Biochimica et Biophysica Acta. 1075(1991) 102-108 © 1991ElsevierSciencePublishersB.V. 0304-4165/91/$03.50 ADONIS 030441659100229X


BBAGEN 23584

Preparation and characterization of polymer coated small unilamellar vesicles M.Y. Ozden and V.N. Hasirci Department of Biological Sciences, Middle East Technical Unirersity Ankara (Turkey)

(Received 12 December1990) (Revisedmanuscriptreceived13 May 1991)

Key words: Smallunilamellarvesicle;Enzymeentrapment;Polymercoated vesicle Glucose oxidase was entrapped in small unilamellar vesicles composed of phosphatidylcholine, dicetyl phosphate and cholesterol. Prediction of the enzyme content of liposomes by calculations based on input concentrations of lipid and protein, dimensions of the lipids and the liposomes yielded one protein per vesicle. The entrapment efficiency was experimentally determined to be about 13%. On the other hand the entrapment efficiency for the small chromate ions was found to be significantly lower (0.1%). The liposomes were then coated with a polymer, poly(1,4-pyridinium diylethylene salt). It was possible to remove the lipoid material from underneath the polymer layer with various techniques. The effect of sonication and treatment with organic solvents (tested for this purpose) on enzyme activity were found to be very significant and Triton X-100 was chosen for this purpose. It was shown that the enzyme within the remaining net has 89% of its original activity.


The great structural versatility of liposomes, their relatively innocuous nature, and ability to incorporate a wide spectrum of biologically active agents have led to the adoption of the system, by numerous workers, as carrier of various substances such as antimicrobial [1] and cancer therapy agents [2], drugs for metal detoxification [3], hormones, enzymes and other drugs which are either unstable or unabsorbable by the gut [4], vaccines [5], agents for activation of macrophages [6], and red blood cell substitutes [7]. There has been considerable progress in liposome technology [8] and these have ensured, in many cases, adoption of the liposome system by the industry [9]. A major disadvantage with liposomes, however, is that they tend to aggregate and fuse [10]. In order to prolong the vesicle lifetime, various approaches such as incorporation of a second surfactant molecule which by itself does not form vesicles (i.e., cholesterol [11]) or

Abbreviations: PC, phosphatidylcholine;DCP, dieetyl phosphate; Chol, cholesterol;UV, ultraviolet. Correspondence: V.N. Hasirei, Department of Biological Sciences, Middle East Technical University,06531-Ankara, Turkey.

modification of the surface of vesicles with charged groups (i.e.,//-amino galactose [12]) have been tried. In the quest to increase the mechanical strength of the fragile liposomes, several groups demonstrated that vesicles could be stabilized by covalent cross-linking of lipid molecules within the lipid bilayer, leading to the formation of 'polymerized vesicles' [13-16]. In order to achieve this kind of stabilization, modified phospholipids containing polymerizable groups (i.e., vinyl [17], acryloylic [18,19], methacryloylic [20,21], butadienic [17,22] and diacetylenic [23-25]) were used. It is also possible to mimic nature and obtain more stable liposomes by surrounding them by a macromolecular coat without introducing any permanent linkage in between. Various routes for the coating of liposomes with polymers have been reported in the literature. These are; adoption of polymer molecules at the liposomal surface by hydrophilic or ionic forces [26-29], fixation of polymer molecules by hydrophobic anchor groups embedded in the lipid matrix [30], preparation of liposomes from amphiphiles which possess a polymerizable group linked to the polar head group by a cleavable spacer after polymerization [31] and fixation of polymerizable, water soluble molecules to the liposomal surface via salt formation, followed by polymerization [32]. A more specific example of this is to use charged

103 lipids with polymerizable counter ions to prepare unsymmetrical polymer membranes with synthetic polyelectrolyte chains or even a net only on one side of the vesicle membranes (Fig. I) [33-35]. In the present study, 4-vinyl pyridine, a water soluble monomer that is known to form salts and polymerize spontaneously in organic solvents or water upon the addition of protic acid~ [32] was used. Liposomal surfaces containing the negative charges of the dicetylphosphate acted as the template to form the salt. With this method the encapsulated enzymes were expected to remain in their native states because inactivation of enzymes observed with other immobilization methods

involving po!ymerization would be avoided by the shielding effect of the iipid biiayer barrier. Polymer coating would also inhibit the fusion of the vesicles. Such vesicles are, therefore, better suited to drug delivery or enzyme replacement studies. The efficiency of encapsulation of enzyme glucose oxidase, and a small, charged species, chromate, the preparative techniques, removal of the inner lipid layer and the effect of the solvents used in this process on the activity of the retained enzyme are discussed, and encapsulation efficiency for glucose oxidase, calculated by using data about lipid areas and input concentrations, is reported.

~q ~9 ~p O-=c~

~,=~ ~ : > 4 3


[~ N


~" _ ~ 9 ~ 9 ~ . ) o





b~6 66~ ~,

= Glucoseoxlde~e O" = Dzcetyl phosphste C~" = Cholesterol or Phosphatid~| choline = 4-Vlh~Ip~r~Ine







~ ~





~ r ~

Fig. 1. Preparation of enzymein a polymernet,


Materials and Methods

Materials Lecithin (from eggs), cholesterol (cholest-5-en-3/3ol), potassium hydroxide, potassium dihydrogen phosphate, and potassium chromate were the products of E. Merck (F.R.G.) and were used without further purification. 4-Vinylpyridine was purchased from E. Merck (F.R.G.), and distilled in the presence of 1,3-dinitrobenzene inhibitor, and stored in the refrigerator until use. Dicetyl phosphate (dihexadecyl phosphate), o-dianisidine (3,3'-dimethoxybenzidine) dihydrochloride, glucose oxidase (/3-o)-glucose; oxygen 1-oxidoreductuse; EC Type Il from Aspergillus niger, Lot. 18F-9505, 25000 units/g solid containing approx. 20%, peroxidase (donor: hydrogen-peroxide oxidoreductase; EC Type II from horseradish, with an activity of 200 purpurogallin units/mg solid, and /3-D(+)-glucose were purchased from Sigma Chemicals (U.S.A.) and was used without further purification. Sephadex G-50 (coarse) and Sephadex G-200 (coarse), dialysis sacks (cellulose; 16 mm diameter), that retain proteins with molecular weight greater than 12000 were obtained from Sigma Chemicals (U.S.A.).

Methods Preparation of liposomes. Preparation of liposomes from a mixture of lipids were carried out according to the methods described previously [36-38]. Phosphatidylcholine (0.6 ml, 116.2 mM in chloroform), dicetyl phosphate (0.6 ml, 33.2 mM in chloroform/methanol mixture, 1:1), and cholesterol (0.6 ml, 16.6 mM in chloroform) were mixed to yield a molar ratio of 7 • 2:1, respectively, and the volume was brought to 6 ml by addition of chloroform. The solvents were driven off by using rotary evaporator under N 2 atmosphere, and the dried lipids were suspended in 6 ml distilled water by gentle shaking. The milky suspension was sonicated for 8 min at 4°C, at 35 W. After the chromatographic separation of the vesicles with Sephadex G-200 the liposome concentration of the eluates were determined by the absorbance at 410 nm [36] using a Hitachi double beam spectrophotometer (Model 220A). Entrapment of glucose oxidase in liposomes. Entrapment of GOD in liposomes was carried out according to the methods previously described [32,36-38]. Solid glucose oxidase (2.4 mg) was dissolved in phosphate buffer (6 ml, pH 6.8, 50 mM) to produce a final concentration of 0.4 mg GOD/ml and this was used to suspend lipids and make the liposomes. The uniformly sized vesicles obtained after the chromatography were then used in the polymer coating process.

Determination of glucose oxidase content in liposomes. Activity of free glucose oxidase was determined spectrophotometrically according to a modified version of the method reported in the Sigma Technical Bulletin [39,40]. The amount of GOD trapped in the liposomes was measured by adding 0.1 ml of Triton X-100 to 0.9 ml of eluate. The samples were incubated in a water bath at 35°C for 2 h and were then separated into two equal portions one of which was placed in a boiling water bath for 5 min (to serve as blank). Both portions were brought to room temperature and 0.1 ml of each was used in the determination of enzymatic activity and percent entrapments were calculated.

Coating of liposomes with poly(1,4-pyridinium diylethylene salt). Poly(1,4-pyridinium diylethylene salt) was coated on the liposomes according to earlier reports [32,33]. Uniform sized liposome fractions (18.5 ml) were mixed with 4-vinyl pyridine solution (275 p.I, 80 mM), and polymerization was initiated with sulphuric acid (55 p.l, 0.18 M) and the process was continued for 24 h at room temperature with continuous stirring. The mixture was resolved by chromatography. The IR spectrum of coated liposome was obtained by using a Phillips (PU 9700 series) infrared spectrophotometer.

Remot:al of the lipids of the polymer coated liposomes. In the removal of the lipids of the coated liposomes, the eluates (18.3 ml) obtained after chromatographic separation were treated with an equal volume of diethyl ether, stored overnight and the aqueous, lower phase was collected. After the volume was reduced to nearly 3 ml by vacuum evaporation, chromatography with Sephadex G-200 was carried out. Entrapment of chromate ions in liposomes. Liposprees were prepared as explained above except that potassium chromate (0.1 mM, 6 ml) was used in the lipid suspension step instead of buffer or GOD solution. Free and latent chromate ions were removed by using Sephadex G-200 column and the chromate concentration in the fractions were determined by measuring their absorbances at 370 nm. In order to determine the entrapment efficiency, aliquots (0.9 ml) of the liposomal fractions were treated with Triton X-100 (0.6 ml, 2%) at 35°C for 2 h and the absorbance at 370 nm was measured, Results and Discussion

Formation of polymer coated liposomes containing glucose oxidase The ultraviolet spectra of the glucose oxidase containing liposomes PC/DCP/chol (7: 2 : 1) is quite similar to that of empty liposomes except for the formation of a new shoulder at 220 nm and the shift of the characteristic peak of the choline moiety of the liposome from 209 nm [34] to 204 nm (Fig. 2) indicating the

105 presence of the enzyme in the vesicles. In the UV spectra of the polymer coated glucose oxidase containing liposomes a major peak at 245 nm (specific for pure polymer) and a shoulder at 265 nm are observed. The presence of this shoulder is the indication of polymer in the presence of lipoidal material as the UV spectra for polymer coated liposomes had exhibited [32]. The disappearance of the absorbance due to enzyme is another sign of presence of the coat on the liposome. Polymer coated glucose oxidase containing liposomes were separated from the contaminants (i.e.. polymer beads and oligomers) by column chromatography using Sephadex 0-200. Upon scanning the eluates at 260 nm, two peaks, one eluting at the void volume (fractions 14-20) and the other eluting much later (fractions 40-60) are observed (Fig. 3). When scanning is done at 410 nm, only the first peak of the 260 nm scan is observed at its original position, it is known that the absorbance at 410 nm is due to turbidity caused by the lipoidal vesicles in their intact form [36], whereas the absorbance at 260 nm is a property of the polymer [34]. It can, therefore, be stated that the sample is composed of two different types of species:



Ii// \~\












Wa~,lef~h (rim)

Fig. 2. UV spectra of glucoseoxidase and various liposomes.Glucose oxidase ( . . . . . ). polymer coated glucose oxidase containing liposome ( - - - - - ) , liposome (,x) and glucose oxidase containing liposome ( )

0 20 40 60 Ftoctlon Number Fig. 3. Elution profiles of the polymer coated liposomes. After polymer coating (second G-200)(D) and after lipid extraction (third G-200) (+).

(i) liposomes containing the polymer coat on their exterior, and (ii) polymer not attached to liposomes (i.e,, beads of pure polymer, oligomers) a n d / o r polymer-lipid aggregates of very small size, possibly resulting from degradation of the polymer coated vesicles during volume reduction (by rotary evaporation). Formation of two peaks in the elution profile is also observed when the lipid component of the coated liposome is removed by extraction and the fractions monitored at 260 nm (Fig. 3). The first peak has a lower absorbance in comparison to the results of the previous chromatography. This is due to the degradative effect observed upon concentrating the !iposomes for chromatography. An important implication of this result is the retention of the 'vesicular structure' by a significant fraction of the coated P C / D C P / C h o l liposprees even after harsh treatments such as vacuum evaporation. The retention of vesicular structure was further substantiated by the observation of diethyl ether extracted coated liposome at its original point in the chromatograph. IR spectra of the glucose oxidase containing coated liposomes are given in Fig. 4. The spectra of polymer coated glucose oxidase containing liposomes and the spectra of the second peak obtained after chromatographic separation (and interpreted above as containing mostly polymer) are basically the same. The reason for not observing any absorbance specific for glucose oxidase is that its major peaks (Amide I and II) are masked by the Ptl,41VN and the lipid peaks.

Determinationsof kbwticparametersfor thefree glucose oxidase In order to calculate the apparent kinetic parameters of the free glucose oxidase, glucose concentrations varying between 1-100 m M were used. From the resultant Lineweaver-Burk plot the apparent K m and ap-



1600 1400 1200 W~numbel (era-))


Fig. 4. IR spectra of the polymer coated enzyme containing liposomes after a series of treatments. (a) In the coating mixture, (b) purified with G-200. (c) lipid extracted (by chloroform), (d) lipid extracted and purified with G-200, and (e) after second G-200, second peak.

parent Vm~~ values were found to be 53.7 mM and 31388 U / g solid, respectively. According to the supplier, the limax had to be 25000 U / g solid under normal conditions and 51500 U / g solid under oxygen saturated conditions at 35°C [39]. Thus, the Vmax determined in this study is in a good agreement with the ones reported above. These differences could be attributed to the variation in the purity of the products, as well as to the variations in the assay methods, such as pH, ionic strength of the medium, and composition of the buffer system. Determination o f the entrapment efficiency Glucuce oxidase. As explained in the previous sections, removal of the free and adsorbed enzymes from the liposomes was carried out by gel permeation chromatography on Sephadex G-200. In order to calculate the entrapment efficiency, the amount of enzyme in the liposomal fractions and in the free enzyme containing (latter) fractions, were determined according to the method described earlier [32,39] using the constructed calibration curve which had a slope of 2 . 1 0 -3 + 4.610 -5 A (tzg/ml) -I. The enzyme was found to be

13.30 + 2.63% in the liposomal peak and 80.50 __.5.75% in the enzyme containing fractions. Since the two values add up to 100% (within the experimental error limits), it is apparent that the entrapment efficiency can be determined by using only the free enzyme containing fractions. In order to check the possibility of enzyme adsorption on tiposome surfaces which might lead to erroneous entrapment efficien6y results, empty liposomes were prepared, mixed with glucose oxidase to give the same proportion as that used above and then chromatographed. Complete recovery of the enzyme in the second peak indicated that under the present conditions no significant adsorption of the enzyme on the liposome took place. In general, entrapment of large macromolecules (i.e., proteins) in small sonicated vesicles results in low entrapment efficiencies [41]. For example, an average encapsulation efficiency of 3.0% is reported for /3fructofuranosidase [42], 5% for amyloglucosidase [43], and 10% for bovine serum albumin [43]. These, however, are significantly lower than the encapsulation efficieneies reported for glucose oxidase in small sonieated vesicles both when the liposome is positively (with stearylamine, 45%) [44], or negatively (with DCP, 50%) charged [45,46]. In order to increase the volume of entrapped water, and thus entrapment efficiency, media of low ionic strength and incorporation of charged lipids are required [47-49]. It is reported that an initial high salt concentration inhibits the binding of proteins to the lipid layer, but it can not reverse the binding once it has occurred [48]. In the glucose oxidase entrapment studies reported above, the initial ionic strength was very low and the separation of the free enzyme from the liposomes was carried out by centrifugation [45,46], or by filtration [44]. Therefore, after formation of the electrostatic interaction, it probably was not possible to separate the adsorbed enzymes from the liposomal fractions by m e a n s of centrifugation or filtration, and this led to high encapsulation efficiency values. The entrapment values obtained in the present study might be low as a result of high initial ionic strength but since the bindings are not strong, the calculated entrapment values are more accurate. Chromate. A n entrapment efficiency of 0.112% is observed with chromate ions (measured at 370 nm). This is a very low value but it is in a good agreement with those reported for chromate ions entrapped in other small sonicated vesicles prepared in a similar way and having identical composition [50]. Release of the anion from those liposomes, whether treated with Triton X-100 or untreated was very rapid. In the first 30 min, the release levels were similar (approx. 40%). This probably contributed to the finding of low encapsulation efficiency values.


Effect of various treatments on enzyme activity Sonication. The effect of sonication on the free enzyme activity was checked, by sonicating stock enzyme solutions for 8 min at 4°C. The slopes of the linear relations observed between the enzyme concentrations and the absorbances at 525 nm for the sonicated samples and the un-sonicated blanks were found to be 1.63" 1{)-3 A ml /zg -~ and 1.97" 10 -~ A ml ttg - l , respectively (Fig. 5). It thus seems that sonication of the free glucose oxidase solution under the present conditions causes a fixed decrease of about 17% in the enzyme activity throughout the range of enzyme concentrations used. Chloroform. Since, the enzymatic activity of glucose oxidase entrapped in liposomes could only be measured after breaking up the vesicles, lysis by organic solvents besides Triton X-100 was tried. Thus, immediately after chromatography, 1 ml aliquots of each fraction were treated with an equal volume of chloroform as mentioned in the Methods and a substantial loss in activity (approx. 50%), was observed in both enzyme peaks (Fig. 6). This is in quite good agreement with the observation of the loss of activity observed with free glucose oxidase, and also with retention of only 41% of activity of alkaline phosphatase after organic solvent treatment [51]. It is highly likely that organic solvents denatures proteins and leads to enzyme inactivation.

Prediction of the entrapment efficiency The size of the liposome and the entrapment efficiency can be predicted by making certain assumptions and using initial concentrations. In the calculation of the entrapment efficiency, a phospholipid surface area of 72 AZ/moleeule, (condensed to 58 ,~z in the presence of cholesterol), and a cholesterol surface area of 38 ~z are assumed [5 l]. The vesicles are assumed to be spherical structures (as

0.32 0.28

030 ~o25



~ o~o o o5

0 I0 20 30 40 50 60 Froch0n Number Fig. 6. Elution profile of glucose oxidase containing liposomes. Before chloroform treatment ( ) and after chloroform treatment (-- -- --).

confirmed with S.E.M. [32]) consisting of bilipid layer membranes enclosing the aqueous solutions. No interaction between the lipid and the encapsulated proteins is taken into account. In the present studY,obY measuring an average liposome diameter of 500 A and using the lipid and enzyme concentrations employed in the entrapment, percent encapsulation efficiency and the number of glucose oxidase molecules per vesicle were calculated. The calculated encapsulation efficiency of 1.64% is in a good agreement with some of the results reported for the other enzymes [42,43], but much lower than it is obtained in this and some other studies (approx. 13%) [44-46]. This difference can be partially explained by the interaction between the enzyme and the lipids, indication of which was observed in the chromatography and centrifugation studies. The presence of one protein molecule per vesicle is generally taken as an initial assumption in vesicle size calculation studies [44] and the predicted value of 0.99 obtained by the above calculation is in a very good agreement with that.

Enzyme activity after polymer coating

E =


1B 0.20 016 o 0.12 .a '= 0.08 004 20








GMcose oxiclor~.[~g/mL] Fig. 5. Effect of sonicationon glucoseoxidase activity. Sonication for 8 rain (•) and no sonication( n ).

When the polymer coated enzyme containing liposprees are used directly as the enzyme source, no indication of the enzyme activity could be found. The activity can only be detected when the lipid layer was removed. The activity was found to be retained to a level of 89%. The inability to determine activity before treatment with solvents of lipids indicates that the vesicles could remain, after various treatments, in intact form as impermeable spheres. Upon disruption (and removal) of the lipid, a permeable, enzyme containing structure is obtained. The presence of structure and not just free enzyme at this stage was also indicated by the chromatographic data presented in Fig. 3.

108 Conclusion

It was possible to entrap enzyme glucose oxidase in polymer coated liposomes and polymer nets. Entrapment efficiencies for G O D and chromate ion were calculated with a high d e g r e e of reproducibility. T h e values for glucose oxidase w e r e not as high as the values r e p o r t e d in the literature for the s a m e e n z y m e . T h e s e liposomes, however, w e r e shown to retain their m o r p h o l o g y and p r e s e r v e up to 89% o f their original e n z y m e activity.

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Preparation and characterization of polymer coated small unilamellar vesicles.

Glucose oxidase was entrapped in small unilamellar vesicles composed of phosphatidylcholine, dicetyl phosphate and cholesterol. Prediction of the enzy...
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