Liposomes and Blood Cells: A Flow Cytometric Study

Figures & data

Figure 1. Interaction of liposomes and platelets detected by flow cytometry. A) CF‐liposomes interacted with washed platelets in buffer. Green fluorescence intensity is shown on the log scale of the x–axis; cell numbers on the y‐axis. The open histogram is the control platelet population; the dark histogram shows the level of fluorescence of the platelet population incubated with CF‐liposomes; the degree of fluorescence conferred by an equal amount of free CF is the gray histogram. B) Fluorescence of mouse platelets in plasma in response to FITC‐PE labeled liposomes is shown. The open histogram is the control platelet population; the dark histogram is the fluorescence of mouse PRP interacted with lipid‐labeled liposomes.

Figure 2. The fluorescence pattern of 10⁶/mL platelets labeled with anti‐CD42b‐R in the presence of CF‐liposomes (lipid 10 nmol/mL, CF 17 pmol/mL) in buffer. The x–axis quantitates red phycoerythrin (PE) florescence and the y‐axis, green CF fluorescence, on a log scale in arbitrary units.

Table 1. Analysis of Fluorescence Associated with the Platelet and Liposome Fractions Separated by Gel Exclusion Chromatography

	Platelet gate (A)		Liposome gate (B)		Platelet gate (A)		Liposome gate (B)
Initial components	% NG event	% G event	% NG event	% G event	% NR event	% R event	% NR event	% R event
CF‐Lip rate < 60 ev/s	0.2	14	0.2	85.6	14	0.4	73.6	12
R18‐Lip rate < 60 ev/s	2.5	0	96.4	1	0.3	2.3	0.4	97
PLATELETS rate > 400 ev/s	97	0	3	0	97	0	3	0
CF‐Lip/PLT rate > 400 ev/s	7	91	0	2	88	10	1.6	0.3
R18‐Lip/PLT rate > 400 ev/s	98.5	0	1.5	0	0	98.5	0	1.5
CF‐Lip + R18‐Lip/ PLT rate > 400 ev/s	11.2	88.2	0	0.6	68.5	31	0.5	0

Events in the specified gate are expressed as a percentage of the total counts in gates A plus B.

% g = (g/(A + B))100.

G green; NG non green; R red; NR non red.

Figure 3. The dose‐response curve of platelets (10⁶/mL) in buffer with liposomes labeled with: CF (solid line) (molar ratio of lipid:CF, 760:1); R18 (broken line) (molar ratio of lipid:R18, 135:1). The x‐axis represents the amount of liposomes (lipid nmol/mL); the y‐axis represents the percentage of fluorescent (red or green) labeled platelets.

Table 2. Liposomes of a Range of Compositions Interact with Platelets in Plasma or Whole Blood

Lipids	%Fluorescence
	Proportions	PRP Platelets	WB Platelets
1. DMPC:DMPG:FITC‐PEa	69:30:01	58.0	66.2
2. EPC:CHOL:FITC‐PEb	54:45:01	52.8	42.9
3. DOPC:DOPG:FITC‐PEc	49:50:01	34.2	26.0
4. EPC:CHOL:DOPA:FITC‐PEc	34:45:20:1	56.0	50.8
5. DOPC:CHOL FITC‐PEd	55:44:01	65.8	49.0
6. DPPC:CHOL:FITC‐PE	55:44:01	55.2	45.4
7. DSPC:CHOL:FITC‐PE	55:44:01	61.4	50.2
8. DMPC:CHOL:FITC‐PE	55:44:01	62.2	48.9

^a Lipid proportions similar to Abelcet ® The Liposome Company.

^b Lipid proportions similar to TLC D‐99, The Liposome Company.

^c Chonn A. PhD Thesis, University of British Columbia, 1991.

^d Oja CD, PhD Thesis, University of British Columbia, 1998.

Figure 4. Interaction of platelets and liposomes in plasma (triangles) or buffer (squares) and in the presence of anti‐CD42b‐R on platelets’ surface in plasma (solid symbols) or in buffer (open symbols). The x‐axis represents the amount of CF‐liposomes, lipid nmol/mL (molar ratio of lipid:CF, 760:1); the y‐axis represents the percentage of fluorescent platelets (10⁶/mL).

Figure 5. Fluorescence of cells in WB induced by the presence of increasing concentrations of CF‐liposomes (molar ratio lipid/CF, 760:1): leukocytes (triangles); red cells (squares); and platelets (circles). The x‐axis represents the amount of CF‐liposomes, lipid nmol/mL (molar ratio of lipid:CF, 760:1); the y‐axis represents the percentage of fluorescent blood cells.

Figure 6. Leukocyte fluorescence in WB. Increasing concentrations of liposome formulations 1 (circles) and 3 (squares) were added and the leukocytes were identified by anti‐CD45‐PC5. The x‐axis represents L of 10 mM liposome lipid added to a platelet population; the y‐axis shows the percentage of the leukocyte population positive for FITC‐PE.

Figure 7. Fluorescence of leukocyte‐platelet aggregates in WB. Increasing concentrations of liposome formulations 1 (circles) and 3 (squares) were added and the leukocyte‐platelet aggregates were selected by anti‐CD45‐PC5, interrogated for CD42b‐R then assessed for the presence of FITC‐PE. The x‐axis represents L of 10 mM liposome lipid added to a platelet population; the y‐axis shows the percentage of the leukocyte population positive for FITC‐PE.

Figure 8. Time course of liposome‐blood cell interaction in WB. Liposomes labeled with CF (solid lines) or R18 (broken lines) were added to whole blood and samples were removed at the indicated intervals. Red cells (closed symbols) and platelets (open symbols) were assessed for fluorescence. Liposomes with PEG₂₀₀₀ (squares) or without (circles) were compared. The x‐axis represents time in seconds; the y‐axis shows the % fluorescence of the cell population.

Figure 9. Intravenous injection of fluorescent liposomes into mice. Different lots of liposomes containing CF (solid lines) or R18 (broken lines) were injected into mice and the fluorescence of platelets and red cells was monitored. The x‐axis shows the times of blood samples taken after the injection; the y‐axes are % florescence of the cell populations: left y‐axis ‐ CF fluorescence; right ‐ R18. The platelet populations are shown by open symbols, and the red cells by closed symbols.