Nanomedicine platform for targeting activated neutrophils and neutrophil-platelet complexes using an α1-antitrypsin-derived peptide motif

Abstract

Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α₁-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet-neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.

Fig. 1 |. Design and characterization of NPs for selective targeting of activated neutrophils.

a, Conceptual framework of targeting NE as a strategy to differentiate activated from resting neutrophils. b, Physiologically, NE recognizes and binds to a 20 amino acid sequence within the reactive centre loop (RCL; magnified in c) of AAT. c, A NEBP was designed from the reactive centre loop of AAT. A cysteine at the P7 position was added for subsequent peptide conjugation to NP lipids. Modelling of docking complex with highlighted reactive centre loop and NEBP was performed using native AAT (Protein Data Bank code 1QLP)^,. d, Solution-phase hydrolysis of NEBP by NE. High-performance liquid chromatography chromatogram showing pure NEBP (black dashed line) and combination of NEBP with mouse (mNE, red line) or human (hNE, blue dashed line) NE reaction mixture. Data are representative of n = 3 individual experiments run in triplicate. e, Surface plasmon resonance of NEBP binding to immobilized mNE. Increasing concentrations of NEBP were injected over a mNE-immobilized CM5 chip. f, To exclusively target activated neutrophils, Ly6G–Fab (blue) was conjugated to DSPE-PEG-carboxy-NHS; NEBP (purple) was conjugated to DSPE-PEG-Mal. To simultaneously target activated platelets and neutrophils, NEBP and PBP were conjugated to DSPE-PEG-Mal to derive PNT-NPs. DSPE-PEG-NHS, N-hydroxysuccinimide terminated polyethylene glycol conjugated distearyl phosphoethanolamine; Mal, maleimide. g, NP size was determined by dynamic light scattering; mean = 200 nm. h, Molecular resolution of NPs using cryogenic transmission electron microscopy; n = 3 individual experiments. Scale, 0.1 μm. i, Kinetic real-time binding of NT-NPs (NT; 0–5 μM) or U-NPs (U; 5 μM) to immobilized hNE; n = 3 individual Langmuir binding models run in triplicate.

Fig. 2 |. NT-NPs selectively bind to activated neutrophils in vitro and in vivo.

a, Schematic representation of targeting ligands on NT-NPs and their binding targets on activated neutrophils. b, Neutrophil viability in the presence of rising concentrations of NT-NPs or U-NPs. Mean ± s.e.m., n = 3 individual experiments run in triplicate, *P = 0.0009, **P = 0.0001, one-way analysis of variance (ANOVA) with Bonferroni correction. NS, not significant. c, Confocal microscopy of static murine neutrophils incubated with NT-NPs or U-NPs, in the absence or presence of fMLP. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) and NE (green). NPs were labelled with RhB (red). Scale, 10 μm; n = 3 independent experiments. d, NP binding on neutrophils shown in c was quantitated by measuring the residual RhB fluorescence on cells following several washing steps; n = 3 independent experiments. Symbols denote high-power fields captured per condition (n = 16). Mean ± s.e.m., *P < 0.0001, one-way ANOVA with Bonferroni correction. e, Wild-type mice (n = 3 per time point) were i.v. administered U-NPs or NT-NPs, and residual NP concentration (conc.) was determined by a RhB fluorescence assay. Mean ± s.e.m., n = 3 independent experiments. f, Mice were i.v. injected with saline or 1 mg kg⁻¹ body weight Escherichia coli O111:B4 LPS. Three hours after, anti-Gr-1 antibody (green) mixed with RhB-labelled NPs (U-NPs or NT-NPs) were i.v. injected and allowed to circulate for 40 min before retinas were excised. Retinal vasculature was visualized with Concanavalin A conjugated to fluorescein isothiocyanate. Square and dotted lines in the middle panels demarcate the magnified retinal regions in the right panels. LPS + NT-NP, n = 6 mice; LPS + U-NP, n = 4 mice; no LPS + NT-NP, n = 5 mice; no LPS + U-NP, n = 6 mice. Scale, 50 μm. g, Representative two-dimensional intensity histograms from retinal images. The y axis represents above-zero red pixel intensity (RhB-labelled NPs). The x axis indicates above-zero green pixel intensity (adhered neutrophils). h, Colocalization analysis of retinal images shown in f. Thresholded Manders’s correlation coefficient values (tM1 = red overlap with green; tM2 = green overlap with red) are shown among groups. Costes probability value ≥ 95% in LPS + NT-NP; Costes probability value = 0% in all other groups. LPS + NT-NP, n = 6 mice; LPS + U-NP, n = 4 mice; no LPS + NT-NP, n = 5 mice; no LPS + U-NP, n = 6 mice. Mean ± s.e.m., *P < 0.000001, **P = 0.000002, ***P = 0.000003, one-way ANOVA with Bonferroni correction.

Fig. 3 |. NT-NPs selectively bind to activated neutrophils and NETs.

a, Freshly isolated human neutrophils were treated with media (inactive) or activated with 100 nM PMA in the absence or presence of 3 × 10⁹ ml⁻¹ U-NPs or (neutrophil-targeted) NEBP-NPs for 2 h. Cells were subsequently stained with DAPI (blue, nuclei) and SYTOX Green (1 μM, green, extracellular DNA); fixed with 4% formalin for 4 min; and washed in PBS but not permeabilized. NPs are inherently red due to RhB labelling. Fluorescent images were obtained using a Leica TCS SP8 confocal microscope at ×10 magnification. Images are representative of n = 6 individual experiments. Scale, 250 μm. b, Quantitation of NP binding to inactive or PMA-activated neutrophils. Mean ± s.e.m., n = 6 individual experiments, *P = 0.001, **P = 0.0022, one-way ANOVA with Bonferroni correction. RFU, relative fluorescence units. c, Purified human neutrophils were treated with media or neutrophil agonists TNF-α (10 ng ml⁻¹), LPS (5 μg ml⁻¹) and fMLP (1 μM). Cells were coincubated with 3 × 10⁹ ml⁻¹ RhB-labelled U-NPs or NEBP-NPs for 1 hour at 37 °C. After several washing and centrifugation steps, cells were fixed and stained with CD66b Alexa Fluor 700 (AF700) and NE Cy5 antibodies. To measure neutrophil–NP binding, cells were first sorted by CD66b positivity, followed by doublet discrimination and gating for hNE. The RhB fluorescence of CD66b and NE double positive single cells was measured. Mean ± s.e.m., n = 3 individual experiments, *P = 0.0015, **P = 0.01, ***P = 0.0046, one-way ANOVA with Bonferroni correction. MFI, mean fluorescence intensity.

Fig. 4 |. Quantitation of NP trafficking by neutrophils.

a, Human neutrophils (1 × 10⁶ ml⁻¹) were resuspended in serum-free Dulbecco’s modified eagle medium/nutrient mixture F-12 (DMEM/F-12) containing 2 mM CaCl₂ and 2 mM MgCl₂, plated in 35 mm glass-bottom dishes and allowed to adhere for 15 minutes. Where indicated, cells were activated with 1 μM fMLP in the absence or presence of 3 × 10⁹ ml⁻¹ U-NPs or (neutrophil-targeted) NEBP-NPs at 37 °C for 2 h. Cells were maintained alive and were not fixed or permeabilized. On completion of the incubation time, cell components were stained and imaged. Scale, 20 μm; n = 3 individual experiments. b,c, For colocalization of NPs with cellular components, thresholded Manders’s correlation coefficient values (tM1) are shown among groups for cell membrane–NP interactions (b) and lysosome–NP interations (c). Costes probability value ≥ 95% in fMLP + NEBP-NP; Costes probability value = 0% in all other groups; n = 3 individual plates scanned; area, 200 μm × 200 μm. For b, mean ± s.e.m., *P = 0.001, **P < 0.001, one-way ANOVA with Bonferroni correction. For c, mean ± s.e.m., *P = 0.018, **P = 0.00092, one-way ANOVA with Bonferroni correction.

Fig. 5 |. Heteromultivalent NPs selectively bind to activated platelet–neutrophil complexes in vitro.

a, Schematic illustration of PNT-NPs. The combined decoration of NPs with a PBP (targeting P-selectin) and NEBP (targeting NE) enables PNT-NPs to bind simultaneously and selectively to activated platelet–neutrophil complexes. b, Freshly isolated human neutrophils and platelets were activated with 1 μM fMLP and 20 nM thrombin prior to being coincubated with RhB-labelled (red) U-NPs or PNT-NPs. Platelets were stained with calcein (green), and nuclei corresponding to activated neutrophils were counterstained with DAPI (blue). Representative images show that PNT-NPs (third row panels) bound to activated platelet–neutrophil aggregates, but undecorated U-NPs (second row panels) did not. Images are representative of n = 10–11 individual experiments run in triplicate. Scale, 100 μm. c, NP retention on activated platelet–neutrophil complexes was measured by quantitating residual RhB fluorescence. U-NP, n = 10 individual samples; PNT-NP, n = 11 individual samples; all run in triplicate. Mean ± s.e.m., **P < 0.01 by Student’s t-test. d, Human neutrophils were either left untreated or activated with fMLP and incubated with U-NPs or PNT-NPs. Representative flow cytograms (left upper and left lower panels) of NP retention on inactive (IN) or activated (AN) neutrophils; n = 5–6 individual experiments run in triplicate. Human platelets were left untreated or activated with 20 nM thrombin, in the absence or presence of U-NPs or PNT-NPs. Representative flow cytograms (right upper and right lower panels) of n = 5–6 individual experiments run in triplicate. AP, activated platelets. e, Cell–NP binding to IN, AN, IP or AP was quantitated by measuring RhB fluorescence intensity; n = 5–6 individual experiments run in triplicate. Data are presented as mean ± s.e.m., **P < 0.0017, ***P = 0.0008, ****P < 0.0001, ordinary two-way ANOVA.

Fig. 6 |. PNT-NPs bind to thromboinflammatory sites and effectively deliver therapeutic cargo to reduce thrombus size.

a, Schematic of microfluidic set-up. Image of 1 μm polystyrene particle streak lines demonstrating vorticle flows recirculating in valve pockets. b, Maximum projections of confocal stack after 30 min of blood flow. NPs are in red, human platelets in green, neutrophils in blue and fibrin(ogen) in cyan. RhB fluorescent intensity within microchannels is shown at the right. Mean ± s.e.m., n = 3 individual experiments run in triplicate, *P = 0.006, two-sided Student’s t-test. c, Neutrophil viability in the presence of free HCQ or HCQ_PNT-NPs. Mean ± s.e.m., n = 3 individual experiments run in triplicate, *P = 0.0079 compared to initial (100%) viability, ordinary two-way ANOVA. d, NE activity of human neutrophils pretreated with AAT, free HCQ or HCQ_PNT-NPs for 2 h prior to stimulation with fMLP. Mean ± s.e.m., n = 3 individual experiments run in triplicate, **P < 0.0001, one-way ANOVA with Bonferroni correction. e, Flow cytometry analysis of NET formation in human neutrophils pretreated with free HCQ or HCQ_PNT-NP prior to stimulation with fMLP. UT denotes untreated neutrophils. Data are presented as relative expression of citrullinated histone H3 (H3-C). Mean ± s.e.m., n = 4 experiments, *P < 0.0001, one-way ANOVA with Bonferroni correction. f, Kaplan–Meier survival analysis in mice i.v treated with saline (control), free HCQ or HCQ_PNT-NPs. g, Schematic representation of deep venous thrombosis model. h, Prior to IVC ligation, mice were i.v. injected with saline (no treatment, n = 5); empty U-NPs (n = 5); empty PNT-NPs (n = 3); HCQ_U-NPs (n = 10); HCQ_PNT-NPs (n = 16); HCQ_NEBP-NPs (n = 13); or HCQ_PBP-NPs (n = 13). Thrombi were harvested at 24 h. Each symbol represents an individual mouse. Mean ± s.e.m., *P = 0.0065, **P = 0.01, ***P = 0.004, one-way ANOVA with Bonferroni correction. Gross images of harvested thrombi from control (top panel) and mice i.v. treated with HCQ_PNT-NPs (bottom panel).