Assembly of protein structures on liposomes by non-specific and specific interactions

Adv.

ASSEMBLY LIPOSOMES INTERACTIONS

OF PROTEIN BY NON-SPECIFIC

ORLIN

Biophys.,

Vol.

34, pp.

139-157

(1997)

STRUCTURES ON AND SPECIFIC

D. VELEV”

Nagayama Protein Array Project, ERATO,JRDC, Tsukuba 300-26, Japan

Tokodai,

Proteins as well as lipids are two of the most important structural components of living matter. Since liposomes were introduced as an artificially created structure, able to mimic natural lipid bilayer membranes (see refs. I, 2), there have been numerous advances in the field of using liposomes as a base for creating complex, functional microparticles for medicine, biology, and industry (2-5). Liposomes can be used as biocompatible microcapsules for drug delivery (2,3, 69). Attempts are being made to combine this function with the introduction of receptors on the liposome surface that would make the liposomes target specific cells or tissues inside a living organism (3,1U, 11). The use of liposomes as matrixes for reconstitution of biological membrane-mediated functions has also been widely investigated (1214). Usually this has been performed with integral or membrane-anchored proteins, like bacteriorhodopsin (15-17) or cholera toxin (18, 19) and human coagulation factor Va (20). There have not been many attempts to artificially target and attach soluble proteins on the outer side of the liposome surface. Streptavidin has been attached to lipo-

*Present Delaware

address: 19716,

Department

of Chemical

Engineering,

U.S.A. 139

University

of Delaware,

Newark,

140

O.D.

VELEV

somes by incorporating biotinylated lipid inside their membrane (22, 22) and vesicle agglutination by lectin-oligosaccharide binding has been studied (23). The exact conformation and structure of the protein layer over the vesicles have not been determined. Another approach to this field has been provided by the use of reconstituted crystalline bacterial surface layers (S-layers) (24-26). It has been demonstrated that the S-layer covered liposomes can be used as a convenient base, to which an outer shell of proteins like ferritin can be attached (27). Most of the liposome-protein structures obtained to date are important mainly from the viewpoint of understanding the basic principles of their assembly and interactions. The accumulation of data and knowledge in the field could lead to exciting new possibilities, principally new medicines and “smart” biotechnological materials (28-30). A variety of interesting structures have been obtained recently (23, 26, 27,3&34), and the liposomes may turn out to be an excellent assembly site for artificial liposome/protein devices. In the present study we investigate the formation of ferritin arrays and shells over the surfaces of liposomes. Ferritin (35) is a water soluble protein that does not exhibit any type of specific affinity with lipids. To assemble the liposome/ferritin structures, we explore a number of alternative colloid interactions - non-specific (electrostatic, Section I), or specific lock-and-key types (avidin-biotin in Section II and lectin-polysaccharide in Section III).

I.

FERRITIN

ARRAY

ASSEMBLY

ON

LIPOSOMES

BY ELECTROSTATIC

IN-

TERACTIONS

The most simple approach to attracting and ordering of the ferritin molecules to the liposome surfaces is to use electrostatic interaction between the ferritin and the charged liposomes. This approach has been widely used for the two-dimensional (2D) crystallisation of different proteins on planar lipid monolayers or charged interfaces (3640). The 2D crystallisation of ferritin on a planar lipid layer was first reported by Fromherz (41). The challenge in our study was to find appropriate liposome composition and environmental conditions for assembling the ferritin onto the spherical vesicles and to work out suitable procedures for preparation of the electron microscopy samples. The liposome suspensions for the experiments were produced by the established extrusion technique (42, 43) at 50°C through stacked polycarbonate membranes with pore diameters of 100 nm. We tried a number of lipid compositions, seeking to obtain liposomes that would

ASSEMBLY

OF

PROTEIN

STRUCTURES

ON

LIPOSOZlES

141

not be rigid and flocculating, but also not too fluid and would preserve their shape during the staining process for the electron microscopy. The best combination found that was used to obtain the results described hereafter was a mixture of purified L-a-lecithin (P-5763 from Sigma) and 1O-20 wt% dipalmitoyl-L-a-phosphatidylcholine (DPPC) (Sigma). The surface charge of ferritin is determined by the pH of the buffered aqueous media. At pH above the isoelectric point of the ferritin molecules (PI= 4.8, ref. 44), the net charge of the protein is negative and positively charged liposomes are required to attract and assemble the protein. Eicosic trimethyl ammonium bromide has been used as a source of positive charge of planar monolayers (41). Fatty amines (like stearylamine) have been reported (45) as appropriate additives for positive charging of lipid vesicles. In our experience, none of these was found to produce fair results with liposomes, due to difficulties with the extrusion, uneven shape and high flocculation of the vesicles. For this reason, we chose as a source of the positive charges a typical cationic surfactant - hexadecyl trimethyl ammonium bromide (HTAB). The assembled structures were studied by transmission electron microscopy (TEM). The micrographs presented hereafter were obtained on a 100 kV JEOL JEMl200EX microscope. Most of the samples were prepared for observation by negative staining. The staining procedure of the delicate liposome/ferritin assemblies required a number of precautions. The carbon film over the grids was carefully hydrophilised by glow discharge to avoid background adsorption of protein. The staining was carried out with a 2% solution of phosphotungstic acid, neutralised to a sodium salt by careful addition of 1 M NaOH until it reached the pH of the liposome suspension. The stained grids were dried in an air chamber of relative humidity lower than 35%.

1.

Effect of pH and HTAB

Concentration

As expected, no coating of the positively charged vesicles was observed at pH = 4 below the isoelectric point of ferritin (citrate-phosphate buffered saline). At pH = 5.8, sustained by 0.02 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer in a 0.08 M NaCl solution, ordered ferritin shells around the vesicles were observed and the data described below were obtained under these conditions. This pH is close to those reported earlier in the 2D crystallisation of ferritin (40). When we increased the pH up to 7 (in phosphate buffered saline), a heavier, but

142

O.D.

Fig. tracted hydrated

1.

EM by

micrographs

electrostatic sample. The

of lecithin

vesicles

interactions. a, negative diameter of each ferritin

with

attached

staining molecule

shell

preparation; is -10 nm.

VELEV

of ferritin b, frozen-

at-

ASSEMBLY

OF PROTEIN

STRUCTURES

ON

LIPOSOMES

143

disordered coating of the liposomes was observed. Using HTAB as a source of positive charges of the liposomes resulted in easily extruded, stable liposome suspensions. In this case one has to account for the solubility of the HTAB in water and the desorption of the HTAB from the liposomes after dilution. We found out that the charge is lost within a few hours if the liposomes are heavily diluted by pure saline. To prevent this, a certain amount of HTAB was always added to the aqueous environment. This turned out to be an easy way of directly controlling the quality of the obtained assemblies. The liposomes were extruded in a suspension of 5 mg/ml in a molar ratio of lipid/HTAB of 15/ 1. This preparation was diluted 100 times with a buffered suspension of 250pg/ml ferritin. When soluble HTAB was added to these samples at a concentration of 15-30 pg/ml, vesicles with ordered ferritin shells were observed in the stained TEM samples (Fig. la). It should be noted, that there is a cooperative effect of complete coating of some of the vesicles, while others remain naked. Decreasing the HTAB concentration below 15 pg/ml resulted in poorly coated liposomes and increasing it above 36 pg/ml led to overloading of the liposomes with disordered protein. The structured shells of ferritin originate from the electrostatic attraction of the protein molecules to the lipid vesicles. This attraction appears to be a short-ranged one, as we proved that incorporation of a non-ionic surfactant with water-penetrating polyoxyethylene chains (Tween 20) in the vesicles totally suppresses the structure formation by sterically screening the liposomes. The short-ranged electrostatic interaction agrees well with the high salinity of the environment - the range of the interaction will be about 2 nm (46), assuming that it is about twice the Debye screening length. Possibly, as suggested earlier (40), the formation of the shells is also assisted by lateral electrostatic interactions between the ferritin molecules. This is supported by the observed cooperative effect. 2. Observation and Properties of the Obtained Samples While pictures of ordered shells of ferritin around the liposomes were obtained by negative staining, we had to prove that these ferritin shells do not result from artifacts in the staining procedure (e.g., sodium tungstate promotes the formation of creatine kinase crystals on cardiolipin layers (47)). Direct proof of the formation of the ferritin structures on the liposomes was provided by cryo-electron microscopy of hydrated liposome/ferritin suspension. The samples for the cryoelectron microscopy were prepared in a thin film over carbon-coated

144

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grids, plunged into a mixture of liquid propane and ethane cooled to about -190°C (48-50). A picture of frozen-hydrated liposomes, covered by ferritin shells is presented in Fig. lb. Partially ordered shells over the liposomes can be seen in the native suspension, even in the absence of phosphotungstate. Further proof of that is provided by the crosslinking procedure described below. Ordered shells over the liposomes were observed only after 24 hr of incubation of the liposome/ferritin mixture. The obtained assemblies appeared to be very delicate and sensitive to mechanical influence. No coated liposomes were obtained in samples that have been subjected to intensive shaking for 30 min or more prior to the staining procedure. When these samples were left for 24 hr at rest, the ordered protein shells over the liposomes reappeared. This provides a proof that only a weak attraction between the protein molecules and the lipid surface is required in order for the molecules to rearrange and form an ordered shell. To make the assemblies applicable for practical purposes, one has to develop a method of fixing the structures. 3. Fixation of the Structures by Crosslinking The structures composed of ordered ferritin shells over the liposomes could be fixed together by using a protein cross-linker like glutaraldehyde (51). In our experience, addition of glutaraldehyde at concentrations IO.4 ~01% did not produce significant results, while higher concentrations led to quick smearing of the protein/liposome structure. To avoid mass-transfer effects (52), and work under milder conditions, we developed a procedure in which 1.6% of glutaraldehyde is mixed for 2 hr with a portion of the initial liposome suspension but without the protein. The ferritin/liposome assemblies are mixed with this glutaraldehyde solution in a 1 : 1 ratio and left to react for 2 hr. This procedure resulted in a routine and controllable formation of fixed assemblies that could be easily stained and observed by TEM (Fig. 2a). The number of liposomes with good coverage in these pictures is greater than in the ones without fixation (except for a few naked liposomes originating from the glutaraldehyde solution). This confirms our conclusions about the delicateness of the structures before the fixation. After fixation, the protein shells could withstand the dissolution of the carrier liposomes by solubilisation of the lipid by non-ionic surfactant (Fig. 2b). In this way one can prepare nanometer-sized protein or liposome/ protein assemblies suspended in an aqueous environment.

ASSEMBLY

OF PROTEIN

STRUCTURES

ON LIPOSOW%

145

Fig. 2. Micrographs of assemblies with crosslinked ferritin shell. a, liposomes are still present below the protein shell; b, liposomes have been extracted by a micellar solution of a non-ionic surfactant (Tween 20).

146 II.

O.D.

FERRITIN

ATTACHMENT

TO

LIPOSOMES

BY AVIDIN-BIOTIN

VELEV

TYPE

OF

BINDING

The challenge for future technologies is to prepare functionalised assemblies which include a number of complementary protein moieties. The non-specific interactions are too rude a tool for such assembly. The aim in our investigations was to find out how to use specific, lockand-key interactions to assemble ferritin shells over liposome templates. While these structures are neither functionalised nor multiprotein, knowledge of the suitable biocolloid interactions could be used in further advances in the field. Our first study was aimed at using the well-known and much investigated avidin-biotin (or streptavidin-biotin) lock-and-key binding (53-58). This type of binding has been used earlier to obtain streptavidin coated liposomes (21, 22) or to assemble higher order structures out of liposomes (29, 30). The problem of evenly coating liposomes with a ferritin shell has not been studied. Specific binding sites on the liposomes were introduced by incorporating 10 mol% of biotinylated lipid (long-spacer lipids Biotin-LC-dipalmitoyl-L-aphosphatidylethanolamine (DPPE) from Pierce or Biotin-LCdioleoyl-L-a-phosphatidylcholine (DOPC) from Avanti Polar Lipids). For coating, we used ferritin-avidin (from Pierce or A-4030 from Sigma) or ferritin-streptavidin conjugates (from Calbiochem, USA). By electrophoresis it has been found that the isoelectric points of the conjugates do not differ significantly from that of pure ferritin (59). The methods for liposome preparation and TEM observation are similar to those described in the previous Section. 1.

Effect of pH and Liposome Charge

It is known that the strength of the avidin-biotin binding may be up to an order of magnitude higher than that of typical non-specific colloid interactions. It is expected that the lock-and-key binding will override any non-specific colloid repulsion that may oppose the formation of the shells over the vesicles. In spite of this, we found that there is almost no binding of the ferritin-avidin conjugate to the biotinylated liposomes at pH = 7. The following hypothesis could be proposed to account for this observation: as mentioned, ferritin and its conjugates are negatively charged at pH = 7, which is above their isoelectric point. On the other hand, biotinylated lipid molecules are carriers of a single negative charge each. The liposomes into which these lipids have been introduced acquire negative charges that repel the protein away from the

ASSEMBLY TABLE

OF PROTEIN

STRUCTURES

ON

147

LIPOSOMES

I

Results position

of Comparative

Experiments

Vesicles

of Ferritin-avidin

Binding

used

to Vesicles

Type

of coating Ferritinavidin conjugate

of Different

Com-

agent Ferritinavidin conjugate + free biotin

Composition

Expected surface charge

Ferritin only

Lipid

+ biotinylated

Negative

N

N

N

lipid Lipid Lipid

+ stearylamine + biotinylated

Slightly Neutral

positive

N N

N

-

C

N

Slightly

positive

N-LC

C

-

LC

C”

LC

lipid/stearylamine

l/1.5

Lipid + biotinylated lipid/stearylamine

l/3.5

Lipid lipid

+ biotinylated

Positive

+ HTAB

The samples N, no coating; a Best coating

were observed 24 hr after preparation. LC, low coating; C, coating. observed.

surfaces. Although the repulsion in the highly saline media is shortranged, it can prevent the even shorter-ranged ligand-receptor interaction from taking place. We were able to prove this hypothesis by studying the effect of liposome composition and pH on the binding process of the ferritinavidin conjugate. First, we fixed the pH at 7 using phosphate buffered saline (0.15 M NaCl) and prepared liposomes with different composition and surface charge by mixing the lipids with either stearylamine or HTAB. In these cases it was also important to distinguish between specific, ligand-receptor type and non-specific, electrostatic or other adsorption. This was achieved by performing three parallel comparative experiments: (i) with pure ferritin non-conjugated to avidin, (ii) experiments with regular ferritin-avidin conjugate, and (iii) experiments with ferritin-avidin conjugate whose binding pockets were previously blocked by the introduction of free biotin in the surrounding environment. The results are summarised in Table I. It is seen that neither of the ferritin types adsorbs or binds to the surfaces of the negatively charged biotinylated liposomes. Non-biotinylated liposomes containing only stearylamine also do not attract the pure protein or the conjugates. Possibly this is a result of the low ionisation of the fatty amine. As described in the previous section, non-biotinylated lipo-

148

O.D.

Fig.

3.

Comparison

contain both biotinylated presaturated

between

specific

biotinylated lipid and vesicles and ferritin-avidin with free biotin.

and

non-specific

stearylamine conjugate;

interactions in

a molar b, same,

VELEV

in vesicles ratio of l/1.5. but the system

that a, is

ASSEMBLY TABLE Summary

OF PROTEIN

STRUCTURES

ON

149

LIPOSOMES

II of the

Biotinylated

Interplay

of Specific

and

Electrostatic

Interactions

in Ferritin

Attachment

to

Vesicles Ferritinavidin or streptavidin conjugate

System Negatively charged vesicles

at

Slightly

positively charged

Ferritin with

pre-treated free biotin

pH=7 pH=4 The

data

C, coating; ~_1

was confirmed LC,

favourable no specific

low

by both

ferritin-avidin

coating;

N, no

electrostatics; binding.

0

coating

or ferritin-streptavidin

conjugates.

or agglutination. unfavourable

electrostatics

(repulsion);

somes with HTAB are strongly positively charged and gather ferritin by non-specific electrostatic attraction, therefore this case is not shown in the table. Much more interesting is the case when the negative charge of the biotinylated lipid is neutralised by the positively charged amine or surfactant molecules, When the molar ratio of biotinylated lipid to stearylamine is close to unity, a strictly specific binding of the conjugate to the liposome surfaces is present (Fig. 3). When the proportion of stearylamine is increased, or when the strongly charged HTAB is used, the lock-and-key binding is enhanced by electrostatic attraction of the ferritin to the liposome surfaces. The best coating is obtained in the case of HTAB-containing vesicles. Still, the specific binding is the primary source of protein attachment. In the subsequent investigations we used only HTAB as a source of positive charges on the vesicles, as the liposomes with stearylamine turned out to be difficult to extrude, with uneven shape and strong flocculation. To check the hypothesis further, we performed experiments with both avidin and streptavidin conjugates, this time varying the pH above and below the isoelectric point of ferritin. The molar ratio of biotinylated lipid to HTAB was fixed at unity. The pH of 4.0 was sustained with the use of a citrate-phosphate buffered saline. The results are summarised in Table II. It is seen that the decrease of pH below the isoelectric point of the conjugates results in a reversal of their binding tendencies: at lower pH, the molecules start to preferably lock onto the negatively charged liposomes. As demonstrated by the experiments with presaturated avidin or streptavidin (last column of Table II), in both cases the observed binding is predominantly specific. Thus

O.D.

Fig. 4. Micrographs using ferritin-avidin

of liposomes with specifically attached ferritin conjugate; b, using ferritin-streptavidin conjugate.

VELEV

shells.

a,

ASSEMBLY

OF PROTEIN

Lipid

STRUCTURES

molecule

Cationic

Biotinylated

151

LIPOSOMES

(zwitterionic)

Surfactant

(poeitively

lipid

(negatively charged)

Ferritin Fig.

ON

5.

Schematics

avidin-biotin

and

- Avidin of the

the

conjugate protein-liposome

electrostatic

interactions

’ structures at pH

assembled

by tailoring

the

= 7.

the hypothesis about the electrostatic interaction being the key to the specific one was verified by reversing both the liposome and the protein charges. Our data is consistent with recent direct measurements of the specific and non-specific contributions to the force between streptavidin-covered lipid layers (58). Although favourable or neutral electrostatic interactions are required for the assembly, the mechanical strength of the obtained liposome/protein complexes is predominantly assured by the strong and specific binding. Typically, the liposomes were surrounded by a heavy coat of disordered ferritin molecules (Figs. 4 and 5). Unlike the case of electrostatic attraction described in the previous section, the mechanical stability of the assemblies was very high, and they could be easily subjected to stirring, mild homogenisation, or centrifugation. By consecutive stirring and centrifugation one could improve the quality of the assembled microspheres, removing the loosely (non-specifically) bound and the unbound conjugates. Thus, once the avidin-biotin binding has occurred, stable structures that do not require further fixation or crosslinking are obtained in one step. On the other hand, the strength and the irreversibility of that binding (together with the poorly defined stoichiometry of the conjugates and the low diffusivity of the biotinylated lipid) are possible reasons for the lack of lateral ordering in the assembled ferritin shell. 2. Tailoring of the Specific and Non-specijic Interactions in the System The experimental data described above demonstrates that nanometer-

152

O.D.

VELEV

sized structures could be assembled only by control of both the nonspecific and unidirectional interactions like the electrostatic, van der Waals, solvation, and hydrophobic (46), and of the directional and specific lock-and-key biorecognition interactions. By tailoring the colloid interactions in a latex particle - emulsion droplet system we were able recently to assemble micrometer-sized ordered supraparticles (34). The combination of specific and non-specific interactions could help in the assembly of much more important and versatile protein and liposome-protein structures.

III.

FERRITIN

ATTACHMENT

BY

LECTIN-OLIGOSACCHARIDE

TYPE

OF

BINDING

A wave of interest has been expressed recently on the lectin-oligosaccharide type of ligand-receptor interaction. This binding is often encountered in living nature and constitutes one of the major bases of cell-cell recognition (60-63). To prepare ferritin/liposome microspheres assembled through this type of interaction, the ferritin molecules were conjugated to Concanavalin A (Con A), a well-known lectin, that binds to terminal mannosyl and glucosyl chains of oligosaccharides or glycoproteins (product C-7898 from Sigma, see also refs. 23, 63-65). To obtain appropriate liposomal templates for binding of the Con A-ferritin conjugate, we used a two-step procedure (65-67). First, liposomes were formed by extrusion by procedures similar to those described above. Then, they were brought into contact with a centrifuged solution of cholesterol-AECM-mannan (Dojindo Labs., Japan), an oligosaccharide, connected to a cholesterol moiety through a spacer arm. Due to the high affinity of the cholesterol to the internal hydrocarbon layer of the lipid membranes, it penetrates inside the liposome membranes, serving as an anchor of the polysaccharide coat (65, 66). The weight ratio of the polysaccharide to the lipid in the samples was sustained at 0.014I.02. All of the experiments were carried out in Tris buffered saline at pH = 7.2. As suggested in the literature (63, 64), 0.1 mM of Mn*+ and 0.2 mM of Ca*+ were added to promote the Concanavalin activity. Three types of comparative investigations were carried out: we mixed the ferritin-Con A conjugate first with uncoated vesicles, then with vesicles coated with an anchored mannan layer and finally with coated vesicles, but in the presence of free mannose that blocks the lectin binding activity. The effect of electrostatics in the case of coated vesicles is either negligible or quite complex, so we chose to study the

ASSEMBLY TABLE Summary

OF PROTEIN

STRUCTURES

ON

153

LIPOSOMES

III of Vesicle

Coating

by Ferritin-Con

A Conjugate System

Vesicle composition Vesicles

Egg Egg

lecithin, lecithin,

DOPC DPPC

low purity high purity

plus

cholesterol

The

samples

LC, tion;

low coating; C, coating; A, agglutination; HA,

a Best

were

coating

observed

only

Mannancoated vesicles (22°C)

Mannan-coated plus free mannose

LC N

C”, LA c, ,4

c, A Cl, A

LC x

N N

c, A c, A

Cl, Cl,

N

24 hr after Cl, high

Mannan-coated vesicles (4°C)

HA HA

N

preparation.

coating worse agglutination;

than in the column on left; LA, N, no coating or agglutination.

low

agglutina-

observed.

Fig.

6.

specifically

Micrograph bound

of lecithin to the

underlying

liposomes

covered

polysaccharide

by layer.

Con

A-ferritin

conjugate,

154

O.D.

Lipid

Vesicle

Lipid

molecule

Cholesterol Binding

site

anchoring

on the polysaccharide Ferritin

Fig. 7. Schematics by Con A-mannan

VELEV

- Lectin

coat conjugate

/

of the protein-polysaccharide-liposome type of binding.

structures

assembled

effect of lipid and polysaccharide composition on the outcome of the process. The data from these experiments are presented in Table III. We observed a heavy coating with ferritin in the samples with lecithin vesicles (Figs. 6 and 7). The much smoother appearance of these assemblies in comparison with those based on avidin-biotin binding possibly originates from the intermediate polysaccharide coat of the vesicles. The specificity of the Concanavalin binding is proven by the lack or low attachment to the liposome surfaces in the absence of mannan layer or in the presence of free mannose. In correspondence with the literature (65) the activity of the lectin is dependent on the environmental temperature, and so is the quality of the obtained assemblies (Table III). A lower temperature evokes higher binding to the liposomes and lower agglutination. Together with attaching the ferritin molecules to the vesicle surfaces, the Con A expressed a significant agglutination activity, binding together the coated vesicles. This feature is particularly obvious in the case of vesicles composed from pure synthetic lipids. The agglutination is lower in the case of natural lipid compositions. Low-purified egg lecithin (P 3556) exhibits the best coating and lower agglutination. This may be a result of specific and non-specific binding to lipopolysaccharides and other natural components present in the nonpurified lecithin vesicles. The major factor influencing the quality of the obtained struc-

ASSEMBLY

OF PROTEIN

STRUCTURES

ON

LIPOSOMES

155

tures appears to be the type of polysaccharide on the liposomes and its specific and non-specific interaction with the ferritin-lectin conjugate.

I\‘.

CONCLUDING

REMARKS

Our study demonstrates that complex microstructured particles could be assembled from liposomes and ferritin using colloid interaction schemes including either non-specific or a combination of non-specific and specific interactions. While the microstructured supraparticles presented above are only model systems suitable for observation by TEM, further development in the area could bring us closer to the fabrication of functional nanometer-sized assemblies which may include combinations of enzymes and other biologically active molecules. The knowledge, control, and multi-step modification of the colloid interactions in the system appear to be the real tool for “smart” particle assembly.

SUMMARl

We investigate different schemes for fabrication of nanometer sized assemblies that consist of a liposome core over which a shell of ferritin is attached. Three distinct interactions were used for this assembly: (i) Electrostatic attraction. The liposomes are charged by the presence of cationic surfactant (HTAB) and at an appropriate pH collect the ferritin molecules into a 2D-ordered ferritin shell. The protein shells can be fixed by glutaraldehyde. Next, the liposomes can be removed by solubilisation, leaving behind ordered ferritin clusters. (ii) Specific avidin-biotin or streptavidin-biotin binding. The ferritin molecules are conjugated to avidin or streptavidin and the liposomes incorporate biotinylated lipid. We found that the specific binding can be completely blocked by unfavourable electrostatic repulsion. To adjust the appropriate liposome charge we include cationic surfactant in the lipid layer. Thus, to accomplish the assembly process, we need to design and modify both the specific and non-specific colloid interactions in the system. The result is liposomes heavily coated with a strongly and specifically attached ferritin layer. (iii) Specific polysaccharide/lectin binding. The liposomes are first coated with a cholesterol-anchored mannan layer. The ferritin molecules are conjugated with Con A that binds to the polysaccharide. A smooth and dense coating with ferritin is obtained (Fig. 3b).

156

O.D.

VELEV

The acquired data can find application in the future fabrication of microstructured, multicomponent, or functionalised protein and liposome/protein assemblies. Acknowledgments This study is performed in collaboration very grateful to Prof. K. Nagayama, Yoshimura for their help and guidance.

with Dr.

M. Ohta. The author is S. Ebina, and Dr. H.

REFERENCES 1 2 3 4 5

D. Papahadjopoulos, Ann. N. Y. Acad. Sk., 308, 1 (1978). D.D. Lasic, “Liposomes: From Physics to Applications,” Elsevier, D. Lasic, Am. Sci., 80, 20 (1992). R.F. Service, Science, 265, 316 (1994). J.H. Fendler, “Membrane Mimetic Chemistry,” Wiley-Interscience,

Amsterdam

New

(1993).

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