2.5.2. Crystallinity

Powder X-ray di ffractometry was used to determine the physical, solid state changes and crystallinity [50] of the model drug PZA, pharmaceutical excipients, optimized drug loaded and placebo formulations. The di ffractograms displayed in Figure 5A–C depict the crystalline nature of pyrazinamide, citric acid and xylitol correspondingly with distinct, high intensity peaks (>30,000 a.u.). Co-polymer polyvinyl alcohol polyethylene glycol is semi-crystalline (characteristic of it make-up), typified with broader band, less intense (<16,000 a.u.) crystalline peak observed at 19.2 (2θ) and multiple blunt regions (Figure 5D), while sodium starch glycolate exhibited a low intensity (<6000 a.u.), undefined topography (Figure 5E), which can be related to its amorphous character, largely contributing to the non-crystalline domains within the entire molecular structure of the optimized formulations. The optimized drug and placebo formulation di ffractograms are considered a sum of individual di ffractions generated by the component excipients and drug [26]. When compared with its drug loaded counterpart, the placebo di ffractogram (Figure 5F) also exemplified the presence of recurrent amorphous and higher intensity crystalline regions (>30,000 a.u.) within its matrix which further validates findings documented from the TGA analysis where the placebo was shown to require higher temperatures to exhibit complete decomposition compared to the drug loaded formulation (Figure 4F,G). The optimized PZA formulation di ffractogram (Figure 5G) irregularly exhibited slightly broader, sparse and lower intensity peaks (<30,000 a.u.) plus an expanse of blunt regions both associated with its crystalline and amorphous states respectively attributed to the excipients and PZA constituents. The presentation of the amorphous segments within the drug formulation structure appears substantial and may be linked with the rapid disintegration characteristic observed upon hydration, as the amorphous state is known to improve the solubility of drug delivery systems, thereby enhancing dissolution and drug absorption within the body [38,41,49]. The crystalline PZA appeared transformed into a partially amorphous state evidenced by the relative loss of its crystalline peaks intensity, particularly that identified at 8.03 (2θ) which was at about 30,000 a.u. and reduced to 10,000 a.u. in the optimized formulation di ffractogram (Figure 5A). These findings also concur with the DSC and XRD analytical outputs.

### 2.5.3. Structural Interpretation

Fourier transform infrared spectroscopy was performed on all pure excipients, PZA, optimized drug loaded and placebo formulations to determine any drug–excipient interactions, and produced spectra are presented in Figure 6. The analysis was focused on identifying vibrational peaks that depict the presence of particular functional groups in the pure drug and pharmaceutical excipients (Figure 6A–G). Pinpointing xylitol peaks related to O-H stretching were observed at 3354 cm<sup>−</sup><sup>1</sup> and 3284 cm<sup>−</sup>1; an intense C-H peak at 1418 cm<sup>−</sup><sup>1</sup> (Figure 6A) while C-H and O-H peaks observed at 2916 cm<sup>−</sup><sup>1</sup> and 3438 cm<sup>−</sup>1, respectively, were specific for sodium starch glycolate xylitol peaks (Figure 6B). Typifying citric acid C-O-H peak was noted at 1372 cm<sup>−</sup>1, C-O vibration at 3282 cm<sup>−</sup><sup>1</sup> and O-H bending at 1172 cm<sup>−</sup><sup>1</sup> (Figure 6C). Specific peaks documented at 1090 cm<sup>−</sup>1, 2902 cm<sup>−</sup><sup>1</sup> and 3296 cm<sup>−</sup><sup>1</sup> correspond to C-O, C-H and O-H stretching, respectively; C-H bend at 1448 cm<sup>−</sup>1, C-H rock at 848 cm<sup>−</sup><sup>1</sup> and -C-C- vibration at 1086 cm<sup>−</sup><sup>1</sup> signify co-polymer polyvinyl alcohol polyethylene glycol—Kollicoat ® IR (Figure 6D) [51–53]. Of note are peaks representing N-H stretch at 3148cm−1, 3292 cm<sup>−</sup>1, 3388 cm<sup>−</sup><sup>1</sup> and 3408 cm<sup>−</sup>1, C-N (ring, stretching) at 1680 cm<sup>−</sup>1, C-C ring stretching at 1436 cm<sup>−</sup>1, C=N at 1162 cm<sup>−</sup>1, C=O at 786 cm<sup>−</sup><sup>1</sup> and C=C at 1704 cm<sup>−</sup>1, which are key to its structural make-up were logged for pure PZA (Figure 6E) [54]. The optimized PZA loaded formulation spectra (Figure 6F) displayed distinctive peaks, appearing within identical vibrational frequency range obtained for PZA but with slight shifts due to the presence of polymeric and non-polymeric additives. Peaks at N-H = 3290 cm<sup>−</sup>1, 3406 cm<sup>−</sup><sup>1</sup> and 3426 cm<sup>−</sup>1, C-O = 1680 cm<sup>−</sup><sup>1</sup> and 1022 cm<sup>−</sup>1, C-C (ring, stretching) = 1434 cm<sup>−</sup>1, typical of the standard PZA chemical backbone, were identified. A presentation of functional groups specific to the pure drug and/or excipients individually reflect in the generated spectra for the optimized drug formulation and exposes the level of structural compatibility amongs<sup>t</sup> these components. This signifies

that despite the relative amorphization noted for formulated PZA, typifying structural peaks remained noticeably unaltered, thereby demonstrating the absence of chemically disruptive interactions during the formulation development process. The placebo FTIR spectra (Figure 6G) showed C-O stretching peaks at 1064 and 1008 cm<sup>−</sup><sup>1</sup> including C-H stretching peak seen at 2916 cm<sup>−</sup><sup>1</sup> accounting for the presence of both poly vinyl alcohol polyethylene glycol and sodium starch glycolate, C-O-H peaks were detected at 1420 cm<sup>−</sup><sup>1</sup> and 1374 cm<sup>−</sup><sup>1</sup> representing citric acid and, vibrational frequencies at 3418 cm<sup>−</sup>1, 3382 cm<sup>−</sup><sup>1</sup> and 3282 cm<sup>−</sup><sup>1</sup> are characteristics of O-H peaks for associated with xylitol, polyvinyl alcohol polyethylene glycol and citric acid chemical backbone structures. These show that the placebo is a homogenous physical blend of the different excipients. Overall, the outcomes of the FTIR analysis indicated that the drug and excipients were well incorporated, compatible and stable with absence of any destructive intermolecular or intramolecular interactions.

**Figure 6.** FTIR spectra of (**A**) xylitol, (**B**) sodium starch glycolate, (**C**) citric acid, (**D**) polyvinyl alcohol polyethylene glycol, (**E**) PZA, (**F**) drug loaded formulation and (**G**) placebo.

### 2.5.4. BET Surface Area and Porosity

The surface areas of optimized drug loaded and placebo formulations were obtained by employing the theory of Brunauer–Emmett–Teller (BET) on nitrogen adsorption isotherms generated from the sample surface measured at 77 K. Typical isotherms are illustrated in Figure 7A,B, respectively. Drug loaded and placebo isotherms show similar trends at relative pressures of 0.2000–0.4000, revealing the existence of micropores in both formulations [55]. Specifically, the micropore volume of the placebo and drug loaded formulations were 0.00001 and 0.00003 cm<sup>3</sup>/g, respectively, and the presence of PZA within the matrix seems to increase the pore volume according to the numerical data. The specific BET surface area of drug loaded formulation (Figure 7C) and placebo (Figure 7D) were 0.0015 and 0.0753 m<sup>2</sup>/g, while the single point surface area measured 0.0020 and 0.1408 m<sup>2</sup>/g, respectively. The surface areas for the drug loaded sample is smaller than that of the placebo. This can be associated with the presence of successfully attached PZA molecules on the optimized formulation matrix. T-plots of the drug loaded (Figure 7D) and placebo (Figure 7E) profiles exhibited similar trends with the same thickness ranging between 0.3000 and 0.5000 nm, which may still be related to the finding that the PZA molecules are well incorporated into the formulation matrix, thus not changing its thickness either PZA-free or PZA-loaded.

**Figure 7.** Graph presentation of Isotherm liner plot of ( **A**) drug loaded and (**B**) placebo, BET surface area plot for ( **C**) drug loaded and ( **D**) placebo and t-plot of (**E**) optimized drug loaded and (**F**) placebo.

### *2.6. Typifying Surface Morphological Features and Transitioning with Hydration*

Scanning electron microscopy was used to examine the differences in the surface morphologies of the anhydrous PZA, optimized drug loaded and placebo formulations as shown in Figure 8A–C, respectively. The pure PZA micrograph (Figure 8A) displayed irregular sized, rod shaped particles with defined edges confirming the crystalline nature of the PZA molecules [47,50]. The optimized PZA formulation surface micrograph (Figure 8B) revealed a uniform distribution of the drug molecules embedded throughout the carrier matrix, whereas the placebo (Figure 8C) showed a relatively plane topography with undulating segments confirming the absence of PZA molecules.

**Figure 8.** Scanning electron micrographs of (**A**) pure PZA, (**B**) optimized PZA loaded formulation, (**C**) placebo formulations. Changes are visible in the surface topographies of the unhydrated and hydrated optimized drug loaded formulation (**D**) before—0 s and after (**E**)10, (**F**) 30, (**G**) 60, (**H**) 90 and (**I**) 120 s of being in contact with simulated saliva under biorelevant conditions (pH 6.8; 37 ± 0.1 ◦C). Images were captured as SEM micrographs at magnification 500× (refer to the blue label "1") and high resolution photographs (refer to blue label "2") at each time point. The circular, red dotted lines shown in the photograph images (labelled "2") represent left over formulation fragments after hydration.

Furthermore, time-dependent disintegration patterns of the optimized drug loaded formulation in preheated simulated saliva (pH 6.8; 37 ± 0.1 ◦C) was investigated using scanning electron microscopy and digital photography. Briefly, images were taken at 0 s (before hydration) and 10, 30, 60, 90 and 120 s after placement in simulated saliva. Captured micrographs and photographs showing changes in formulation surface morphology as it collapsed upon hydration and subsequently released PZA molecules over time are illustrated side-by-side (labelled 1 and 2) in Figure 8D–I. At time 0 s before hydration (Figure 8D), formulation matrix morphology was intact and displayed a wide coverage with PZA molecules (defined edge particles as per micrograph) as also discussed above. As formulation wetting progressed with time, gradual matrix collapse and breakdown occurred, and progressive migration of embedded PZA molecules ensued as a result of matrix disintegration (Figure 8D–I). Initial matrix collapse and drug molecule (visible as embedded particles with defined edges) migration into simulated saliva started at about 10 s (Figure 8E) and was more pronounced at 30 s (Figure 8F), followed by visible matrix erosion, dissolution and continuous outward movement of incorporated drug molecules as time elapsed (Figure 8G–I). These outcomes agree with the low disintegration time (34.98 s) recorded for the drug loaded formulation and is considered an indication of the desired rapid matrix fragmentation, which makes it an attractive and potentially suitable orodispersible delivery system for use in children. In essence, we were able to use the information provided from visualizing microscopic disintegration processes to validate macroscopic level formulation breakdown events.

### *2.7. Organoleptic Properties of Optimized Drug Loaded Formulation*

Preliminary evaluation of organoleptic properties was based on color, texture (general appearance) and acceptability. Results showed that the orodispersible formulations placed on the tongue dispersed quickly—under 60 s in the presence of saliva (required no water for swallowing) and produced a generally satisfying taste as described by the panel. On the average, the volunteers rated the formulation 3.5, implying that the remaining bitterness was minor and mostly considered adequately taste masked/tasteless by them. The panelists also described the formulation color as acceptable, texture as satisfactory and easy to handle. Outcomes of this preliminary qualitative investigation makes the developed orodispersible preparation potentially attractive for pediatric use.

### *2.8. Cytobiocompatibility Evaluation*

In vitro cytotoxicity assay was performed to determine the potential biocompatibility of the optimized drug loaded orodispersible formulation relative to the placebo and pure PZA using hepatocyte cell line (Hep2G) as a model. HepG2 cell line is commonly used to examine the toxicity of antitubercular drugs and respective formulations [56–58]. Cell viability was measured with the 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) and neutral red (NR) assays at di fferent xenobiotic concentrations ranging from 0.0005—5 mg/mL over a treatment period of 24 h. Cytotoxicity levels were presented as mean percentage cell viability and standard error of mean (SEM) for both MTT and NR assays (Figure 9). For the MTT assay, it was observed that cell viability generally increased as sample concentration decreased (Figure 9A–C). The optimized drug loaded preparation (Figure 9A) showed cell viability 60% and higher for all test concentrations while the placebo formulation displayed lowest cell viability <20% at 5 mg/mL, which consistently increased between 0.5 and 0.005 mg/mL and then insignificantly dropped to 77.09% at the lowest concentration of 0.0005 mg/mL (Figure 9B). Pure pyrazinamide, on the other hand (Figure 9C), expressed highest cell proliferation at lowest concentration 0.0005 mg/mL (96.29%) and the reverse at 5 mg/mL (32.40%). A combination of PZA and excipients in the formulation seemed to promote biocompatibility and cell growth in comparison to outputs captured for the placebo and PZA only. Interestingly, the neutral red analyses showed no cytotoxicity for all three test samples with values majorly greater than 100% for all concentration levels (Figure 9D–F). The only slight decrease in cell viability was noted for PZA at 5 mg/mL and it was statically insignificant. Hence, the NR uptake assessment revealed that drug formulation, placebo and PZA supported cell division and growth, an indication of cytobiocompatibility. These findings may be as a result of the di fferences in the biochemical reactions of both assays. The MTT assay is based on cellular respiration or mitochondrion cell metabolic activities while the NR analysis measures dye uptake and concentration within the lysosomes thus measuring staining capacity of live cells [3,59]. Consequently, a reduction in MTT-based cell viability can represent a decrease in metabolic activity. The fact that similar trends are not identified for the NR assay at all test concentrations, the cells

can be considered to exhibit cytostatic effects at some point. This may mean that the introduction of the test compounds may have inhibited cell growth but does not necessarily promote cell destruction or death—as it is with the case of cytotoxicity. Identified cellular responses resulting from cell exposure to test samples can be termed as dose-dependent and two-phased, an occurrence related with hormesis which, is a two-way adaptive reaction of cells (biological systems) to external stress such as xenobiotics, environmental changes (e.g., pH, temperature) [60]. For both implemented assays, the optimized PZA orodispersible formulation demonstrated no significant reduction in cell viability.

**Figure 9.** HepG2 cell biocompatibility levels measured by MTT analysis (**A**) drug loaded formulation, (**B**) placebo and (**C**) pyrazinamide and, neutral red assay (**D**) drug loaded formulation, (**E**) placebo and (**F**) pyrazinamide. Results represent mean ± SEM and statistical significance (*p* < 0.05) indicated with an asterisk (\*)**.**

### *2.9. Drug Formulation Stability under Changing Storage Conditions*

Pyrazinamide formulation stability under varying environmental storage conditions was evaluated over 12 weeks. Briefly, drug formulations were placed in airtight, glass jars containing desiccant bags and stored in: (a) a dark enclosure (23 ± 3 ◦C/65 ± 5% RH), (b) refrigerator (4 ± 2 ◦C) and (c) under regular room conditions (24 ± 3 ◦C/70 ± 5% RH). Tests were performed in triplicate per storage condition and the stability indicators quantified were inner and outer diameters, disintegration time, dissolution pH, weight, and drug content using earlier described methods. Results were reported as average ± standard deviation. Freshly prepared control formulations were tested immediately (time = 0 weeks) and stability indicators recorded in three replicates. At the 12-week time-point, samples stored in dark enclosures (23 ± 3 ◦C/65 ± 5% RH) and refrigerator (4 ± 2 ◦C) retained their physical shape, color and showed minimal variation in stability indicators compared to values recorded

at the starting point. Samples stored under regular room conditions (24 ± 3 ◦C/70 ± 5% RH) with fluctuating light exposure were considerably unstable, evidenced by their physical discoloration and documented stability indicators relative to the control samples (Table 3). Summarily, the suggested storage conditions for the optimized PZA containing orodispersible pharmaceutical formulation would be in airtight vessels containing desiccant bags kept away from direct or fluctuating light sources and under ambient or refrigerator settings.


**Table 3.** Stability indicators recorded under different storage conditions.

**\* Note**: **I**—refrigerator (4 ± 2 ◦C); **II**—dark enclosure (23 ± 3 ◦C/65 ± 5% RH); **III**—regular room conditions (24 ± 3 ◦C/70 ± 5% RH).

### **3. Materials and Method**
