Macrophage-like nanoparticles concurrently absorbing endotoxins and proinflammatory cytokines for sepsis management

The murine J774 cell line was purchased from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% FBS (HyClone) and 1% penicillin–streptomycin (pen–strep) (Invitrogen). Plasma membrane was collected according to a previously published centrifugation method (40). Specifically, cells were grown in T-175 culture flasks to full confluency and detached with 2 mM EDTA (USB Corporation) in PBS (Invitrogen). The cells were washed with PBS three times (500 × g for 10 min each) and the cell pellet was suspended in homogenization buffer containing 75 mM sucrose, 20 mM Tris⋅HCl (pH = 7.5, MediaTech), 2 mM MgCl₂ (Sigma-Aldrich), 10 mM KCl (Sigma-Aldrich), and one tablet of protease/phosphatase inhibitors (Pierce, Thermo Fisher Scientific). The suspension was loaded into a Dounce homogenizer and the cells were disrupted with 20 passes. Then the suspension was spun down at 3,200 × g for 5 min to remove large debris. The supernatant was collected and centrifuged at 20,000 × g for 25 min, after which the pellet was discarded and the supernatant was centrifuged at 100,000 × g for 35 min. After the centrifugation, the supernatant was discarded and the plasma membrane was collected as an off-white pellet for subsequent experiments. Membrane protein content was quantified with a Pierce BCA assay (Life Technologies).

MΦ-NPs were formulated in two steps. In the first step, ∼80-nm polymeric cores were prepared using 0.67 dL/g carboxyl-terminated 50:50 PLGA (LACTEL absorbable polymers) through a nanoprecipitation method. The PLGA polymer was first dissolved in acetone at a concentration of 10 mg/mL. Then 1 mL of the solution was added rapidly to 3 mL of water. For fluorescently labeled PLGA cores, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD, excitation/emission = 644 nm/665 nm; Life Technologies) was loaded into the polymeric cores at 0.1 wt%. The nanoparticle solution was then stirred in open air for 4 h to remove the organic solvent. In the second step, the collected macrophage membranes were mixed with nanoparticle cores at a membrane protein-to-polymer weight ratio of 1:1. The mixture was sonicated with a Fisher Scientific FS30D bath sonicator at a frequency of 42 kHz and a power of 100 W for 2 min. Nanoparticles were measured for size and size distribution with DLS (ZEN 3600 Zetasizer, Malvern). All measurements were done in triplicate at room temperature. Serum and PBS stabilities were examined by mixing 1 mg/mL of MΦ-NPs in water with 100% FBS and 2× PBS, respectively, at a 1:1 volume ratio. Membrane coating was confirmed with transmission electron microscopy (TEM). Briefly, 3 μL of nanoparticle suspension (1 mg/mL) was deposited onto a glow-discharged carbon-coated copper grid. Five minutes after the sample was deposited, the grid was rinsed with 10 drops of distilled water, followed by staining with a drop of 1 wt% uranyl acetate. The grid was subsequently dried and visualized using an FEI 200 kV Sphera microscope.

MΦ-NPs were purified from free vesicles, membrane fragments, and unbound proteins by centrifugation at 16,000 × g. Macrophage cell lysates, membrane vesicles, and MΦ-NPs were mixed with lithium dodecyl sulfate (LDS) loading buffer to the same total protein concentration of 1 mg/mL as determined with a Pierce BCA assay (Life Technologies). Electrophoresis was carried out with NuPAGE Novex 4–12% Bis-Tris 10-well minigels in Mops running buffer with an XCell SureLock Electrophoresis System (Invitrogen). Western blot analysis was performed by using primary antibodies including rat anti-mouse CD14, rat anti-mouse CD126, rat anti-mouse CD130, rat anti-mouse CD284, Armenian hamster anti-mouse CD120a, Armenian hamster anti-mouse CD120b, and Armenian hamster anti-mouse CD119 (BioLegend). Corresponding IgG-horseradish peroxidase (HRP) conjugates were used for the secondary staining. Films were developed with ECL Western blotting substrate (Pierce) on a Mini-Medical/90 Developer (ImageWorks).

To study whether LPS binding with MΦ-NPs was dependent on LBP, CD14, or TLR4, the mixture of MΦ-NPs (1 mg/mL) and FITC-LPS (from E. coli O111:B4, 125 ng/mL; Sigma) in 1× PBS was added with FBS (10% as the source of LBP), anti-CD14 (10 μg/mL; BioLegend), or anti-TLR4 (10 μg/mL; Invivogen), respectively. The solution was incubated at 37 °C for 30 min. Following the incubation, MΦ-NPs were spun down with ultracentrifugation (16,000 × g). The fluorescence intensity from FITC-LPS remaining in the supernatant was measured. The fluorescence intensity from a FITC-LPS solution of 125 ng/mL served as 100%. The mixtures without adding FBS or antibodies were used as the controls. An equivalent amount of MΦ ghost (protein mass) was used as a control to assess the loss of membrane function during coating. The mixture added with nonspecific IgG from human serum was also included as a negative control to exclude the effect of the nonbinding domains of the antibody that may contribute to LPS inhibition. All experiments were performed in triplicate.

To quantify LPS removal with MΦ-NPs, MΦ-NPs (0.4 mg, 4 mg/mL) were mixed with LPS from E. coli K12 (Invivogen) with varying amount of 5, 10, 25, and 50 ng (50, 100, 250, and 500 ng/mL), respectively, in 1× PBS containing 10% FBS. In a parallel experiment, the removal was studied by fixing LPS amount at 50 ng (250 ng/mL) but varying the amount of MΦ-NPs at 0.1, 0.2, 0.3, and 0.4 mg (0.5, 1, 1.5, and 2 mg/mL), respectively. In both cases, the mixtures were incubated for 30 min and then spun down at 16,000 × g for 15 min to pellet the nanoparticles. The free LPS content in the supernatant was quantified by using limulus amebocyte lysate (LAL) assay (Thermo Fisher Scientific) per manufacturer’s instructions. All experiments were performed in triplicate.

To determine MΦ-NP binding with cytokines, including IL-6, TNF-α, and IFN-γ, 100 µL of MΦ-NP samples (1 and 4 mg/mL) mixed with IL-6 (2,000 pg/mL), TNF-α (370 pg/mL), or IFN-γ (880 pg/mL) in PBS containing 10% FBS were incubated at 37 °C for 30 min. Following the incubation, the samples were centrifuged at 16,000 × g for 15 min to pellet the nanoparticles. Cytokine concentrations in the supernatant were quantified by using ELISA (BioLegend). All experiments were performed in triplicate.

Murine TLR4 reporter cells (HEK-Blue mTLR4 cells, Invivogen) were first used to determine LPS neutralization by MΦ-NPs. Cells were cultured in DMEM supplemented with 10% FBS, 1% pen–strep, 100 μg/mL normocin, 2 mM l-glutamine, and 1× HEK-Blue selection (Invivogen). In the study, 2.5 × 10⁴ cells were seeded in each well of a 96-well plate with 160 μL HEK-Blue detection medium, followed by adding 20 μL of 100 ng/mL LPS in PBS. Then 20 μL of nanoparticle solution of MΦ-NPs, RBC-NPs, or PEG-NPs (all at a concentration of 10 mg/mL), was added into each well. Control wells were added with 20 μL PBS. Cells without any treatment served as the background. The mixture was incubated for 12 h. SEAP was quantified by measuring the absorbance at 630 nm with an Infinite M200 multiplate reader (Tecan). All experiments were performed in triplicate.

Production of iNO was also used to evaluate LPS neutralization with MΦ-NPs. Briefly, 2 × 10⁴ J774 cells were seeded in each well of a 96-well plate. The cells were incubated with 10 μM of 2′, 7′-dichlorofluorescin-diacetate (DCFH-DA) (Sigma) in culture medium for 1 h and then washed three times with the culture medium. Then the wells were added with 180 μL of medium containing 10 ng/mL of LPS. Then 20 μL of nanoparticle solution of MΦ-NPs, RBC-NPs, or PEG-NPs (all at a concentration of 10 mg/mL), was added into each well. Twenty microliters of PBS was added to control wells. Cells without any treatment served as the background. The plate was incubated at 37 °C for 5 h. The production of iNO was quantified by measuring the fluorescence intensity at 520 nm using an excitation wavelength of 485 nm (Infinite M200 multiplate reader, Tecan). All experiments were performed in triplicate.

LPS neutralization with MΦ-NPs was further evaluated by examining E-selectin expression on HUVECs. Specifically, HUVECs were cultured to confluence in a 96-well plate. Then 200 μL of LPS (250 ng/mL) mixed with MΦ-NPs, RBC-NPs, or PEG-NPs (4 mg/mL) in culture medium was added to the cells and the plate was incubated at 37 °C. Cells added with LPS and PBS were used as controls. Three wells were used per sample. After 1, 2, 3, and 4 h of incubation at 37 °C, medium was removed and cells were washed with PBS. Then the cells were fixed with 4% paraformaldehyde (Sigma) at room temperature for 15 min. Following the fixation, cells were washed twice with PBS and blocked with 1% BSA (Sigma). Subsequently, the reagent was decanted and 50 μL of primary antibody (mouse anti-human E-selectin, 1:10 dilution in 1% BSA; BioLegend) was added to each well and incubated at 37 °C for 45 min. Wells were then rinsed three times with 1× PBS before the addition of 50 μL of secondary antibody (HRP-conjugated anti-mouse IgG, 1:10 dilution in 1% BSA; BioLegend) followed by an incubation for 45 min at 37 °C. After this, wells were again rinsed three times with 1× PBS and after the final rinse, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added to each well. The plate was incubated at 37 °C followed by measuring the absorbance at 450 nm.

To visually examine E-selectin expression, cells following the same treatment as the above experiment were incubated at 37 °C for 4 h and rinsed twice with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.2% Triton X-100 (Sigma) in buffer for 10 min, and then incubated with 1% BSA in PBS for 30 min. Cells were then stained with mouse anti-human E-selectin for 1 h, washed three times with 1× PBS, and then incubated with anti-mouse IgG Alexa 488 conjugates in 1% BSA in PBS for 1 h. Cell nuclei were stained with DAPI (1 mg/mL stock solution; Thermo Fisher Scientific). Fluorescence images were taken with an EVOS fluorescence microscope (Thermo Fisher Scientific).

All animal studies were approved under the guidelines of the University of California San Diego (UCSD) Institutional Animal Care and Use Committee. Mice were housed in an animal facility at UCSD under federal, state, local, and NIH guidelines for animal care. In the study, no inflammation was observed at the sites of injection.

The experiments were performed on 6-wk-old male ICR mice (Harlan Laboratories). To determine the circulation half-life, 150 μL of DiD-labeled MΦ-NPs (3 mg/mL) was injected i.v. through the tail vein. At 1, 15, and 30 min, and 1, 2, 4, 8, 24, 48, and 72 h postinjection, one drop of blood (∼30 μL) was collected from each mouse via submandibular puncture with heparin-coated tubes. Then 20 μL of blood was mixed with 180 μL PBS in a 96-well plate for fluorescence measurement. Pharmacokinetic parameters were calculated to fit a two-compartment model. For biodistribution study, 150 μL of DiD-labeled MΦ-NPs (3 mg/mL) was injected i.v. through the tail vein. At 24, 48, and 72 h postinjection, organs including the liver, kidneys, spleen, brain, lungs, heart, and blood were collected from six randomly selected mice. The collected organs were weighed and then homogenized in PBS for fluorescence measurement. All fluorescence measurements were carried out with an Infinite M200 multiplate reader (Tecan).

The efficacy of MΦ-NPs in neutralizing LPS was first evaluated with a mouse endotoxemia model with 6-wk-old male BALB/c mice (Harlan). To evaluate the efficacy through cytokine production, mice were injected with 5 μg/kg LPS through the tail vein. After 15 min, MΦ-NPs, RBC-NPs, or PEG-NPs were injected at 200 mg/kg. Following the injections, blood samples (<30 μL) were collected at predetermined time points via submandibular puncture. Untreated mice and mice injected with LPS alone were used as controls. Cytokines, including IL-6 and TNF-α, in the plasma were quantified by ELISA as described above. In each group, six mice were used. To evaluate efficacy through survival, mice were first sensitized with d-galactosamine hydrochloride (Sigma-Aldrich) via i.p. injection at a dosage of 800 mg/kg. After 30 min of sensitization, LPS and nanoparticles were injected intravenously. Ten mice were used in each group.

LPS neutralization efficacy was also evaluated with a mouse bacteremia model. Specifically, 6-wk-old female C57BL/6 (The Jackson Laboratory) mice were injected intraperitoneally with 1 × 10⁷ cfu of uropathogenic E. coli (UPEC) CFT073 suspended in 100 μL of 1× PBS. After 30 min, mice were randomly placed into two groups (n = 10), and each mouse was injected with 500 μL of MΦ-NPs at a concentration of 10 mg/mL in 10% sucrose solution intraperitoneally. Mice were killed 4 h after the injection. Blood and organs were collected and homogenized with a Mini Beadbeater-16 (BioSpec) in 1 mL of PBS. Proinflammatory cytokines in the blood, including IL-6, TNF-α, and IFN-γ, were quantified by a cytometric bead array per manufacturer’s instructions (BD Biosciences). For bacterial enumeration, homogenized samples were serially diluted with PBS (from 10- to 10⁷-fold) and plated onto agar plates. After 24 h of culture, bacterial colonies were counted. To evaluate efficacy through survival, the same experimental procedure was carried out and survival was monitored over a period of 60 h (n = 10).