MPEG-SHz

Dil-labelled MPEG-SHz liposome dispersion (25 xL, lipid concentration 100 mM) was diluted up to 500 pL with PBS (+) and incubated at 37 °C for a predetermined period to allow PEG to be released from the liposome surface. This dispersion was mixed with 500pL of the above-obtained PMN suspension. After incubation at 37 °C for the predetermined period, the mixture was centrifuged to sediment PMN-associated liposomes, leaving the free liposomes in the supernatant. To the resultant pellet of PMNs (approx. 50 pL) was added 1 mL of PBS containing 2 w/v% Triton. The mixture was sonicated for 1 min with a probe sonicator (35 W) to dissolve Dil in the PMN-associated liposomes. 

On the basis of these results, the progressive hydrazo bond formation introducing MPEG-CHO to the liposome surface was estimated by the partition measurement. The effects of different reaction conditions such as temperature, time, nominal concentration and molar ratio of the liposomes over MPEG-CHO, on the partition coefficient of MPEG-SHz liposomes were investigated (data not shown). It was found that the conditions for hydrazo bond formation between SHz liposomes and MPEG-CHO could be optimised (as in MATERIALS AND METHODS). 

Changes in the physicochemical properties of MPEG-SHz liposomes incubated in PBS at 37 C were evaluated with time, and compared with those of liposomes with stably linked PEG, namely, stabilised MPEG-SA liposomes and MPEG-DSPE liposomes. The lipid composition and linkage between PEG and the liposome surface of the liposomes tested here are shown in Table 1. 

These results indicate that the decrease in the PEG content on the MPEG-SHz liposomes is attributable to the unstable bonding between PEG and the liposome surface, but not to the physical extraction of the PEG-lipid conjugate from the liposomal membrane. 

Figure 1. Time course of the partition coefficient changes of MPEG-SHz liposomes incubated in PBS at 37 °C.

The amount of liposomes that adhered to PMNs immediately after mixing, increased with preincubation time. The rate of further adherence after mixing appeared to follow the relationship. This result suggests that the affinity of MPEG-SHz liposomes for the cells increased as the PEG was released from the liposome surface with time. In other words, liposome surface substantially changed from being bioinert into bioreactive with time. 

This discrepancy may be due to the aggregation tendency of the PEG-releasing liposomes, especially in the evaluation system of the affinity for PMNs. It is plausible that divalent cations and/or proteins secreted from PMNs could induce the aggregation of MPEG-SHz liposomes as the incubation time is prolonged, resulting in an increase in the particle size. It... 

Figure 5. Time course of the liposome adhesion to PMNs. MPEG-SHz liposomes were preincubated in PBS (+) at 37 °C for 0 ( ), 1 (O), 2 (A), 7 (O), or 20 (X) h, then mixed with PMNs and incubated. The incubation with PMNs was stopped just after mixing (1st plot of each symbol) and continued for 0.5 (2nd), 1.5 (3rd) and 3.0 (4th) h.

Figure S. Schematic illustration of the time-dependent interaction between MPEG-SHz liposomes and biocomponents.

Abbreviations Choi, cholesterol Dil, l,l -dioctadecyl-3,3,3, 3 -tetramethylindocarbocyanine perchlorate DPPC, L-a- dipalnritoylphosphiatirtylcholine MPEG-CHO, mono-methoxypoly (ethylene glycol)5000-aldehyde MPEG-DSPE, monomethoxypoly (ethylene glycol)2(XX)-succinyl phosphatidyl-ethanolanune PBS, phosphate bulferedsahne PMN, polymorphonuclear leukocyte SA, stearylamine SHz, stearic hydrazide. 

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