Targeting of phagolysosomes containing conidia of the fungus Aspergillus fumigatus with polymeric particles

Abstract  Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens. Key points • Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes. • These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages. • Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-12287-1.


Synthesis of PLGA-DY-550
The dye-labelled PLGA (PLGA-DY-550) was synthesized according to a carbodiimide method as reported elsewhere (Reul et al 2012). In a two necked flask 1 g (83 µmol) of PLGA, 30 mg (156 µmol) of EDCхHCl and 18 mg (156 µmol) of HOSu were added. Vacuum was applied and the flask was re-filled with argon (thrice). The solids were dissolved in 15 mL of dry CH 2 Cl 2 and 64 µL (460 µmol) of triethylamine were added. The solution was stirred for 2 h at room temperature. Separately, 1 mg (1.5 µmol) of DY-550 was dissolved in 1 mL of dry DMF and added into the reaction mixture. The reaction mixture was stirred at room temperature overnight, followed by a dilution with 20 mL of CH 2 Cl 2 and was washed with 50 mL of distilled water. The organic phase was dried over Na 2 SO 4 . The solution was filtered and concentrated under reduced pressure. The residual solution was precipitated in cold methanol (-20°C) and dried in vacuo to receive the labelled polymer as a red powder. Yield = 66% (658 mg). The 1 H NMR spectrum [ppm] measured with an 300 MHz Bruker Avance I spectrometer, (Bruker BioSpin GmbH, Rheinstetten, Germany) in deuterated dimethyl sulfoxide (DMSO-d 6 ),: δ = 1.37 -1.64 (br, 3 H, CH 3 ), 4.77 -5.08 (br, 2 H, CH 2 ), 5.08 -5. 42 (br, 1 H, CH) ppm. The size-exclusion chromatography (SEC) measurements were performed on an Agilent 1200 series system (Polymer Standards Service GmbH (PSS), Mainz, Germany), equipped with a PSS degasser, a G1310A pump and a Techlab oven set to 40°C. A G1362A refractive index detector and a DAD G1315D detector were utilized for data acquisition. The used eluent was a solution of 0.21% (w/v) of LiCl in N,N-dimethylacetamide (DMAc) at a flow rate of 1 mL min -1 . A PSS GRAM guard, a PSS GRAM 30 and a PSS GRAM 1000 column were used in series. The obtained dye labelled PLGA revealed a molar mass of M n = 20.0 kg mol -1 with a dispersity (Ð) of 1.94. These values were calculated from a calibration curve prepared with poly(methyl methacrylate) (PMMA) using standards obtained from PSS (Polymer Standards Service GmbH, Mainz, Germany) of narrow dispersity (Ð). The fluorescence spectrum of the dye was recorded on a Jasco FP-8300 instrument (Jasco, Pfungstadt, Germany) using a spectroscopy-grade solvent (DMSO) and quartz cuvettes (1 cm pathway). The device was measuring from 540 to 800 nm with a scan speed of 100 nm min -1 and a data interval of 1 nm.

Determination of PVA content
To determine the PVA content in the final formulations, 20 µL of NaOH (1 M) were added into 90 µL of the PP suspension and mixed for 15 min using a microplate shaker at 850 rpm at room temperature. Afterwards, 20 µL of a 1 M HCl solution, 60 µL of boric acid solution (0.65 M) and 10 µL of Lugol's solution (0.01 M elemental iodine and 0.016 M of potassium iodide) (Spek et al 2015) were added. This sample was then subjected for absorbance (λ = 650 nm) measurement using the TECAN Infinite M200pro microplate reader (Tecan, Crailsheim, Germany). Regression method from standard calibration curve was then used for final PVA content (c = 0.023 to 3.0% w/v, R 2 = 0.97) (Table S1, Fig. S1, in the SI).
Transfection of cells and live cell imaging RAW 264.7 macrophages were incubated in 8-well µ-slides and transfected with 50 CellLight Lamp1-GFP viral particles per cell. The cells were incubated overnight to allow adherence at 37°C and 5% (v/v) CO 2 under humidified atmosphere. A. fumigatus conidia were stained with CFW (100 µg mL -1 ) and added to the transfected cells at an MOI of 5 in DMEM containing 2% (v/v) of FCS. The slide was centrifuged for 5 min at 100 g to synchronize the uptake. The cells were incubated to allow for phagocytosis of conidia for 2 h. Then, extracellular conidia were stained with 0.04% (v/v) Trypan blue for 1 min (Liesche et al 2015), the cells were washed twice with PBS, and the PPs were added to a final concentration of 10 µg mL -1 . Non-transfected cells were stained with the membrane staining dye CellMask Deep Red according to the manufacturer's recommendations, prior to adding FITC-labelled conidia and PPs to the cells. The uptake of PPs by macrophages containing conidia was monitored by CLSM over 6 h.
Transmission electron microscopy RAW 264.7 macrophages were cultivated overnight in a 6-well plate at a density of 3×10 6 cells per well. The cells were infected with conidia at a MOI of 5 for 2 h and treated with PPs at a final concentration of 100 µg mL -1 for 4 h. After incubation, the cells were rinsed with PBS and detached with 0.5 mL TrypLE per well. The cell suspension was then centrifuged for 2 min at 600 g at 4°C. The supernatant was removed and the cells were re-suspended in 4% (v/v) of glutaraldehyde in PBS for 2 h at 20°C for fixation. Samples were washed thrice with Dulbecco's phosphate buffered saline (DPBS) for 5 min. After each washing step, the cells were centrifuged for 2 min at 134 g. The samples were fixed with a 1% (w/v) osmium tetroxide/DPBS solution for 1 h. After fixation, the samples were washed twice with DPBS for 15 min each, followed by dehydration of the samples using a graded ethanol series with 50%, 70%, 90% and 100% (v/v) ethanol content. Each step involved treatment of the samples for 10 min in the respective ethanol solutions and centrifugation at 134 g for 2 min. Thereafter, the samples were infiltrated with Embed812 resin first diluted in a ratio of 2:1 with ethanol for 1 h at RT. The samples were centrifuged for 8 min at 134 g and the diluted resin was exchanged by Embed812 mixed with 18 µg mL -1 of 2,4,6-tris(dimethyl aminomethyl)phenol (DMP-30) and incubated overnight at RT. Incubated samples were again centrifuged at 134 g and incubated further with fresh Embed812 and DMP solutions for another 2 h. The cells were then transferred into BEEM® capsules (Plano) and subjected to a fresh Embed812 and DMP solution and were then cured at 60°C for 24 h. An RMC PowerTome PT-PC Ultramicrotome (Boeckeler Instruments) diamond knife was used to obtain 100 nm thick slices of the cured, embedded samples. These 100 nm slices were collected on carbon-coated TEM grids (Quantifoil) and were stained with a 4% (w/v) uranyl acetate solution for 20 min and an additional 5 min with Reynold's lead citrate. These were then subjected to transmission electron microscopy (TEM) using a FEI Tecnai G² 20 equipped with LaB 6 filament operated at an acceleration voltage of 120 kV. Images were acquired on a 1376×1024 pixels MegaView CCD camera (Olympus Soft Imaging Solutions).

Image editing
For immunofluorescence, live cell imaging and TEM, the raw images obtained were cropped and contrast enhanced in order to increase visibility and to concentrate on the important details. This was performed with the ImageJ software (v. 1.53c, National Institutes of Health, Bethesda, USA, https://imagej.nih.gov/ij/).
Evaluation of cytotoxicity RAW 264.7 macrophages (ATCC TIB-71) were cultivated in flat-bottom 96-well plates at a concentration of 2×10 4 cells per well in DMEM supplemented with 10% (v/v) of fetal calf serum (FCS), 2 mM of L-glutamine and 27.5 µg mL -1 of gentamycin at 37°C with 5% (v/v) CO 2 under humidified atmosphere, and were allowed to adhere to the wells and grow overnight. Next, 100 μL of PP suspensions prepared in DMEM (without phenol red) with 2% (v/v) of FCS in concentrations of 1, 10, 50, and 100 µg mL -1 were added to the cells. The cells were incubated for 24 h at 37°C and 5% (v/v) CO 2 under a humidified atmosphere. Thereafter, resazurin was added to each well to reach a final concentration of 20 µg mL -1 . The fluorescence of resorufin, the product of reduction of resazurin using NADH+H + (O'Brien et al 2000), was recorded after 4 h of incubation by TECAN Infinite M200pro microplate reader (Tecan, Crailsheim, Germany), excitation with a 560 nm wavelength and measuring emission at 590 nm; these measurements were executed in triplicate.
Cytokine measurement RAW 264.7 macrophages were cultivated in 24-well plates at a concentration of 3×10 5 cells per well in DMEM supplemented with 10% (v/v) of FCS, 2 mM of L-glutamine and 27.5 µg mL -1 of gentamycin at 37°C with 5% (v/v) CO 2 under humidified atmosphere, and were allowed to adhere to the wells and grow overnight. The macrophages were infected with A. fumigatus conidia in medium with 2% (v/v) FCS at an MOI of 5; after a brief centrifugation of 5 min at 100 g the cells were incubated for 2 h at 37°C with 5% (v/v) CO 2 under humidified atmosphere. The particles were added to infected and non-infected cells at a concentration of 100 µg mL -1 . The cells were incubated for 6 h more at 37°C with 5 % (v/v) CO 2 . As a control for cytokine release, cells were treated with 500 ng mL -1 of LPS. At the end of the incubation, the supernatant was collected, centrifuged with 11,000 g for 5 min at 4°C, to remove particles and cell debris. Then, the supernatant was stored at -80°C for further analysis. The collected supernatant was tested by enzyme-linked immunosorbent assay (ELISA) to detect the mouse cytokine TNF-α (ELISA MAX™ Deluxe Set, Biolegend).  Table S2. Significance testing to determine whether the slope of the fitted logistic function, Eq (1), is different from zero in the dynamics that can be seen in Fig. 2