i08 BBAMI-M 7(1671 BBA Report Biochimica et Bioph;'sic'el Acta, 1062 {1991 ) 108-112 ' 1991 Elsevier Science Puhlishers B.V. 0005-2736/91/$03.50 A DONIS 0005273691001(134 Size and stability of dipalmitoylphosphatidylcholine/cholesterol unilamellar vesicles are affected by interaction with proteins L. Dini ~, A. Di Giulio 2, A, Pavan -~, G. Ravagnan 4 and G. Mossa 4 t DIpartimenm di Bwlogia, Unit'ersit~ di Rmna 'T~Jr Vergata', Roma (ltalr), " Dipartmtettto di Scien-e e Tecnologie Btomediche e Bwmetria. Unit'ersit?l di L'.4quila. Roma (ira[v), ¢ Dtparnmento di Medk'ina Sperimentale. Unit,ersith di Roma 'La Sapwnza'. Roma (ha(v) and 4 lstituto di Medicina Sperimentale de! C ,¥. R., Boma (italr) (Received 4 July 1990} (Revised manuscript received 15 November 19901 Key words: Dipalmitoylphosphatidylchotine: Lipasome: Ascorbate oxidase: Protein-lipid interaction: Freeze-fracture The effec! of entrapping the enzyme ascorbate oxidase into dipalmitoylphosphatidylcholine/cholesterol vesicles, was studied by conventional transmission electron microscopy and freeze-fracture. The freeze-fracture technique has definitely demonstrated the unilamellar nature of empty and enzyme-loaded vesicles. Images of freeze-fractured and label-fractured liposomes also indicate that the observed reduction of vesicles volume could be related to the localization of ascorbate oxidase across the membrane. The membrane localization of ascorbate oxidase may explain the oxidation of externally added ascorbate by intact enzyme-loaded liposomes. Finally, the ageing of liposomes appears to be accelerated in the presence of proteins. Unilamellar vesicles are being used as targetable vectors for several molecules such as drugs [I], enzymes 12] and genetic material [3]. However. little attention has been devoted in the past to the localization of the carried material within lipid vesicles. In fact, DNA and proteins may interact in several way~; with lipids. In particular, proteins may be entrapped inside the vesicle, may interact with the inner and/or outer lipid leaflet or may span the lipid bilayers. These different interactions are fundamental in the liposome-cell fusion process and eventually in the re- lease of the carried material inside the target cells. Previous work carried out in many laboratories showed that the survival time of liposomes and the ability of fusing with target cells are influenced by size 14]. surface charge [5], lipid composition [61 and stability 17]. Ahhreviations: AAO. ,scorbic acid oxidase: BSA, b~wine serum al- bumin: CLIO. chole:,tcrol; DPPC, l-,,dipalmitoylphosphatidyl- ch~line; PBS. pht~sphate-buffered saline; TEM, transmission dectron micro~,~:opy. ('orrc,~pondct~cc: L. l)ini, Dipartimento di Biologia Univcrshh di Rtmaa "Tot Vergata', Via O. Raimor~do, 00173 Roma. Italy. Our research deals with the entrapment of the plant enzyme ascorbate oxidase (EC 1.10.3.3.) into dipalmi- toylphosphatidylcholine/cholesterol liposomes in order to deliver it to cells. In a recent study [8], we have reported the effect of loading iiposomes with ascorbate oxidase on their physico-chemical properties and on the catalytic activity of the entrapped enzyme. In that paper we gave evidence that some enzyme was adsorbed on the membrane of liposomes. Since the presence of ascorbate oxidase outside the liposomes may prevent their use as carrier,, for this enzyme, we have further investigated by conventional transmission electron mi- croscopy and freeze-fraclure, the interaction of ascor- bate oxidase with liposomes. The influence of the pro- tein on the overall morphology and stability of the vesicles was also studied. Information on the used chemicals, on the liposome preparation and on the enzyme activity determination is reported elsewhere 18]. Gel filtration of vesicles was performed as follows. A liposome suspension in PBS (4 ml), containing 50 #moles of total lipids, was carefully layered onto a Sephadex G-25 (LKB. Bromma, Sweden) column (1.2 x 25 cm). The column was eluted, at room temperature, using PBS at a flow rate nf 6 ml/h and 1 ml volume fractions were collected. The fractions were analyzed for the turbidity