*2.3. pH Effects over PPN*

The polymerization of pyrrole with FeCl3 and PVA results in acidic PPN suspensions. Accordingly, the pH values of the PPN suspensions must be tuned to be used in biological applications. Therefore, it is necessary to evaluate the influence of pH on the properties of the PPN. The pH values studied were 4.2 (pH obtained from synthesis), 6, 7.4, 8, and 10; adjusted using solutions of 0.1 M NaOH.

### *2.4. Characterization of PPN*

Hydrodynamic size, polydispersity index (PDI), and zeta potential (ζ) were measured using a Zetasizer (Zetasizer Nano ZS Malvern Instruments Ltd., Worcestershire, UK). Hydrodynamic size measurements of PPN were performed by the Dynamic Light Scattering technique (DLS). A refractive index of 1.33 and water as a dispersant were used in the analysis. Each sample was measured three times with at least 10 runs at 25 ◦C for the size and ζ analysis. The ζ was obtained by using the laser Doppler electrophoresis technique. The morphology of PPN was observed with a field transmission electron microscopy (TEM) JEOLTEM-2010F with an operating voltage of 200 kV (JEOL, Ltd., Tokyo, Japan). For TEM observations, a drop of the sample solution was spread on a smooth film of a carbon-coated copper TEM grid and dried. After that, 1% phosphotungstic acid was applied on the grid for 1 min; the excess was absorbed using filter paper. All samples were subsequently dried in a vacuum before observation. The optical absorption spectra of PPN were acquired in a UV6300PC spectrophotometer (VWR, Radnor, PA, USA). The characteristic absorption band of PPN in the NIR region was verified. A standard curve of PPN suspensions was built at 808 nm to determine the PPN concentration. All experiments were performed in triplicate.

#### *2.5. Photothermal Evaluation of PPN*

The temperature variations of PPN suspensions irradiated with an NIR laser beam with an output power of 1.5 W centered at 808 nm (Changchun New Industries Optoelectronics Tech Co., Ltd., Model No. PSU-III-LED, Changchun, China) were measured. PPN suspensions were placed into a rectangular quartz cuvette cell and irradiated with the NIR laser. The experimental setup for the analysis and data acquisition is shown in Figure 2. The temperature variations were measured using a K-Type thermocouple connected to a multimeter that acted as a data acquisitor. Suspensions of PPN from 0 to 30 μg/mL and the pH of nanoparticle suspensions from 4.2 to 10 were analyzed. As a control, the temperature of a nonirradiated cuvette cell with water was measured simultaneously to calculate the temperature increments of the suspensions; that is, to adjust to the ambient temperature variations.

**Figure 2.** Scheme of heating experiments with the laser irradiation, indicating the dimensions of the quartz cuvette cell and the suspension volume (**left**), and a frontal view with the approximate position of the laser spot and the thermocouple (**right**).

#### *2.6. Spatiotemporal Temperature Distribution Analysis*

The spatiotemporal temperature distribution of irradiated PNP suspensions was mathematically analyzed by modeling PPN suspensions inside a quartz cuvette. The modeled PNP suspension volume is comprised of two regions: the laser region, where the laser irradiates and the heat is produced, then the suspension region, where the heat is transported only by heat conduction. The energy equation in the laser region is:

$$
\rho \mathcal{C}\_P \frac{\partial T}{\partial t} = k \nabla^2 T + \eta \frac{I}{V} \left( 1 - 10^{-OD} \right) + S\_W \tag{1}
$$

where the term on the left side is the thermal energy accumulation. The first term on the right-hand side is the heat conduction, and the second term represents the photothermal heating from laser irradiation. This second term *η <sup>I</sup> V* <sup>1</sup> <sup>−</sup> <sup>10</sup>−*OD* consists of heat generation due to the PPN. Furthermore, the heat generation due to the water absorption is considered in the third term of the right side (*Sw*). The energy equation for the suspension region is

$$
\rho \mathcal{C}\_P \frac{\partial T}{\partial t} = k \nabla^2 T \tag{2}
$$

The equation shows the accumulation term on the left-hand side, and on the right-hand side, only the conduction term appears.

In these equations *<sup>ρ</sup>* (kg/m3) is the density and *CP* (J/kg·K) is the heat capacity of the suspension, respectively, *k* is the thermal conductivity (W/m·K), *η* counts for the photothermal transduction efficiency, *I* (W) is the laser power, *V* (m3) stands for the irradiated volume, *OD* is the optical density of the suspension and *SW* (W/m3) is the water heat generation in the irradiated volume.

To solve Equations (1) and (2), the following boundary condition was applied to the cell walls and the air/suspension interface:

$$
\log \sigma = \mathcal{U} \left( T\_f - T \right) + \epsilon \sigma \left( T\_\infty^4 - T^4 \right) \tag{3}
$$

where *q* is the heat flux at the system boundaries (W/m2), *U* is the overall heat transfer coefficient (W/m2·K), *Tf* is the temperature of media surrounding the cell (air) (K), *<sup>T</sup>* is the suspension temperature (K), *ε* is the emissivity of either the cell wall or the air/solution interface, *σ* is the Stefan–Boltzmann constant, and *T*∞ is the temperature of the radiation sink on the exterior of the domain (K). The overall heat transfer coefficient *U* incorporates the thermal resistance of the cell wall and the external free convection thermal resistance. Furthermore, the bottom of the quartz cuvette was considered adiabatic, resulting in the following boundary condition:

$$q = 0 \, at \, z = 0 \,\tag{4}$$

The main physical parameters used in the mathematical model were obtained from the experimental setup and the literature [27], as presented in Table 1. The model was numerically solved using Ansys Fluent R3 [20,21] using a hexahedral mesh of 85,536 elements. A semi-implicit method for pressure-linked equations was used with a time step of 0.4 s and 3750-time elements.

**Table 1.** Main physical parameters used in the mathematical model.


\* The emissivity of glass at 300 K is between 0.90 and 0.95; the emissivity of water at 300 K is 0.96 [27].

Determination of Model Parameters: *η*, *U*, and *SW*

The values of parameters *η*, *U* and *SW* were obtained by fitting the model temperature predictions to the experimental measurements during laser heating. A two-step approach was followed: first, the order of magnitude *η* and *U* was obtained by analyzing a hypothetical case of the adiabatic cell and using a heat transfer correlation, respectively. The second step consisted of minimizing the sum of squares of differences between the experimental and predicted temperature increments using the heat model.

The order of magnitude of *η* was determined from the maximum temperature in the irradiated system (analyzed by considering a hypothetical case of an adiabatic cell), where all the energy absorbed by the system is accumulated, resulting in the maximum possible temperature at the given laser power and time of irradiation. The value of *η* defined as baseline must satisfy two conditions at a specific PPN concentration. The first condition is that the temperature increment in the adiabatic case be higher than the experimental one. The second condition is that the initial heating rate be faster than the experimental heating rate (Figure S1).

The order of magnitude of *U* was obtained using a correlation for free convection on a vertical surface. The properties of air used were estimated from an average temperature between the temperature of the surrounding media and the temperature measured by the thermocouple in the heating plateau (Figure S2).

The second step for determining *η* and *U* was implemented as follows: a set of values of *U* for each concentration and a single value of *η* equal to or greater than the baseline values were used to solve the model. Then the model was solved using the parameters and conditions described above. Next, the sum of squares was calculated for the values following Equation (5). The process was repeated at least three times to find the values of *η* and *U* and the minimum sum of squares. The term *SW* was obtained by fitting the heat model to the experimental data at a concentration of 0 μg/mL nanoparticles.

$$
\sigma^2 = \sum\_{i=1}^n \frac{\left(\Delta T\_{\exp} - \Delta T\_{\sin}\right)^2}{n - k} \tag{5}
$$

#### *2.7. Statistical Data Analysis*

The statistical analysis for PPN hydrodynamic size, PDI, and ζ measurements was performed by the software OriginPro (ver. 9). Results were analyzed by ANOVA followed by Tukey's HSD test (α = 0.05). The differences were considered statistically significant when the *p*-values were minor or equal to 0.05.

#### **3. Results and Discussion**

### *3.1. Characterization of PPN Synthesis*

PPN was synthesized by chemical oxidizing polymerization with hydrodynamic average sizes of 98 ± 2 nm from three independent preparations, verifying the method's reproducibility. Furthermore, the DLS results show a narrow size distribution with a PDI of 0.04 ± 0.02 (as shown in Figure 3A). Different sizes can be obtained by varying the concentrations and molecular weight of PVA and the molar ratio between FeCl3 and pyrrole monomer [16]. Furthermore, using a different stabilizer in the chemical oxidizing polymerization could influence the PPN sizes. The literature has reported polyvinylpyrrolidone (PVP) as a stabilizer, resulting in PPN sizes ranging from 151.5 to 93.5 nm in the function of the PVP concentration increment [28]. The ζ distribution of PPN that resulted from the synthesis shows a single peak with a narrow width and a positive value, as seen in Figure 3B. This value, obtained from three independent preparations of PPN, was 20.0 ± 2.1 mV, indicating good nanoparticle stability due to electrostatic particle repulsion and preventing further particle aggregation [29]. The positive charge of these PPN is attributed to the presence of amino groups in the polypyrrole polymeric chain [30]. Other authors using a similar synthesis method of PPN report ζ values of 14.0 ± 0.4 mV for particles of 106 ± 1 nm [31]. The slight differences in the ζ values may be due to the concentrations of PVA used in the synthesis since the PPN surface covered by this stabilizer changes with the concentration used in the synthesis. Then, as the PVA concentration increases in the synthesis, the PPN surface-exposed hydroxyl groups increase, producing variations in the PPN surface charge.

**Figure 3.** PPN properties were obtained from the synthesis (pH value from the synthesis of 4.2). (**A**) Diameter size distribution from DLS measurements. Means ± SD, *n* = 3. (**B**) ζ distribution. Means ± SD, *n* = 3. (**C**) TEM image of PPN with an insert of a single particle. (**D**) Histogram of the size distribution of PPN from TEM images (analysis with a number of particles >700).

The PPN morphologies and sizes can be observed in Figure 3C,D, respectively. The PPN has a uniform quasi-spherical morphology, with diameters of ~60 nm, as shown in Figure 3C and the insert. These morphologies and sizes agree with previous literature reports [32]. Furthermore, the excellent monodispersity can be confirmed by the narrow histogram distribution presented in Figure 3D, obtained from TEM pictures using the free software ImageJ version 1.53e. The difference in the sizes obtained from the DLS technique and the TEM analysis, as known, is related to the hydrodynamic size of PPN in the suspension (DLS) and the actual size of dried particles (TEM). Furthermore, in the literature, it was found that PPN synthesized with different methods showed similar amorphous structures [33,34].

The optical properties of the aqueous PPN suspensions were analyzed by UV-vis spectroscopy. The UV-vis absorption spectra were recorded at different PPN concentrations in the wavelength range of 300–1000 nm, as shown in Figure 4. PPN exhibits the typical π–π\* transition band of the polypyrrole around 420 nm, in agreement with the literature [35,36]. At the same time, the observed band above ~650 nm is attributable to the sizeable π-conjugated structure of polypyrrole chains [37]. As expected, the absorbance increased proportionally to the PPN concentration. Similarly, Zha et al. reported that the absorbance increases linearly as the concentration of PPN in water is elevated, indicating the excellent dispersity of the aqueous PPN solution [15]. Furthermore, a calibration curve was obtained from this linear behavior, with a resulting extinction coefficient (ε) of 0.0559 a.u./(μg/mL·cm) and a determination coefficient (R2) of 0.994 (Figure S3). It should be mentioned that the absorption spectra of PPN also depend on the nature of the solvent [38].

**Figure 4.** Optical absorption spectra of PPN suspensions at pH values of 4.2 and different concentrations of particles.

#### *3.2. pH Effects over PPN*

The pH effect over the hydrodynamic particle size, ζ, and optical spectra of PPN suspensions are presented in Figure 5. The pH values of PPN suspensions were: 4.2, 6.0, 7.4, 8.0, and 10.0. In this range of pH, the PPN size presents a quadratic behavior with respect to the pH of the suspension (Figure 5A). However, the size differences are not significant between the pH values except for a pH of 10, where a significant difference resulted, compared with all the other pH values. This behavior is similar to the PDI values obtained, the results of which indicate monodisperse particles (PDI < 0.1) at pH values below 10 (Figure 5A). As observed for the size results, the only significant difference in PDI was at a pH of 10, compared with all the other PDI values. The ζ results for the different pH suspensions could be described with a quadratic relationship, as presented in Figure 5B. The ζ values for suspensions with pH of 4.2 and 6.0 do not present significant variations, with values around 20 mV. However, when the pH value of the suspension increases to 7.4 and 8.0, the ζ decreases to values of 9.8 ± 0.4 and 9.3 ± 1.1 mV, correspondingly, with significant differences for the pH values of 4.2 and 6.0.

Nevertheless, the ζ differences between pH 7.4 and 8.0 are not significant. The ζ values of suspensions of PPN at pH 10 resulted in −4.7 ± 1.3 mV, with substantial differences from all the other ζ values. The changes in the ζ of PPN are related to the deprotonation process that suffers the polymeric chain of PPN as the pH rises [39,40]. This deprotonation affects the surface charge due to changes in the PPN surface composition. Other authors have reported that iron nanoparticles coated with polypyrrole change from a positive ζ value to a negative one at a pH higher than 9 [41,42]. The significant differences observed in size and PDI measurement results could be attributed to the change in the surface charge character of the PPN, from positive to negative, indicating that some disturbance was obtained by passing throughout the isoelectric point of PPN (~pH of 9.4).

**Figure 5.** PPN properties at different pH values. (**A**) Diameter size distributions and PDI of PPN; Squares represent nanoparticle diameter sizes, the continuous line represents the quadrating fitting to the diameter size, and circles represent the polydispersity index. Means ± SD, *n* = 3. (**B**) ζ of PPN; squares represent nanoparticle ζ, and the continuous line represents the quadrating fitting. Means ± SD, *n* = 3. (**C**) Optical absorption spectra of PPN suspensions; insert shows the color of suspensions at different pH values, ranging from 4.2 to 10.

The optical spectra of PPN at different pH values are presented in Figure 5C, with wavelength scans ranging from 300 to 1000 nm. The characteristic optical spectra bands of PPN suspensions are similar for all the pH values. However, when the pH of the PPN suspension increases, a shift to a shorter wavelength is produced in the spectra of PPN. Furthermore, a change in the color of the PPN solutions is observed, from a black-green color in acidic conditions to a light blue in alkaline conditions (insert of Figure 5C). These wavelength shifts are related to the surface charge of the PPN, as described in Figure 5B. As the PPN is deprotonated (pH rises), the hydroxyl groups cover the surface, and a shift to shorter wavelengths is observed.
