(T1430-03-18) Dual Drug-Loaded Cubosome Nanoparticles for Pulmonary Tuberculosis Treatment

Purpose: Tuberculosis (TB) is a highly infectious and potentially fatal disease caused by the bacillus mycobacterium tuberculosis (M.tb). TB mainly affects the lungs and treatment is challenging as the M.tb bacteria reside within the alveolar macrophages leading to formation of granulomas. The current TB treatments include oral dosages of delamanid (DLD) and bedaquiline (BDQ) but suffer from low local bioavailability, resistance, and systemic side effects. Hence, we propose formulating DLD-BDQ co-loaded cubosomes. The combination of antibiotics decreases the likelihood of M.tb. bacteria developing resistance. Cubosomes are novel, lipid nanocarriers with a cubic inner structure that improves drug aqueous solubility and facilitates efficient uptake of drugs into macrophages to inhibit the growth of bacteria. The encapsulation of TB antibiotics in nanoparticles increases overall drug delivery by enhancing penetration into the granuloma due to their small size ( < 200 nm).

Methods: The effect of single drugs in M.tb bacteria (H37Ra, ATCC 25177^TM) was confirmed by determining the minimum inhibitory concentration (MIC) using resazurin microtiter assay (REMA). The dual-drug effect of DLD and BDQ in combination was determined using a checkerboard assay which determined the MIC and optimal ratio of drug combination. The interaction between DLD and BDQ when administered in combination was determined using the fractional inhibitory concentration index (FICI), which is a ratio of the drugs MIC’s individually and when in combination. A single emulsion solvent evaporation method was used to prepare the phytantriol (PHY) cubosomes before rehydrating the lipid premix with a dilute surfactant (Kolliphor® P 407). The formulation was then subjected to probe sonication to further reduce the particle size. In order to encapsulate the optimized drug ratio, different drug ratios were added to the cubosomes, and the final encapsulated drug ratio was determined. The cubosomes were characterized for particle size, poly dispersity index (PDI), and zeta potential using Malvern Zetasizer Nano ZS. The solid-state characteristics were analyzed using differential scanning calorimetry (DSC) to ensure the formulations complete encapsulation of the co-therapy. Further, the cellular internalization of the cubosomes into macrophages (THP-1, ATCC TIB-202^TM) was confirmed using microscopy imaging of coumarin 6-loaded cubosomes.

Results: The MIC for DLD and BDQ in M.tb bacteria was determined to be 0.250 μg/mL (Fig.1A) and 0.063μg/mL (Fig.1B), respectively. However, in combination, the MIC for DLD and BDQ was 0.063μg/mL and 0.016μg/mL (Fig.1C) respectively, showing a 2-fold decrease in both MIC values as compared to the individual drugs. Based on the MIC values of the combination drugs, we determined the optimized drug ratio to be 1:4 BDQ:DLD. Furthermore, the FICI calculated for the optimized drug ratio showed a borderline additive and synergistic effect with a FICI of 0.5 (Fig.1D), allowing for a reduction in the effective concentration of antibiotics and overall systemic side effects. When formulating the combination therapy, initially adding 1:4 BDQ:DLD resulted in a final encapsulated drug ratio below 1:4. However, an initial drug ratio of 1:6 BDQ:DLD resulted in the optimized drug ratio (1:4) being encapsulated (Fig.2A). The optimized drug ratio was successfully loaded into the cubosomes with an encapsulation efficiency of 71.79±2.8% w/w and 97.58±1.95% w/w for DLD and BDQ, respectively. Further, the cubosomes formulation showed an average particle size of 193.5±6.5 nm with a PDI of 0.17 and a zeta potential of -8.73±6.4 mV (Fig.2B). The complete encapsulation of the combination drugs is proven by the disappearance of the signature melting peaks for each drug in the cubosomes thermogram (Fig.2C). The effective internalization of the cubosomes was proven (Fig.3C) by the localization of coumarin-6 dye around the nuclei, in the cytoplasm of the macrophages within 2 h of the treatment. The increased uptake into the macrophages is due to the small size, < 200nm, allowing them to undergo phagocytosis and release the drug once internalized into the macrophage.

Conclusion: The DLD-BDQ combination therapy showed an overall additive effect against M.tb bacteria and was also successfully loaded at an optimized ratio into PHY cubosomes nanocarriers. Our ongoing studies include assessing the minimum inhibitory concentration of the cubosome formulation and evaluating the long-term stability of the cubosomes. Further, based on our previous experience we will also study the potential for delivering the cubosomes via nebulization.

Minimum inhibitory concentration of (A) Delamanid and (B) Bedqualine were estimated using REMA. (c) Minimum inhibitory concentration of DLD-BDQ estimated using checkerboard assay and visualized by bacterial viability heatmap.

(A) Expected and Actual Ratios of DLD:BDQ in cubosomes. (B) Characterization for the optimized formulation including encapsulation efficiency, particle size, PDI and zeta potential. (C) Differential scanning calorimetry thermograms for blank cubosomes, DLD, BDQ and combination drug loaded cubosomes.

Internalization of coumarin 6-loaded cubosomes (green) into macrophages (blue nuclei) as seen with EVOS M5000 fluorescent microscope.