*2.7. Stability Evaluations*

#### 2.7.1. Short-Term Stability Testing under Different Environmental Conditions

The physicochemical stability of the RDS formulation (500.0 mg ± 2 mg per test) was monitored under different storage conditions over four months. A set of samples were placed in an enclosed glass holder that was transferred into the stability tester (Labcon PSIE RH 40 Chamber Standard Incubator, Laboratory Marketing Services, Maraisburg, South Africa) fixed at 30 ◦C ± 2 ◦C and a relative humidity of 65% ± 3% adapted from the WHO stability testing scheme for pharmaceutical products containing well established drug substances [28]. Another sample group was stored in airtight, opaque glass vials under standard room conditions (temperature: 25 ◦C ± 5 ◦C and humidity: 55% ± 5%). The last sample set was stored in airtight, opaque glass vials and placed in the refrigerator (Sanya Labcool Pharmaceutical Refrigerator, MPR-720R, Sanyo Electrical Biomedical Co. Ltd, CA, USA) at 4 ◦C ± 1 ◦C. Formulation stability was monitored at 0, 1, and 4 month intervals for changes in particle size, polydispersity index, zeta potential, and drug content. Samples were also physically examined for any changes in physical appearance or color changes. All tests were conducted in triplicate.

### 2.7.2. In Vitro Aqueous Stability Assessment

The stability of the RDS formulation in aqueous solution (mimicking the re-constitution process) was evaluated. RDS samples (500 ± 2 mg) were placed in airtight, opaque containers and dispersed in 50 mL sterile deionized water. Hydrated samples were prepared in triplicate per test conditions. Test conditions included placement under ambient conditions (25 ± 5 ◦C), as well as in the refrigerator (4 ± 2 ◦C) for 11 days. Hydrated samples (2 mL) were collected from each test vessel for isoniazid content quantification at 0, 1, 5, and 11 days using UVspectrophotometry, as described earlier. Samples were manually agitated daily and before collection to ensure uniform re-dispersion. All tests were carried out in triplicate.

### *2.8. Preliminary Formulation Toxicity Assessment*

The human breast adenocarcinoma (MCF-7) cell line was used for preliminary evaluation of the RDS formulation biocompatibility. The sample was dissolved in double-distilled water to a final stock concentration of 2 mg/mL, filter-sterilized through a 0.22 μm Cameo acetate membrane filter (Millipore Co., Bedford, Massachusetts), and stored at 4 ◦C until used. The negative control was 1% DMSO while 100 μM camptothecin, a known chemotherapeutic agent, was used as the positive control. The MCF-7 cell line was routinely maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), at 5% carbon dioxide and 37 ◦C ± 0.1 ◦C. Cells were seeded into 96-well plates at a final concentration of 15,000 cells/well. Test samples were added at final well concentrations of 100, 50, 25 μg/ml, and incubated for 24 h. Thereafter, the spent medium was replaced with 200 μL of 0.05 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) solution and incubated at 37 ◦C ± 0.1 ◦C for 3 h. The MTT was then aspirated from the cells and replaced with 200 μL DMSO to dissolve the formazan crystals. The absorbance was read at a maximum wavelength of 540 nm against DMSO as a blank using a microtiter plate reader (BioTek Powerwave XS, Winooski, VT, USA). The absorbance readings obtained were used to compute the number of viable cells present in the media. All results are presented as the mean reading ± standard deviation, and the statistical significance of all experimental data was evaluated using the GraphPad Prism 7 software (GraphPad, San Diego, CA, USA) using a two-way Analysis of Variance (ANOVA). The number of viable cells in both test and control samples were determined using Equation (3), and all tests were conducted in triplicate.

$$\% \text{Cell viability} = \frac{\text{Number of viable test cells}}{\text{Number of viable control cells}} \times 100\tag{3}$$

### **3. Results and Discussions**

### *3.1. Formulation Synthesis and Yield*

The isoniazid-loaded RDS formulation was developed using a combination of the direct dispersion emulsification technique, lyophilization, and dry milling. A cream-white, free flowing isoniazid loaded RDS powder with an average yield of 87.43% ± 0.13% was obtained.

#### *3.2. Size, Polydispersity Index, and Zeta Potential Determination and Morphology*

The RDS powder formulation showed an average particle size of 1.63 ± 0.20 μm, indicating its intrinsic micro-structure. The PDI can be described as a ratio that provides information about homogeneity of particle size distribution as it relates to a particular system serving as a useful reflection of the quality of the particulate system/dispersion ranging from 0.0–1.0 with values ≤0.1 relating to the highest quality of particulate dispersion, ≤0.3 as optimum, ≤0.5 as generally acceptable [24]. A generally acceptable PDI value of 0.37 ± 0.04 was recorded for the RDS powder, indicating that the particles were mostly homogenous and well dispersed within the formulation. The measured ZP was −41.10 ± 5.57 mV, showing a stable system, where a ZP value of ±30 mV is considered a stable and satisfactory formulation [24,29]. Representative graphs based on an independent measurement of the zeta potential and particle size distribution are shown in Figure 1a,b. Researchers have shown that TEM imaging is an effective and powerful tool for characterizing the morphology of nano- and micro-structured biomaterials and drug carriers as it uses more powerful beams to produce higher resolution images with more details and information [30,31]. The TEM micrographs showed minimally aggregated, dispersed RDS particles that appeared as darker areas relative to the background, with rounded outer morphologies (Figure 1c,d).

**Figure 1.** Representative graphs displaying: (**a**) particle size distribution, (**b**) zeta potential distribution, as well as TEM micrographs showing different surface topographies and characteristics of the reconstitutable dry suspension (RDS) particles at different scales: (**c**) 1μm and (**d**) 5 μm, respectively.

## *3.3. Thermal Behavior*

Salient changes in the thermal behavior of isoniazid relative to that of the RDS formulation were studied using conventional DSC methods. The recorded melting peak of pure, unformulated isoniazid was 171.7 ◦C, which compared well to literature [32]. The melting peak of isoniazid (Figure 2a) was characterized by its sharp and defined geometry confirming its purity and crystalline nature. The RDS thermogram was typified by the presence of multiple distinct sharp or broad peaks showing its physicochemically stable and multi-component state. The RDS formulation showed an intermittent appearance of sharp and blunt peaks representing its semi-crystalline nature (Figure 2b,c). The isoniazid melting peak identified on the RDS formulation thermogram (171.1 ◦C) was similar to that of the pure drug (171.7 ◦C), showing its stability within the excipient matrix. However, the formulated isoniazid peak presented as a broad band, which can be related to physical transitioning from crystalline into amorphous structures attributable to solvation and encapsulation of drug molecules into the processed polymeric carrier. A significant change in heat flow from −36.4 mW (unformulated isoniazid) to −3.7 mW (isoniazid in RDS) (Figure 2b,c) was seen, further supporting the likely drug encapsulation in the amorphous state, coupled with stable molecular dispersions within the polymeric chains [24]. Overall, the thermograms (Figure 2a–c) demonstrate the absence of any destructive/irreversible physicochemical interactions between isoniazid and the excipients during the RDS preparation process.

**Figure 2.** Differential scanning calorimetry (DSC) thermograms of (**a**) unformulated isoniazid, (**b**) RDS formulation, and (**c**) an expanded segmen<sup>t</sup> of the RDS thermograms showing the transitions that occurred with formulated isoniazid.

## *3.4. X-ray Di*ff*ractometry*

The changes in matrix crystallinity between pure isoniazid and the RDS formulation was further confirmed using X-ray diffraction analysis (XRD) with recorded diffractograms presented in Figure 3a,b. Diffractograms recorded for pure isoniazid showed high intensity, well-defined sharp peaks between 9.3θ and 32.3θ, with intensities as high as 39,885.7 validating the crystalline nature of isoniazid (Figure 3a). This trend differs from that observed for the RDS formulation, which presents broader, not so well-defined peaks between 13.9θ and 28.1θ at a maximum intensity of 11,142.9, which is much lower relative to the isoniazid diffractogram. Furthermore, low intensity (less than 4288.1) plateau-like, broad-banded sections were also identified between 5.3θ—14.6θ and 28.5θ—90.1θ, further confirming that the RDS formulation consists of more than one component, characterized with intermittent, mostly amorphous and minimal crystalline domains, i.e., a semi-crystalline nature (Figure 3b). These findings agree with the outcomes of the DSC analysis.

**Figure 3.** X-ray diffractograms of (**a**) unformulated isoniazid and (**b**) isoniazid loaded RDS formulation.
