Materials

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was purchased from Avanti Polar Lipids Pharmaceutical Co., Ltd (Shanghai, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)] (DSPE-PEG-Mal, 3400 Da) was purchased from Xi’an Ruixi Biotech Co., Ltd (Xian, China). 1,19-Dioctadecyl-3,3,39,39-tetramethylindodicarbocyanine perchlorate (DiD) was supplied by Sigma-Aldrich (Carlsbad, CA, USA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Invitrogen (Carlsbad, CA, USA). 4′,6-Diamidino-2-phenylindole (DAPI, C1002) was obtained from Beyotime Biotechnology Co., Ltd (Nantong, China). Accustain Trichrome Stain (Masson) kit and Percoll were obtained from Sigma-Aldrich. Matrigel was obtained from Corning (Corning, NY, USA). Recombinant human TNFα (ab259410) and ELISA kits for TNFα (ab208348), IL1β (ab197742), IL6 (ab222503), and CXCL2 (ab184862) were purchased from Abcam (Cambridge, MA, USA). BCA Protein Assay Kit (P0010) was purchased from Beyotime Biotechnology. TRIzol reagent and RT-PCR Kit were obtained from Invitrogen. Anti-CD34 antibody (sc-53511) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-TNFα antibody (ab183218), anti-IL1R antibody (ab106278), anti-IL6R antibody (ab222101), anti-LFA1 antibody (ab13219), and anti-CXCR2 antibody (ab225732) were purchased from Abcam. Anti-β-actin antibody (AA128), HRP-labeled goat/anti-mouse IgG (H + L, A0216) and HRP-labeled goat/anti-rabbit IgG (H + L, A0208) were obtained from Beyotime Biotechnology. Anti-PH3 antibody (ab321073), anti-Ki67 antibody (ab16667), anti-CD31 antibody (ab222783), anti-F4/80 antibody (ab6640), anti-CD206 antibody (ab125028), and anti-cardiac troponin T antibody (ab8295) were all obtained from Abcam.

Animal

Male C57BL/6 mice (aged 8–12 weeks, 20–25 g in weight) and male ICR mice (aged 8–12 weeks, 30–35 g in weight) were purchased from Shanghai SLAC Laboratory Animal Ltd. All animal experimental procedures were approved by the Animal Care and Use Committee of Zhongshan Hospital, Shanghai, People’s Republic of China and were in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council (US) Institute for Laboratory Animal Research.

Neutrophil collection

Fresh human peripheral blood neutrophils were obtained from Zhongshan Hospital. Neutrophil isolation was performed according to previous studies with some modifications [30]. Briefly, neutrophils were purified by density gradient centrifugation. Leukocyte-rich plasma was placed over a three-layer Percoll gradient of 78%, 69%, and 52% and centrifuged at 1000×g for 30 min. The interface containing neutrophils was between the 69% and 78% gradient layers and the upper part of the 78% layer. The isolated neutrophils were rinsed with PBS, suspended in serum-free RPMI media, and then stimulated with recombinant human TNFα (50 ng/mL) for 2 h at 37 °C. Next, the stimulated cells were resuspended in serum-free RPMI and cryopreservation medium and stored at − 80 °C.

Neutrophil membrane derivation

First, neutrophils were suspended in lysing buffer containing 225 mM d-mannitol, 75 mM sucrose, 30 mM Tris–HCl (pH 7.5), 0.2 mM EGTA, and a protease and phosphatase inhibitor cocktail. Then, the cells were disrupted using a Dounce homogenizer with a tight-fitting pestle (20 passes). Next, the homogenized solution was centrifuged at 20,000×g for 30 min at 4 °C. Then, the supernatant was collected and centrifuged at 100,000×g for 40 min at 4 °C. Subsequently, membranes were collected as sedimentation at the bottom of the centrifuge tube. Membrane content was quantified using a BCA kit. Approximately 100–200 million neutrophils generated 1 mg of membrane protein. Neutrophil membrane was suspended in 0.2 mM EDTA to a protein concentration of 2 mg/mL and stored at − 80 °C for subsequent studies.

Synthesis of nanoparticles

Neu-LPs were synthesized via a lipid film rehydration process [14]. Briefly, 9 mg of DOPC and 1 mg of DSPE-PEG-Mal were dissolved in 10 mL of chloroform and dried in a flask by rotary evaporation for 1 h. Then, the thin lipid film was hydrated with 10 mL of distilled water at 37 °C for 15 min and sonicated at a frequency of 52 kHz and a power of 100 W for 30 min. For membrane coating, the neutrophil membrane was mixed with the liposome cores at a weight ratio of 30:1 (polymer-to-membrane protein). The mixture was then sonicated with a bath sonicator for 10 min. The particles were then extruded through membranes with pore sizes of 400, 200, and 100 nm using an extruder. Fluorescent dye-labeled Neu-LPs were prepared by the same method as Neu-LPs except 25 μg of DiD (Neu-LPs-DiD) were added to the lipid solution. Then free unlabeled fluorescence DiD was separated by centrifugation at 100,000g for 30 min and determined by using a microplate reader (SpectraMax® M5, Molecular Devices). The fluorescence label efficiency was calculated as follows:

$$mathrm{Label , efficiency }=frac{mathrm{Total ,amount }-mathrm{ The ,amount ,in ,supernatant}}{mathrm{Total ,amount }}times 100mathrm{%}$$

The DiD label efficiency of Neu-LP was 91.2 ± 1.4%.

Nanoparticle characterization

The hydrodynamic diameters and zeta potentials of Neu-LPs were measured by DLS (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK). The morphology of Neu-LPs was visualized by TEM (H-600, Hitachi, Tokyo, Japan). To evaluate the stability of Neu-LP, LP or Neu-LP were suspended in water at a concentration of 1 mg/mL. At set timepoints over the course of 7 days, the sizes of the samples were measured by DLS to test for aggregation.

Membrane colocalization assay

A membrane colocalization study was performed as previously described with some modification [14]. NMVs were labeled with DiI (excitation/emission = 549/565 nm), and LPs were labeled with DiD (excitation/emission = 644/665 nm). Free dyes were removed by centrifugation at 100,000g for 30 min and the fluorescence label efficiency was calculated as mentioned above. The DiI label efficiency of NMV was 92.5 ± 2.2% and DiD label efficiency of LP was 93.2 ± 1.8%. Then, samples were prepared by extrusion or simple pipetting and thereafter were visualized under a confocal microscope (Leica Microsystems, Wetzlar, Germany).

Cell culture

HUVECs, cardiomyocytes (H9C2), and macrophages (RAW 264.7) were purchased from American Type Culture Collection. HUVECs were cultured in human endothelial cell growth medium (ECM), while H9C2 cells and RAW 264.7 cells were cultured in high glucose culture medium. Cells were maintained at 37 °C in a 5% CO2 environment. All media were supplemented with 10% (v/v) FBS as well as penicillin and streptomycin (100 U/mL).

Inflammatory factors neutralization assay

Proinflammatory factors (TNFα, I1β, IL-6, and CXCL2) were mixed with Neu-LPs to final concentrations of 0–4 mg/mL. The mixtures were incubated for 6 h at 37 °C and then centrifuged at 15,000×g for 20 min to remove the Neu-LPs. The cytokine concentrations in the supernatant were quantified by ELISA kits. Linear fitting of the proinflammatory factor curves were performed in GraphPad Prism 7.

In vitro myocardial inflammation model

Macrophages (5 × 104 cells) were seeded in the outer edges of a glass-bottom dish, while H9C2 cells (1 × 105 cells) were seeded in the center region of the dish. Then, the culture chamber was continuously flushed with (5% CO2 and 95% N2) for 6 h at 37 °C to obtain an anoxic solution. The co-culture was then replaced in a CO2 incubator (5% CO2, 95% air, 37 °C) for reoxygenation. After this process, the levels of inflammatory cytokines in the hypoxia-reoxygenation conditioned medium (H/R-CM) were significantly increased [31]. The co-cultured cells were treated with supplemented media mixed with H/R-CM in a 1:1 ratio and incubated with PBS, LPs, or Neu-LPs for another 6 h. Changes in inflammatory cytokine (TNFα, IL1β, IL6, and CXCL2) levels were detected by ELISA assays.

The TUNEL assay

H9C2 cells were seeded in 12-well plates (5 × 104 cells per well) at 37 °C to permit cell adhesion and reach 80–90% confluence. The culture medium was replaced with supplemented RPMI media mixed with H/R-CM in a 1:1 ratio and the cells were incubated with PBS, LPs, or Neu-LPs. After 6 h of treatment, cardiomyocyte apoptosis rates were measured using a TUNEL Apoptosis Assay Kit and the cells were observed using an inverted fluorescence microscope (ZEISS Group)..

Real-time PCR analysis

Changes in gene expression levels were determined by real-time PCR (RT-PCR). RNA was isolated from mouse cardiomyocytes, macrophages, endothelial cells, or ischemic myocardium tissue by TRIzol and reverse-transcribed into cDNA using the First-Strand Synthesis System for RT-PCR Kit. SYBR Green-based quantitative RT-PCR was performed using the Mx3000 Multiplex Quantitative PCR System. The relative expression was calculated using the comparative CT method (2−ΔΔCt). Triplicate samples were used for each experimental condition to determine relative expression levels. Primer sequences used are listed in Table 1.

Table 1 Primer sequences used for PCR analysis

Neutrophil chemotaxis assay

Neutrophils (1 × 104 cells/well) were seeded in the upper chamber of a transwell system, while supplemented media mixed with H/R-CM in a 1:1 ratio and PBS, LPs, or Neu-LPs were added to the lower chamber. After incubation for 6 h, the chambers were removed. The cells were fixed with 4% paraformaldehyde and stained with 0.25% crystal violet for 10 min. After three rinses with PBS and air drying, the chambers were inverted on a glass slide and the number of chemotaxis-migrated neutrophils was counted under a microscope.

Flow cytometric analysis

Cell suspensions of macrophages were stained with FITC-conjugated anti-F4/80 antibodies and PE-conjugated anti-CD86 antibodies (1:100 dilution) for 40 min at 4 °C. Flow cytometric analysis was performed on a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (TreeStar, Inc., San Carlos, CA).

Cell proliferation assay

EdU assays were performed to investigate cell proliferation. Briefly, HUVECs were seeded into the lower chamber of a transwell system, while RAW 264.7 cells were added to the upper chamber in supplemented media mixed with H/R-CM in a 1:1 ratio and incubated with PBS, LPs, or Neu-LPs. After 6 h of treatment, HUVECs were stained with anti-CD31 antibody and EdU-488 kit according to the manufacturer’s protocol and then observed under an inverted fluorescence microscope. The number of positive cells was analyzed by ImageJ (US National Institutes of Health).

Endothelial tube formation assay

An endothelial tube formation assay was performed as previously described with some modification [32]. Matrigel (200 μL) was first added to each well of a 48-well plate to evenly cover the bottom, and then the plate was placed in an incubator at 37 °C for 2 h until the Matrigel solidified. HUVECs (2 × 104 cells/well) were seeded in the prepared 48-well plates and incubated at 37 °C for 4 h. Tube formation was observed under an inverted fluorescence microscope and capillary length was analyzed using ImageJ.

Assay for chemotactic migration across a vascular barrier

An in vitro vascular intimal barrier model was constructed with HUVECs using a transwell cell culture system. Briefly, HUVECs (1 × 105 cells/well) were seeded in the upper chamber and H9C2 cells (1 × 105 cells/well) were added to the bottom chamber and cultured in medium containing 10% (v/v) FBS. After 6 h of hypoxia, DiD-labeled LPs (0.5 mg/mL, 200 μL) or DiD-labeled Neu-LPs (0.5 mg/mL, 200 μL) were added to the upper chamber and incubated for 3 h. The fluorescence intensities of Neu-LPs in the supernatant, intracellular, and filtered compartments were determined using a microplate reader (SpectraMax® M5, Molecular Devices).

Mouse model of MI/R

Male C57 mice were subjected to transient ligation of the left anterior descending coronary artery for 60 min followed by reperfusion. Successful acute MI/R injury was confirmed by visual inspection of the left ventricle color and changes in the electrocardiogram. One day post ischemia–reperfusion, animals were randomized into three treatment groups (n = 6 mice per group): intravenous injection of: (1) 200 μL PBS, (2) LPs (0.5 mg/mL, 200 μL), or (3) Neu-LPs (0.5 mg/mL, 200 μL).

ELISA assay

To investigate the in vivo neutralizing effects of Neu-LPs, the expression levels of inflammatory factors (TNFα, IL1β, and IL6) in ischemic hearts were measured by ELISA assays according to the manufacturer’s instructions.

Assessment of Neu-LP targeting and biodistribution

The biodistribution of Neu-LPs was assessed according to a previous article [33]. One day after reperfusion, PBS, LPs, or Neu-LPs (0.5 mg/mL, 200 μL) were intravenously injected into MI/R model mice. At predetermined intervals (3 h, 1 day, and 3 days), the mice were sacrificed. Their hearts and other organs were harvested and imaged using an in vivo imaging system (IVIS, PerkinElmer). The fluorescence intensity of DiD was analyzed using Living Image Software. After imaging, all hearts were cryosectioned (5 μm) and imaged using a confocal microscope.

Cardiac function assessment

The left ventricular function of MI/R model mice with various treatments (PBS, LPs, or Neu-LPs) were analyzed by echocardiography (Vevo 770, Visual Sonics, Toronto, ON, Canada) 4 h post reperfusion and 4 weeks after treatment. The model mice were anesthetized with low-dose isoflurane for echocardiographic examination. Two-dimensional targeted M-mode traces were obtained at the level of papillary muscle. LVEF, FS, LVEDV, and LVESV were measured in at least three consecutive cardiac cycles.

Histochemical and immunohistochemical assessments

At various timepoints (3, 7, 14, and 28 d) after systemic administration of PBS, LPs, or Neu-LPs, heart tissues were harvested and cut into 5-μm paraffin-embedded sections or cryosections. Fibrous heart tissue was identified by staining sections with Masson’s Trichrome reagent according to the manufacturer’s instructions. Fibrosis was imaged under an inverted fluorescence microscope. Apoptotic cells were identified by staining sections with TUNEL staining kits. Cardiomyocytes were stained with mouse anti-cardiac troponin T primary antibodies. Transcriptional regulation was identified by staining sections with rabbit anti-PH3 primary antibody. Cell proliferation was identified by staining sections with rabbit anti-Ki67 primary antibody. Vasculogenesis was identified by staining sections with rabbit anti-CD31 primary antibodies. To examine macrophage polarization, mouse anti-F4/80 and rabbit anti-CD206 primary antibodies were used. Neutrophils were identified by rabbit anti-MPO primary antibodies. FITC (488 nm) or Texas-Red (594 nm) secondary antibodies were conjoined with the related primary antibodies. DAPI was utilized to visualize cell nuclei in the sections. Images were taken with a confocal microscope and quantified using ImageJ. Flow cytometry was also employed to detect the macrophage polarization. Experimental procedures are the same as previously described and PE-anti-779 F4/80 (565410), PE-Cy7-anti-CD86 (25-0862-82), APC-anti-CD206 (17-2062-82) were used to perform Flow cytometry.

Safety evaluation

To evaluate the safety of Neu-LPs, healthy ICR mice aged 6 weeks were intravenously injected with PBS (200 μL), LPs (0.5 mg/mL, 200 μL), or Neu-LPs (0.5 mg/mL, 200 μL) every other day for one week (n = 6). Blood samples were collected from the mice and biochemical indexes tests were performed. ELISA were adopted to detect the concentration of IgM in MI/R mice after PBS, LPs or Neu-LPs treatment according to the manufacturer’s instruction. For histological analyses, the major organs (heart, liver, spleen, lung, and kidney) were embedded in paraffin and stained with H&E.

Statistical analysis

All results are expressed as mean ± standard deviation (SD). Comparisons between two groups were performed with Student’s t-test. One-way analysis of variance (ANOVA) was employed for comparisons among more than two groups. P-values less than 0.05 were considered statistically significant.

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