Preparation and characterisation of polymerised proteovesicles

Colloids and Surfaces,

57 (1991)

Elsevier Science Publishers

197-204 B.V., Amsterdam

197

Note

Preparation and characterisation proteovesicles

of polymerised

M.D. Reboiras’ and M.N. Jones Department of Biochemistry Ml39PT, UK

(Received

16 October

and Molecular

Biology,

1990; accepted 9 November

University

of Manchester,

Manchester

1990)

Abstract The cationic double-chain surfactant, hexadecyl[ 1 l- (methacryloyloxy)undecyl] ammonium bromide in combination with dipalmitoylphosphatidylethanolamine (DPPE) derivatised with maleimidobenzyl-N-hydroxysuccinimide (MBS) has been used to make polymerisable vesicles which can be conjugated with a protein (lysozyme) to form polymerised proteovesicles. Polymerisation was carried out at room temperature using Fenton’s reagent (hydrogen peroxide and ferrous ions). It was found that unpolymerised proteovesicles can be prepared by this procedure but such pre-formed proteovesicles cannot be polymerised without initiating precipitation; however if polymerisation precedes protein conjugation, proteovesicles can be prepared in the dispersed state. In contrast to unpolymerised proteovesicles, polymerised proteovesicles form small aggregates in solution together with physically adsorbed lysozyme, suggesting that the rigidity of the underlying vesicle bilayers significantly influences the physical adsorption process.

INTRODUCTION

Polymerised vesicles are currently of potential interest as drug carriers and have the advantage in overcoming the problems of instability on storage or in vivo associated with conventional (non-polymerised) vesicles [ 11. Although targeting of conventional vesicles by covalently attaching site-directing macromolecules, such as antibodies and lectins, to their surfaces continues to be extensively studied [ 2-51, polymerised vesicles having covalently linked surface protein have not been studied to any extent. Regen et al. [ 61 appear to be the first to describe the preparation of polymerised vesicles with covalently attached protein. They prepared vesicles from a methacryloyl phospholipid derivative (1,2-bis (12- (methacryloyloxy)dodecanoyl-DL-3-glycero(N-3,3diethoxypropyl)phosphocholine) and covalently attached cr-chymotrypsin to ‘Present address: Department0 Madrid, 28049, Spain.

0166-6622/91/$03.50

de Quimica,

0 1991-

Facultad

de Ciencias,

Elsevier Science Publishers

Universidad

B.V.

Autonoma

de

198

the vesicle surface via a Schiff base reaction between the phospholipid surface aldehyde groups generated by acidification of the diethyl acetal and the amino groups of the protein. The extent of protein conjugation achieved was high (290 g mol-’ of lipid) and suprisingly increased on reduction of the Schiff base linkage with borohydride. In principle, polymerised proteovesicles could be produced by conjugation of unpolymerised vesicles with protein followed by polymerisation or by conjugation of prepolymerised vesicles. The choice of route will depend on the chemistry used for conjugation and polymerisation. For polymerisation of lipids or surfactants with methacryloyl groups, free-radical initiators such as azobisisobutyronitrile which require a high temperature (6040” C) [ 71 are unsuitable for polymerisation of pre-formed proteovesicles as protein denaturation may occur. To overcome such problems it has been shown that Fenton’s reagent (hydrogen peroxide and ferrous ions) is a suitable initiator for the polymerisation of methacryloyl surfactant vesicles at low temperatures [ 81. We now report a study on the use of Fenton’s reagent for the production of polymerised surfactant proteovesicles. The polymerisable vesicles were prepared from a mixture of cationic methacryloyl surfactant (hexadecyl [ ll- (methacryloyloxy)undecyl]ammonium bromide) and dipalmitoylphosphatidylethanolamine (DPPE ) derivatised with maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (I) and conjugated with lysozyme derivatised with N-succinimidyl-S-acetylthioacetate (SATA) (II) according to the following reaction scheme: 0

Vesicle(DPP-NHCO-Ph-N

+

Protein-

NHCOCH2SCI

0 I-

Veslcle(DPP-NHCO-Ph-N S-CH$ONH-Protein 0

The results described relate to both the polymerisation of pre-formed proteovesicles and to the conjugation of polymerised surfactant vesicles. EXPERIMENTAL

Materials

The cationic surfactant hexadecyl [ ll- (methacryloyloxy )undecyl] ammonium bromide prepared as described by Regen et al. [9] was a gift from Dr.

199

I.G. Lyle, Unilever Research, Port Sunlight Laboratory, U.K. L-cw-Dipalmitoyl phosphatidylethanolamine (DPPE), product No. P-0890, and lysozyme product No. L 6876 were obtained from Sigma Chemical Co., Poole, Dorset, U.K. N-Succinimidyl-S-acetylthioacetate (SATA) was from Calbiochem, Cambridge, U.K. and n-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS ) was from Pierce Warriner Ltd., Chester, U.K. [ 3H] -DPPC was from Amersham International, U.K. All other reagents were of analytical grade and aqueous solutions were made up with doubly distilled water. METHODS

The MBS derivative of DPPE was prepared as previously described [ 5 1. The SATA derivative of lysozyme was prepared by the method of Duncan et al. [lo] using a molar ratio of SATA to lysozyme of 5 : 1. The derivatised lysozyme was separated from unreacted SATA by gel filtration on a Sephadex G50 column. The extent of derivatisation was determined from the sulphydryl content after deacetylation with hydroxylamine as described by Ellman [ 111. Vesicles were prepared by sonication of hydrated films of surfactant and DPPE-MBS containing 0.015-0.02 g of surfactant 1.5-2 mg DPPE-MBS and [ 3H] -DPPC (100-200 ~1 of stock (0.2 &i per 100 ml) ) in a total volume of 4.6 ml isotonic phosphate buffered saline pH 7.4 (PBS). After preparation the vesicles were fractionated by gel filtration on a Sepharose 4B column and suitable fractions were taken for conjugation with derivatised lysozyme. The resulting proteovesicles were separated from unreacted lysozyme by gel filtration using either a Sephadex G-200 or a Sepharose 4B column. The lipid and protein contents of the resulting proteovesicle fractions were determined from the [3H] -DPPC counts and Lowry assay [ 121 respectively. The vesicles were polymerised either before or after conjugation with protein by addition of a mixture of hydrogen peroxide and ferrous sulphate in a molar ratio 1: 1 at a ratio of lipid to initiator of 30: 1 as previously described [8]. (The hydrogen peroxide concentration was approximately 10d4 M.) The vesicles and proteovesicles were characterised in terms of size distribution by photon correlation spectroscopy using a Malvern autosizer model RR146. The instrument gives the equivalent normal weight distribution of particle size (diameter) from which the weight average diameter &. and standard deviation CT were computed. RESULTS AND DISCUSSION

Two routes for the production of polymerised proteovesicles were investigated. In the first route the proteovesicles were pre-formed and an attempt was made to polymerise them. In the second route the vesicles were polymerised and then conjugated with derivatised lysozyme. It was important to demon-

200

strate that proteovesicles could be formed from the cationic surfactant incorporating the reactive lipid (DPPE-MBS ) and that the resulting vesicles could be satisfactorily conjugated by the chosen reaction scheme (see Introduction). Figure 1 shows the elution profile of a proteovesicle preparation from a Sephadex G-200 column after conjugation of vesicles prepared from a mixture of methacryloyl surfactant and DPPE-MBS (weight ratio 10 : 1) with derivatised lysozyme. The lysozyme was derivatised at a molar ratio of SATA to protein of 5 : 1 which introduced 1.2 SH groups per lysozyme molecule. The co-elution of lipid and protein clearly separated from unreacted protein confirms the formation of proteovesicles. The characterisation data for the proteovesicles are shown in Table 1. The proteovesicles had relatively wide size distributions, the standard deviation of the equivalent weight distribution of particle diameters (6) was on average 235 nm. The weight average diameter of the proteovesicles was larger than that of the vesicles from which they were formed, suggesting some degree of aggregation, although fractionation of the original vesicle distribution will occur. The weight average numbers of protein molecules per vesicle ( (p,) were relatively high although the areas per protein molecule on the vesicle surfaces indicate that the protein was not close packed. The lysozyme molecule has an average diameter of 3.4 nm [ 131 which gives a projected area of 9.08 nm’ compared with the average area of 59 nm2 occupied by lysozyme molecules on the proteovesicle surface. Attempts to polymerise pre-formed proteovesicles prepared as described were not successful; the addition of initiator (H,O,-FeSO,) resulted in proteovesicle precipitation at pH 7.4 (PBS). The second route to polymerised proteovesicles in which the vesicles were 700 600

100 - 90

-

- 60

-

70

- 60

20

Fraction

30

no. (2ml)

Fig. 1. Elution profile from a Sephadex G-200 column of unpolymerised methacryloyl surfactant vesicles (initial&= 280 nm, o= 179 nm) DPPE-MBS (weight ratio 10: 1) in phosphate-buffered saline pH 7.4 after conjugation with derivatised lysozyme (1.2 moles SH per mole ); 0, concentration; 0, disintegrations per minute (DPM) from tracer lipid [ 3H-DPPC].

lysozyme

201

TABLE 1 Characterisation of proteovesicles and polymerised proteovesicles composed of methacryloyl factant and DPPE-MBS (weight ratio 10 : 1) conjugated with lysozyme Fraction

no.

Lysozyme : lipid” molar ratio

&,(ka)b (nm)

PW

Surface area per protein (nm*)

3.479.10-3 4.046*10-3

505 k 258

13 110

425 f 246

10 980

61 52

Unpolymerised 10 11 12

4.756*10W3

339_+217

8 400

43

13

2.701.10-3

387 k 220

6 060

78

Polymerised 12 13 14

0.0274 0.157 0.256

606 k 347 669 ?c378 977 _+702

0.153*106 1.07-106 3.92. lo6

“Lipid refers to methacryloyl surfactant plus DPPE-MBS. bc& is the weight-average diameter and u the standard deviation

0 3 8 -;_ k 0

2400

L

2200

-

2000 1600

-

1600 1400

-

1200 1000 600

-

600

-

7.5 1.3 0.76

of the weight distribution.

Oooooooo\ I 0

1 ‘0

I

\9

%

9

o/

._a -I

0

sur-

5

10

15

20

Fraction

25

30

35

40

45

50

no. (Zml)

Fig. 2. Elution profile from a Sephadex 4B column of polymerised methacryloyl surfactant vesicles (initial d-,= 285 nm, CJ= 176 nm) incorporating DPPE-MBS (weight ratio 10: 1) in 0.1 M sodium [lipid] = 7.04 mM, chloride pH 7. The polymerisation conditions were as follows: [H,0,]=[Fe2+]=0.239mA4.

polymerised prior to conjugation was more satisfactory. Figure 2 shows the profile of polymerised vesicles on a Sephadex 4B column on elution in 0.1 M sodium chloride. Gel filtration not only serves to fractionate but also to remove initiator from the polymerised vesicles. Peak fractions were taken for assessment of polymerisation and for conjugation. Figure 3 shows the effect of addition of ethanol on the absorbance of polymerised and unpolymerised vesicles.

202 0.350

0

10

20 %(v/v)

30

40

50

Ethanol

Fig. 3. Absorbance at 400 nm of methacryloyl surfactant vesicles incorporating DPPE-MBS (weight ratio 10: 1, initial lipid concentration 0.219 mM) as a function of ethanol concentration: 0, polymerised vesicles, 0, unpolymerised vesicles. (The absorbance has been corrected for dilution effects as previously described [ 81.)

Previous work [8] showed that vesicle stability in ethanol was a convenient test of polymerisation. The addition of ethanol to unpolymerised vesicles results in an absorbance peak as the vesicles fuse and/or aggregate prior to disruption. In contrast the peak is generally higher and broader for polymerised vesicles and the absorbance remains high because the vesicles cannot disrupt at high ethanol concentrations. Figure 4 shows the results of conjugation of polymerised vesicles to form proteovesicles. The co-elution of lipid and protein distinct from that of free protein confirms the formation of proteovesicles. The lipid and protein peak maxima do not, however, coincide. Table 1 gives the characterisation data for the proteovesicles in the fractions, from which it is clear that not only are the sizes of the proteovesicles substantially larger than those of the polymerised vesicles from which they were formed (d;, = 306 nm, o= 195 nm) but also that the apparent surface density of the protein greatly exceeds that which is physically realistic, taking the projected area of the lysozyme molecule as 9.08 nm2. Since polymerised vesicles must remain intact it follows that the particles eluting from the column must be aggregates together with excess lysozyme. The displacement of the protein peak relative to that of the lipid most likely arises because of the presence of non-covalently bound lysozyme within the aggregates. Direct evidence for non-covalently bound lysozyme comes from the protein-to-lipid molar ratios of the polymerised vesicles which exceed the ratio of reactive (DPPE-MBS) to total lipid, which was 0.064 for both the unpolymerised and polymerised proteovesicles in Table 1. Thus if all the DPPE-MBS was conjugated with protein, the protein-to-lipid molar ratio cannot exceed

203

250 I

150

200 -

150 -

OB 0

V

5

10

15

20

Fraction

25

30

55

40

45

50'

no. (2ml)

Fig. 4. Elution profile from a Sephadex 4B column of polymerised methacryloyl surfactant vesicles (initial d-,=306 nm, G= 195 nm) incorporating DPPE-MBS (weight ratio 10: 1) in 0.1 Msodium chloride, pH 7 after conjugation with derivatised lysozyme (1.2 moles SH per mole): 0, lysozyme concentration; 0, distintegrations per minute (DPM) from tracer lipid [ 3H] -DPPC.

0.064. It follows that from the protein-to-lipid molar ratios for unpolymerised proteovesicles only 4-7% of the DPPE-MBS has reacted with the protein, which is very similar to values found previously for vesicle conjugated using this reactive lipid [4,5]. CONCLUSIONS

These observations show that Fenton’s reagent can be used to produce proteovesicles provided polymerisation precedes conjugation. In contrast to unpolymerised proteovesicles polymerisation results in significant non-specific physical adsorption of protein, suggesting that the rigidity of the underlying bilayer is an important factor in the adsorption process. Physical adsorption of lysozyme on non-polymerised phospholipid vesicles has been previously observed to vesicles of opposite charge to the protein [ 141. Here both lysozyme (p1 11) and vesicles have a net positive charge under the conditions of the experiments. (Attempts to link covalently a negatively charged protein (wheat germ agglutinin) to the cationic vesicles, either unpolymerised or polymerised, always resulted in precipitation. ) It is noteworthy that the previous report [ 61 on the preparation of polymerised proteovesicles using the Schiff base linkage resulted in relatively high protein-to-lipid molar ratios (0.01-0.02) which significantly increased on reduction of the Schiff base linkage. No explanation was given for the increase but in the light of the present study it may be a consequence of an increase in non-specific binding since reduction of preformed proteovesicles could not lead to further specific (covalent ) binding.

204 ACKNOWLEDGEMENT

We thank the D.G.I.C.Y.T.,

Spain, for financial

support for M.D.R.

REFERENCES 1 2 3 4 5 6 7 a 9 10 11 12 13 14

S.L. Regen, in M.J. Ostro (Ed.), Liposomes: From Biophysics to Therapeutics, Marcel Dekker, New York, 1987, Chapter 3. L. Leserman and P. Machy, in M.J. Ostro (Ed.), Liposomes: From Biophysics to Therapeutics, Marcel Dekker, New York, 1987, Chapter 5. F.J. Martin, T.D. Heath and R.R.C. New, in R.R.C. New (Ed.), Liposomes, A Practical Approach, IRL Press, Oxford, 1990, Chapter 4. F.J. Hutchinson, SE. Francis, LG. Lyle and M.N. Jones, Biochim. Biophys. Acta, 978 (1988) 17. F.J. Hutchinson and M.N. Jones, FEBS Lett., 234 (1988) 493. S.L. Regen, M. Singh and N.K.P. Samuel, Biochem. Biophys. Res. Commun., 119 (1984) 646. D. Bolikal and S.L. Regen, Macromolecules, 17 (1984) 1287. M.D. Reboiras, G.A. Morris and M.N. Jones, Colloids Surfaces, 49 (1990) 385. S.L. Regen, B. Czech and A. Singh, J. Am. Chem. Sot., 102 (1980) 6638. R.J.S. Duncan, P.D. Weston and R. Wrigglesworth, Anal. Biochem., 132 (1983) 68. G.L. Ellman, Arch. Biochem. Biophys., 82 (1959) 70. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R. Diamond and D. Phillips, Brookhaven Protein Data Bank, Entry 3LYZ. Q. Yang and P. Lundahl, J. Chromatogr., 512 (1990) 377.