Promising chitosan-alginate combination for rifampicin dry powder inhaler to target active and latent tuberculosis

Materials

Materials used in this research included rifampicin (Luohe Nanjiecun, China), chitosan (Bio Chitosan, Indonesia), sodium alginate (Shandong Jiejing, China), and other chemicals of analytical grade.

Method

Preparation of rifampicin DPI

Rifampicin DPI was prepared by the spray drying technique using a B-290 mini spray dryer with a nozzle size of 0.7 mm (BÜCHI, Germany), using a combination of chitosan and alginate as carrier excipients. The formulation of rifampicin DPI was shown in Table 1. Briefly, chitosan and rifampicin were dissolved separately in 1% acetic acid of pH 2–3 and then mixed. After the mixture was dispersed homogeneously, the pH was adjusted with NaOH 1N to pH 4–5. Sodium alginate was dispersed in deionized water and then dripped into a chitosan-rifampicin solution while stirring until homogenous. The mixture was continuously stirred and sprayed with a spray dryer with an inlet temperature of 150°C and a spraying rate of 25 ml/minute. Dry powders were stored in an airtight container in a desiccator.

Physical characterization

The organoleptic properties of rifampicin DPI were observed, including its shape, color, taste, and smell. Particle morphology was analyzed using a scanning electron microscope (SEM) (SSX-550, Shimadzu, Japan). The moisture content of rifampicin DPI was measured with a moisture balance (Adam Equipment AMB 50, UK) at 105°C. Aerodynamic particle size was analyzed by a cascade impactor (Andersen, Japan) and presented as mass median aerodynamic diameter (MMAD).

Drug content and entrapment efficiency

The drug content of DPI was analyzed by dissolving 50 mg DPI in 25.0 ml methanol. The solution was then diluted in a phosphate buffer of pH 7.4, and its absorbance was measured using a spectrophotometer (UV-1800, Shimadzu, Japan) at 473 nm. Drug content was expressed as rifampicin concentration in each gram DPI. Entrapment efficiency was calculated by comparing the drug amount in DPI and its theoretical amount, as described in the following formula:

$\%\mathrm{Entrapment}\mathrm{efficiency}=\frac{\mathrm{Drug}\mathrm{content}\mathrm{measured}\mathrm{in}\mathrm{powder}\mathrm{(}\mathrm{mg}\mathrm{)}}{\mathrm{Theoretical}\mathrm{drug}\mathrm{content}\mathrm{in}\mathrm{each}\mathrm{formulation}\mathrm{(}\mathrm{mg}\mathrm{)}}\times 100\mathrm{\%.}$

Drug release profile

The drug release study was performed on DPI (equal to 25 mg of rifampicin), using a dissolution tester (Electrolab TDT-08L, India) basket type. This study was performed in 900 ml of simulated lung fluid (containing a phosphate buffer of pH 7.4 with 0.05% SLS) and 900 ml of simulated macrophage fluid (containing a phthalate buffer of pH 4.5). The dissolution medium was maintained at 37°C ± 0.5°C with a stirring speed of 100 rpm. Six milliliters of the samples was taken at 5, 15, 30, 45, 60, 75, 90, and 120 minutes. The samples were filtered using a 0.45 µm filter and then measured using a spectrophotometer (UV-1800, Shimadzu, Japan) at the maximum absorbance of rifampicin at 473 nm.

In vitro cytotoxicity

An in vitro cytotoxicity study was carried out toward cell line A549 (epithelial cell carcinoma of the lung). Cells were grown with a concentration of 5,000 cells in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) until reaching 50% confluency. Rifampicin and rifampicin DPI were dispersed in the DMEM/F-12 to obtain a final active ingredient concentration of 0.01, 0.1, 1, 2, and 5 mg/ml and were then added to the cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed by adding 10 µl MTT (5 mg/ml) per well and incubation for 4 hours at 37°C. Formazan crystal was dissolved in ethanol, and the absorbance was measured at a wavelength of 595 nm (Chvatal et al., 2018).

Table 1. Formulation of rifampicin DPI. [Click here to view]

Physical properties of rifampicin DPI

As depicted in Figure 1, the spray drying process of rifampicin DPI produced a fine and orange-colored dry powder. Observation of DPI using the SEM showed different shapes and morphologies of the rifampicin powder and rifampicin DPI. As depicted in Figure 2, the rifampicin powder showed various nonspherical shapes within its powder. F1 (RIF-Chi) showed more spherical powder, while F2 (RIF-Alg) had a rough surface. The rough fibrous surface of F2 was due to the alginate which did not perfectly dissolve in the medium; thus, the spray drying process was not optimal. The combination of chitosan and alginate in DPI (F3, F4, and F5) also produced a rough-surfaced particle.

Figure 1. The physical appearance of rifampicin DPI: F1 (A), F2 (B), F3 (C), F4 (D), and F5 (E). [Click here to view]

Figure 2. Morphology of Rifampicin (A), F1 (B), F2 (C), F3 (D), F4 (E), and F5 (F) with 5,000× magnification. [Click here to view]

The spray drying process of rifampicin DPI produced a powder with a moisture content of 7.05%–12.97% (Table 2). Alginate-containing DPIs (F3, F4, and F5) showed higher moisture content. DPI F2 which contained alginate only as a carrier showed the highest moisture content. This phenomenon was due to the hygroscopic properties of the alginate and its high ability to absorb water (Rowe, 2009). The higher moisture content of the alginate-containing DPI was also due to the high viscosity of the alginate solution, which caused a challenge in the drying process of these formulas. The drying process was performed with the same parameters for all formulas (inlet temperature of 150°C and a spraying rate of 25 ml/minute). However, with the high viscosity of the alginate solution (in DPIs F2, F3, F4, and F5), it was more difficult for water to be evaporated from the DPIs, thus resulting in a powder with a higher moisture content.

The moisture content of the DPI was further correlated to its particle size. As shown in Table 2, DPI F1 which contains chitosan only as the carrier possessed the lowest moisture content, as well as the smallest aerodynamic particle size. On the other hand, DPIs F2–F5 with a higher moisture content also showed a bigger aerodynamic particle size. As discussed above, the high viscosity of the alginate solution (in DPIs F2, F3, F4, and F5) caused difficulty for water evaporation from the DPIs; thus, more water was still entrapped inside the powder. A high moisture content may also decrease the flowing capacity of powders due to stickiness properties (Lechanteur and Evrard, 2020), thus increasing aerodynamic particle size.

Among those formulas containing the combination of chitosan and alginate, DPI F3 (RIF-Ch-Alg 2:1:1) showed the smallest aerodynamic (MMAD) particle size. Measurement of the aerodynamic particle size of these DPIs was based on the deposition of powders in the cascade impactor (which represent the trachea, bronchus, and bronchioles of the lung), which is also affected by its morphology, density, and moisture content (de Boer et al., 2017).

A particle with MMAD 1–5 µm can be expected to be deposited in the deep lung. Therefore, since the aerodynamic particle size of all formulas, as shown in Table 2, was above 5 µm, it is assumed that these powders were deposited in the upper airway (bronchus or bronchioles). For further study, it is important to optimize the formula and improve the preparation and drying process of the DPIs to produce DPIs with lower moisture content and smaller aerodynamic particle size (5–10 µm) which can deposit in the deep lung.

Table 2. Physical properties of rifampicin DPI. [Click here to view]

Drug content and entrapment efficiency of rifampicin DPI

Drug content and entrapment efficiency of the DPIs were measured, and the results were presented in Table 3. The drug content of DPIs was 3.227–12.153 mg/g powder. The entrapment efficiency of DPIs was still lower than 50% (12.826%–48.107%). It was due to the suspension form of the polymers, and rifampicin was not perfectly dissolved in it, and thus much of the rifampicin was not well entrapped by the carrier. In the above formula, F3 (RIF-Ch-Alg 2:1:1) showed the lowest drug content and entrapment efficiency. It was probably due to the forming of the polyelectrolyte complexes of chitosan-alginate, which were less dissolved in the solution and thus less capable of entrapping the loaded drug (Huang et al., 2021; Li et al., 2008).

Drug release profile of rifampicin DPI

The drug release profile of rifampicin DPI was studied in the phosphate buffer of pH 7.4 with SLS 0.05% and in the phthalate buffer medium of pH 4.5 to represent simulated lung fluid and simulated macrophage fluid, respectively, to ensure the intended drug release profile in both conditions is achieved. A dissolution test on the rifampicin powder itself was also performed.

As shown in Table 4 and Figure 3, we found no significant difference in the dissolution of the rifampicin powder in simulated lung fluid (medium pH 7.4) and that in simulated macrophage fluid (medium pH 4.5). The rifampicin powder is dissolved more in an acidic medium (19.20 ± 1.57 mg/ml in pH 2.06) than in a basic medium (0.85 ± 0.13 mg/ml in pH 7.06) (Mariappan et al., 2007). However, simulated lung fluid of pH 7.4 contained additional SLS 0.05% to mimic the surfactant in lung fluid and thus can increase the solubility of rifampicin.

The dissolution profiles of the rifampicin DPIs were different compared to the rifampicin powder. This difference was due to the different characteristics of each excipient in the simulated lung or macrophage fluid. Formulating rifampicin in the chitosan carrier (F1) provides a higher dissolution rate in the medium of pH 4.5 (40.246% ± 1.054% in 2 hours) than in the medium of pH 7.4 (34.691% ± 1.086% in 2 hours). On the contrary, the dissolution of rifampicin from the alginate carrier (F2) was higher in the medium of pH 7.4 (98.827% ± 1.164% in 2 hours) than in the medium of pH 4.5 (53.082% ± 0.996% in 2 hours). This phenomenon was related to the solubility of chitosan, which is more soluble in the acid medium, and sodium alginate, which is more soluble in the neutral and base medium (Rowe, 2009).

Table 3. Drug content and entrapment efficiency of rifampicin DPI. [Click here to view]

Table 4. Dissolution of rifampicin DPI. [Click here to view]

Figure 3. Drug release profile of rifampicin powder and rifampicin DPI (F1 – F5) in phthalate buffer medium pH 4.5 (A) and in phosphate buffer medium pH 7.4 + SLS 0.05% (B). Data is presented as average ± standard deviation (n=3). [Click here to view]

Table 5. Viability of A549 lung epithelial cell lines after being exposed to the powder. [Click here to view]

The combination of chitosan and alginate as a carrier for the rifampicin DPIs (F3, F4, and F5) resulted in higher dissolution of rifampicin in the medium of pH 7.4. Among those three formulas, F3 (RIF-Ch-Alg 2:1:1) showed higher rifampicin dissolution in both media (41.356% ± 1.259% in pH 4.5 in 2 hours; 78.301 ± 1.331% in pH 4.5 in 2 hours) as compared to F4 and F5. This result was suggested to be correlated with the formation of chitosan-alginate polyelectrolyte complexes. F3 which contained chitosan-alginate 1:1 formed more polyelectrolyte complexes which facilitate the dissolution of drugs from the DPIs (Huang et al., 2021; Li et al., 2008).

The development of this DPI was aimed at the treatment of active and latent TB. Therefore, among chitosan-alginate-containing DPIs, F3 (RIF-Ch-Alg 2:1:1) was considered as the best formula since it can provide a higher rifampicin concentration in the simulated lung medium (to target active TB) and in the simulated macrophages medium (to target latent TB). However, further studies on macrophage uptake of this DPI should be performed to support the goal. Furthermore, it has potential to be further studied in vivo, to explore its efficiency in targeting active and latent TB.

In vitro cytotoxicity of rifampicin DPI

DPI F3 (RIF-Ch-Alg 2:1:1) was selected for in vitro cytotoxicity toward A549 lung epithelial cells, since it resulted in a satisfactory dissolution profile. A cytotoxicity test on the rifampicin powder was also performed as a comparison to the F3 DPI. Table 5 shows the viability of A549 lung epithelial cell lines after being exposed to the rifampicin DPI. The results revealed that DPI F3 was less toxic than the rifampicin powder. At a concentration of 0.1 mg/ml, the viability of A549 cells was still higher after being exposed to DPI F3 (89.73%) than that after being exposed to the rifampicin powder (51.32%). This might be due to the hydrophilicity of the particle. The particle of DPI F3 was considered more hydrophilic than the rifampicin powder due to the hydroxyl groups of chitosan and alginate as a carrier. The more hydrophilic the particles are, the less the particle would be recognized as a foreign particle; thus, it was less toxic to the lung epithelial cells (Chvatal et al., 2018). This result suggested that the application of chitosan-alginate as a carrier in the DPI is considered beneficial to increase the safety of the powder toward lung cells.