Preparation and characterization of liposomes with incorporated Neisseria gonorrhoeae protein ib and amphiphilic adjuvants

Journal

of Controlled Release, 7 (1988) 123-132

Elsevier Science Publishers

B.V., Amsterdam

-

123 Printed

in The Netherlands

PREPARATION AND CHARACTERIZATION OF LIPOSOMES WITH INCORPORATED Neisseria gonorrhoeae PROTEIN IB AND AMPHIPHILIC ADJUVANTS Frans van Dalen’, Crommelin’*” ‘Department

Gideon Kersten2, Tom Teerlink2, E. Coen Beuvery2 and Daan J.A.

of Pharmaceutics,

University of Utrecht, Croesestraat

79,3522

AD Utrecht (The Netherlands)

‘Departments of Bacterial Vaccines and Inactivated Viral Vaccines, National institute of Public Health and Environmental Hygiene (RIVM), P.O. Box 1, 3720 BA Bilthoven (The Netherlands) (Received May 19, 1987; accepted in revised form December 9, 1987)

Liposomes were prepared according to a three-step procedure. Octylglucoside, lipid and optionally protein (outer membrane protein IB from N. gonorrhoeae), lipid A or dimethyldioctadecylammonium bromide (DDA) containing mixed micelle dispersions were diluted, then dialysed and finally filtrated. The liposome preparations were characterized for their particle size (both freshly prepared and after storage) and the contents of the different constituents. Data on the orientation of protein IB in the bilayer were collected. Stable, well-defined liposomes could be obtained with eggphosphatidylcholine/cholesterol bilayers containing optionally DDA or lipidA with or without protein IB. For dipalmitoylphosphatidylcholine/cholesterol combinations a charge-inducing agent (DDA or dipalmitoylphosphatidylglycerol [DPPG]) was required to stabilise the liposomes which further contained (optionally) lipid A (only with dipalmitoylphosphatidylcholine/cholesterol/ DPPG) with or without protein IB. In general, the uptake of all constituents into the bilayer was almost quantitative. Enzymatic degradation experiments showed that protein IB had the same orientation and surface exposure as in the bacterial outer membrane.

INTRODUCTION

Several preparation techniques for liposomes as carriers for antigens such as proteins have been described [l-9]. Apart from the antigens also adjuvants (e.g. lipid A, dimethyldioctadecylammonium bromide [ DDA] ) can be incorporated into the bilayer structure to stimulate the immune response [ 10-141. Product characterization and stability is essential to guarantee the reproducibility of the immune response stimulation in vivo. However, little attention has been paid to characterize the product properties in terms of their physico*To whom correspondence

016%3659/88/$03.50

should be addressed.

0 1988 Elsevier Science Publishers

chemical and chemical nature and to study the stability against aggregation and fusion. Here we describe a simple method based on the detergent removal principle to prepare well-defined protein-containing liposomes. The method consists of a sequence of steps, i.e. dilution of a mixed micellar solution, dialysis to remove detergent and filtration to obtain a narrow particle-size distribution. The applicability of this technique was tested under different conditions. Variables under investigation were the composition of the bilayer, the temperature during liposome formation and the ionic strength of the medium. Variation of the bilayer structure included, apart from variation of the phospholipid contents, also the in-

B.V.

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corporation of amphiphilic proteins such as the outer membrane protein IB of N. gonorrhoeae and amphiphilic adjuvants such as lipid A and DDA. The monitored parameters were: (1) particle size of the vesicles after finishing the preparation process and on storage, (2) recoveries of phosphate, cholesterol, protein, octylglucoside and DDA. We also collected data on the orientation of the liposome-incorporated membrane protein. The non-ionic detergent octylglucoside was used for several reasons. Complete removal can be achieved by dialysis because of its high critical micelle concentration (CMC) [ 151. Besides, it has proven to be a suitable detergent for the reincorporation of several other proteins in liposomes without affecting their biological activities [ 16,171.

MATERIALS

AND METHODS

Materials

Egg yolk L-cx-phosphatidylcholine type V-E (PC), dipalmitoyl-L-cu-phosphatidyl choline (DPPC ), dipalmitoyl-DL-a-phosphatidyl-Lglycerol ammonium salt (DPPG), cholesterol (CHOL), n-octyl-P-D-glucopyranoside (octylglucoside ), n-decyl-P-D-glucopyranoside (decylglucoside), bromotrimethylsilane (BTMS), bis (trimethylsilyl) trifluoroacetamide (BSTFA) with 1% trimethylsilane added, and chymotrypsine were purchased from Sigma Chemical Company (St. Louis, MO., U.S.A.). Dimethyldioctadecylammonium bromide (DDA) was supplied by Eastman Kodak (Rochester, N.Y., U.S.A.). Lipid A was obtained from Calbiochem-Behring, La Jolla, Ca., U.S.A. Lipid A contains 0.6 pmol P/mg (determined by phosphate analysis). Tetrabromophenol sulphonephthaleine was obtained from Merck, Stuttgard, F.R.G. All other reagents or solvents were of analytical grade. The gonococcal protein IB (strain C3 ) was isolated and purified according to the procedure described by

Jiskoot et al. [lo]. Protein IB was stored at 4’ C in buffer containing 50 mM tris(hydroxymethyl)aminomethane (Tris), 200 mA4 sodium chloride, 10 mM EDTA, 0.05% zwittergent 23-14 (Serva, Heidelberg, F.R.G.) and 0.02% sodium azide. Preparation

of mixed micelles

PC, DPPC or DPPG were dissolved in a 1:l chloroform/methanol mixture, DDA and cholesterol were dissolved in chloroform. In a pearshaped flask the phospholipid(s), CHOL and DDA (if used) were mixed and dried on a rotary evaporator at 30°C. The film was left under vacuum (3 kPa) for l-2 hours. An appropriate amount of a solution of 150 mM octylglucoside in solution A (10 mM Tris, 0.9% sodium chloride adjusted to pH 7.4 with diluted HCl), solution B (demineralized water adjusted to pH 7.4 with diluted NaOH), solution C (10 mM Tris, 0.9% sodium chloride adjusted to pH 3.0 with diluted HCl) or solution D (demineralised water adjusted to pH 3.0 with diluted HCl) was added. Solutions B or D were used to obtain a medium with low ionic strength; solutions C or D were used when both the positively charged DDA and protein IB had to be incorporated into the micelle. Solubilization was accomplished by gently shaking and warming of the flask to 45’ C in a water bath until the film was completely solubilized. Lipid A (if used; molar ratio lipid A/phospholipids 0.0075) was incorporated by the procedure described by Schuster et al. [ 181. A concentrated aqueous suspension (5 mg/ml in 0.5% triethylamine) was introduced into the flask and dried under a nitrogen stream with regular addition of chloroform. When dry, the other lipids were brought into the flask as described above. To incorporate protein IB it had to be precipitated first from the stored solution by adding 96% ethanol to a final concentration of 80% (vol/vol). After centrifugation (10 min, 4000 rpm, Hettich/Rotanta 3500, Tiittingen,

125

F.R.G. ) the precipitate was dried under a nitrogen stream to remove traces of ethanol. Protein IB was resolubilized in 150 mM octylglucoside in solution A, B, C or D at room temperature.

cording to Peterson 1211 and modified f22] by means of an extra washing procedure with diethylether/ethanol3:1 (v/v) to remove interfering substances.

Preparation of liposomes: Diiution-dialysisfiltration technique

~cty~lucoside and CHOL ~o~entrutio~ were determined by a gas chromatographic method. Analysis was performed by using the silylation procedure described by Jiskoot et al. [ 231. With decylglucoside as an internal standard and with the standard addition technique. An amount of 1~1 of the derivatized samples was injected in a Hewlett-Packard 5710 A gas chromatograph equiped with a HP 5702 B oven temperature programmer and flame ionisation detection (detector temperature 350’ C ) . The stationary phase was 3% OV-1 on Chromosorb WHP and the mobile phase was nitrogen gas (20 ml/min ). The injector temperature was 250°C. A temperature program controlling the column temperature was started at the moment of injection: 3 minutes isothermal at 23O”C, then the column was warmed up at a rate of 15 ‘C per minute until the column temperature reached 300’ C. Octylglucoside and CHOL contents were calculated from peak heights (relative to decylglucoside peak heights) of the samples with and without octylglucoside and CHOL standards.

The desired amounts of the mixed micelles were combined in a water-jacketed glass chamber at 25” C or 45” C to yield 2.0 ml of mixed micelles with a phospholipid concentration of 11.4 mM and an initial ratio of about 55 ,ugprotein to 1 pmol phospholipid..The mixed micelle dispersions were ma~etically stirred. After 5 minutes the mixed micelles were diluted 11 times with 20.0 ml solution A, B, C or D in 16 seconds by means of a automatic titration unit (Multidosimat E415, Metrohm, Herisau, Switzerland). The dispersion was kept at the temperature of preparation for at least 5 minutes before the pH was adjusted to 7.4 with diluted NaOH (if necessary). An amount of 18 ml of the liposomal dispersion was dialysed for 66 hours at 4°C protected from light against 260 ml of solution A or B in a shaking bath (120 strokes per minute, Grant Industries Ltd., Cambridge, U.K.). The solution was refreshed every 22 hours. High-permeability cellulose membranes (M, cut-off 10,000, Diachema, Riischlikon, Zurich, Switzerland) with an exchange area of 10 cm2 were used. After dialysis the liposomes were filtered first through a 0.6 pm and then through a 0.2 ,um polycarbonate membrane filter (Nuclepore Corp., Pleasanton, Ca. ) under 0.7 MPa nitrogen. Analytical methods

Phospholipid concentrations were determined by inorganic phosphate analysis after destruction with perchloric acid at 180°C according to Bartlett [ 191 in a modification of the method of Bijttcher et al. [ 20 1. Protein co~entratio~

were determined ac-

The DDA concentration was determined with an ion-pair extraction technique with tetrabromophenolsulphonephthalein (bromopheno1 blue) followed by spectrophotometric determination of the extracted blue complex [ 24,251. We modified the method with a cleanup procedure because choline-based phospholipids and lipids interfered with the ion-pair formation. The mean hydrodynamic diameters of the liposomes were calculated from the mean diffusion coefficients determined by dynamic light scattering measurements (System 4600, Malvern Instruments Ltd., Worcestershire, U.K., equipped with a 27 mW He-Ne laser, NEC Cor-

126

poration, Tokyo, Japan) followed by on-line analysis of the autocorrelation function with a 64-channel lin-log correlator (particle analysis processor type 7027, Malvern Inst~ments Ltd., Worcestershire, U.K.). Apart from the calculated mean hydrodynamic diameter, a parameter related to the polydispersity of the dispersion was obtained. Measurements were carried out in a temperature-controlled scattering cell holder at 25.0” C and a scattering angle of 120”. Unless otherwise stated the size measurements were performed within two hours after filtration. Rate and extent of chymotrypsin-induced degradation of protein IB

In order to determine the orientation of protein IB in the bilayer of “gel” and “fluid” state liposomes prepared with the dilution-dialysisfiltration method, these dispersions were exposed to chymotrypsin. The rate and final extent of protein IB degradation in liposomes was compared with protein IB in zwittergent micelles and with protein IB in a natural environment, that is, in outer membrane complexes (OMC ) produced by iV. gonorrhoeae. DPPC:CHOL:DPPG (7:2:1) and PC:CHOL (7:2) liposomes were prepared as described above. Protein IB in zwittergent 23-14 was the same dispersion as mentioned in the ~~~eriu~ section, OMC were isolated as described by Jiskoot et al. [lo]. The chymotr~sin/protein IB ratio was 0.04 (weight basis). The protein IB concentration was about 35 lug/ml in all preparations. Samples were taken prior to addition of the chymotrypsin solution (0.5 mg/ml), and 2.5,lO and 60 min after addition incubation was performed at room temperature. The samples were heated for one min at 100°C to stop digestion, cooled in ice, and analysed with SDS-PAGE according to Laemmli [ 261. A 16% acrylamide gel was used; this gel was stained with Coomassie Brilliant Blue. Intact protein IB, banding at the 35 kD position, was quantified relative to non-

incubated samples by densitometric with a laser densitometer (Ultrascan Bromma, Sweden 1.

scanning XL, LKB,

RESULTS Physic~~hemical liposomes

characteristics

of

the

In Figs. 1-6 an overall view is given of the particle size of the different liposomal dispersions that were prepared by varying the temperature, the ionic strength of the medium, and the charge on the particles, in the presence or absence of protein IB or adjuvants (lipid A or DDA). The average size was determined both immediately after filtration and after storage in the refrigerator for 4 weeks. All dispersions were prepared at least in duplicate. Mean hydrodynamic diameters CHOL (“gel” state) liposomes

of the DPPCf

No stable neutral DPPC:CHOL 7:2 liposomes could be prepared at 45’ C and an ionic strength (I) of 0.16 A4 (Fig. 1). After every preparation step aggregates appeared immediately. Introduction of a charged constituent (DPPG or DDA ) in the mixed micelles resulted in non-aggregating liposomes with a low polydispersity level after the described three-step process. The negatively charged liposomes had a diameter of about 0.28 pm and the positively charged of about 0.34 pm. Incorporation of protein IB increased the diameters of the DPPGcontaining liposomes to 0.33 pm and of the DDA-containing liposomes to 0.56~m. The size of negatively charged liposomes with lipid A was not affected by incorporation of protein IB. A reduction of the preparation temperature from 45°C to 25°C (1=0.16 M) resulted in smaller negatively charged liposomes in presence of protein IB. Only the DDA-containing liposomes with protein IB increased in size with

127

GEL STATE LIPOSOMES

T = 45% AND I

q

0.6

0.6

size in

size in

pm

pm

1

0

‘4

F

S

F

S

F positive

S

negative

F

S

GEL STATE LIPOSOMES

T = 45% AND I

GEL STATE LIPOSOMES

0.6

size in

size in F

pm

/I

S

F

S

negative

T

q

25;

AND I

A

LOW

q

F

0

0 F

s

neutral

F

S

F

positive

S

negative

F

FLUID STATE LIPOSOMES

T

.F

S

F

S

S

negative

F

FLUID STATE LIPOSOMES

0.6

in

size in

T

q

S

negative lipid

0.6

pm

F positive

A

25 “c AND I = 0.16M

q

S

neutral

negative lipid

size

F

:

lipid

0.6

pm

F

A

LOW

q

S

25% AND I = 0.16M

q

positive

negative lipid

T

GEL STATE LIPOSOMES

0.16M

25;

A

AND I = LOW

pm

0

0 F

S

neutral

F positive

S

F

S

neutral lipid A

F

S

neutral

F positive

S

F

S

neutral lipid A

Figs. 1-6. Liposome diameters as a function of bilayer composition, protein incorporation and ageing. F: liposomes after filtration; S: liposomes stored at 4°C for 4 weeks; -: liposomes without protein IB incorporated; +: liposomes with protein IB incorporated; neutral: PC:CHOL (7:2) or DPPC:CHOL (7:2) liposomes; positive: PC:CHOL:DDA (7:2:1) or DPPC:CHOL:DDA (7:2:1) liposomes; negative: DPPC:CHOL:DPPG (7:2:1) liposomes; lipid A: liposomes with lipid A incorporated, Z=low: hydration medium B or D; Z=O.16 M hydration medium A or C.

128

0.08 pm. The polydispersity was not influenced by the reduction of the temperature. Reduction of the ionic strength by excluding sodium chloride and Tris from the medium (I= low) substantially reduced the size from the charged as well as the uncharged liposomes. Non-aggregating liposomes could again only be produced after introducing a charged constituent. The diameter of DPPG-containing liposomes was about 0.08 pm. This small diameter was independent of the temperature of preparation, incorporation of lipid A and/or protein IB. The DDA-containing liposomes again were slightly larger. No definite trend could be observed when the temperature was lowered or protein IB incorporated. In low ionic strength media, the systems tended to have higher polydispersity levels than comparable liposomes prepared in high ionic strength media. DDAcontaining liposomes in particular showed relatively large polydispersity factors, indicating that the dispersions were heterogeneous with respect to particle size. Mean hydrodynamic diameters CHOL (“fluid” state) liposomes

of the

PC/

No charge-inducing agent was necessary to stabilize PC:CHOL 7:2 liposomes, prepared by diluting mixed micelles in a medium with an ionic strength of 0.16 M at 25’ C. Incorporation of protein IB with or without lipid A or DDA induced at most a 25 nm reduction in diameter. The presence of DDA had no effect on the polydispersity of the system as it had with DPPCcontaining liposomes. Reducing the ionic strength of the medium did not result in an extensive decrease in particle size. Reductions between 0 and 15% were observed. Recovery

of liposomal

constituents

The phosphate recoveries were about 90% in the negatively charged DPPG-containing liposomes, independent of temperature. In DDA-

containing liposomes recovery was 15% lower at I=O.16 M, independent of protein IB incorporation. In uncharged DPPC:CHOL 7:2 liposomes only 50% was recovered at both temperatures. Reduction of the ionic strength increased the recoveries in the positively charged and uncharged liposomes with 15 to 20%. The recoveries of the DPPG-containing liposomes were not affected. The protein, cholesterol and octylglucoside recoveries were corrected for the loss in phosphate during the preparation process with [X,il/X,,] x [Pdil/ Pa1 x 100% ] where P stands for the amount of phosphate and X for the amount of another constituent present in the dispersion after dilution (dil) or filtration (fil). The protein recoveries in both the PC/CHOL and DPPC/ CHOL liposomes ranged from 90 to 100% (the final ratio of protein IB to phospholipid varied from 35 to 40 ,ug protein/pmol phospholipid). The recoveries obtained from DDA-containing liposomes were lo-15% higher. No clear trends were observed when ionic strength or temperature of preparation were changed or when lipid A was incorporated. In all cases the molar ratio octylglucoside/ phospholipid was less than 0.006 after filtration; 0.006 equals the lowest detectable ratio under these circumstances. The actual octylglucoside/phospholipid ratio might be even lower. For DPPG-, DDA- and PC-containing particles, the temperature of preparation, ionic strength and presence of protein IB had no distinct effect on the cholesterol recouery. The reranged from 90 to 125%. In covery DPPC:CHOL 7:2 liposomes, the absence of a charge resulted in high recoveries in media with high ionic strength and low recoveries (around 55%) in media with low ionic strength. The variation in the recoveries between the different liposome dispersions was more pronounced in “gel” state than in “fluid” state liposomes. Nearly all DDA was recovered from DPPCcontaining liposomes (85-120% ). There was a tendency for recoveries to increase in case of

129

low preparation temperatures and if protein IB was incorporated. The recoveries for PC-containing liposomes were much lower (40-75% ) . Stability

of the liposomes

In general, the mean hydrodynamic diameter of the particles increased after storage for 4 weeks at 4°C. This diameter growth was negligible (about 4% ) for the “fluid” state liposomes, and did not depend on ionic strength or liposome composition for the liposome types studied. The polydispersity level of the systems did not change. The size of the “gel” state liposomes without DDA increased about 8% in buffered saline media and about 20% in the medium with low ionic strength. A 30% growth was observed in DDA-containing liposomes prepared at 25 “C. The polydispersity factor also tended to increase. In almost all DDA-containing liposomes white flakes appeared after storage for 3 to 4 weeks. This flocculation phenomenon did not depend on the state of the bilayer, protein IB incorporation, preparation temperature or ionic strength of the medium. Liposome dispersions prepared in media with low ionic strength were unstable on dilution at room temperature with 0.9% NaCl solution; the particle sizes increased within 45 minutes to 3 pm. Dilution with 5% glucose solution appeared to have the same effect, but larger particles were formed at a much slower rate. The

mean diameter of the dispersions increased from 0.12 pm to 0.24 ,um within 80 minutes. This phenomenon was independent of the temperature of preparation, incorporation of protein IB or the selected bilayer constituents. Within 24 hours “particle sizes” of 0.7 pm were reached. Orientation

of the protein I6 in liposomes

In the bacterial outer membrane protein IB has both termini embedded in the membrane, leaving a central region exposed at the surface [ 271. This exposed region is susceptible to proteolysis. Upon cleavage with chymotrypsin the protein is split into two fragments with molecular weights of 20 and 15 kD which both remain associated with the membrane [ 271. If the protein is inserted into the liposomal bilayer in the correct orientation, a similar degradation pattern should be observed. Protein IB molecules that are inserted in the wrong orientation will have their surface-exposed region inside the liposome. Therefore, incorrectly inserted protein IB is expected to resist proteolysis by chymotrypsin. Table 1 shows a comparison of the extent of degradation of protein IB incorporated in liposomes and as present in the outer membrane. In all preparations 90% of the protein IB could be degraded. SDS-PAGE analysis revealed the presence of the expected degradation products of 20 and 15 kD in all preparations. Apart from

TABLE 1 Chymotrypsin-mediated degradation of protein IB incorporated into protein-detergent in the native outer membrane; experimental details are given in the text

micelles, liposomes

and as present

(% )

Incubation time (min)

Protein

Protein-detergenta micelle

OMCb

PC/CHOL 7:2

DPPC/CHOL/DPPG 7:2:1

2.5 10 60

49 73 88

82 82 88

88 84 90

86 94 95

IB degradation

*Protein-detergent micelle: protein IB/zwittergent 23-14 = 1:14. bOMC: outer membrane complexes from N. gonorrhoeae.

130

the conclusion that protein IB was readily available for degradation and thus was mainly exposed to the exterior phase, the results also indicate that no major conformational changes occurred during isolation and incorporation. Chymotrypsin is a rather non-specific protease; fully denatured protein IB is degraded by this enzyme in several low molecular weight fragments.

DISCUSSION With the exception of lipid A all liposomal constituents were determined without the use of radioactive tracer techniques. A determination of lipid A recovery by means of a GLC method is at present under investigation. Phospholipid recovery varied. For most dispersions less than 20% was lost during the preparation procedure. As a rule the protein, cholesterol and DDA were completely taken up by the liposomes. The recovery of DDA from the “fluid” state liposomes was - for unknown reasons far from complete. In the present work it was shown that it is possible to prepare small and practically detergent-free vesicles by dilution of a solution consisting of mixed micelles, followed by detergent removal by dialysis and a filtration step. The reproducibility and stability of the physical characteristics of the liposomes depended on the bilayer components. Critical parameters were bilayer fluidity and the presence of charge-inducing agents in the bilayer. By a proper selection of experimental conditions and bilayer constituents stable liposomes could be produced reproducibly. It was difficult to prove the unilamellar character of the vesicles by means of 31P-NMR [ 231. DDA interferes with the broadening agent Mn2+ for unknown reasons [ 231, and the pore protein IB probably allows Mn2+ to pass through the bilayer. Concentrated dispersions of liposomes consisting of DPPC:CHOL:DPPG (7:2:1) prepared in media with high ionic

strength showed no interference and proved to be unilamellar (data not shown). The mean diameters of the “gel” state liposomes (1=0.16 M) exceeded the 1.0 pm level after dilution (results not shown). These dispersions were also highly polydisperse. Filtration dramatically reduced both the diameter and polydispersity of these dispersions. For the “gel” state liposomes prepared under low ionic strength conditions and for all “fluid’ state liposomes filtration only caused a minor (or no) size reduction (cf. Ref. [23] ). No clear relationship between size reduction and phospholipid recovery could be established. Therefore, this size reduction for “gel” state liposomes (I=O.16 M) is - at least partly - caused by extrusion of the oversized vesicles or deaggregation of clusters of vesicles or a combination of these effects. A number of studies were published dealing with the effects of experimental conditions (e.g. ionic strength, temperature and dilution rate) and bilayer composition (e.g. presence of charge-inducing agents, proteins) on the size and stability of the dispersions prepared via the detergent removal method [23,28-391.It is clear that the rigidity of the bilayer and electrostatical repulsion are important parameters. The theoretical models predict that a low bilayer rigidity and high electrostatical repulsion will provide small, highly curved, liposomes. However, the experimental results in this study are often in conflict with these general rules. One can only speculate about the factors that are responsible for these deviations. A symmetrical orientation of the protein in the liposome bilayers with the protein equally extending from the inner and outer bilayer surface, has been observed if the detergent remethod was used. Symmetrical moval distribution of proteins over both sides of the bilayer were reported for example for the dalanine carboxypeptidase from Bacillus stearothermophilus [ 401, the spike proteins of Sendai virus [41] and the haemagglutinin of influenza A [ 42 1.An asymmetrical orientation,

131

however, where the protein is exposed to the exterior of the vesicle, is more often encountered, i.e. with the spike proteins of Semliki Forest virus [ 341, the glycoprotein of vesicular stomatitis virus [ 431, the membrane-bound penicillinase of Bacillus licheniformis [ 441, the glycoprotein of rabies virus [ 451, the gp85 glycoprotein of Friend murine leukemia virus [ 461, the phage Ml3 coat protein [47], the acetylcholine receptor of Torpedo californica [48] and the human leucocyte antigens [ 491. Gonococcal protein IB has probably two hydrophobic moieties, both embedded in the bilayer of the OMC in which it is asymmetrically oriented with its surface-exposed region facing outwards [ 271. The results of the enzymatic degradation experiment with a neutral “fluid” state and a negatively charged “gel” state liposome show that protein IB associated with liposomes was mainly exposed to the exterior phase. Our results, however, do not allow to draw definite conclusions on the exact orientation of protein IB in the bilayers of both types of vesicles. It is known that the physical state of the bilayer (below or above the transition temperature T, ) can strongly affect the protein-bilayer interaction [ 471. The model proposed by Helenius et al. [50] predicts the observed asymmetrical protein IB orientation in the liposome obtained after fast removal of detergent by dilution. This would mean that protein IB exists in the mixed micellar dispersion predominantly as oligomerit detergent-protein complexes and not as detergent-protein-lipid micelles. The model proposed by Jackson and Litman [ 511 predicts a symmetric orientation of protein IB in liposomes if the detergent is removed very fast from the mixed micellar dispersion by dilution. Wickner 1471 found that the efficiency of protein incorporation depended on the temperature. Above the T, protein uptake was limited in DMPC (dimyristoylphosphatidylcholine ) and DLPC (dilauroylphosphatidylcholine ) vesicles. Below these temperatures much more protein was incorporated. We did not observe this temperature effect on protein IB uptake.

It is quite remarkable that stable “gel” state liposomes could be formed below the transition temperature. This finding is in contrast with REV and MLV preparation procedures where the bilayer should be in the “fluid” state to produce liposomes. This result opens the possibility to produce protein-containing liposomes at temperatures where heat-sensitive proteins do not denature. This can be useful for the preparation of vaccines based on immunogenicityenhancing phospholipids with a high T, [ 52,531. To summarize, the described method produces stable, detergent-free, negatively or positively charged “gel” state liposomes both above and below the transition temperature range and neutral “fluid” state liposomes. All these liposomes can incorporate amphiphilic substances with high recoveries for all constituents (phosphate, protein and CHOL). The results are not dramatically affected by preparation temperature or the incorporation of protein IB or lipid A. Limited control of particle sizes can be achieved by manipulating the ionic strength of the medium. The preparation procedure has the potential to produce dispersions with optimum antigen-presenting properties which can be used in vaccines. REFERENCES H. Tamauchi, T. Tadakuma, T. Yasuda, T. Tsumita and K. Saito, Immunology, 50 (1983) 605-612. D. Gerlier, 0. Bakouche and J.F. Dore, J. Immunol., 131 (1983)485-490. N.F. Pierce, J.B. Sacci, Jr., C.R. Alvingand E.C. Richardson, Rev. Infect. Dis., 6 (1984) 563-566. L. Huang and S.J. Kennel, Biochemistry, 18 (1979) 1702-1707. N.F. Moore, Y. Barenholz and R.R. Wagner, J. Virol., 19 (1979) 126. M. Trudel, F. Marchessault and P. Payment, J. Virol. Meth., 3 (1981) 187-192. P. Casali, J.G.P. Sissons, R.S. Fujinami and M.B.A. Oldstone, J. Gen. Virol., 54 (1981) 161-171. P.T. Naylor, H.S. Larsen, L. Huang and B.T. Rouse, Infect. Immunol., 36 (1982) 1209-1216. J.R. North, A.J. Morgan, J.L. Thompson and M.A. Epstein, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 75047508.

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