Materials
Poly(D,L-lactic-co-glycolic) acid (Resomer RG 502 H, copolymer composition of 50:50, 7–17 kDa, acid terminated) was purchased from Evonik Industries (Germany). Partially hydrolyzed PVA (Mowiol 4–88), acetone (> 99%) and dimethyl sulfoxide (DMSO > 99%, spectroscopic grade) were all purchased from Sigma-Aldrich (Germany). The dye DY635 was purchased from Dyomics (Jena, Germany). The covalent coupling of PLGA polymer with the dye DY635 amine was performed according to a standard procedure with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and was provided by SmartDyeLivery (Jena, Germany). The acetalation of dextran was done according to an adapted procedure (Mw of parent dextran 60 kDa), degree of substitution (DS) 2.97 (DScyclic acetal = 1.98, DSacyclic acetal = 0.99) [19]. BRP-187 was synthesized according to an established protocol [42]. Deuterated and non-deuterated lipid mediator standards for UPLC-MS–MS quantification were purchased from Cayman Chemical/Biomol (Germany). For further materials, see specific experimental section.
Acdex-rhodamine B synthesis
Rhodamine B was coupled to Acdex (Mw 9 to 11 kDa, Sigma Aldrich) according to an adapted procedure [43]: 1 g Acdex (6.17 mmol anhydroglucose unit, DSacyclic acteal = 0.56, DScyclic acetal = 2.14) and 13 mg rhodamine B isothiocyanate were dissolved in 15 mL anhydrous pyridine and heated to 80 °C for 72 h under argon. The reaction mixture was precipitated in 150 mL distilled water, and centrifuged; the pellet was lyophilized. The resulting pink-colored powder was purified via gel permeation chromatography using BioBeads S-X1 in tetrahydrofuran (THF) to remove the free dye (yield 57%). Size-exclusion chromatography was performed in dimethylacetamide (DMAc) and 0.21% lithium chloride with a UV–VIS detector measuring at λ = 562 nm to prove the conjugation of the dye to the polymer.
Nanoparticle formulation
Particles were prepared by nanoprecipitation using a syringe pump (Aladdin AL1000-220, World Precision Instruments, Berlin, Germany) with a flow rate of 2 mL min−1. First, 25 mg polymer (Acdex or PLGA) were dissolved in 5 mL acetone. For the drug solution, a 10 mg mL−1 stock of BRP-187 in DMSO was prepared and sonicated in an ultrasound bath for 15 min at room temperature. Subsequently, 75 µL of the drug solution were mixed with the polymer solution. For the aqueous phase, 40 mL of 0.3% (w/v) PVA solution were prepared. Further, the polymer (or polymer-drug) solution was injected into the aqueous solution, while stirring at 800 rpm at room temperature. After nanoprecipitation, the samples were stirred for 24 h in a fume hood to evaporate the acetone. The particles were washed once, using a Rotina 380 R centrifuge (Hettich Lab Technology, Germany) at 12.851×g for 60 min at 20 °C. After removing the supernatant, NPs were redispersed in 2.5 mL pure water, vortexed 5 to 10 s and then sonicated in an ultrasound bath for 30 min. For the Acdex NPs, 100 μL of 0.01% triethylamine (TEA) of pH 9 were added to the suspension. The NPs were stored at 4 °C overnight to allow complete dispersion in water. The concentration of the particle dispersions was determined by freeze-drying up to six aliquots of 100 or 200 μL NPs dispersion. The mass of the NPs was accurately weighed (Radwag Waagen, MYA 11.4Y, Germany) and an average was calculated for the NPs concentration.
Dynamic light scattering (DLS)
The size, polydispersity index and the zeta-potential of the particles was measured using a Zetasizer Nano ZS with a laser wavelength of λ = 633 nm (Malvern Instruments, Germany). The measurements were performed at 173˚ backscatter angle with the following settings: Five repeated measurements at 25 °C, each measurement with five runs of 30 s and a 30 s equilibration time. The zeta-potential of the lyophilized NPs was measured at 25 °C with three repeated measurements. The NPs were characterized after purification (10 µL NP dispersion diluted in 1 mL pure water), and after lyophilization (100 µL of NP dispersion were lyophilized and redispersed in 1 mL pure water). The intensity size distribution is reported as the hydrodynamic diameter (dH) of the NPs.
The degradation behavior of Acdex and PLGA NPs was determined by monitoring the mean count rate (kcps). The NP samples were measured using fixed settings (37 °C, measurement position 4.65, and attenuator 7). The Acdex NPs were measured for 15 h: 180 measurements (delay between measurements 240 s), where each measurement consisted of three runs with 20 s run durations. The PLGA NPs were measured every day over a period of 60 days: Five measurements (with no delay between measurements), where each measurement consisted of one run with 30 s run duration.
UV–VIS spectroscopy
For the calculating the encapsulation efficiency (EE) and loading capacity (LC) of the drug in the NPs, aliquots of 200 µL of the washed NP dispersion were lyophilized. The dry NP powder was dissolved in 200 µL DMSO (spectroscopic grade). The polymer-drug solution was measured at λ = 316 nm with 3 × 3 multiple reads per well and 2000 µm well border using the Infinite M200 Pro platereader (Tecan Group, Switzerland). For all EE measurements, a Hellma Quartz flat-transparent plate with 96 wells was used. A calibration curve of BRP-187 was obtained for each batch in the concentration range of 0.24 to µg mL−1 with R2 = 0.9997. Equations 1 and 2 were used to calculate EE and LC
$$\varvec{LC} = \frac{{\varvec{mass}\, \varvec{of} \varvec{drug} \,\varvec{recovered}}}{{\varvec{mass}\, \varvec{of} \varvec{particle} \,\varvec{recovered}}} \times 100$$
(1)
$$\varvec{EE} = \frac{{\varvec{LC}}}{{\varvec{LC \,theoretical}}} \times 100$$
(2)
PVA assay
Determination of PVA in the NPs (%, w/w) was performed using UV–VIS spectroscopy. PVA forms a complex with iodine, which absorbs light at λ = 650 to 690 nm. In an adapted protocol, Lugol solution was used as iodine source [44]. Lyophilized NPs were redispersed in pure water (3 mg mL−1) and 90 µL were pipetted into a 96-well plate. Then, 20 μL of 1 M sodium hydroxide was added to each NP-containing well, and the solutions were mixed for 15 min at 850 rpm at room temperature. Next, 20 μL 1 M hydrochloric acid, 60 μL 0.65 M boric acid and 10 μL of Lugol solution were added to each well. Measurements on the plate reader were done at λ = 650 nm 15 min after the addition of the Lugol solution. The experiments were repeated three times for each NP formulation.
Scanning electron microscopy (SEM)
Electron microscopy imaging was performed with a Sigma VP Field Emission Scanning Electron Microscope (Carl-Zeiss, Jena, Germany) using an InLens detector with an accelerating voltage of 6 kV. The samples were coated with a thin layer of platinum (4 nm) via sputter coating (CCU-010 HV, Safematic, Switzerland) before the measurement. ImageJ was used to measure the particle sizes from the images acquired by the SEM, where a mean diameter was deduced by measuring 150 to 200 NPs/image.
Degradation study
The degradation behavior of loaded and unloaded NPs was tested at 37 °C in 0.05 M Tris–HCl buffer of pH 7.4 and 0.05 M acetate buffer of pH 4.8. Lyophilized NPs were dispersed in pure water (concentration of 3 to 7 mg mL−1). Next, NPs were mixed with buffer solution incubated at 37 °C and analyzed by DLS. Acdex NPs were measured for 15 h, whereas PLGA NPs were measured over 60 days (see Sect. “Fluorescence dye-labeled nanoparticle uptake in PMNL”). The degradation of the NPs was analyzed by monitoring the change in the mean count rate, size and PDI in DLS.
Cell isolation and cell culture
Leukocyte concentrates were prepared from peripheral blood obtained from healthy human adult donors that received no anti-inflammatory treatment for the last 10 days (Institute of Transfusion Medicine, University Hospital Jena, Germany). The approval for the protocol was given by the ethical committee of the University Hospital Jena and all methods were performed in accordance with the relevant guidelines and regulations. To isolate PMNL and monocytes, the leukocyte concentrates were mixed with dextran (dextran from Leuconostoc spp. MW ~ 40,000 g mol−1, Sigma Aldrich, Taufkirchen, Germany) for sedimentation of erythrocytes; the supernatant was centrifuged on lymphocyte separation medium (Histopaque®-1077, Sigma Aldrich). Contaminating erythrocytes in the pelleted PMNL were removed by hypotonic lysis using water. The pelleted PMNL were subsequently washed twice in ice-cold phosphate-buffered saline pH 7.4 (PBS) and finally resuspended in PBS. The peripheral blood mononuclear cell (PBMC) fraction on top of lymphocyte separation medium was washed with ice-cold PBS and seeded in cell culture flasks (Greiner Bio-one, Nuertingen, Germany) for 1.5 h (37 °C, 5% CO2) in PBS with Ca2+/Mg2+ (0133 g L−1/0,1 g L−1) to isolate monocytes by adherence. For differentiation and polarization of monocytes to M1 and M2 macrophages, we followed published procedures [39]. To obtain M1 macrophages, adherent monocytes were treated with 20 ng mL−1 granulocyte macrophage-colony stimulating factor (GM-CSF) Peprotech, Hamburg, Germany) for six days in RPMI 1640 supplemented with 10% fetal calf serum (FCS), 2 mmol L−1l-glutamine, penicillin (100 U mL−1) and streptomycin (100 µg mL−1) for differentiation and were further incubated with 100 ng mL−1 lipopolysaccharide (LPS) and 20 ng mL−1 interferon-γ (Peprotech) for 48 h. M2 macrophages were obtained by treatment of monocytes with 20 ng mL−1 M-CSF (Peprotech) for 6 days, followed by 20 ng mL−1 IL-4 (Peprotech) for 48 h. Correct polarization and purity of macrophages was routinely checked by flow cytometry (FACS Canto Plus flow cytometer, BD Biosciences, Heidelberg, Germany) as reported [45] using the following antibodies: FITC anti-human CD14 (2 µg/test, clone M5E2, BD Biosciences), PE anti-human CD54 (1 µg/test, clone HA58, BD Biosciences), APC-H7 anti-human CD80 (0.25 µg/test, clone L307.4, BD Biosciences), PE-Cy7 anti-human CD163 (2 µg/test, clone RM3/1, Biolegend, San Diego, CA, USA), PerCP-eFluor710 anti-human CD206 (0.06 µg/test, clone 19.2, BD Biosciences, San Diego, CA, USA).
Determination of 5-LO product formation in PMNL
For evaluation of the effects on 5-LO product formation in human PMNL, cells (5 × 106 mL−1) were pre-incubated with BRP-187 or NPs (Acdex[blank], PLGA[blank], Acdex[BRP-187], PLGA[BRP-187]) for the indicated times (15 min up to 5 h) at 37 °C. Cells were then stimulated with 2.5 µM Ca2+-ionophore A23187 (Cayman, Ann Arbor, USA) for 10 min, and then the incubation was stopped with 1 mL ice-cold methanol containing 200 ng mL−1 PGB1 as internal standard. Samples were subjected to solid phase extraction and formed 5-LO products were separated and analyzed by RP-HPLC as described [46].
Determination of prostaglandin E2 formation in human macrophages
Human monocyte-derived M1 macrophages (2 × 106 cells) were seeded in 6-well-plates and preincubated for 15 min, 5 or 20 h with BRP-187 or NPs (Acdex[blank], PLGA[blank], Acdex[BRP-187], PLGA[BRP-187]) at 37 °C. The macrophages were subsequently incubated with pathogenic E. coli [O6:K2:H1] for 90 min. The reaction was stopped with ice-cold methanol containing deuterium-labeled internal standards (d8-5S-HETE, d4-LTB4, d5-LXA4, d5-RvD2, and d4-PGE2; 500 pg each). Samples were kept at − 20 °C for one day to allow protein precipitation. After centrifugation (2000×g, 4 °C, 10 min), 8 mL acidified water was added (final pH = 3.5) and samples were subjected to solid phase extraction using RP-18 columns and PGE2 was analyzed by UPLC-MS-MS exactly as described before [45].
Lactate dehydrogenase assay
The release of LDH from cells was analyzed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega GmbH, Mannheim, Germany). Briefly, 1 × 106 M1 or M2 macrophages per well, suspended in RPMI-medium containing 10% FCS, penicillin/streptomycin and l-glutamine, were seeded in a 24-well plate. Lysis control and 0.2% triton X-100 were added to the cells and incubated for 45 min; compounds and control (0.1% DMSO) were added and incubated for 24 h at 37 °C. Stop solution was added, the plate was centrifuged (250×g, 4 min, room temperature) and 50 μL of supernatant from each well was transferred in a 96-well plate. Afterwards, 50 μL of substrate mixture was added and incubated for 30 min at room temperature in the dark. To finally stop the reaction, 50 μL of stop solution were added. The photometric measurement was performed at 490 nm using a Multiskan Spectrum plate reader (Thermo Fischer).
Fluorescence dye-labeled nanoparticle uptake in PMNL
Time-dependent uptake of fluorescence dye-labeled NPs by PMNL was analyzed by flow cytometry and confocal laser scanning microscopy. Adherent PMNL (2 × 106) were incubated for the indicated time points with 0.5 mg mL−1 labeled NPs (PLGA-DY635[BRP-187] or Acdex-RhoB[BRP-187]). For flow cytometry, cells were washed once with PBS containing 0.5% BSA and incubated with PBA-E (PBS with 0.5% BSA, 2 mM EDTA and 0.1% sodium azide) containing 0.4% lidocaine for detaching. PMNL containing fluorescently stained NPs were analyzed by flow cytometry using BD LSR Fortessa (BD Bioscience). The red laser (644 nm) in combination with 670|14 filters for DY635 labeled NPs and the violet laser (405 nm) in combination with 655|8 filters for rhodamine B labeled NPs were used for flow cytometric analysis. Data were analyzed using FlowJo X Software (BD Bioscience).
For confocal laser scanning microscopy, PMNL were (i) washed once with PBS after incubation with the respective NPs for the indicated time points as described above for flow cytometry, (ii) submerged with phenol red-free RPMI 1640 and (iii) subsequently subjected to microscopic analysis. CLSM images were acquired using a Zeiss LSM 880 (Carl Zeiss, Oberkochen, Germany) with following settings: PLGA NPs labeled with DY635: λEx = 633 nm, λEm = 638 to 759 nm and transmission signal with PMT detector; Acdex NPs labeled with rhodamine B: λEx = 514 nm, λEm = 531 to 703 nm and transmission signal with PMT detector. Images were captured with an iLCI Plan-Neofluar 63 × objective using identical settings for image acquisition within experimental groups.