Fig 2: HPLN particles displaying Peptide M become internalized by CHO cells expressing mouse CD177-HA.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.

Fig 3: Flow cytometry confirms Peptide H-HPLN binding to CD177-positive neutrophils in a pool of purified human leukocytes.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. A. Forward and side scatter plot of human leukocytes (and remaining red blood cells) with the neutrophil population shown in a circle. 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). C. 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). D. 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.

Fig 4: Peptide H-HPLN particles bind CD177-expressing human neutrophils in whole blood.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.

Fig 5: Some Peptide H-HPLN particles co-localize with LAMP2 after prolonged incubation, suggesting delivery into the lysosomes.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.