Joke Regts

TRANSFER AND EXCHANGE OF PHOSPHOLIPID BETWEEN SMALL UNILAMELLAR LIPOSOMES AND RAT PLASMA HIGH-DENSITY LIPOPROTEINS - DEPENDENCE ON CHOLESTEROL CONTENT AND PHOSPHOLIPID-COMPOSITION

Abstract We investigated the ability of small unilamellar liposomes of various lipid compositions to maintain their integrity in the presence of rat plasma or plasma fractions. Liposomal damage was determined in terms of release of an entrapped water-soluble marker, carboxyfluorescein, and, simultaneously, loss of liposomal 14C-labeled phospholipid. Complete retentions of carboxyfluorescein during a 30-min incubation with plasma could be attained with liposomes containing at least 40 mol% cholesterol. By substituting sphingomyelin for phosphatidylcholine a considerable prolongation of solute retention was achieved. Sphingomyelin/cholesterol (molar ratio 3 : 2) liposomes retained nearly all of the entrapped dye during a 20-h incubation with plasma, while phosphatidylcholine/cholesterol liposomes lost as much as 40%. Incorporation of cholesterol also reduces the transfer of 14C-labeled phospholipid from liposomes to plasma HDL. While transfer of phosphatidylcholine was partially reduced, transfer of sphingomyelin was completely inhibited under the same conditions. Isolated HDL was unable to bring about the extent of carboxyfluorescein release and phospholipid transfer which was observed with whole plasma, but required the addition of lipoprotein-free plasma. The subfraction of HDL that is rich in apolipoprotein E (HDL1) plays at most a minor role in liposome-plasma interactions. Native HDL phospholipids were biosynthetically labeled with 32P and isolated HDL was incubated with 14C-labeled liposomes in presence of lipoprotein-free plasma. In this way we demonstrated that, whereas phosphatidylcholine transfer from cholesterol-poor liposomes is mainly an unidirectional process leading to excessive liposome degradation, phosphatidylcholine transfer from cholesterol-rich liposomes involves an exchange process and is attended by almost complete retention of entrapped solute.

Action of phospholipases A2 on phosphatidylcholine bilayers. Effects of the phase transition, bilayer curvature and structural defects.

We examined the action of porcine pancreatic and bee-venom phospholipase A2 towards bilayers of phosphatidylcholine as a function of several physical characteristics of the lipid-water interface. 1. Unsonicated liposomes of dimyristoyl phosphatidylcholine are degraded by both phospholipases in the temperature region of the phase transition only (cf. Op den Kamp et al. (1974) Biochim. Biophys. Acta 345, 253--256 and Op den Kamp et al. (1975) Biochim. Biophys. Acta 406, 169--177). With sonicates the temperature range in which hydrolysis occurs is much wider. This discrepancy between liposomes and sonicates cannot be ascribed entirely to differences in available substrate surface. 2. Below the phase-transition temperature the phospholipases degrade dimyristoyl phosphatidylcholine single-bilayer vesicles with a strongly curved surface much more effectively than larger single-bilayer vesicles with a relatively low degree of curvature. 3. Vesicles composed of egg phosphatidylcholine can be degraded by pancreatic phospholipase A2 at 37 degrees C, provided that the substrate bilayer is strongly curved. The bee-venom enzyme shows a similar, but less pronounced, preference for small substrate vesicles. 4. In a limited temperature region just above the transition temperature of the substrate the action of both phospholipases initially proceeds with a gradually increasing velocity. This stimulation is presumably due to an increase of the transition temperature, effectuated by the products of the phospholipase action. 5. Structural defects in the substrate bilayer, introduced by sonication below the phase-transition temperature (cf. Lawaczeck et al. (1976) Biochim. Biophys. Acta 443, 313--330) facilitate the action of both phospholipases. The results lead to the general conclusion that structural irregularities in the packing of the substrate molecules facilitate the action of phospholipases A2 on phosphatidylcholine bilayers. Within the phase transition and with bilayers containing structural defects these irregularities represent boundaries between separate lipid domains. The stimulatory effect of strong bilayer curvature can be ascribed to an overall perturbation of the lipid packing as well as to a change in the phase-transition temperature.

Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition

We investigated the intrahepatic distribution of small unilamellar liposomes injected intravenously into rats at a dose of 0.10 mmol of lipid per kg body weight. Sonicated liposomes consisting of cholesterol/sphingomyelin (1:1), (A); cholesterol/egg phosphatidylcholine (1:1), (B); cholesterol/sphingomyelin/phosphatidylserine (5:4:1), (C) or cholesterol/egg-phosphatidylcholine/phosphatidylserine (5:4:1), (D) were labeled by encapsulation of [3H]inulin. The observed differences in rate of blood elimination and hepatic accumulation (A much less than B approximately equal to C less than D) confirmed earlier observations and reflected the rates of uptake of the four liposome formulations by isolated liver macrophages in monolayer culture. Fractionation of the liver into a parenchymal and a non-parenchymal cell fraction revealed that 80-90% of the slowly clearing type-A liposomes were taken up by the parenchymal cells while of the more rapidly eliminated type-B liposomes even more than 95% was associated with the parenchymal cells. Incorporation of phosphatidylserine into the sphingomyelin-based liposomes caused a significant increase in hepatocyte uptake but a much more substantial increase in non-parenchymal cell uptake, resulting in a major shift of the intrahepatic distribution towards the non-parenchymal cell fraction. For the phosphatidylcholine-based liposomes incorporation of phosphatidylserine did not increase the already high uptake by the parenchymal cells while uptake by the non-parenchymal cells was only moderately elevated; this resulted in only a small shift in distribution towards the non-parenchymal cells. The phosphatidylserine-induced increase in liposome uptake by non-parenchymal liver cells was paralleled by an increase in uptake by the spleen. Fractionation of the non-parenchymal liver cells in a Kupffer cell fraction and an endothelial cell fraction showed that even for the slowly eliminated liposomes of type A endothelial cells do not participate to a measurable extent in the elimination process, thus excluding involvement of fluid-phase pinocytosis in the uptake process.

INTRAHEPATIC UPTAKE AND PROCESSING OF INTRAVENOUSLY INJECTED SMALL UNILAMELLAR PHOSPHOLIPID-VESICLES IN RATS

Small unilamellar vesicles consisting of sphingomyelin, cholesterol and phosphatidylserine in a molar ratio of 4:5:1 containing [3H]inulin as a marker of the aqueous space or [Me-14C]choline-labeled sphingomyelin as a marker of the lipid phase were injected intravenously into rats. After separation of the non-parenchymal cells into a Kupffer cell fraction and an endothelial cell fraction by elutriation centrifugation analysis of the radioactivity contents demonstrated that Kupffer cells were actively involved in the uptake of the vesicles whereas endothelial cells did not contribute at all. Uptake by total parenchymal cells was also substantial but, on a per cell base, significantly lower than that by the Kupffer cells. By comparising the fate of the [3H]inulin label and the [14C]sphingomyelin label it was concluded that release of liposomal lipid degradation products especially occurred from Kupffer cells rather than from parenchymal cells. In both cell types, however, substantial proportions of the 14C-label accumulated in the phosphatidylcholine fraction, indicating intracellular degradation of sphingomyelin and subsequent phosphatidylcholine synthesis. Treatment of the animals with the lysosomotropic agent chloroquine prior to liposome injection effectively blocked the conversion of the choline-labeled sphingomyelin into phosphatidylcholine in both cell types. This observation indicates that uptake of the vesicles occurred by way of an endocytic mechanism.

BIODISTRIBUTION OF LIPOSOMES CONTAINING SYNTHETIC GALACTOSE-TERMINATED DIACYLGLYCERYL-POLY(ETHYLENEGLYCOL)S

We describe the synthesis of biodegradable poly(ethyleneglycol)-coupled galactolipids in which the galactose moiety is separated from a diacylglyceride lipid anchor by poly(ethylene glycol) chains of 10, 20 or 40 oxyethylene residues (PEG10/20/40). These Gal-PEG lipids (Gal-PEG-Lip) were incorporated in the bilayer of liposomes. The surface exposure of the galactose was investigated by aggregation experiments with ricinus communis agglutinin 120. Only the liposomes containing the PEG10 galactolipid aggregated with the lectin. Therefore liposomes were prepared containing Gal-PEG10-Lip and a trace amount of [3H]cholesteryl oleyl ether with an average diameter of approximately 100 nm and injected intravenously into rats. The Gal-PEG10-Lip liposomes were cleared from plasma with a T1/2 of 0.3 h. Identically sized and composed control liposomes without the Gal-PEG10-Lip had a T1/2 of approximately 12 h. The rapid plasma elimination of the Gal-PEG10-Lip liposomes could be attributed entirely to increased uptake by the liver amounting to more than 90% of injected dose. Uptake by the spleen was decreased to less than 1% of injected dose. A single injection of N-acetylgalactosamine 1 min prior to Gal-PEG-Lip liposome administration reduced the initial rate of plasma clearance to control levels. The increased liver uptake was almost entirely attributable to increased uptake by the Kupffer cells. Incorporation of PEG-DSPE in the Gal-PEG10-Lip liposomes only partially reversed the effect of the galactolipid with respect to liver and spleen uptake as well as intrahepatic distribution. These experiments demonstrate that liposome surface-exposed galactose residues, even if attached at the distal end of a poly(ethyleneglycol) chain anchored in the liposomal bilayer are effectively recognized by the galactose particle receptor on the Kupffer cells but fail to achieve significant targeting to the asialoglycoprotein receptor on the hepatocytes.

On the mechanism of hepatic transendothelial passage of large liposomes

Liposomes of 400 nm in diameter can cross the 100‐nm fenestrations in the endothelium of the hepatic sinusoid, provided they contain phosphatidylserine (PS) but not phosphatidylglycerol (PG) [Daemen et al. (1997) Hepatology 26, 416]. We present evidence indicating that (i) the PS effect does not involve a pharmacological action of this lipid on the size of the fenestrations, (ii) fluid‐type but not solid‐type PS liposomes have access to the hepatocytes and (iii) the lack of uptake of PG liposomes by hepatocytes is not due to a lack of affinity of the hepatocytes for PG surfaces. We conclude that the mechanism responsible for the uptake of large PS‐containing liposomes by hepatocytes in vivo involves a mechanical deformation of these liposomes during their passage across the endothelial fenestrations.

Immunization strategy against cervical cancer involving an alphavirus vector expressing high levels of a stable fusion protein of human papillomavirus 16 E6 and E7

We are developing immunization strategies against cervical carcinoma and premalignant disease, based on the use of recombinant Semliki Forest virus (SFV) encoding the oncoproteins E6 and E7 from high-risk human papilloma viruses (HPV). Thus far, protein-based, as well as genetic immunization studies have demonstrated low to moderate cellular immune responses against E6 and E7. To improve these responses, we modified the structure and expression level of the E6 and E7 proteins produced by the SFV vector. Specifically, a construct was generated encoding a fusion protein of E6 and E7, while furthermore a translational enhancer was included (enhE6,7). Infection of cells with recombinant SFV-enhE6,7 resulted in the production of large amounts of the E6,7 fusion protein. The fusion protein was more stable than either one of the separate proteins. Immunization of mice with SFV-enhE6,7 resulted in strong, long-lasting HPV-specific cytotoxic T lymphocyte responses. Tumor challenge experiments in mice demonstrated that immunization with SFV-enhE6,7 resulted in prevention of tumor outgrowth and subsequent protection against tumor re-challenge.

Toxicity of doxorubicin entrapped within long-circulating liposomes

We studied the effect of doxorubicin entrapped within long-circulating liposomes (Dox-LCL) on the phagocytic capacity and bacterial blood clearance capacity of rat liver macrophages. Dox-LCL (125 nm in diameter) were composed of egg phosphatidylcholine (PC), cholesterol (CH) and poly(ethyleneglycol)distearoylphosphatidylethanolamine (PEG-PE) (55:45:5 molar ratio; MW PEG 1900), and loaded with doxorubicin by means of a trans-membrane pH gradient. The doxorubicin/lipid ratio was 0.36:1 (mol/mol). At different time-points after one, two or three intravenous injections of Dox-LCL, radiolabeled negatively charged test liposomes (egg PC, CH, and phosphatidylserine in a 4:5:1 molar ratio) were injected. After 2 h, liver macrophages were isolated and the amount of macrophage-associated radioactivity was determined. Twenty-four hours after a single injection of 5 mg/kg Dox-LCL, no significant effect was observed. However, 48 h after injection, phagocytic activity was reduced significantly (49%). Recovery of phagocytic capacity of the liver macrophages took 8 days after two injections of Dox-LCL (2 x 5 mg/kg). Seventy-two hours after the last of two injections of Dox-LCL, bacterial blood clearance was significantly reduced as compared to clearance in control rats and in rats injected twice with doxorubicin combined with placebo liposomes. When comparing these Dox-LCL data with previous data on the effects of Dox-L, Dox-LCL appears less toxic than Dox-L for the liver macrophage population following i.v. administration both with respect to specific phagocytic activity and cell numbers. Due to the delay in onset of toxic effects and the faster recovery from Dox-LCL treatment as compared to Dox-L treatment, it is conceivable that therapeutic protocols can be designed with Dox-LCL that circumvent long-term impairment of the liver macrophage population.

Eradication of established HPV16-transformed tumours after immunisation with recombinant Semliki Forest virus expressing a fusion protein of E6 and E7

Previously, we described the efficacy of immunisation with recombinant Semliki Forest virus (SFV), expressing the human papillomavirus 16 (HPV) oncoproteins E6 and E7, in inducing HPV-specific CTLs and anti-tumour responses. Recently, we developed a novel recombinant SFV construct encoding a relatively stable fusion protein of HPV16 E6 and E7 under control of a translational enhancer derived from the SFV capsid protein. In the present study we demonstrate that immunisation of tumour-bearing mice with this improved vector results in the regression and complete elimination of established tumours. We furthermore demonstrate that a long-term high level of CTL activity, up to 340 days, accompanies the anti-tumour response. Thus, immunisation with recombinant SFV particles encoding increased levels of a fusion protein of HPV16 E6 and E7 efficiently induces CTL activity and CTL memory resulting in a potent therapeutic anti-tumour effect.

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles.

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24 degrees C. At 37 or 15 degrees C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296--307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37 degrees C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35 degrees C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24 degrees C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed. The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization. When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly dissolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed. It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253--256).