Surface modification of poly (L-lactic acid) films to improve electrical conductivity by surface entrapment/in situ polymerization methods

Surface properties of polymeric biomaterials can play a vital role in determining the outcome of biomaterial-living tissue interactions [1–5]. Electrical conductivity serves an important function in the stimulation of tissues such as brain, bone, heart and skin [6, 7]. In 1997, McCaig and his colleagues reported the production of electric fields during cellular activities such as cell division, proliferation, and migration. As a result, cellular activity can be controlled by electrical signals. The neurons, muscles, fibroblasts and osteoblasts in the body are mostly under electrical stimulation. These electrical signals can differentiate cells into cardiovascular and neural cells. Conductive polymers provide the physical and chemical properties of organic polymers and the electrical characteristics of metals for cell growth and tissue regeneration [8–10]. The major problem with conductive polymers in medical applications is their long degradability and low strength in vivo. To eliminate this problem, the scientists made a composite of biodegradable conductive polymers by mixing conductive polymers and biodegradable polymers. Polymer blends and composites based on conducting polymers and biodegradable polymers have been explored for biomedical applications [11]. Poly (L-lactic acid) (PLLA) is a biodegradable polymer widely used as a biomaterial, especially tissue engineering [12, 13]. One of the main problems of polylactic acid is the lack of chemical reaction and biological activity and it cannot communicate well with cells. The hydrophobicity of the polylactic acid surface prevents its proper interaction with cells and many studies have been done to increase its surface hydrophilicity and bioactivity [12]. The modified PLLA surface by conductive polymer could be a suitable candidate for tissue engineering applications. Much research has been done in the past decade to produce PLA/PANI composites. In several papers, PANI was blended into the PLA polymer structure to form conductive nanofibers, which enables electrical stimulation, viability and maturation of cardiac cells [14–20]. In addition, Wang et al used these fibers as cardiomyocyte-based 3D bioactuators [20]. Combining polyaniline with biodegradable polymers such as PLA can improve its performance in nanofibers production. Therefore, most studies have focused on balk modification of PLA, and fewer articles have investigated surface modification of PLA. Due to the high importance of biomaterials in exposure to cells, modification of the PLA surface by conductive polymers and the preservation of its block properties require further studies. By adding PANI chains to the surface, it can maintain the proper strength and biodegradability of the primary polymer.

Many surface modification methods have been used to modify PLLA such as graft copolymerization, chemical conjugation using wet chemistry, plasma treatment, photo grafting, entrapment, coating, migratory additives [21–23]. Surface entrapment (SE) is a kind of physical surface modification used to incorporate modifying species into a swelling region of PLLA substrate to entangle with polymer chains [21, 24–27]. In this method, PLLA films are exposed to a solvent/non-solvent mixture to allow the modifying species to be diffused in to the gel layer of the PLLA surface [28]. Swelling surface is rapidly collapsed by immersion in the non-solvent for incorporating the modifiers [29]. Modifying polymers can be easily deposited on the PLLA surface by chemical in situ polymerization [30].

Polyaniline (PANI) is one of the conducting polymers with three oxidation states, from completely reduced leucoemeraldine to completely oxidized pernigraniline states [31–33]. The electrical conductive form of PANI is the emeraldin salt (ES) [27]. Polyaniline can be doped by protonic acid such as hydrochloric acid (HCL), camphor sulfonic acid (CSA), p-toluene sulfonic acid (PTSA), or by an oxidation agent, either chemically or electrochemically [34–36].

In this study, PANI was used to modify PLLA films by surface entrapment and surface entrapment in situ polymerization (SEIP) methods. The modified films were characterized by attenuated total reflection Fourier transformer infrared (ATR-FTIR) Spectroscopy, UV-visible analysis, scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) mapping analysis. In addition, electrical conductivity of the PLLA surfaces was measured using the 4-probe technique.

2.1. Materials

2.2. Film preparation

2.3. Polyaniline synthesis

2.4. Entrapment process

2.5. Surface characterization

Attenuated total reflection Fourier transformer infrared (ATR-FTIR) spectra of the control and modified PLLA films were determined by a spectrophotometer (Bruker EQUINOX 55) with the incidence angle of 45 degree and the spectra were collected in the range of 500 to 4000 cm⁻¹.

The FT-IR spectra of the EB form PANI were recorded by preparing polyaniline-KBr pellets using 1:20 mass ratio of polyaniline KBr.

The transparent thin PLLA film was formed on the glass slide. Then the slide was modified with doped and dedoped polyaniline. The UV–vis Spectra of the modified PLLA films were recorded by UV–vis spectrophotometer (CARY 100 Conc) in the range of 290–900 nm.

The morphologies of the polyaniline entrapped PLLA surfaces and the unmodified surfaces were studied using a (VEGA 2 TESCAN) scanning electron microscope (SEM). The surfaces were coated with a thin layer of gold. For the cross sectional images, the samples were fractured in liquid nitrogen.

Energy dispersive x-ray mapping analysis (EDX Mapping) was provided in addition to the conventional SEM image to provide a meaningful picture of the element distribution of a surface or cross section.

The electrical conductivity of the PLLA surfaces was measured using the 4-probe technique. In this method, the four probes were located in the same distance from each other. Then a constant current was applied to the outer probes and the potential difference was determined using internal probes by a voltmeter [31]. The conductivity was calculated as follows:

$$\begin{eqnarray*}&&{\boldsymbol{\sigma }}={\bf{1}}/\left({\boldsymbol{\pi }}t/{\bf{ln2}}\right)\left({\boldsymbol{v}}/{\boldsymbol{I}}\right)\end{eqnarray*}$$

where σ is conductivity in s/cm, t is the thickness of films in cm, I is the current in mA, and V is the voltage in mV [37].

The presence of the PANI on the PLLA surfaces was confirmed by comparing the ATR-FTIR spectra of the unmodified and modified PLLA films (figure 1). In the ATR-FTIR spectra of PLLA film (figure 1(a)), the peaks could be observed at 1752 cm⁻¹ , 1454 cm⁻¹ , 1083 cm⁻¹ , 2998 cm⁻¹ and 2947 cm⁻¹. They corresponded to the –C=O stretching vibration (ester carbonyl group), $-{{\rm{CH}}}_{3}$ bending vibration, –C–O– stretching vibration , $-{{\rm{CH}}}_{2}-$ asymmetric and symmetric stretching vibrations, respectively [38, 39].

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In the FTIR spectra of EB form PANI (figure 2), the peak at 3400 cm⁻¹ was attributed to N–H stretching of an aromatic amine. The peaks at 1593 cm⁻¹ and 1499 cm⁻¹ were assigned to the stretching vibrations of the quinoid rings and the stretching vibrations of benzenoid rings, respectively. The peak at 1303 cm⁻¹ corresponded to the C–N stretching. Aromatic CH stretching vibration appeared in 3026 cm⁻¹. The peaks at 1165 cm⁻¹ and 830 cm⁻¹ corresponded to the C–H in-plane bending vibration and the out-of-plane vibration in the 1,4-disubstituted benzene ring, respectively [40].

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An additional peak corresponding to PANI could be observed in the modified films by SE and SE IP methods (figures 1(b), (c)). Thus, the presence of PANI was demonstrated on the surface of PLLA films.

UV–vis Spectra of doped and dedoped polyaniline on the surface of the PLLA film are shown in figure 3. Polyaniline has a conjugated pi bond on its backbone that is responsible for its novel electrical properties [41]. In polyaniline, the large number of interacting pz-orbitals leads to the formation of a fully occupied π-valence band (VB) and an empty π*-conduction band (CB). Electrons can be easily jumped from VB to a higher energy level in CB [42]. Electric properties of polyaniline are similar to those of semiconductors [43]. The electrical conductivity of polyaniline results in charge carriers, which are obtained from the doping process in electron system. Doping can induce charge in polyaniline by taking electrons from the VB (p-doping). Polyaniline is made conductive by the protonation of backbone nitrogen site [44, 45].

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Absorption wavelength of 330 nm and 630 nm (figure 3(a)) corresponded to π → π* transition in the benzene and quinone rings, respectively [40]. The absorption band at 380 nm (figure 3(b)) was attributed to the π → π* transition in benzenoid units of the polymer chains [40]. The absorption band at 330 nm for dedoped polyaniline was shifted to 380 nm for the doped polyaniline. The π → π* transition in the doped polyaniline occurred at higher wavelengths and lower energy was required for this mutation. In other words, the gap between the valence band and the conduction band was less and electron transfer occurred more easily. Therefore, the intensity of the absorption band at 630 nm was decreased by adding small amounts of protonation and new absorption bands appeared at 430 and 730 nm. New absorbance peaks in the curve (b) could be taken as evidence showing polaron transition by doping [40]. As a result, doping could lead to a decrease in the energy gap between the conduction band and the valence band and the electrical conductivity obtained in the doped surface.

The SEM micrograph of the unmodified PLLA film is shown in figure 4(a). The unmodified PDMS film had a smooth surface. The SEM micrographs of the modified PLLA films by SE are shown in figure 4. More considerable presence of PANI on the PLLA surfaces was observed with increasing the concentration from 1% (w/v) to 5% (w/v). SEM micrograph of PLLA film, after immersion in a mixture of the solvent and the non-solvent without the addition of PANI, is shown in figure 4(d). After being exposed to the solvent/non-solvent mixture, the amorphous regions on the surface of the films were dissolved much faster than the crystalline regions. Thus, a porous surface was observed after the entrapment of PANI on the surface [46]. Moreover, crystalline regions were disappeared and the thickness of the film was reduced with increasing the exposure time.

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The SEM micrographs of the modified PLLA films in different concentrations of aniline by SEIP method are shown in figure 5. At high concentrations of monomers, the rate of polymerization in the solution was found to be higher than that on the surface of PLLA films. It could be because of the more difficult migration of monomers in the solution adjacent to the surface [47] (figure 5(c)). The polymerization rate was lower at low concentrations (figure 5(a)) and polymer formation on the surface of the films wasn't good in a specified range of time. The optimal concentration can be seen in figure 5(b).

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As can be seen in figure 5, the structural order was increased with decreasing the temperature. At low temperatures (figure 5(b)), the polymerization started slowly and polymer growth was slower and more regular. Thus, there was more possibility of polymer formation on the surface of the films, while at higher temperatures (figures 5(e), (g)), the polymer growth was found to be faster. At higher temperatures, the products of the polymerization could be mostly short polymer chains or oligomers because the color of the modified films looked brownish [41, 48, 49]. On the other hand, PLLA films were softer at higher temperatures during polymerization and lost their form.

The effect of dopant/monomer ratio (CSA:Ani) on the surface morphology is shown in figures 5(b), (d). Camphor sulfonic acid (CSA) was a protonic surfactant containing a polar head and a long non-polar chain which functioned as a dopant.The hydrophilic head ($S{O}_{3}^{-}$) protonated the –NH group of aniline. The hydrophobic tail of CSA was closer to the PLLA surface by van der Waals forces, leading to better adhesion on the PLLA surface [50, 51]. When the dopant ratio was increased, more polymerization centers were formed on the surface of the film and the polymerization took a longer time. Therefore, more polymers were formed on the surface of PLLA films. The hydrochloric acid (HCL) molecules were more hydrophilic than CSA and tended to remain in the aqueous solution instead of the surface of PLLA films (figure 5(h)).

In the same conditions, the polymer was not completely formed at lower time with the change of time and it tended toward the best layers formed (figures 5(h), (f)).

EDX mapping images of C, O and N elements and EDX spectrum from the cross section of the modified PLLA films by Both SE and SEIP methods are shown in figures 6 and 7. EDX analyses were used to determine the spatial distribution of the elements. The corresponding C and O maps showed the homogenous distribution of these elements. EDX mapping images of the N atoms indicated the increased concentration of N element on the surface of the films. The presence of N element proved the formation of PANI in the surface of PLLA films. As can be seen in the images, the thickness of the PANI layer was around 10 μm. The morphology of the PANI consisted of irregular fibers in the SE method (figure 4); thus PANI formation on the surface was not quite compact (figure 6). On the other hand, the presence of sulfur element confirmed PANI doping by CSA (figure 6).

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The penetration of aniline monomers into the top thin layer of the PLLA films and formation of PANI can be observed in figure 7. It is believed that these steps could be considered as follows: Firstly, the films were immersed in chloroform and the top thin layer on the surface was swelled. Then, the films were placed into the aniline solution and it penetrated into the swollen layer. Afterward, the polymerization was started and PANI formed in that layer after addition of the oxidant solution. In the SEM cross sectional micrographs of the modified PLLA films by SEIP method (figure 7), the surface was completely covered by PANI because polymerization began by monomer at a low temperature.

The electrical conductivity of modified PLLA films by SE is shown in table 2. As can be observed, the electrical conductivity was increased with an increase in the concentration of PANI from 1% (w/v) to 5% (w/v). The electrical conductivity was also increased with increasing the immersion time from 0.5 h to 1.5 h, whereas the electrical conductivity was decreased after 3 h due to the reduction of the surface uniformity of the films by time. Based on the results from solvent/non-solvent ratios, lower amounts of surface gelation were observed at lower ratios of chloroform in the mixture and thus, lower amounts of PANI were entrapped on the surface. Thus, the electrical conductivity was decreased to 1.2 × 10⁻³ S cm⁻¹ at the ratio of 50:50. Accordingly, at the ratio of 30:70, the amount of entrapped PANI was not significant.

The electrical conductivity of the modified PLLA films by SEIP is shown in figures 8–10. The electrical conductivity was studied by both CSA and HCL as the dopant. The electrical conductivity of the doped films by CSA was higher than that by HCL, because the polymerization rate was lower and a more regular structure was formed. On the other hand, CSA was an organic compound showing a better interaction with the polymer chains. Thus, as other researchers have suggested, the electrical conductivity and morphology of PANI depend on the amount of doping and the type of doping [52].

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According to the results in figure 8, the electrical conductivity was increased with time. But the changes were not significant at times more than 24 h. Before 24 h, the electrical conductivity was increased. Therefore the best time for polymer formation was 24 h at 0 °C.

The highest electrical conductivity at 24 h was observed when the concentration of aniline in solution was 2.5% (figure 9). PANI polymerization rate was dependent on the aniline concentration. Increasing the monomer concentration (5%) caused defects in the polymer chain and the electrical conductivity was decreased. At low concentrations (1.66%), the polymerization time was not enough and the electrical conductivity was low.

The optimal concentration and time were selected in 2.5% and 24 h and the temperature was changed at three points (0 °C, 25 °C and 37 °C) (figure 10). At lower temperatures, the polymerization rate was lower and polymer growth had a high order. Therefore, the electrical conductivity was increased with decreasing the temperature. Beginning of the polymerization was observable by a change in the color of the solution. Since the rate of polymerization was increased at higher temperature; the possibility of long chain formation, and consequently the electrical conductivity decreased at 37 C.

With increasing the amount of CSA as the dopant, the electrical conductivity was changed from 0.21 S cm⁻¹ to 0.22 S cm⁻¹. At the first glance, the electrical conductivity was expected to be increased with increasing CSA, but because CSA was a nonconductive material, increasing the amount of CSA did not change electrical conductivity some much. The results of other studies revealed that parallel with increasing the ratio of CSA to aniline, the conductivity increases but this study showed that excessive doping reduces electrical conductivity [52]. In other words, a nonconductive component was inserted into the structure of PLLA surface.

In this research, the effect of monomer concentration, doping process, time and temperature of polymerization on the film morphology and electrical conductivity in SEIP method were discussed. At high concentrations of monomers due to lack of migration of monomers to the surface and at low concentrations of monomers due to lack of time, polymerization is not fully performed. After optimizing the monomer concentration, the effect of temperature was investigated. The best morphology was obtained at water bath (0 °C) on the surface and the highest electrical conductivity was achieved at the same temperature. Comparing CSA with HCL as doping agents with the same polymerization conditions, HCL only performs polyaniline doping and does not correlate between PLLA surface and polyaniline. Therefore, removing polyaniline from surfaces that are dipped in HCL is easier and faster. CSA is a better option for the doping process. CSA-doped samples result in higher electrical conductivity due to lower polymerization speed and optimum structural order. On the other hand, excessive CSA reduces electrical conductivity. By comparing the obtained results, the highest electrical conductivity in SEIP method was achieved by polymerization of the optimum concentration of monomer at 24 h and 0 °C and the use of CSA as doping agent. The electrical conductivity obtained in this method was much higher than the composites made of conductive polymer and degradable polymer. The reported electrical conductivity in the composite samples was about 0.04 S cm⁻¹ [53–58] but with the polyaniline coating on the surface in this method, we achieved 0.22 S cm⁻¹.

The biomaterial surface plays an important role in their interaction with cells and hence the modification of the level of biodegradable scaffolds has been of great importance in tissue engineering. Conductive polymers have high biological activity and the ability to transmit electrical signals stimulates cells to grow. Therefore conductive polymers are an ideal choice as coatings on the scaffold surface. Their mechanical strength is one of the other weaknesses of conductive polymers that influence their use as a balk of scaffolds. In addition, they cannot be coated on the surface alone due to their low strength and the possibility of being removed from the surfaces. Therefore, in this research, a composite coating on the surface is suggested. In other words, with polymer entrapment or monomer polymerization between the polylactic acid chains in the surface layer, a kind of interpenetrating polymer network (IPN) is formed of biodegradable polymer and conductive polymer which is much stronger than conventional coating methods. In this study, PANI was successfully used to modify the PLLA surfaces via SE and SEIP methods. The electrical conductivity of the modified PLLA surfaces was greatly increased. The electrical conductivity of PLLA surface, uniformity and density of PANI on surface in the SEIP method were higher than those obtained in the SE method. Since the solubility of PANI in organic solvents is low, its application as a coating on the surface of biodegradable polymers is suggested. Thus, the use of conductive polymer on the surface firstly eliminates the problem of biodegradability for tissue engineering application and secondly, by coating on the surface of the PLA, it enhances bioactivity and better interaction with cells. On the other hand, its electrical conductivity is much higher than composites made of biodegradable polymers and conductive polymers. In composite scaffolds, the presence of a significant percentage of nonconductive polymer decreases the electrical conductivity of the structure. On the other hand, as the conductive component increases, the mechanical strength of the structure and its degradability are affected. Therefore, with conductive coatings on the scaffold surface, in addition to maintaining the integrity and useful properties of the base polymer, it provides access to the properties required for biomaterial and cell interaction in tissue engineering.

Maximum electrical conductivity in this research was observed in modified PLLA films by CSA dopant, 2.5% aniline, after 24 h at water bath (0 °C). We demonstrated the surface entrapment in situ polymerization (SEIP) could be a facile and efficient route to modify the surfaces of PLLA films by the entrapment of PANI.