Transfer of MPEG19oo-DSPE from micellar phase to pre-formed liposomes imparts long in vivo circulation half-life to an otherwise rapidly cleared lipid composition. MPEG19oo-DSPE transfers efficiently and quickly in a time and temperature dependent manner. There is negligible content leakage and a strong correlation between assayed mol% MPEG19oo-DSPE, liposome diameter increase, and pharmacokinetic parameters such as distribution phase half-life. Since a biological attribute (liposome clearance rate) can be modified by the insertion process, it suggests a simple and economical way to impart sitespecific targeting to a variety of liposome delivery systems. This method is also a convenient way to measure the 'brush' thickness of such conjugates directly.

Summary This study tested the therapeutic efficacy of doxorubicin hydrochloride in two formulations: free in saline suspension and encapsulated in polyethylene glycol-coated, long-circulating liposomes. The drug formulations at a dose level of 3 mg doxorubicin per kg body weight were injected intravenously to treat the human pancreatic carcinoma AsPC-1, implanted s.c. into nude Swiss mice. Liposomeencapsulated doxorubicin was significantly more effective in inhibiting tumour growth and in effecting cures, and had only minor systemic toxic side-effects, indicated by a transient weight loss. Confocal laser scanning microscopy was used to determine the tumour uptake and the clearance of doxorubicin in the free and in the liposomal forms. The liposome-encapsulated doxorubicin entered the tumour in greater quantity, and remained in the tumour longer, than the free drug. The liposome formulation produced a sixfold or greater increase in doxorubicin at the disease site. It is probable that increased penetration into the tumour, and long presence with slow drug release from liposomes in the tumour, account for the enhanced therapeutic effect when the drug was encapsulated in polyethylene glycol-coated liposomes.

The biodistribution and pharmacokinetics of111In-DTPA-labelled pegylated liposomes in tumour-bearing nude mice was studied to examine possible applications of pegylated liposome-targeted anti-cancer therapies. Nude mice received an intravenous injection of 100 μl of111In-DTPA-labelled pegylated liposomes, containing 0.37–0.74 MBq of activity. The t 1/2α and t 1/2β of111In-DTPA-labelled pegylated liposomes were 1.1 and 10.3 h, respectively. Tumour uptake was maximal at 24 h at 5.5 ± 3.0% ID g–1. Significant reticuloendothelial system uptake was demonstrated with 19.3 ± 2.8 and 18.8 ± 4.2% ID g–1at 24 h in the liver and spleen, respectively. Other sites of appreciable deposition were the kidney, skin, female reproductive tract and to a lesser extent the gastrointestinal tract. There was no indication of cumulative deposition of pegylated liposomes in the lung, central nervous system, musculoskeletal system, heart or adrenal glands. In contrast, the t 1/2α and t 1/2β of unencapsulated111In-DTPA were 5 min and 1.1 h, respectively, with no evidence of accumulation in tumour or normal tissues. Incubation of111In-DTPA-labelled pegylated liposomes in human serum for up to 10 days confirmed that they are very stable, with only minor leakage of their contents. The potential applications of pegylated liposomes in the arena of targeted therapy of solid cancers are discussed. © 2000 Cancer Research Campaign

Abstract. A conventional fluorescence microscope was modified to observe the sites of resonance energy transfer (RET) between fluorescent probes in model membranes and in living cells. These modifications, and the parameters necessary to observe RET between membrane-bound fluorochromes, are detailed for a system that uses N-4-nitrobenzo-2-oxa-l,3-diazole (NBD) or fluorescein as the energy donor and sulforhodamine as the energy acceptor. The necessary parameters for RET in this system were first optimized using liposomes. Both quenching of the energy donor and sensitized fluorescence of the energy acceptor could be directly observed in the microscope. RET microscopy was then used in cultured fibroblasts to identify those intracellular organelles labeled by the lipid probe, N-SRh-decylamine (N-SRh-C~). This was done by observing the sites of RET in cells doubly labeled with N-SRh-C~ and an NBD-labeled lipid previously shown to label the endoplasmic reticulum, mitochondria, and nuclear envelope. RET microscopy was also used in cells treated with fluorescein-labeled Lens culinaris agglutinin and a sulforhodamine derivative of phosphatidylcholine to examine the internalization of plasma membrane lipid and protein probes. After internalization, the fluorescent lectin resided in most, but not all of the intracellular compartments labeled by the fluorescent lipid, suggesting sorting of the membrane-bound lectin into a subset of internal compartments. We conclude that RET microscopy can colocalize different membrane-bound components at high resolution, and may be particularly useful in examining temporal and spatial changes in the distribution of fluorescent molecules in membranes of the living cell.ESONANCE energy transfer (RET) ~ is characterized by the transfer of photon energy from one fluorochrome (the donor) to another molecule (the acceptor) that is in close physical proximity. The donor fluorescence is quenched and, if the acceptor is an appropriate fluorescent molecule, it will fluoresce as if excited directly (2). Because RET decreases in proportion to the inverse sixth power of the distance between the two probes, this phenomenon is effective only when the donor and acceptor molecules are within 100 /~ of each other (31). RET therefore provides a convenient method for probing inter-and intramolecular distances in proteins (for a review, see reference 30) and in membranes.1. Abbreviations used in this paper: BHK, baby hamster kidney; (C6-NBD)-PA, 1,2-(oleoyl, NBD-aminocaproyl) phosphatidic acid; (C6-NBD)-PC, 1,2-(acyl, NBD-aminocaproyl) phosphatidylcholine; (C6-SRh)-PC, 1,2-(acyl, SRh-aminocaproyl) phosphatidylcholine; (C6-tBOC)-PC, 1,2- (acyl, tert-butoxycarbonyl-aminocaproyl) phosphatidylcholine; DOPC, dioleoyl phosphatidylcholine; FITC-LCA, fluorescein isothionyl-Lens culinaris agglutinin; HMEM, 18 mM Hepes-buffered Eagle's minimal essential medium without indicator, pH 7.4; NBD, N~-nitrobenzo-2-oxa-1,3-diazole; N-NBD-PE, N-NBD-dioleoyl phosphatidylethanolamine; N-SRh-C~0, N-SRh-decylamine; N-SRh-PE, N-SRh-...

SummaryThe relationship between tumour size and uptake of 111 In-DTPA-labelled pegylated liposomes has been examined in a human head and neck cancer xenograft model in nude mice. The mean tumour uptake of 111 In-labelled pegylated liposomes at 24 hours was 7.2 ± 6.6% ID/g. Liposome uptake for tumours < 0.1 g, 0.1-1.0 g and > 1.0 g was 15.1 ± 10.8, 5.9 ± 2.2 and 3.0 ± 1.3% ID/g, respectively. An inverse correlation between tumour weight and liposome uptake was observed by both Spearman's rank correlation test (r s = -0.573, P < 0.001) and Pearson's correlation coefficient (r s = -0.555, P < 0.001). For 18 tumours with macroscopic central necrosis, the ratio of uptake in the tumour rim relative to the necrotic tumour core was 11.2 ± 6.4. Measurement of tumour vascular volume for tumours of various sizes revealed an inverse correlation between tumour weight and tumour vascular volume (Spearman's rank correlation test, r s = -0.598, P < 0.001), consistent with poor or heterogeneous vascularization of larger tumours. These data have important implications for the clinical application of pegylated liposome targeted strategies for solid cancers which are discussed in detail.

Polylysine promoted extensive membrane mixing of liposomes only if the buffer pH was below the pKa of the lysyl residues. This observation suggested that fusion could be regulated in a physiological pH range if the homopolymer of L-histidine was substituted as fusogen. Microgram quantities of polyhistidine were added to liposomes composed of soybean phospholipids, or to defined phospholipid-cholesterol mixtures which simulate the lipid composition of plasma membranes. A quantitative resonance energy transfer assay determined the extent of lipid phase mixing related to fusion. No fusion was detected at pH 7.4, but when the pH was lowered to 6.5 or below, fusion was rapid and substantial. The extent of membrane mixing increased with progressive acidification of the vesicle-fusogen suspension. The charge density of each polyhistidine molecule, not the total cationic charge per vesicle, influenced the extent of fusion. The kinetics of the fusion reaction were rapid, as membrane mixing was completed within 1 min. If the vesicle suspension was acidified before fusogen addition, the rate of membrane mixing slowed 4-fold. This, as well as a slight increase in light scattering noted whenever polyhistidine was added at pH 7.4, suggests an enhancement of fusion kinetics by preaggregation of vesicles at neutral pH. The lipid composition, regulation of membrane mixing by pH in a physiological range, and rapid kinetics suggest that this model of liposome fusion may be pertinent to understanding some biological fusion events.

Vincristine is used clinically for the treatment of various types of cancer. Recent significant therapeutic improvements obtained by entrapping anthracyclines in sterically stabilized liposomes raised the question whether the therapeutic index of vincristine can be similarly increased by formulation into such long-circulating liposomes. Encapsulation of vincristine in sterically stabilized liposomes (SL-VCR) prolonged the drug's distribution phase plasma half-life in rats from 0.22 to 10.5 hr. There was no significant difference in LD50 (> < or = 2.5 mg/kg, i.v.), but mice given sublethal doses of SL-VCR experienced significantly less weight loss than those given the same dose of free drug. Compared to free drug, SL-VCR was most effective against i.p. or s.c. implanted tumors. However, i.v. tumor inoculation nullified the therapeutic advantage of encapsulation. A single i.v. 2 mg/kg dose of SL-VCR increased the life span of mice bearing i.v. implanted P388 cells by only 44%, while the life span of i.p. P388 implanted mice was increased by 199%. In an s.c. implanted murine colon carcinoma, multiple doses of free drug did little to slow the growth of the tumors, but SL-VCR was able to produce long-term survivors in several dose regimens. These results indicate that prolonged circulation time increases the therapeutic index of VCR entrapped in liposomes against s.c. or i.p. implanted tumors, but does not improve the drug's activity against rapidly growing i.v. disseminated leukemias.

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