Assessment of Pulmonary Administration through Dry Powder Inhaler (DPI) Using Ethambutol-Loaded Soli

Preparation of EMB-Loaded SLNs and Formulation of DPI

Ethambutol hydrocloride (EMB) is an anti-tuberculosis drug, which is commonly used as a protection agent against of unrecognized resistance to other drugs employed to treat this disease. Since oral form of EMB has some side effects and cellular toxicity, direct administration of EMB into lungs seems to be an attractive and reasonable option in order to overcome these side effects. Our main goal in this study was assessment of pulmonary administration through dry powder inhaler (DPI) using EMB-loaded solid lipid nanoparticles (SLNs). We prepared EMB-loaded SLNs using two techniques (hot homogenization and ultrasonication). DPI formulations were made by spray drying of EMB-loaded SLNs with and without mannitol. For investigation of flowbility of the prepared powders, Carr’s index and Hausner ratio, and for in vitro deposition of the powders, Next Generation Impactor (NGI) analysis were used. The encapsulation efficiency and particle size of obtained particles were higher than 98% and sub-100 nm, respectively. Toxicity investigation of EMB-loaded SLNs via MTT assay showed biocompatibility and non-toxicity of the SLNs.

Results of flowability and aerodynamic traits assessment of EMB-loaded SLN DPI powder confirmed the suitability of prepared powders. Overall, the attained results showed that EMB-loaded SLN DPI has high potential for direct treatment of tuberculosis.

EMB is one of the various WHO introduced drugs for the TB therapy and is used to prevent or delay resistant strains (31). Anti-TB drugs’ direct administration through the lung can be of interest in the treatment of TB (32). The inhaled administration of EMB can be very effective because the lung is the primary portal of entrance for TB cause mycobacteria and this way couldavoid drug oral administration side effects. There are few studies about SLNs applications as pulmonary drug delivery systems for TB therapy. Pandey et al. formulated rifampin, isoniazid, and pirazinamide drugs by solvent emulsion technique in SLNs and tested the inhaled nebulized form in Indian guinea pigs (15,33).

In 2016, Marty et al. formulated rifampin in SLNs using emulsion melting method, and after freeze drying, they successfully prepared SLNs and created DPI of them (34). Furthermore, SLNs have also been used for the pulmonary delivery of erlotinib (29), alendronate (25), mometasonefuroate (35), budesonide, and other compounds (36). So, the incorporation of EMB to inhalable formulations could be of interest in TB treatment. The size of nanoparticles is an important factor in internalizing them in the body. In the SLNs preparation, the particle size reduction leads to an increase in the surface,which causes a monotonic distribution of drug among alveoli(37). Various researchers demonstrated that the preparationmethods and the composition of used materials have a significant influence on the SLNs physicochemical properties like the size and EE (38).

Preparation and Physicochemical Evaluation of EMB-Loaded SLNs

To prepare SLNs, various techniques have been used depending on the type of work or medication; thus, in the present study, the appropriate technique has been selected for drug formulation improvement. It has been reported that the combination of homogeneization and ultrasound techniques lead to production of particles with small size (39,40). Thus, the same techniques were applied for formulation of EMB in SLNs, which results in nanoparticles with a size below 100 nmin all prepared formulations. In addition to the SLNs preparation techniques, Tween 80 application as lipid phase surfactant is effective in attaining particles with size below 100 nm.

The EE of prepared nano-formulations were very high and in the best formulation reached 99.04% and low PDI value (0.253) demonstrates a narrow distribution of particle size. EMB-loaded SLNs were stored at 4°C for 4 weeks in the physical stability test. The obtained results of PDI, size, and EE% did not demonstrate considerable change during this period. The sizes of nanoparticles at the synthesis time and after 4 weeks of storage were 57.65 and 56.79 nm, respectively. A slight decrease in the nanoparticles size may be due to the transfer of saturated lipid crystal, which can be due totemperature and storage. The PDI of nanoparticles changed from 0.253 to 0.349. The drug loading at the synthesis time of the nanoparticles and after 4 weeks of storage was 3.74 and 3.69, respectively that showed partial withdrawal of drug fromSLNs. These values indicated that EMB-loaded SLNs at low temperature can be stable up to 4 weeks.

The EMB release rate from SLNs was compared with release of free EMB using dialysis bag method. Slow release profile of EMB from SLNs indicates its suitability for drug delivery. The graph analysis demonstrates that in the first 7 h, the release rate of free EMB and EMB-loaded SLNs were about 40%, and 25%, which indicates slow release of drug from SLNs as compared to free drug. Therefore, it can be reduced the dose and minimized the drug side effects by its formulation in SLNs.

Low toxicity is an essential factor for numerous drug delivery nanosystems application in biomedical sciences (41). For that reason, EMB-loaded SLNs in vitro cytotoxicity wastested on A549 cell line via treatment of them with different concentrations of EMB and EMB-loaded SLNs. The MTT assay results did not show any considerable cytotoxicity.Therefore, this formulation can be applied as remedy after testing on animal specimens.