Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.     

Capillary electrophoretic separation of high-molecular-weight poly(ethylene glycol)-modified proteins

This study was designed to demonstrate the utility of capillary electrophoresis (CE) for separating high-molecular-weight poly(ethylene glycol) (PEG)-conjugated proteins. As a CE method, sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was applied to analyze interferon alpha (IFN) modified with branched and trimer-structured PEG molecules. Five mono-PEG-IFN conjugates prepared with two branched PEGs (MW 20 and 40 kDa) and three trimer-structured PEGs (MW 23.5, 43.5, and 47 kDa) were purified by cation-exchange chromatography and their masses were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The SDS-CGE method showed high separation capacity by differentiating PEG-IFN conjugates with small differences in molecular size, such as PEG_40K-, PEG_43.5K-, and PEG_47K-IFNs, and it was useful for checking the purity of each mono-PEG-IFN. This study shows that SDS-CGE can well be utilized in the development and quality control of PEGylated proteins prepared with various types of PEG.     

Size-exclusion reaction chromatography (SERC): a new technique for protein PEGylation

A new, widely applicable process that combines reaction and separation in a single unit operation is described. The process, size-exclusion reaction chromatography (SERC), simultaneously allows control of the extent of reactions in which molecular size is altered and the separation of products and reactants. In SERC, a moving reaction zone is formed by injection of reactants onto a size-exclusion chromatography column. Reactants and products are partitioned differently within the mobile phase, resulting in different linear flow rates through the column. The products are therefore removed selectively from the reaction zone, minimizing their residence time in the reaction zone and allowing their separation in the downstream section of the column. For reactions such as protein PEGylation, in which successive addition of PEG groups to the protein results in significant molecular size increases, SERC potentially offers a method by which a dominant final PEGylated protein size can be produced at high yield. The SERC PEGylation of two model proteins, alpha-lactalbumin and beta-lactoglobulin, is demonstrated and results show that simultaneous reaction and separation was obtained.     

Difference in microchip electrophoretic mobility between partially and fully PEGylated poly(amidoamine) dendrimers

The objective of this study was to investigate the difference in electrophoretic mobility between partially and fully poly(ethylene glycol)-conjugated poly(amidoamine) dendrimers (part-PEG-PAMAM and full-PEG-PAMAM, respectively) using a microchip capillary gel electrophoresis (MCGE). While MCGE allowed size-based separation of PEG-PAMAMs prepared with monomethoxy PEG-nitrophenyl carbonate, full-PEG-PAMAMs migrated slower than part-PEG-PAMAMs that were similar in size or larger. When the measured molecular weights obtained from MCGE analysis and the calculated molecular weights were plotted, each part-PEG-PAMAM and full-PEG-PAMAM showed correlation coefficients greater than 0.98. This study indicates that MCGE would be useful for characterizing PEG-PAMAMs with different PEGylation degrees.     

Enzymatic preparation of streptavidin-immobilized hydrogel using a phenolated linear poly(ethylene glycol)

Hybrid gels constructed from proteins and polymers have attracted a wide range of attention in the field of biomedicine and bioengineering. We report herein the enzymatic preparation of polymer–protein hybrid hydrogels composed of terminally bis-functionalized linear poly(ethylene glycol) (PEG) and streptavidin (SA). PEG was conjugated with tyramine to introduce terminal phenolic hydroxyl (Ph-OH) groups. A peptide tag containing a tyrosine residue (G₄Y-tag) was genetically introduced at the C-terminus of SA. The Ph-OH-modified PEG and G₄Y-tagged SA (SA-G₄Y) were treated by horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) to yield (PEG-Ph-OH)–(SA-G₄Y) hybrid gels. Biotinylated enhanced green fluorescent protein (biotin-EGFP) was selectively captured in the obtained hybrid gels, indicating that SA-G₄Y retained its biological function. The amount of biotin-EGFP immobilized in the hybrid gels depended on the concentration of SA-G₄Y. In addition, biotinylated bacterial alkaline phosphatase (biotin-BAP) was immobilized in the hybrid gel. The immobilized biotin-BAP exhibited more than 95% of the initial activity after 5 rounds of recycling. The results suggest the facile functionalization of the hybrid gel with a variety of biotinylated functional molecules.     

Pharmacokinetic, biodistribution, and biophysical profiles of TNF nanobodies conjugated to linear or branched poly(ethylene glycol)

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins has been used to prolong in vivo exposure of therapeutic proteins. We have examined pharmacokinetic, biodistribution, and biophysical profiles of three different tumor necrosis factor alpha (TNF) Nanobody-40 kDa PEG conjugates: linear 1 × 40 KDa, branched 2 × 20 kDa, and 4 × 10 kDa conjugates. In accord with earlier reports, the superior PK profile was observed for the branched versus linear PEG conjugates, while all three conjugates had similar potency in a cell-based assay. Our results also indicate that (i) a superior PK profile of branched versus linear PEGs is likely to hold across species, (ii) for a given PEG size, the extent of PEG branching affects the PK profile, and (iii) tissue penetration may differ between linear and branched PEG conjugates in a tissue-specific manner. Biophysical analysis (R(g)/R(h) ratio) demonstrated that among the three protein-PEG conjugates the linear PEG conjugate had the most extended time-average conformation and the most exposed surface charges. We hypothesized that these biophysical characteristics of the linear PEG conjugate accounts for relatively less optimal masking of sites involved in elimination of the PEGylated Nanobodies (e.g., intracellular uptake and proteolysis), leading to lower in vivo exposure compared to the branched PEG conjugates. However, additional studies are needed to test this hypothesis.     

Charaterization and hydrodynamic behaviour of modified gelatin: II. Characterization by high performance size exclusion chromatography — comparison with dextrans and proteins

Gelatin samples obtained by chemical modification (succinylation) are studied by SEC on silica based chromatographic supports. The influence of the pH of eluent mixtures (potassium phosphate added to NaCl) in the range 7-3.3 shows that the void volume peak (VVP) is lowered or even vanishes at pH 3.3 with the 3000 SW (TSK) gel. A process using an ultrasound treatment before injection is reported in order to determine accurately the molecular parameters of gelatin onto TSK gel with a minimal VV P. This peak is attributed to molecular aggregation of a part of the modified gelatin. After disaggregation by ultrasound or heat treatment the results are in good accordance with those obtained by other methods. It is demonstrated that with proteins and dextrans the TSK 3000 SW gel does not agree with the universal calibration curve (log[ν] · versus K_d as reported previously. A single calibration curve is obtained when the Stokes radius is plotted versus K_d. Gelatin fractions are eluted at pH 7 close to this calibration curve. This plot shows that gelatin fractions at pH 3.3 are not eluted by a pure size exclusion mechanism on 3000 SW gel. It is concluded that hydrophobic interactions between fractions of gelatin and the gel explain the high retention of these samples.     

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.     

Transfer of a lipophilic drug (temoporfin) between small unilamellar liposomes and human plasma proteins: influence of membrane composition on vesicle integrity and release characteristics

Abstract

The introduction of PEG lipid conjugates into lipid bilayers leads to long circulating liposomes with improved pharmacokinetics and pharmacodynamics characteristics. The concentration range of PEG-lipids is limited by their micelle forming properties. We investigated two phosphatidyl oligoglycerols as potential alternatives to PEG-lipid conjugates and compared their micelle forming properties after incorporation of increasing amounts of oligoglycerols into gel-phase liposomes via cryo-transmission electron microscopy. The incorporation of highly hydrophobic drugs into liposomes makes water soluble formulations possible and improves the therapeutic properties of the drug. We incorporated the hydrophobic photosensitizer temoporfin into liposomes varying in membrane fluidity and nature of surface modifying agents. The main purpose of this study was the investigation of liposome integrity and temoporfin incorporation stability in the presence of plasma. After incubation of temoporfin-loaded liposomes with human plasma for different time intervals, liposomes and the single lipoprotein fractions were separated via size-exclusion chromatography. Liposome stability and temoporfin distribution profile over the lipoprotein fractions were determined with the help of a non-exchangeable ³H-lipid label and ¹⁴C-labeled temoporfin. The results demonstrate that both oligoglycerols are suitable alternatives to PEG-lipid conjugates because of the lack of micelle forming properties, comparable liposome stability, and a reduced temoporfin transfer rate compared to PEG-lipids. Furthermore, the incorporation stability of temoporfin is – at least to some extent – influenced by membrane fluidity, indicating that fluid membranes may be better suited for retention of lipophilic drugs.     

Co-lyophilization of bovine serum albumin (BSA) with poly(ethylene glycol) improves efficiency of BSA encapsulation and stability in polyester microspheres by a solid-in-oil-in-oil technique

The effect of the co-lyophilization of bovine serum albumin (BSA) with poly(ethylene glycol) (PEG) on the BSA encapsulation efficiency and formation of soluble BSA aggregates upon solid-in-oil-in-oil (s/o/o) encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres was investigated. Suspension of the lyophilized BSA-PEG formulations in methylene chloride produced small protein powder particles of less than 1 m diam. and this afforded high encapsulation efficiencies of typically 90% ameliorating one of the problems in s/o/o encapsulation. Formation of soluble BSA aggregates upon s/o/o encapsulation followed by 24 h in vitro release was between 5% and 22%, much lower than values of 59% reported for BSA without stabilizing excipients. Therefore, PEG also afforded BSA stabilization during s/o/o encapsulation. Sustained release occurred over ca. 2 months and was complete.     

Carrier design: Synthesis and conformational studies of poly (L-lysine) based branched polypeptides with hydroxyl groups in the side chains

In the present study the development of a new series of branched polypeptides that contain hydroxyl groups in side chains is reported. Serine or threonine were attached by 1-hydroxy-benzotriazole catalyzed active ester method to the N-terminals of oligo (DL -alanine) chains grafted to a polylysine backbone resulted in poly[Lys-(Ser₁-DL -Ala_m)] (SAK) and poly-[Lys-(Thr_i-DL -Ala_m)] (TAK). Ser was coupled also directly to the η-amino groups of polylysine followed by polymerization of N-carboxy-DL -alanine anhydride resulting oligo (DL -Ala) chain terminals. In this way a reverse sequence was built up in the side chain corresponding to the poly[Lys-(DL -Ala_m-Ser_i)] (ASK). The number of hydroxyl groups in the polymer was increased by the synthesis of a branched polypeptide with oligo (DL -serine) branches instead of oligo (DL -alanine) ones—poly[Lys-(DL -Ser_m)] (SK). Classification of solution conformations of branched polypeptides was carried out by CD spectroscopy performed in water solution of various pH values and ionic strengths. Incorporation of single Ser residues in poly[Lys-(X_i)]-type polypeptides markedly promotes the formation of ordered structure without resulting precipitation even in high salt concentration. The presence of branches with multiple DL -Ser residues resulted in a slightly decreased ability of the polypeptide backbone to adopt an ordered conformation. Comparison of the CD properties of the SAK-ASK pair demonstrates that these compounds are similar, showing an increased tendency to form an ordered spatial arrangement in solution at elevated pH or ionic strength; however, differences in their CD spectra suggest that SAK has higher capability to form regular conformation under comparable conditions. The replacement of Ser by the Thr residue in poly[Lys-(X_i-DL -Ala_m)] induced a conformational transition and TAK exhibited a more helical structure. These results might indicate that not only hydrophobic or ionic attraction, but also H-bond interaction, can play a role in the formation and/or stabilization of ordered conformation of branched polypeptides. Findings with the hydroxyl group containing polymers reported in this paper can also explain their prolonged shelf stability and high water solubility. © 1997 John Wiley & Sons, Inc. Biopoly 42: 719–730, 1997     

Preparation and characterisation of Cibacron Blue F3G-A poly(ethylene) hollow-fibre affinity membranes

Poly(ethylene) hollow-fibre membranes with immobilised Cibacron Blue F3G-A were obtained in four different ways from epoxy-activated fibres. Membranes with a maximum capacity of 26 mg lysozyme ml^–1and a dye density of 52 mol ml^–1were obtained when ammonia was used to open the epoxy group before dye immobilisation. Pure water flux of the modified membranes at 1 bar pressure was 1.0 cm min^–1, thus meaning only a reduction of 1.5-fold with regard to the unmodified membranes. The support-dye bond was stable as judged by the unmodified capacity of the membranes and the negligible amount of dye leaked after 520 h of exposure to 6 M urea in 0.5 M NaOH.     

Preparation and characterization of poly(ethylene glycol)-g-chitosan with water- and organosolubility

A new scheme was proposed for synthesizing poly(ethylene glycol)-g-chitosan (PEG-g-CS), where methoxy poly(ethylene glycol) iodide (MPEG-I) (M_n 2000) was used for N-substitution of triphenylmethyl chitosan (TPM-CS) in organic medium. The graft copolymers were obtained by subsequent removal of protecting groups with dichloroacetic acid. By varying PEG-I/TPM-CS feed ratio, the grafting levels (GL) of PEG can be adjusted. The chitosan derivatives were characterized by FTIR, ¹H NMR, ¹³C NMR and DSC. All the copolymers were soluble in water over wide pH range. Furthermore, organosolubility of the hybrids in DMF and DMSO was also achieved when the DS value more than 24%. The lysozyme degradation rate of the copolymers in aqueous neutral medium decreased with the increase of GL value.     

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Low expression and instability during isolation are major obstacles preventing adequate structure‐function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C‐terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C‐termini (C_in) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C‐termini (C_out) to C_in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C_out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein‐detergent complex was identified using an extended fluorescence‐detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure‐function studies. Five MPs were successfully cleaved from the GFP tag by site‐specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C_out topology, yielding sufficient protein suitable for structure‐function studies and are superior to expression and purification in the absence GFP fusion tagging.     

Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol)

The presence of poly(ethylene glycol) (PEG) at the surface of a liposomal carrier has been clearly shown to extend the circulation lifetime of the vehicle. To this point, the extended circulation lifetime that the polymer affords has been attributed to the reduction or prevention of protein adsorption. However, there is little evidence that the presence of PEG at the surface of a vehicle actually reduces total serum protein binding. In this review we examine all aspects of PEG in order to gain a better understanding of how the polymer fulfills its biological role. The physical and chemical properties of the polymer are explored and compared to properties of other hydrophilic polymers. An evidence based assessment of several in vitro protein binding studies as well as in vivo pharmacokinetics studies involving PEG is included. The ability of PEG to prevent the self-aggregation of liposomes is considered as a possible means by which it extends circulation longevity. Also, a dysopsonization phenomenon where PEG actually promotes binding of certain proteins that then mask the vehicle is discussed.     

Highly efficient gene transfer with degradable poly(ester amine) based on poly(ethylene glycol) diacrylate and polyethylenimine in vitro and in vivo

BACKGROUND: Polyethylenimine (PEI) is toxic although it is one of the most successful and widely used gene delivery polymers with the aid of the proton sponge effect. Therefore, development of new novel gene delivery carriers having high efficiency with less toxicity is necessary. METHODS: In this study, a degradable poly(ester amine) carrier based on poly(ethylene glycol) diacrylate (PEGDA) and low molecular weight linear PEI was prepared. Furthermore, we compared the gene expression of the polymer/DNA complexes using two delivery methods: intravenous administration as an invasive method and aerosol as a non-invasive method. RESULTS: The synthesized polymer had a relatively small molecular weight (MW = 7980) with 25 h half-life in vitro. The polymer/DNA complexes were formed at an N/P ratio of 9. The particle sizes and zeta-potentials of the complexes were dependent on N/P ratio. Compared to PEI 25K, the newly synthesized polymer exhibited high transfection efficiency with low toxicity. Poly(ester amine)-mediated gene expression in the lung and liver was higher than that of the conventional PEI carrier. Interestingly, non-invasive aerosol delivery induced higher gene expression in all organs compared to intravenous method in an in vivo mice study. Such an expressed gene via a single aerosol administration in the lung and liver remained unchanged for 7 days. CONCLUSIONS: Our study demonstrates that poly(ester amine) may be applied as an useful gene carrier.     

Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase

PEG-modified recombinant mammalian urate oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 ± 2.1 mg/dl (mean ± SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 ± 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.     

Control of hyperuricemia in subjects with refractory gout, and induction of antibody against poly(ethylene glycol) (PEG), in a phase I trial of subcutaneous PEGylated urate oxidase

PEG-modified recombinant mammalian urate oxidase (PEG-uricase) is being developed as a treatment for patients with chronic gout who are intolerant of, or refractory to, available therapy for controlling hyperuricemia. In an open-label phase I trial, single subcutaneous injections of PEG-uricase (4 to 24 mg) were administered to 13 such subjects (11 had tophaceous gout), whose plasma uric acid concentration (pUAc) was 11.3 +/- 2.1 mg/dl (mean +/- SD). By day seven after injection of PEG-uricase, pUAc had declined by an average of 7.9 mg/dl and had normalized in 11 subjects, whose mean pUAc decreased to 2.8 +/- 2.2 mg/dl. At doses of 8, 12, and 24 mg, the mean pUAc at 21 days after injection remained no more than 6 mg/dl. In eight subjects, plasma uricase activity was still measurable at 21 days after injection (half-life 10.5 to 19.9 days). In the other five subjects, plasma uricase activity could not be detected beyond ten days after injection; this was associated with the appearance of relatively low-titer IgM and IgG antibodies against PEG-uricase. Unexpectedly, these antibodies were directed against PEG itself rather than the uricase protein. Three PEG antibody-positive subjects had injection-site reactions at 8 to 9 days after injection. Gout flares in six subjects were the only other significant adverse reactions, and PEG-uricase was otherwise well tolerated. A prolonged circulating life and the ability to normalize plasma uric acid in markedly hyperuricemic subjects suggest that PEG-uricase could be effective in depleting expanded tissue stores of uric acid in subjects with chronic or tophaceous gout. The development of anti-PEG antibodies, which may limit efficacy in some patients, is contrary to the general assumption that PEG is non-immunogenic. PEG immunogenicity deserves further investigation, because it has potential implications for other PEGylated therapeutic agents in clinical use.     

The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles

Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4′-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent ‘donor’ and 1-(4′-carboxyethyl)-6-(4″-nitro)diphenyl-trans-1,3,5-hexatriene as ‘acceptor’. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20°C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of l-α-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with < 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.