Binding of Adriamycin to Liposomes as a Probe for Membrane Lateral Organization

• Published in: Biophysical journal 1999-02, Vol.76 (2), p.896-907
• Main Authors: Söderlund, Tim, Jutila, Arimatti, Kinnunen, Paavo K.J.
• Format: Article
• Language: English
• Subjects: Binding Sites; Biochemistry; Calorimetry, Differential Scanning; Doxorubicin - metabolism; Drugs; Fluorescent Dyes; Kinetics; Lipids; Liposomes - metabolism; Membranes; Membranes - chemistry; Phosphatidylcholines - chemistry; Phosphatidylcholines - metabolism; Phosphatidylglycerols - chemistry; Phospholipids - chemistry; Spectrometry, Fluorescence; Static Electricity; Temperature
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/S0006-3495(99)77253-X

A stopped-flow spectrofluorometer equipped with a rapid scanning emission monochromator was utilized to monitor the binding of adriamycin to phospholipid liposomes. The latter process is evident as a decrease in fluorescence emission from a trace amount of a pyrene-labeled phospholipid analog (PPDPG, 1-palmitoyl-2-[(6-pyren-1-yl)]decanoyl- sn-glycero-3-phospho-rac-glycerol) used as a donor for resonance energy transfer to adriamycin. For zwitterionic 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) liposomes, fluorescence decay was slow, with a half-time t 1/2 of ∼2 s. When the mole fraction of the acidic phospholipid, 1-palmitoyl-2-oleoyl- sn-glycero-3-phospho-rac-glycerol (POPG), was increased to X PG ≥ 0.04, the decay of fluorescence became double exponential, and an additional, significantly faster process with t 1/2 in the range between 2 and 4 ms was observed. Subsequently, as X PG was increased further, the amplitude of the fast process increased, whereas the slower process was attenuated, its t 1/2 increasing to 20 s. Increasing [NaCl] above 50 mM or [CaCl 2] above 150 μM abolished the fast component, thus confirming this interaction to be electrostatic. The critical dependence of the fast component on X PG allows the use of this process to probe the organization of acidic phospholipids in liposomes. This was demonstrated with 1,2-palmitoyl- sn-glycero-3-phosphocholine (DPPC) liposomes incorporating PPDPG ( X PPDPG = 0.03), i.e., conditions where X PG in fluid bilayers is below the required threshold yielding the fast component. In keeping with the presence of clusters of PPDPG, the fast component was observed for gel-state liposomes. At ∼34°C (i.e., 6° below T m), the slower fluorescence decay also appeared, and it was seen throughout the main phase transition region as well as in the liquid-crystalline state. The fluorescence decay behavior at temperatures below, above, and at the main phase transition temperature is interpreted in terms of thermal density fluctuations and an intermediate state between gel and liquid-crystalline states being involved in the phospholipid main phase transition. This is the first observation of a cluster constituted by acidic phospholipids controlling the membrane association of a drug.