA, respectively).

The electrospun fibers were collected on a target, which was placed at different distances (10–20 cm) from the syringe tip (inside diameter of 0.84 mm). The voltage was varied between 10 and 30 kV and applied to the collecting target using a high-voltage supply (Gamma High Voltage Research, ES30-5W). The polymer solutions were delivered via a KDS100 infusion syringe pump from KD Scientific to control the mass-flow rate (from 0.5 to 10 mL h⁻¹). All electrospinning experiments were carried out at room temperature.

Composition, morphology and properties of electrospun polylactide–poly(3-thiophene methyl acetate) mixtures

¹H-NMR spectra were acquired with a Bruker AMX-300 spectrometer operating at 300.1 MHz. Chemical shifts were calibrated using tetramethylsilane as an internal standard. Deuterated chloroform was used as the solvent.

Infrared absorption spectra were recorded with a Fourier Transform FTIR 4100 Jasco spectrometer in the 4000–600 cm⁻¹ range. A Specac model MKII Golden Gate attenuated total reflection (ATR) with a heated Diamond ATR Top-Plate was used.

Optical morphologic observations were performed using a Zeiss Axioskop 40 microscope. Micrographs were taken with a Zeiss AxiosCam MRC5 digital camera.

Inspection of the morphology of electrospun samples was conducted by scanning electron microscopy using a Focus Ion Beam Zeiss Neon 40 instrument (Carl Zeiss, Germany). Carbon coating was accomplished by using a Mitec K950 Sputter Coater fitted with a film thickness monitor k150×. Samples were visualized at an accelerating voltage of 5 kV. Diameter of electrospun fibers was measured with the SmartTiff software from Carl Zeiss SMT Ltd.

Calorimetric data were obtained by differential scanning calorimetry with a TA Instruments Q100 series equipped with a refrigeration cooling system (RCS). Experiments were conducted under a flow of dry nitrogen with a sample weight of approximately 5 mg and calibration was performed with indium. Heating runs were carried out at a rate of 20 °C min⁻¹ with both electrospun and samples slowly cooled (10 °C min⁻¹) from the melt state.

Thermal degradation was studied at a heating rate of 20 °C min⁻¹ with around 5 mg samples in a Q50 thermogravimetric analyzer of TA Instruments and under a flow of dry nitrogen. Test temperatures ranged from 50 to 600 °C.

X-ray powder diffraction patterns were obtained with a PANalytical X'Pert diffractometer with CuK_α radiation (λ = 0.1542 nm) and a silicium monocrystal sample holder.

Electrochemical characterization

In order to assess the electrochemical behavior of the PLA–P3TMA scaffolds cyclic voltammetry (CV) studies were conducted with an Autolab PGSTAT302N galvanostat equipped with the ECD module (Ecochimie, The Netherlands). Measurements were performed on fiber mats, which were deposited by electrospinning on both sides of steel AISI 316 sheets of 1 × 1 cm². All electrochemical assays were performed using a three-electrode one compartment cell under nitrogen atmosphere and at room temperature.

The cell was filled with 50 mL of phosphate buffered saline (PBS, pH = 7.4) with 0.1 M LiClO₄ as supporting electrolyte. Steel sheets were used as working electrode, while an Ag|AgCl electrode containing KCl saturated aqueous solution was the reference electrode (offset potential versus the standard hydrogen electrode, E⁰ = 0.222 V at 25 °C). Steel AISI 316 sheets of 1 × 1 cm² were used as counter electrode.

Electrochemical measurements were carried out i from −0.4 V to 1.1 V, at scan rate of 50 mV s⁻¹.

Five consecutive oxidation–reduction cycles were conducted to assess the loss of electrochemical activity, which was determined as:

where ΔQ is the difference of anodic voltammetric charge between the first cycle and the last cycle and Q₁ is the anodic voltammetric charge corresponding to the first cycle.

Wettability

Contact angle measurements were performed using the water drop method at room temperature. Images of 0.5 μL distilled water drops were recorded after stabilization (30 s) using a OCA 20 (DataPhysics Instruments GmbH, Filderstadt). The contact angle values were obtained as the average of eight independent measures for each sample. The software SCA 20 was used to analyze the images and acquire the contact angle values.

Cellular adhesion and proliferation assays

Vero cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 2 mM L-glutamine at 37 °C in a humidified atmosphere with 5% CO₂ and 95% air. The culture medium was changed every two days and, for sub-culture, the cell monolayers were rinsed with phosphate buffered saline (PBS) and detached by incubation with trypsin–EDTA (0.25%) for 2–5 min at 37 °C. Cell concentration was established by count with the Neubauer camera using 4% trypan-blue as dye vital. The detached cells with viability ≥95% were used for cultures following the conditions for biocompatibility assays.

PLA–P3TMA electrospun nanofibers were collected on circular coverslips (diameter 1.5 cm). These samples were placed into the wells of a multiwell culture plate and then sterilized by UV-radiation for 15 min in a laminar flux cabinet. For fixing the samples on the well, a small drop of silicone (Silbione® MED ADH 4300 RTV, Bluestar Silicones France SAS, Lyon, France) was used as adhesive. Samples were incubated with 1 mL of culture medium during 30 min under culture conditions to equilibrate the material. Finally, the medium was aspired and the material was evaluated for cell adhesion and proliferation by exposing cells to direct contact with the material surface.

For the cellular adhesion assay, aliquots of 50–100 μL containing 5 × 10⁴ cells were seeded onto the electrospun samples placed in each well. The plate was incubated under culture conditions for 30 min to allow cellular attachment onto the material surface. Then, 1 mL of the culture medium was added to each well, and the plate was incubated during 24 h. Finally, the cell viability was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. The controls were realized by cells culture on self polystyrene surface of the plate (TCPS).

For the cellular proliferation assay, the procedure was similar to the adhesion assay, but the aliquot of 50–100 μL contained 2 × 10⁴ cells. Cultures were maintained during 7 days to allow the cellular growth and an adequate cellular confluence in the well. The media were renewed each two days; and finally, the viability was determined by the MTT assay.

Each sample was evaluated using five replicates, results being averaged and graphically represented. The statistical analysis was performed by one-way ANOVA test to compare the means of all groups. The t-test was applied to determine a statistically significant difference between different groups. The tests were performed with a confidence level of 95% (p < 0.05).

Results and discussion

Electrospinning of polylactide–poly(3-thiophene methyl acetate) mixtures

In order to select the most appropriate conditions to obtain continuous PLA–P3TMA microfibers, several solvents and binary mixtures were tested at different voltages, flows, polymer concentrations and needle tip-collector distances. Although the chemical structure (Fig. 1) of both polymers was quite different, their solubility characteristics were similar and, consequently, a common solvent could be selected for the electrospinning process. In fact, solvent plays a fundamental role for the continuous micro/nanofiber production^26,27 and, in general, a relatively high polymer concentration is required to avoid the formation of droplets and electrospun beads when a good solvent is selected.²⁸ Both PLA and P3TMA are highly soluble in chloroform²¹ and, therefore, this solvent was selected as a starting point in the optimization of processing conditions. However, quality of fibers improved over a wide range of polymer compositions when chloroform–acetone mixtures were employed. Although chloroform seems more appropriate for PLA considering the reported Hildebrand solubility parameters²⁹ (i.e. 18.83 MPa^0.5, 19.98 MPa^0.5 and 17.64 MPa^0.5 for chloroform, acetone and PLA, respectively), the best results were achieved when a small percentage of acetone was added. In fact, Hansen parameters³⁰ determined for dispersion, polar and hydrogen-bonding components suggests that the addition of acetone improves polar interactions (Table 1). Thus, a chloroform–acetone (70 : 30 v/v) mixture had closer parameters to PLA than the individual solvents and gave good electrospun fibers for all assayed PLA–P3TMA polymer blends. In fact, this solvent mixture was also previously selected as the best solution for electrospinning other PLA/polymer mixtures.³¹

Fig. 1 Chemical structures of poly(3-thiophene methyl acetate) and polylactide.

Table 1 Hansen parameters of polylactide and selected electrospinning solvents

Sample	δd (MPa^0.5)	δp (MPa^0.5)	δh (MPa^0.5)	δT (MPa^0.5)
Chloroform	17.8	3.1	7.0	18.9
Acetone	15.0	10.4	5.5	19.9
Chloroform–acetone 70 : 30 v/v	17.0	5.3	6.5	19.2
Polylactide	17.6	5.3	5.8	19.3

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The low molecular weight of P3TMA precluded its processability into micro/nanofibers since chain entanglements were insufficient to stabilize the jet. Therefore spraying of droplets that coalesced into ill-defined shapes was observed (data not shown). PLA was essential to both render a scaffold with biodegradable properties and improve processability by increasing the average molecular weight of the polymer mixture. In fact a PLA content higher than 50% was necessary even in the most favourable electrospinning conditions to completely avoid the formation of droplets. Fig. 2 illustrates the optimization process when a deposition distance of 12 cm was chosen according to a first screening. It can be observed that big drops corresponding to the conducting polymer were obtained when a relatively high flow (i.e. 10 mL h⁻¹) and low voltage (i.e. 15 kV) were employed. At an intermediate voltage (25 kV) the drop size decreased and at a high voltage (30 kV) beads were characteristic. The decrease of the flow up to 4 mL h⁻¹ improved considerably the morphology and, specifically, a low voltage led to small beads whereas continuous and homogeneous size fibers could be attained at an intermediate voltage. The selected electrospinning conditions for the different conducting/biodegradable polymer mixtures are summarized in Table 2.

Fig. 2 Optical micrographs showing typical morphologies obtained by electrospinning a PLA–P3TMA 67 mixture from a chloroform–acetone (70 : 30 v/v) solution and a deposition distance of 12 cm.

Table 2 Optimal electrospinning conditions for the different studied samples^a

Sample	Voltage (kV)	Flow rate (mL h^−1)
a The applied parameter is indicated in bold characters when a range of spinnability is given. In all cases the optimal distance between syringe tip and collector was 12 cm.
PLA–P3TMA 100	15–20	4–10
PLA–P3TMA 83	20–25	4–10
PLA–P3TMA 67	20–25	4
PLA–P3TMA 50	20–25	4

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Morphology of polylactide–poly(3-thiophene methyl acetate) nanofibers

Fig. 3 shows representative SEM micrographs of electrospun samples with different compositions. In general, long micro/nanofibers with a cylindrical morphology and randomly distributed in the fibrous mats could be attained. Fibers were adhered to each other forming a dense but porous structure. Diameter distribution was relatively wide. This feature was particularly remarkable for the PLA–P3TMA 50 sample, in which significant amounts of fibers with diameters so different as 250 nm and 2 μm were clearly distinguished. However, in all cases the most predominant size was in the 600–900 nm range. Fig. 4 shows the monomodal distributions observed for the samples prepared under the optimized conditions. It is interesting to note that the increase on the P3TMA ratio in the electrospinning mixture led to a slight decrease on the average diameter (i.e. values progressively decreased from 864 to 633 when P3TMA content increased from 0 to 50 wt%).

Fig. 3 SEM micrographs taken at low (top) and high (bottom) magnification of electrospun nanofibers of PLA–P3TMA samples obtained from a chloroform–acetone (70 : 30 v/v) solution using optimized concentration, voltage, needle-collector distance and flow.

Fig. 4 Diameter distribution of electrospun nanofibers of PLA–P3TMA samples obtained from a chloroform–acetone (70 : 30 v/v) solution using optimized concentration, voltage, needle-collector distance and flow.

Surface texture of fibers changed also gradually with composition as depicted in the high magnification images of Fig. 3. It is clear that the typical rough/porous surface of PLA fibers progressively became smooth as the P3TMA content increased (e.g. an almost completely smooth texture was observed for PLA–P3TMA 50). Nanofibers were cut with the focused ion beam in order to evaluate their homogeneity through the visualization of the generated cross sections. Fig. 5a clearly shows that PLA nanofibers had an irregular shape as expected from their rough surface texture and also that the section was relatively homogeneous. However, the cross sections of fibers prepared from polymer mixtures (i.e. PLA–P3TMA 50 shown in Fig. 5b) were completely different since in some cases a relative thick and bright outer part could be clearly distinguished. It is possible that the inner part was constituted by some clusters richer in a conductive P3TMA phase, giving place to the observed contrast. In order to verify that fibers were not hollow, a water jet was headed towards their cross section centre. The inset of Fig. 5b illustrated the apparition of a small hole caused by the impact of the jet and consequently demonstrated a compact fibre structure. It should be pointed out that cross sections taken at different places of the microfibers were rather variable since homogeneous and heterogeneous distributions were detected, suggesting that the indicated aggregates are randomly distributed along the micro/nanofibers.

Fig. 5 High magnification SEM micrographs showing the cross section of electrospun PLA (left) and PLA–P3TMA 50 (right) nanofibers. The inset shows the corresponding cross section after being exposed to a water jet.

Characterization of electrospun polylactide–poly(3-thiophene methyl acetate) mixtures

NMR and FTIR spectroscopies were used to assess the composition of the electrospun hybrid scaffolds. Thus NMR spectra revealed the presence of characteristic signals of each homopolymer (Fig. 6), the corresponding areas being in agreement with the theoretical composition of the sample. Spectra always showed the characteristic quadruplet at 5.21, 5.19, 5.17 and 5.15 ppm associated to the proton of the PLA methine group and the doublet at 1.60 and 1.58 ppm associated to the protons of the PLA methyl group. In addition complex signals, which reflect sequence sensitivity, were also observed and attributed to P3TMA. Thus, thiophene protons gave rise to an isolated multiplet (7.18–7.15 ppm) whereas signals of methylene and methyl lateral groups appeared overlapped as a double duplet (3.82–3.80 ppm and 3.62–3.60 ppm) and a duplet (3.77 and 3.72 ppm), respectively. Splitting of the CH₃ signal reflects the head-to-head (HH) and head-to-tail (HT) dyads produced during the chemical polymerization as has been previously reported.²⁵ Areas of dyad signals were practically identical, demonstrating a non-regioregular structure with a statistical disposition of the monomer units. HT and HH dyads corresponding to the methylene protons were splitted again demonstrating triad sensitivity (e.g. splitting of HH dyad gave rise to HHT and THH triads, whereas THT and HHT triads were derived for the HT dyad). The complex signal associated to the CH group can be interpreted in a similar way, the highest chemical shifts corresponding to the TH dyad.

Fig. 6 NMR spectra of a representative electrospun PLA–P3TMA 83 sample.

FTIR spectra of P3TMA samples coming directly from synthesis and from electrospinning were identical and showed a single C=O signal at 1732 cm⁻¹ (Fig. 7). Therefore, ester groups were not cleaved during processing, supporting the interpretation of the complex NMR spectra on the basis of sequence sensitivity. Furthermore, a broad band associated to OH groups could not be detected in the 3300–2500 cm⁻¹ FTIR region (not shown). The spectra of the conducting polymer showed also typical signals at 1435 cm⁻¹ (thiophene ring stretching), 1322 cm⁻¹ (methyl deformation), 1198 and 1167 cm⁻¹ (asymmetric and symmetric C–O stretching), 1012 cm⁻¹, 839 cm⁻¹ (aromatic CH out of plane deformation) and 741 cm⁻¹ (methyl rocking).

Fig. 7 FTIR (1850–650 cm⁻¹) spectra of electrospun PLA (a), PLA–P3TMA 67 (b), PLA–P3TMA 50 (c) and P3TMA (d). For comparison purposes the spectrum of a blend containing 50 wt% of each homopolymer is shown in (e).

The spectra of the scaffold samples were highly similar to that corresponding to the neat PLA (Fig. 7), the C=O streching vibration at 1759 cm⁻¹ and the asymmetric and symmetric C–O stretching at 1183 and 1082 cm⁻¹, respectively, being the most intense bands. The intensity of the characteristic signals of P3TMA decreased with its ratio in the scaffold (e.g. bands at 1322 and 839 cm⁻¹). It should be pointed out that the strong P3TMA band at 1167 cm⁻¹ could not be observed in the spectra of scaffolds even for the highest P3TMA content. This suggests a change in the environment of the ester groups when the P3TMA was processed together with PLA. However, it is also significant that the transmittance measured at 1167 cm⁻¹ was clearly lower for the hybrid scaffold than for the neat PLA (see blue arrows in Fig. 7), even though this wavenumber still corresponded to a transmittance maximum. For the sake of completeness, Fig. 7e shows the spectrum of a powder mixture composed by 50 wt% of each polymer where the two strong PLA bands at 1183 and 1082 cm⁻¹ can be clearly observed together with the strongest P3TMA band. However, the latter appears slightly shifted due to its overlapping with a medium intensity PLA band at 1129 cm⁻¹. Differences between the spectra of electrospun samples and polymer mixtures are evident and suggest the occurrence of some specific interactions in the processed samples.

Table 3 contains the main calorimetric data (i.e. glass transition, cold crystallization and melting temperatures as well as crystallization and melting enthalpies) obtained from the heating run of all electrospun scaffolds, whereas heating traces of representative samples can be seen in Fig. 8. Several features deserve attention:

Table 3 Selected calorimetric data from the heating scan performed with the different PLA–P3TMA electrospun samples

Sample	Tg (°C)	Tc (°C)	ΔHc (J g^−1)	Tm (°C)	ΔHm (J g^−1)	ΔHm–ΔHc (J g^−1)	Xc^a
a Degree of crystallinity referred to the PLA content and using a heat of fusion of 106 J g^−1 for a 100% crystalline sample.^32 Values on the left correspond to the crystallinity of as electrospun samples whereas those on the right correspond to the crystallinity attained during the heating scan.b Commercial sample included for comparison purposes.
PLA^b	60.0	—	—	149.8	33.4	33.4	31.5
PLA–P3TMA 100	61.7	96.6	17.4	146.2	22.0	4.6	4.3, 20.7
PLA–P3TMA 83	59.5	96.4	14.0	147.2	17.5	3.5	4.0, 19.9
PLA–P3TMA 67	59.4	96.5	14.3	147.5	17.8	3.5	4.9, 25.1
PLA–P3TMA 50	60.4	94.1	12.0	145.7	16.2	4.2	7.9, 30.6
P3TMA	67.2	—	—	111.3	11.6	11.6	—

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Fig. 8 DSC heating (a) and cooling (b) scans performed with electrospun PLA–P3TMA 100 (a and b) and PLA–P3TMA 67 (c) samples obtained under the corresponding optimized processing conditions. For comparison purposes the heating scan of a P3TMA powder sample is shown in (d).

(a) A broad exothermic peak (70–130 °C) corresponding to the cold crystallization of PLA is always observed. The high molecular orientation attained in the electrospinning process facilitated the PLA crystallization, as previously reported.³² On the contrary, amorphous samples (Fig. 8c) were always attained when samples were slowly cooled from the melt due to the lack of orientation and the difficulty of PLA to crystallize.

(b) The degree of crystallinity referred to the PLA content tends to increase when nanofibers are obtained from mixtures with a higher P3TMA content, which is probably due to the higher orientation attained when the fibre diameter decreased. This trend was observed in both samples from direct electrospun (i.e. calculated through the difference between melting and crystallization enthalpies) and samples obtained after cold crystallization (i.e. considering only the melting enthalpy).

(c) All samples show a clear glass transition, as could be presumed for amorphous samples, and a typical relaxation endothermic peak, which indicates that metastable PLA glassy material achieves equilibrium thermodynamic conditions with a lower specific volume, enthalpy and entropy.³³

(d) Incorporation of P3TMA has a scarce influence on the characteristic glass transition, crystallization and melting temperatures of the final scaffold. In fact, neither the glass transition nor the melting peak of P3TMA (Fig. 8d) were detected in the heating runs of the resulting scaffolds.

It is interesting to note that P3TMA obtained from chemical polymerization was semicrystalline despite the random disposition of its repeating unit. The X-ray diffraction profile (Fig. 9) is characterized by a strong and well defined peak at 1.17 nm that is related to the interchain distance (inset of Fig. 9). Profiles of electrospun samples showed only amorphous halos without Bragg reflections associated to any of the two homopolymers. However, crystallization took place when the sample was heated up to 130 °C and the diffraction profile clearly showed the two strongest reflections of the α-form of PLA (i.e. those appearing at 0.542 and 0.472 nm that correspond to the (200) + (110) and (203) indices, respectively).^34,35 Therefore, crystallization involved only the PLA since the main reflection of P3TMA can only be guessed.

Fig. 9 X-ray power diffractograms of a P3TMA powder sample and an electrospun PLA-P3TMA 50 scaffold before and after being heated up to 130 °C. Inset shows a scheme of the molecular arrangement where for the sake of simplicity only a head-to-head distribution has been represented.

Scaffolds were thermally stable up to more than 200 °C, as deduced from TGA and DTGA curves (Fig. 10). Incorporation of P3TMA slightly modified the degradation process, leading to a regular increase of both the char yield at 600 °C and the onset degradation temperature. Thermal degradation of PLA has previously been explained according to a complex reaction process with the participation of at least two different mechanisms.³⁶ The DTGA curve of PLA is clearly asymmetric showing a shoulder in the low temperature region, which is associated with a minor degradation process. This shoulder disappears in the DTGA curves of scaffolds suggesting that the first degradation step is hindered by the presence of P3TMA. It is also clear that the DTGA maximum is slightly shifted to a lower temperature, which coincides with one of the two degradation processes observed also for P3TMA. In addition, the scaffold has a hardly observed minor decomposition process that corresponds to the higher temperature degradation step of P3TMA.

Fig. 10 TGA degradation curves of the different electrospun PLA–P3TMA samples. Inset compares DTGA curves of PLA, PLA–P3TMA 50 and P3TMA.

Electrochemical characterization of electrospun polylactide–poly(3-thiophene methyl acetate) mixtures

The low molecular weight of P3TMA makes unfeasible to get electrospun mats and to carry out the corresponding CV measurements from the neat polymer. Processability has been demonstrated to be possible when a high molecular weight polymer (e.g. PLA) is incorporated as it was also demonstrated in previous works concerning the electrochemical characterization of P3TMA nanomembranes prepared by spin coating.^21,22

The control voltammogram recorded for PLA fibers (Fig. 11a) shows an oxidation shoulder with anodic potential E^a_p(O₁) of 0.86 V and an oxidation peak with E^a_p(O₂) higher than 1.1 V. Both peaks have been assigned to the formation of irreversible polarons and bipolarons. Besides, the cathodic scan shows a weak reduction shoulder R₁ with a cathodic peak potential E^c_p(R₁) of −0.08 V. The voltammogram recorded for the PLA–P3TMA 83 hybrid was highly similar to that obtained for PLA (Fig. 11a, curves 3 and 4), but interestingly the hybrids with a lower PLA content (i.e. PLA–P3TMA 50 and 67 samples) showed clearly improved electrochemical properties (Fig. 11a, curves 1 and 2). Thus, E^a_p(O₁) and E^a_p(O₂) anodic potentials shifted to 0.75 and 1.40 V for the 67 wt% sample and 0.82 and 1.08 V for the 50 wt% sample, respectively, and pointed out an increased irreversibility for the oxidation process. Furthermore, the weak R₁ reduction shoulder was always observed (i.e. cathodic peak potentials of −0.04 V and −0.05 V for the PLA–P3TMA 50 and 67, respectively). Current densities (j) determined at 1.10 V were 0.152 mA cm⁻² and 0.394 mA cm⁻² for PLA–P3TMA 83 and 50, samples, respectively.

Fig. 11 (a) Control voltammograms collected using a scan rate of 50 mV s⁻¹ for electrospun mats of PLA (curve 4) and PLA–P3TMA with 50 wt% (curve 1), 67 wt% (curve 2) and 83 wt% (curve 3) in a PBS solution containing 0.1 M LiClO₄. Curve 5 corresponds to a steel sheet used as control. (b) Cyclic voltammograms of the same species after 5 consecutive oxidation–reduction cycles.

The electroactivity, which refers to the ability to store charge, was evaluated by integrating the cathodic and anodic areas of the voltammograms displayed in Fig. 11. Specifically, the electroactivity increases with the similarity between such areas. Results indicate that the electroactivity increases significantly upon the incorporation of P3TMA, especially when the concentration of conducting polymer is higher than 30% wt. Thus, the electroactivity measured for PLA–P3TMA 83, 67 and 50 is 17%, 190% and 289% higher than those of PLA. In order to investigate the electrochemical stability of the samples, five consecutive cycles were applied by varying the potential in the interval defined by −0.4 V (initial and final potential) and 1.1 V (reversal potential) at 50 mV s⁻¹. The electroactivity decreases with the oxidation and reduction areas of consecutive voltammograms. The voltammograms recorded after such five cycles, which are shown in Fig. 11b, indicate that the oxidation–reduction peak distributions are highly similar. As it can be seen, the reduction of the cathodic and anodic areas, which reflects a loss of electroactivity, depends on the P3TMA content. More specifically, the LEA determined for PLA–P3TMA 50 and PLA–P3TMA 83 samples is 27% and 38%, respectively, evidencing that the electrostability increases with the P3TMA content.

Table 4 summarizes the electrochemical behavior for all samples including the values of the current densities measured at 1.10 V for the first and fifth cycles. Major differences in the electrochemical behavior were found when the integrated area and the current density were considered. Thus, independently of the number of cycle, charge and current density increased when the P3TMA content did. Results indicate that the incorporation of P3TMA into the PLA matrix provided fibers with interesting electrochemical features since an increase of both the electroactivity and electrostability were clearly observed.

Table 4 Electrochemical behavior of PLA and PLA–P3TMA 83 wt%, 67 wt%, and 50 wt% samples: current density at 1.1 V and charge for the 1^st cycle and the 5^th oxidation–reduction cycles, and loss of electrochemical activity after 5 consecutive cycles (LEA)

Sample	j at 1.1 V (mA cm^−2)		Q (mC)		LEA (%)
	1^stcycle	5^thcycle	1^stcycle	5^thcycle
PLA	0.138	0.102	2.9	2.0	31
PLA–P3TMA 83	0.152	0.098	3.1	1.9	38
PLA–P3TMA 67	0.355	0.223	8.4	5.5	34
PLA–P3TMA 50	0.394	0.290	11.3	8.3	27

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Wettability of polylactide–poly(3-thiophene methyl acetate) mixtures

Fig. 12 compares the contact angle measured for the different systems studied in this work. The average value of the contact angle determined for PLA (133.4° ± 1.5°) and P3TMA (88.6° ± 2.6°) reflects the hydrophobic character of the polyester and the slightly hydrophilic nature of the polythiophene derivative. It should be noted that the contact angle found for PLA is similar to that recently reported by Liu et al. for PLA nanofibers prepared using a similar technique.³⁸ The very low wetting ability of electrospun PLA nanofibers should be attributed to the combined effect of the surface roughness and morphology for these particular nanostructures. Addition of P3TMA to PLA does not produce relevant changes in the contact angle of the latter, values obtained for PLA–P3TMA 83, 67 and 50 mixtures being 134.5° ± 2.4°, 135.1° ± 1.6° and 128.7° ± 0.8°, respectively. The similarity between the contact angles of PLA and PLA–P3TMA combined with the electrochemical results displayed in the previous subsection suggest that the behavior of the mixtures as supportive matrix for the cell growth should be better than that of the homopolymer. Thus, the incorporation of P3TMA to the PLA matrix is expected to mainly affect the hydrophilicity of the surface, which in fact remains practically unaltered, and the ability of exchange ions across cell membranes, which was shown to be enhanced (see previous subsection).

Fig. 12 Graphical representation of the contact angles measured for PLA, P3TMA and PLA–P3TMA mixtures with 83 wt%, 67 wt% and 50 wt%. Tukey test, p < 0.05; a and b vs. other materials.

Cell adhesion and growth in the PLA–P3TMA hybrid scaffolds

SEM images of VERO cells adhered and grown on the PLA (control) and PLA–P3TMA matrices were qualitatively very similar (Fig. 13). The VERO cells cultured during 24 h on the 3D surfaces led always to a homogeneous distribution and rounded morphologies (Fig. 13a and c), which should correspond to individual cells or small groups of a few cells (insets of Fig. 13a and c). Cells appeared also adhered to the fibers by small filopodia. Cells cultured during 7 days on the mats surface were able to form monolayers (Fig. 13b and d), a typical indicator of cell viability and proliferation. The surface colonization progressed through a suitable cellular extension, which apparently was facilitated and guided by the fiber array (inset in the Fig. 13b and d).

Fig. 13 SEM images of VERO cells adhering (a and c) and growing (b and d) on electrospun fibers of PLA (a and b) and PLA–P3TMA 50% w/w (c and d). The arrows indicate morphological details of the cellular extensions. Quantitative data of the relative adhesion (e) and proliferation (f) of cells onto the fibers mats. * p < 0.05 vs. others fibers mats and control (tissue culture plate), Tukey test for n = 4 replicates.

In quantitative terms, the P3TMA content in the fibers did not affect the cell adhesion (Fig. 13e) but interestingly the cellular proliferation was slightly improved in the scaffolds with a higher P3TMA content (Fig. 13f). In this way, the new electrospun micro/nanofibers constituted by the hybrid materials may improve the electrochemical properties and the cellular response (frequently mediated by ionic channels) to several physiological processes such as membrane depolarization during cell division.³⁷

In conclusion, PLA–P3TMA micro/nanofibers were well suited to provide a good substrate for cell adhesion and cell proliferation and offered an appropriate 3D environment. Furthermore, cells developed in the PLA–P3TMA matrices showed always a healthy morphology.

Conclusions

Mixtures of PLA and P3TMA can be effectively electrospun to render hybrid micro/nanofibers that combine the biocompatibility and electrochemical properties of each homopolymer. Processing conditions (solvent, concentration, flow, voltage, collector distance) were optimized to allow incorporating up to 50 wt% of P3TMA and keeping continuous fiber morphology. Increasing contents of the conducting polymer gave rise to smoother fiber surfaces, smaller diameters, higher orientation of PLA chains and the sporadic formation of cluster aggregates inside the fibers.

Incorporation of P3TMA into the PLA matrix provided an increase of the electroactivity and electrostability, both charge and current intensity clearly increasing with the P3TMA content. Current density determined at 1.10 V was so high as 0.394 mA cm⁻² for fiber mats containing 50 wt% of P3TMA while the electroactivity of such hybrid was 289% higher than that of PLA. The electrostability of samples was probed since the average charge loss was less than 27% after performing five consecutive oxidation–reduction cycles. New hybrid scaffolds were good substrates for cell adhesion and cell proliferation and even the last was clearly enhanced respect to the parent PLA scaffolds. Cellular response to physiological processes seemed to be improved by the incorporation of P3TMA.

Acknowledgements

Authors are indebted to supports from MICINN and FEDER (MAT2012-36205 and MAT2012-34498) and the DIEU of the Generalitat de Catalunya (2009SGR925 and 2009SGR1208). M.M.P.-M. thanks financial support through a FPI-UPC grant. Support for the research of C.A. was received through the prize “ICREA Academia” for excellence in research funded by the Generalitat de Catalunya.

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