Nano-in-Microparticles for Aerosol Delivery of Antibiotic-Loaded, Fucose-Derivatized, and Macrophage-Targeted Liposomes to Combat Mycobacterial Infections: In Vitro Deposition, Pulmonary Barrier Interactions, and Targeted Delivery

Abstract

Nontuberculous mycobacterial infections rapidly emerge and demand potent medications to cope with resistance. In this context, targeted loco-regional delivery of aerosol medicines to the lungs is an advantage. However, sufficient antibiotic delivery requires engineered aerosols for optimized deposition. Here, the effect of bedaquiline-encapsulating fucosylated versus nonfucosylated liposomes on cellular uptake and delivery is investigated. Notably, this comparison includes critical parameters for pulmonary delivery, i.e., aerosol deposition and the noncellular barriers of pulmonary surfactant (PS) and mucus. Targeting increases liposomal uptake into THP-1 cells as well as peripheral blood monocyte- and lung-tissue derived macrophages. Aerosol deposition in the presence of PS, however, masks the effect of active targeting. PS alters antibiotic release that depends on the drug's hydrophobicity, while mucus reduces the mobility of nontargeted more than fucosylated liposomes. Dry-powder microparticles of spray-dried bedaquiline-loaded liposomes display a high fine particle fraction of >70%, as well as preserved liposomal integrity and targeting function. The antibiotic effect is maintained when deposited as powder aerosol on cultured Mycobacterium abscessus. When treating M. abscessus infected THP-1 cells, the fucosylated variant enabled enhanced bacterial killing, thus opening up a clear perspective for the improved treatment of nontuberculous mycobacterial infections.

Keywords: air-liquid interfaces; bedaquiline; liposomal dry powders; particle tracking; pulmonary surfactants.

Scheme 1

Biological barriers in pulmonary drug delivery. Targeted liposomes and liposomal dry‐powder formulations loaded with bedaquiline (BDQ) or levofloxacin (LVX), respectively, were developed to overcome such barriers. A) Interaction of liposomes with epithelial cells and pulmonary macrophages in the presence of mucus and surfactant. B) Receptor‐mediated internalization of targeted liposomes into myeloid cells under submerged and air–liquid interface conditions and in the presence of pulmonary surfactant. C) Assessing antimycobacterial activity against extracellular and intracellular Mycobacterium abscessus. Abbreviations: MDMs: Peripheral blood monocyte‐derived macrophages, dTHP‐1: differentiated THP‐1 cells.

Figure 1

Size distribution of fucosylated liposomes in the presence of LecB after overnight incubation at a 2:1 molar ratio measured by a) particle tracking analysis and b) dynamic light scattering. c) Microfluidic assay with LecB covalently attached to the bottom of the flow chamber. Following a 10‐min flush with TargoSphere liposomes and a 30‐min rest, excess CLR‐TargoSpheres were removed by flushing the chamber for 15 min with buffer. Images are representative for nontargeted liposomes (left), targeted liposomes (center), and targeted liposomes after an additional flushing step with competitive inhibitor (right). d) Quantification of fluorescence intensity. Error bars indicate means ± SD (n = 12–15, N = 3). Significance was defined as *** (p < 0.001).

Figure 2

Release profiles of a) bedaquiline (BDQ) and b) levofloxacin (LVX) from fucosylated (Lipo_fuco) and plain liposomes (Lipo_plain) in the presence and absence of pulmonary surfactant Alveofact, respectively. LVX was released more rapidly in the presence of pulmonary surfactant (PS) and compared to BDQ. The release of BDQ did not exceed 10% after 96 h and was not detectable when PS was present in the donor medium. Error bars represent means ± SD (n = 9, N = 3). Significance was defined as ***/^### (p < 0.001) and **/^## (p < 0.005).

Figure 3

Quantification of liposome uptake by flow cytometry under submerged conditions at a concentration of 50 µg mL⁻¹ after 2 h incubation at 37 °C in volumes of 300 µL per condition. Fucosylated liposomes showed a higher uptake than plain liposomes. Increased uptake into human dTHP‐1 cells, peripheral blood monocyte‐derived macrophages (MDM) and lung tissue macrophages was reduced in the presence of free l‐fucose (10 × 10⁻³ m) as a competitive inhibitor. At 4 °C, the internalization was drastically reduced due to the inhibition of active uptake, thus indicating the proportion of cell‐associated liposomes in this specific case. Data represent means ± SD (n = 6–9, N = 3 for dTHP‐1 and MDM, and n = 3–5 with N = 2 for lung macrophages). Significance was defined as *** (p < 0.001), ** (p < 0.005), and * (p < 0.05).

Figure 4

Quantification of liposome uptake by flow cytometry after aerosol deposition at a dose of 15 µg/well after 2‐h incubation at 37 °C in the presence or absence of pulmonary surfactant, Alveofact, respectively. When pulmonary surfactant was present in the interface, the uptake into dTHP‐1 cells was reduced, which was not observed for blood‐monocyte derived macrophages (MDM) and macrophages from human lung tissue. Data represent mean ± SD (n = 6–9, N = 3 for dTHP‐1 and MDM and n = 3–5 with N = 2 for lung macrophages). Significance was defined as * (p < 0.05).

Figure 5

a) Aerodynamic properties of microparticles and their deposition in different stages of the next‐generation impactor and b) scanning electron microscopy images of empty lactose‐leucine microparticles (MP) and of MP containing fucosylated or plain liposomes, respectively. No differences in the fine‐particle fraction were found between the different MP variants. Scale bars: 2 µm. Data represent means ± SD from three independent replicates (n = 9, N = 3). Cut‐off sizes for the Next Generation Impactor at a flow rate of 60 L min⁻¹ are 8.06, 4.46, 2.82, 1.66, 0.94, 0.55, and 0.34 µm for stages 1–7, and MOC for micro‐orifice collector, respectively.^{[ 27 ]}

Figure 6

a) The initial size of the liposomes increases during spray‐drying and remains constant upon six weeks of storage. b) BDQ‐loaded liposomes before and after spray‐drying (and redispersion) have similar sizes with intact lipid bilayers as observed by cryo‐transmission electron microscopy. Data represent means ± SD (n = 6–8, N = 3) with significance defined as *** (p < 0.001).

Figure 7

Targeting function of liposomes is preserved after spray‐drying as demonstrated by the a) LecB binding assay and b) in microfluidic chambers. c) Uptake of plain (MP_lipo_plain) and fucosylated (MP_lipo_fuco) BDQ‐loaded nano‐in‐microparticles following redispersion. Uptake after 2‐h incubation at 37 °C by dTHP‐1 cells evidenced by flow cytometry confirmed that active targeting was preserved. d) Representative confocal images analyzed for dissolved dry powders containing fucosylated liposomes show the intracellular localization of liposomes. Nuclei were stained with DAPI (blue), actin with phalloidin (green), and liposomes with PE‐Texas red. Data represent means ± SD (n = 9, N = 3) with significance defined as *** (p < 0.001).

Figure 8

a) A slide with deposited tracheobronchial mucus was placed into stage 2 of the next generation impactor and analyzed by video microscopy after deposition of the dry powder to study the mobility of plain (MP_lipo_plain) and fucosylated (MP_lipo_fuco) liposomes released from microparticles. b) The slope of individual mean squared displacement (MSD) curves discriminates between mobile (black line, α > 0.5) and immobile (red line, α ≤ 0.5) particles. Percentages indicate the mobile/immobile fraction of all tracked particles. c) The distribution of log (MSD) values at a timescale of t = 0.5 s indicates a higher mobility of fucosylated liposomes, as also signified in the representative trajectory. Three independent experiments were performed with at least >150 particles/frame.

Figure 9

Antimycobacterial activity of free bedaquiline (BDQ), BDQ‐loaded liposomes and dry powders of BDQ‐loaded liposomes on extra‐ and intracellular M. abscessus under submerged and air–liquid interface conditions. a) Efficacy of the formulations against extracellular bacteria at different BDQ concentrations at submerged conditions and b) at a fixed concentration of 500 ng mL⁻¹ after dry powder aerosol deposition at air‐interface conditions. Intracellular infection of dTHP‐1 cells treated with free BDQ, BDQ liposomes, and the respective powders with and without fucose targeting after c) 24 h and d) 72 h. Data represent means ± SD (n = 9, N = 3) with significance defined as *** (p < 0.001), ** (p < 0.01), and * (p < 0.05).