**2. Results**

Films of PPY loaded with an anionic drug (either DMP or MER, see Figure 1) were deposited onto the surface of indium tin oxide (ITO)-coated glass electrodes via electropolymerization. The electrochemical oxidation (at a potential of 1.0 V) and polymerization of pyrrole on the anode (the ITO-coated glass electrode) yielded a film where the positive charges on the backbone of the polypyrrole were counterbalanced by the presence of the anionic dopants from the electrolyte during electropolymerization (in this case one of the anionic drugs, DMP or MER).

The successful deposition of polypyrrole films onto the surface of the ITO electrodes was easily observable by eye (i.e., presence of a black film on the surface of a clear and colorless ITO electrode), and confirmed by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) data, as shown in Figure 2 (SEM and EDX imaging of DMP-doped films), Figure 3 (SEM and EDX imaging of MER-doped films), and Figure 4 (EDX data for DMP-doped and MER-doped films).

SEM revealed the surfaces of the films to have μm-scale roughness characteristic of electropolymerized PPY, with the features having a broad distribution of sizes, from very small particles of ca. 100–200 nm to much larger 1–20 μm "cauliflower-like" structures, akin to the features reported for films prepared via analogous electropolymerization methodologies in the literature. There were concomitant differences in electrical properties (i.e., impedance/resistance) relative to the surface-area-to-volume ratio of the materials [16,46]. EDX analysis [47] showed elemental signals characteristic of Au (instrumental background, characteristic Kα 2.123 keV; data not shown), Si (glass electrode, characteristic Kα 1.74 keV), and the elements associated with the drug-loaded polymer films: C and N (characteristic of polypyrrole) [48], F, O, and P (characteristic of DMP), and O and S (characteristic of MER).

**Figure 2.** Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) images of a DMP-doped PPY film: (**A**) SEM image of a DMP-doped PPY film; (**B**) EDX layered image from a DMP-doped PPY film; (**C**) Kα emission of C; (**D**) Kα emission of F; (**E**) Kα emission of N; (**F**) Kα emission of O; (**G**) Kα emission of P; (**H**) Kα emission of Si. Scale bars represent 100 μm.

**Figure 3.** SEM and EDX images of an MER-doped PPY film: (**A**) SEM image of an MER-doped PPY film; (**B**) EDX layered image from an MER-doped PPY film; (**C**) Kα emission of C; (**D**) Kα emission of P; (**E**) Kα emission of F; (**F**) Kα emission of O; (**G**) Kα emission of N; (**H**) Kα emission of Si. Scale bars represent 100 μm.

The EDX maps in Figures 2 and 3 demonstrate the elemental composition of the films to be homogeneous over the surface of the films. Analysis of the EDX data for DMP-doped or MER-doped films (Figure 4A,B, respectively) showed elemental signals characteristic of carbon (Kα at 0.277 keV) and nitrogen (Kα at 0.392 keV) found on polypyrrole. For the DMP-doped films, there were peaks at 0.677 keV, 0.525 keV, and 2.014 keV, characteristic of the Kα of F, O, and P, respectively (Figure 4A). For the MER-doped films, there were peaks at 0.525 keV, 2.308 keV, and 2.622 keV, characteristic of the Kα of O, S, and Cl, respectively (indicative of the hydrochloride salt of MER, Figure 4B).

**Figure 4.** (**A**) EDX data from a DMP-doped PPY film; (**B**) EDX data from an MER-doped PPY film.

Examination of the films using Fourier-transform infrared (FTIR) spectroscopy in attenuated total reflection (ATR) mode also confirmed the presence of the drugs (DMP and MER) doping the PPY (Figure 5). The FTIR spectra of the PPY films showed absorptions at ca. 1480 cm<sup>−</sup><sup>1</sup> and ca. 1535 cm<sup>−</sup>1, corresponding to the symmetric and asymmetric ring-stretching modes, respectively [49–53]. The FTIR spectra of DMP and DMP-doped PPY films showed absorptions at 989 cm<sup>−</sup><sup>1</sup> and 1197 cm<sup>−</sup>1, corresponding to the characteristic symmetric and asymmetric stretching vibrations of the phosphate groups, while the absorption band at 1641 cm<sup>−</sup><sup>1</sup> corresponded to the C=O stretching vibration of DMP (Figure 5A,B). The FTIR spectra of MER and MER-doped PPY films showed absorptions characteristic of stretching vibrations of the C=O bond in the β-lactam ring of MER, located at 1863 cm<sup>−</sup><sup>1</sup> (Figure 5C,D).

**Figure 5.** Fourier-transform infrared (FTIR) spectra collected in attenuated total reflection (ATR) mode: (**A**) DMP; (**B**) DMP-doped PPY film; ( **C**) MER; ( **D**) MER-doped PPY film.

X-ray diffraction (XRD) analysis of the DMP-doped and MER-doped films revealed some interesting structural information confirming the inclusion of the drugs in the films (Figure 6). PPY has a relatively amorphous structure with a broad peak in the region of 2θ = 20–30◦ in the XRD patterns [54] which is associated with the closest distance of approach of the planar aromatic rings of pyrrole (e.g., face-to-face pyrrole rings) [55]. Interestingly, doping PPY with DMP led to a peak shift to the region of 2θ = 15–28◦, confirming that addition of DMP alters the packing of the PPY chains in the film [56]; likewise, doping PPY with MER also led to a peak shift to the region 2θ = 15–38◦. Interestingly, some of the crystalline peaks of pure MER (which appear at 12.6◦, 16.6◦, 18.2◦, 19◦, 20◦, 21.6◦, 22.3◦, 22.7◦, 23.3◦, 25.2◦, 26.7◦, 28.2◦, 29◦, 30◦, 31.7◦, 34.5◦, 37.7◦, 39◦, and 44◦ [57]) can be identified in the XRD pattern (appearing, albeit weakly, at 2θ = 17.9◦, 21.9◦, 23.7◦, 34.5◦, and 38.0◦).

**Figure 6.** X-ray diffraction (XRD) data. (Black line) DMP-doped PPY film. (Gray line) MER-doped PPY film.

The electrochemical properties of the DMP-doped PPY and MER-doped PPY films were studied via cyclic voltammetry (CV, Figure 7), and electrochemical impedance spectroscopy (EIS, Figure 8). Cyclic voltammograms of DMP-doped PPY and MER-doped PPY films (1st, 5th, 10th, 15th, 20th, 25th, 30th, 35th, and 40th cycles) are displayed in Figure 7A,B, respectively. As evident from the CV curves, the charge-storage capacities of the films decreased steadily on repeated cycling, and the areas under the curves of the 35th and 40th cycles were almost the same. The currents evolved in the MER-doped PPY were higher than those in the DMP-doped PPY, and the oxidation and reduction peaks were more prominent. Occurrences of reduction and oxidation peaks correspond to the de-doping of the films (i.e., release of drug molecules), and the films were subsequently re-doped by other anions (either the anionic drug, or anions from the PBS buffer: H2PO4− and HPO4<sup>2</sup><sup>−</sup>, and Cl−).

EIS measurements were conducted to investigate the electron-transfer resistance (Ret) of the drug-doped PPY films, and the respective Nyquist plots are displayed in Figure 8. All plots have a semi-circular arc in the high-frequency range, followed by a vertical line along the imaginary axis corresponding to a diffusion process. The diameter of the suppressed semicircle gives the value of the electron-transfer resistance (Ret), which was the most directive and sensitive parameter reflecting the changes at the electrode–solution interfaces, and could be evaluated from the difference in the real part of the impedance between low frequency and high frequency [58]. The Ret values were 21.18 Ω and 76 Ω for DMP-doped PPY and MER-doped PPY films, respectively. The Ret of MER-doped PPY films was higher than that of DMP-doped PPY films, indicating that the electron transfer was more easily achieved at the DMP-doped PPY film interfaces. The diffusion resistance of DMP-doped PPY films was shorter than that of MER-doped PPY films, indicating a shorter ion-diffusion path length of the [Fe(CN6)]<sup>3</sup>−/4<sup>−</sup>, H2PO4<sup>−</sup>, HPO4<sup>2</sup><sup>−</sup>, and Cl− ions into the interior of the film. Importantly, the EIS results are in good agreemen<sup>t</sup> with the CV results.

**Figure 7.** Cyclic voltammetry (CV) data of the films in phosphate-buffered saline (PBS; pH = 7.4) at a scan rate of 50 mV·s<sup>−</sup>1: (**A**) DMP-doped PPY film; (**B**) MER-doped PPY film.

**Figure 8.** Nyquist plots derived from electrochemical impedance spectroscopy (EIS) data of the films in PBS (pH = 7.4). (Black line) DMP-doped PPY film. (Gray line) MER-doped PPY film.

The release of drugs doped into the PPY films was studied using UV spectroscopy (Figure 9), where the release was either passive (i.e., in the absence of an electrochemical stimulus) or electrochemically triggered (i.e., in the presence of one or more rounds of electrochemical stimulation—30 s of stimulation at a reducing potential of 0.6 V, followed by 10.5 min of rest (Figure 9A)). The quantity of the drug in solution was quantified at various time points, and the data are reported as cumulative release as a percentage of the total mass of the drug in the film (films were individually weighed; DMP-doped PPY films contained 12 wt % of DMP, and MER-doped PPY films contained 4 wt % of MER), and compared to passive drug release from unstimulated films every 11 min.

**Figure 9.** Electrochemically enhanced delivery of drugs from films in PBS (pH = 7.4) as determined by UV spectroscopy: (**A**) Electrical stimulation paradigm: three cycles of 30 s on, 10.5 min off; (**B**) cumulative release of DMP from DMP-doped PPY films, passive release (black bars), electrically stimulated release (gray bars); (**C**) cumulative release of MER from MER-doped PPY films, passive release (black bars), electrically stimulated release (gray bars).

For both DMP-doped and MER-doped films, the drugs were observed to passively diffuse from the films, as is the norm for drug-loaded polymers. The passive diffusion of DMP from the films was ca. 10–15% of the total DMP content of the films over the course of the experiment, whereas the passive diffusion of MER from the films was ca. 10–30% of the total MER content of the films over the course of the experiment. The amount of drug released from the electrically stimulated films was observed to be higher than that for the passive control samples at each time point measured. For the DMP-doped films (Figure 9B), there was an increase of ca. 10–15% in the amount of drug released at various time points; whereas for the MER-doped films (Figure 9C), there was a an increase of ca. 15–30% in the amount of drug released at various time points after each round of electrical stimulation.

Some physical descriptors (constitutional and electronic) for DMP and MER were calculated using the Molecular Operating Environment (MOE) software (version 2014.0901, Chemical Computing Group Inc., Montreal, QC, Canada). The selected descriptors were the dipole moment, LogP (octanol/water), molecular globularity, number of H-atom donors and acceptors, and molecular flexibility (Table 1). The dipole of DMP was lower than that of MER (1.7033 vs. 9.2305, respectively), as was the number of hydrogen-bond acceptors (8 vs. 9, respectively), hydrogen-bond donors (5 vs. 7, respectively), and flexibility (5.3661 vs. 8.8623, respectively). The globularity of DMP was higher than that of MER (0.1110 vs. 0.0265, respectively), as was the LogP (1.2640 vs. −0.5960, respectively).


