CD177-mediated nanoparticle targeting of human and mouse neutrophils

<div><p>Neutrophils are the most abundant white blood cells, with a vital role in innate immune defense against bacterial and fungal pathogens. Although mostly associated with pathological processes directly related to immune defense, they can also play a detrimental role in inflammatory conditions and have been found to have a pro-metastatic role in the spread of cancer cells. Here, we explore ways to temporarily suppress these detrimental activities. We first examined the possibility of using siRNA and antisense oligonucleotides (ASOs) for transient knockdown of the human and mouse C5a receptor, an important chemoattractant receptor involved in neutrophil-mediated injury that is associated with myocardial infarction, sepsis, and neurodegenerative diseases. We found that siRNAs and ASOs transfected into cultured cell lines can eliminate 70–90% of C5a receptor mRNA and protein within 72 h of administration, a clinically relevant time frame after a cardiovascular event. Targeted drug delivery to specific cells or tissues of interest in a mammalian host, however, remains a major challenge. Here, using phage display technology, we have identified peptides that bind specifically to CD177, a neutrophil-specific surface molecule. We have attached these peptides to fluorescent, lipid-based nanoparticles and confirmed targeting and delivery to cultured cells ectopically presenting either human or mouse CD177. In addition, we have shown peptide-nanoparticle binding specifically to neutrophils in human and mouse blood. We anticipate that these or related tagged nanoparticles may be therapeutically useful for delivery of siRNAs or ASOs to neutrophils for transient knockdown of pro-inflammatory proteins such as the C5a receptor.</p></div

Flow cytometry confirms Peptide H-HPLN binding to CD177-positive neutrophils in a pool of purified human leukocytes.

<p>Leukocytes were incubated on ice in the presence or absence of anti-CD177 antibody, washed and incubated on ice in the presence or absence of Peptide H-HPLN and Alexa Fluor 488 goat anti-mouse antibody. <b>A.</b> Forward and side scatter plot of human leukocytes (and remaining red blood cells) with the neutrophil population shown in a circle. <b>B.</b> FL-1 histogram of CD177 expression. Left panel: Total cell population in the absence and presence of anti-CD177 antibody (1° Ab) and Alexa Fluor 488 goat anti-mouse antibody (2° Ab). Right panel: Gated neutrophil population in the absence or presence of anti-CD177 antibody (1° Ab) and Alexa Fluor 488 goat anti-mouse antibody (2° Ab). <b>C.</b> FL2 plot showing Peptide H-HPLN binding. Total cell population in the absence or presence of Peptide H-HPLNs (left panel) and gated neutrophil population in the absence and presence of Peptide H-HPLNs (right panel). <b>D.</b> FL1-A and FL2-A plot showing fluorescence of the total cell population (left panel) and gated neutrophil population (right panel). The experiment was carried out twice with similar results. The blood sample shown had a higher percentage of CD177-positive neutrophils than the blood sample from the other donor.</p

HPLN particles displaying Peptide M become internalized by CHO cells expressing mouse CD177-HA.

<p>CHO cells expressing mouse CD177 containing an HA-tag were incubated with 75 μg/ml Peptide M-HPLN particles for 2 h on ice to allow binding. Cells were then washed and kept on ice or warmed to 37°C for 1 h. Cells were then treated with subtilisin to remove surface-bound Peptide M-HPLN particles, or treated with buffer alone. After fixation and permeabilization, mouse CD177-HA was stained with a mouse anti-HA antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. Most cells out of more than 100 cells visualized on the slides had a similar staining pattern as those seen in these micrographs. Scale bar, 50 μm.</p

HPLNs displaying Peptide H bind human CD177 on cells.

<p>CHO cells expressing human CD177 were incubated for 3 h at 37°C with 75 μg/ml Peptide H-HPLNs. Cells were fixed and permeabilized, and CD177 was detected using a mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The HPLN particles are fluorescent red. As a negative control, HPLN particles displaying a scrambled peptide were used. The experiment was carried out twice, with at least 100 cells examined in each experiment. A direct correlation between the expression level of CD177 (based on fluorescence intensity) and the Peptide H-HPLN signal could be observed. The cell in the lower panel shows a typical cell distribution of CD177 and Peptide H-HPLNs after 3 h incubation. In contrast, the two cells shown in the upper panel are characteristic of the CD177 staining in the absence of Peptide H-HPLNs and in the presence of Scrambled Peptide H-HPLNs. Scale bar, 50 μm.</p

Some Peptide H-HPLN particles co-localize with LAMP2 after prolonged incubation, suggesting delivery into the lysosomes.

<p>CHO cells expressing human CD177 were incubated for 3 h and 17 h at 37°C with 75 μg/ml Peptide H-HPLN particles. Cells were fixed and permeabilized, and the lysosomes were stained with a mouse anti-hamster LAMP2 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once. The selected micrographs are representative of more than 100 cells with similar labeling patterns. Scale bar, 50 μm.</p

Quantitative phage peptide binding assay.

<p>Quantitative phage peptide binding assay.</p

Time course of CD177 and Peptide H-HPLN redistribution in human neutrophils.

<p>Purified human neutrophils were incubated with Peptide H-HPLNs at 37°C with aliquots taken at 0, 15, 30, 60 and 120 min. Cells were rinsed with PBS, centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. The experiment was carried out once using blood from a donor with a high CD177 expression level. Hundreds of cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.</p

Peptide H-HPLN particles bind CD177-expressing human neutrophils in whole blood.

<p>Peptide H-HPLN particles were incubated with whole human blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with mouse anti-human CD177 antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody. Scrambled Peptide H-HPLN particles were used as a negative control. The experiment was carried out four times using blood from four different donors. In each case, slides with several hundred cells were examined and the micrographs are representative of these results. Scale bar, 10 μm.</p

Peptide M-HPLN particles bind CD177-expressing mouse neutrophils in whole blood.

<p>Peptide M-HPLN particles were incubated with whole mouse blood for 1 h at 37°C. The white blood cells were collected from the buffy coat and the red blood cells were lysed. The white blood cells were centrifuged onto glass slides, fixed in methanol and stained with rabbit anti-mouse CD177 polyclonal antibody and Alexa Fluor 488 goat anti-rabbit secondary antibody. Peptide H-HPLN particles were used as a negative control. The experiment was carried out twice. The micrographs represent the results from >100 cells. Scale bar, 10 μm.</p

Amino acid sequences of peptides that bind human and mouse CD177.

<p>Amino acid sequences of peptides that bind human and mouse CD177.</p