*3.6. Drug Content*

The percentage content of isoniazid encapsulated within the RDS formulation matrix was computed relative to the theoretical drug content, totaling 94.12% ± 2.10%. This indicates that there was minimal drug loss during the RDS manufacturing and processing phases.

## *3.7. Drug Release Behavior*

To understand the in vitro drug release behavior and kinetics, dissolution studies were carried out on the RDS formulation containing an equivalence of 100 mg isoniazid in pH 7.4, 6.8, and 1.2 buffered solution over two hours under biorelevant conditions. Percentage cumulative drug release (%CDR) was calculated as the total amount of isoniazid liberated from the RDS formulation matrix,

with an increase or decrease in %CDR representing a respective rise or decline in the release rates. %CDR varied for each dissolution media (pH 1.2 = 67.88% ± 1.88%, pH 6.8 = 60.18% ± 3.33%, and pH 7.4 = 49.36% ± 2.83%). Isoniazid release decreased as media pH increased. In other words, a reduction in the pH of the aqueous micro-environment seemed to impact the processes of formulation wetting, pore formation and closure, water penetration, phase transitioning, drug-excipient dissolution and degradation, changes in drug-excipient physical interaction and solubility process, and eventual diffusion of drug molecules coupled with gradual matrix geometry transitions (erosion or swelling) [33]. The RDS formulation demonstrated the potential to stabilize and release the isoniazid molecules in different dissolution media with significant differences in the percentage of drug released under these different conditions.

In addition, isoniazid release at pH 7.4 was selected for further mathematical model fitting to understand the RDS formulation release kinetics over a period when 100% drug liberation was achieved (Figure 5). In vitro isoniazid release from the RDS formulation at pH 7.4 was characterized with an initial burst at 5 min (7.51 ± 0.72 %), followed by a relatively consistent increase in drug release over time, until complete release (100%) was recorded at approximately 300 min.

**Figure 5.** Graphical profile showing 100% isoniazid release from the RDS formulation over time.

The generated profile (Figure 5) was further analyzed using mathematical kinetic models employing the KinetDS, version 3.0 open source freeware. Release profile analysis and model of best fit selection was based on a combination of robust validation quantities, namely the correlation coefficient (R2) closest to one and lowest Akaike Information Criterion (AIC) numerical value (Table 2) [31]. On this basis, the zero order kinetic model provided the best fit parameters (R<sup>2</sup> = 0.976 and AIC = 74.080) for the isoniazid release data depicted in Figure 5. This indicates that isoniazid release from the RDS formulation is consistent over time, irrespective of the initial drug concentration. In principle, zero order drug release mechanism is beneficial for achieving continuous drug plasma and biological fluid levels, reducing dosing frequency, and improving patient compliance, aiding desired pharmacotherapeutic efficacy [34].

**Table 2.** Representative mathematical models and their respective fit parameters.


### *3.8. Short-Term Formulation Stability Assessment*

### 3.8.1. Stability Evaluation under Varying Storage Conditions

Evaluation of formulation stability was performed in triplicate per sample under multiple storage conditions: Stability tester—30 ◦C ± 2 ◦C and 65% ± 3%; Room—25 ◦C ± 5 ◦C and 55% ± 5%; and Refrigerator—4 ◦C ± 1 ◦C; over four months employing particle size, polydispersity index, zeta potential, and drug content as indicators of stability. Results showed minimal numerical di fferences in indicators measured, indicating that the isoniazid loaded RDS formulation can be described as stable under the prescribed environmental storage conditions (Table 3). A slight alteration in physical appearance relating to a color change from white to cream-white was observed at four months, under accelerated storage in the stability chamber (30 ◦C ± 2 ◦C; 65% ± 3%) and room conditions (25 ◦C ± 5 ◦C; 55% ± 5%). Despite the fact that stability indicators remain closely related, the slight color change is undesirable, considering patient acceptance, making these environments unsuitable for the storage of the RDS formulation. Therefore, the most ideal storage setting for the RDS formulation is, thus, at 4 ± 1 ◦C as stability indicators are comparable with no color change observed (Table 3).


**Table 3.** Stability indicators measured under the di fferent storage conditions.

**a** Particle Size (standard deviation ≤ 0.13 μm in all cases), **b** Polydispersity Index (standard deviation ≤ 0.03 in all cases), **c** Zeta Potential (standard deviation ≤ 1.03 mV in all cases), **d** Drug Content (standard deviation ≤ 0.99% in all cases).

### 3.8.2. Formulation Stability in an Aqueous Environment

The stability of the drug loaded RDS formulation in an aqueous medium was investigated under select storage conditions (room and refrigeration) over 11 days, mimicking storage duration for commonly reconstituted antibiotic solutions/suspensions. Percentage drug content was chosen as the hydrostability indicator, and overall, there were insignificant changes in its numerical values under room or refrigerated storage conditions, with no visible color changes (Table 4). Summarily, the RDS formulation is stable in the aqueous medium over 11 days, either refrigerated or stored under ambient conditions, suggesting that the RDS formulation is a potentially useful preparation for re-constitution purposes, especially in pediatrics.

**Table 4.** Hydrostability indicators at di fferent time-points, under ambient and refrigerated conditions.


### *3.9. Cell Viability Assessment*

Preliminary assessment of the effects of the RDS formulation on viability was performed on MCF-7 cell lines. Tests were conducted over 24 h using different concentrations of the RDS ranging from 0 μg/mL (negative control) to 100 μg/mL. This cell line is routinely used as a prototype for assessing the biocompatibility of drugs, delivery systems, and biologicals that are non-specific for treating carcinomas [35–37]. Graphical representations of the impact of different test formulation concentrations on cell viability relative to camptothecin (positive control) and the negative control are presented in Figure 6. The RDS lowest concentration (25 μg/mL) caused significant MCF-7 cell proliferation (*p* < 0.0500: *p* = *0.0001*). The increased viability may signify that the RDS formulation supported cell growth at a low concentration (25 μg/mL) and can be considered an indication of biocompatibility. This may be attributed to the drug or excipient concentration or a combination of both. In contrast, the 50 and 100 μg/mL reduced cell viability, although not significantly (*p* > 0.0500: 50 μg/mL—*p* = *0.0056*; 100 μg/mL—*p* = *0.0001*). Nevertheless, higher concentrations of the RDS (50 and 100 μg/mL) did not reduce cell viability as much as the camptothecin that decreased it to 48.93% ± 3.99%. In general, the RDS was well-tolerated by the MCF-7 cells at all test concentrations. Cellular responses following exposure to the formulation can be described as biphasic and dose-dependent, a phenomenon associated with hormesis (a two-phased adaptive response of cells and organisms to increasing or decreasing amounts of external stress, e.g., drug, chemical substances, and disease state) [38,39]. It is not unusual for cells to exhibit hormetic effects as they are biological systems known to be dynamic and constantly evolving. These initial findings form a baseline for future work on understanding the biocompatibility of the RDS formulation and its components (active drug and excipients) in vitro and in vivo.

### **4. Conclusions and Future Work**

In this study, isoniazid loaded reconstitutable dry suspension was prepared using the direct dispersion emulsification technique, coupled with lyophilization and dry milling. The direct dispersion technique produced a relatively high yield (87.43% ± 0.13%) of RDS particles with good drug loading capabilities (94.12% ± 2.10%). The RDS formulation showed no significant evidence of toxicity supported by outcomes from viability studies in the MCF-7 cells. The formulation was physicochemically stable, mostly amorphous, with marginal intermittent crystalline domains, and had

no irreversible alterations in its backbone chemical structure. It demonstrated the ability to regulate isoniazid release in a controlled, zero order manner, and was environmentally stable under common storage conditions, either as a dry powder or in the hydrated form. The findings from this work may contribute towards improving flexible pediatric dosing for tuberculosis drug treatment, considering the current global shortage of such preparations, especially for the first-line anti-tubercular drugs.

To establish a course for further investigations, we identified the need to extend biocompatibility testing for the RDS formulation and its components to normal cells and tissue isolates from animal models (e.g., mice, rabbit, or pigs) employing cytotoxicity assays and histopathological techniques, respectively. Further preclinical evaluation of pharmacokinetics, e fficacy, and eventual optimization of isoniazid dosing and absorption from the RDS formulation, in animal models similar to humans, such as pigs, are important next steps. Considering the hydrophilic/hydrophobic and particulate nature of the RDS, we anticipate that it can find extensive use as an e ffective carrier for other anti-tubercular drugs (e.g., pyrazinamide, rifampicin) suitable for pediatrics irrespective of their solubilities and molecular weights. For implementation purposes, a range of these bioactives would need to be tested in vitro/in vivo and optimized to ensure desirable drug loading, controlled release, and absorption for the intended pharmacotherapeutic application. Overall, the RDS formulation reported herein has the potential for improving tuberculosis treatment within the pediatric population.

**Author Contributions:** Conceptualization, O.A.A.; methodology, O.A.A., R.K.H., H.D.; formal analysis, O.A.A., R.K.H.; investigation, O.A.A., R.K.H., H.D.; writing-original draft preparation, O.A.A.; writing, review and editing, O.A.A., R.K.H., H.D.; project administration, O.A.A., R.K.H.; resources, O.A., R.K.H., H.D.; funding acquisition, O.A.A., R.K.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the South African National Research Foundation/Department of Science and Technology (Grant Number-85110 and 113143) and Sefako Makgatho Health Sciences University DHET Research Development Grant (Grant Number: D112-RDG).

**Acknowledgments:** We thank the National Centre for Nano-structured Materials, Council for Scientific and Industrial Research, South Africa, for sample characterization. We thank the CSIR National Centre for Nano-structured Materials for sample characterization. Opinions, findings and conclusions or recommendations expressed in this publication are that of the authors. The funders accept no liabilities whatsoever in this regard.

**Conflicts of Interest:** The authors declare no conflict of interest.
