Poly(ethylene glycol)-Modification of the Phospholipid Vesicles by

The PEG-lipids have to shove the PEG brush in order to incorporate into the membrane. The size of the PEG-lipids, namely the length of the acyl chains...
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Bioconjugate Chem. 2000, 11, 372−379

Poly(ethylene glycol)-Modification of the Phospholipid Vesicles by Using the Spontaneous Incorporation of Poly(ethylene glycol)-Lipid into the Vesicles Keitaro Sou, Taro Endo, Shinji Takeoka, and Eishun Tsuchida* Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan. Received October 12, 1999; Revised Manuscript Received January 14, 2000

The critical micelle concentrations of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5000)] (PEG-DPPE) and its distearoyl analogue (PEG-DSPE) were 70 and 9 µM, respectively, in buffer solutions ([Tris] ) 20 mM, [NaCl] ) 140 mM, pH 7.4) at 37 °C. When these PEG-lipid micelle dispersions were mixed with the dispersions of phospholipid vesicles comprised of a C16 membrane, of which the carbon number is 16, or a C18 membrane, the PEG-lipid micelles were dissociated into monomers and then spontaneously incorporated into the surface of the preformed vesicles. The incorporation rates and the enthalpy changes during incorporation were measured with an isothermal titration microcalorimeter. The incorporation rate of PEG-DPPE was faster than that of PEG-DSPE, because the dissociation rate of the PEG-DPPE micelles was faster than that of PEGDSPE micelles. The incorporation equilibrium constant of PEG-DSPE was larger than that of PEGDPPE due to its slow dissociation rate from the membrane, caused by the stronger hydrophobic interaction. The combination of PEG-DSPE and the C18 membrane was the most thermodynamically stabilized pair. Furthermore, the dispersion stability of the surface-modified vesicles prepared by this spontaneous incorporation was analyzed by using the critical molecular weight of the polymer for the aggregation of vesicles. The aggregation of the vesicles was successfully supressed with an increase in the molecular weight of the PEG in the PEG-lipid and its incorporation ratio.

INTRODUCTION

It is well-known that phospholipids spontaneously assemble to form vesicles (or liposomes) in aqueous dispersions. The degree of interaction force has been measured as experimental values such as phase transition temperature, flip-flop rate, the magnitude of fusion between vesicles, and fluorescence anisotropy representing segmental motion (1). The findings provided the possibility that some amphiphiles with an appropriate hydrophilic-hydrophobic balance could be incorporated into the vesicles by simple mixing as a result of coassembly. Poly(ethylene glycol) (PEG) is a physiologically stable water-soluble polymer and prevents the access of plasma proteins by its steric hindrance; it has been extensively used to modify not only the surface of phospholipid vesicles but also proteins, drugs, or immobilized materials such as an artificial blood vessel (2, 3). The vesicles of which the surface was modified with PEG chains are used to prevent aggregation and fusion and to prolong blood circulation (4, 5). The PEG modification and physical properties of PEG-lipid/phospholipid mixed systems have been extensively studied (6-11). We have studied vesicles encapsulating concentrated hemoglobin as artificial red cells and applied the PEG-modification in order to stabilize the dispersion state and prolong their life in blood circulation (12). The PEG-lipid is generally incorporated into the surface of vesicles by mixing it with components in common organic solvents before dispersing them into an aqueous solution. In this case, both sides of the bilayer membrane * To whom all correspondence should be addressed.

are modified. The PEG chains extending from the inner surface should reduce the interior volume for encapsulation because of their exclusion volume effect. Furthermore, they do not take part in the steric stabilization of the vesicle dispersion even if they are important from the standpoint of the thermodynamic stabilization (13). Modification only to the outer surface was therefore carried out to couple PEG with the phosphatidylethanolamine incorporated into preformed vesicles (14) or to mix the PEG-lipid with a vesicle dispersion, followed by the insertion of PEG-lipids into the preformed vesicles (15, 16). However, the detailed study about the incorporation kinetics and equilibrium states has not been reported yet. In this paper, we observed the spontaneous incorporation of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamineN-[monomethoxy poly(ethylene glycol)] or 1,2-distearoylsn-glycero-3-phosphatidylethanolamine-N-[monomethoxy poly(ethylene glycol)] into vesicles using an isothermal titration calorimeter and 1H NMR. The kinetics and thermodynamics of the coassembling phenomenon were discussed relating to the hydrophobic interaction force. Furthermore, we clarified the effect of the molecular weight of the PEG on preventing the vesicle aggregation. EXPERIMENTAL PROCEDURES

Materials. The poly(ethylene glycol) (standard) [PEG (Mw 640, 1040, 1530, 2060, 3070, 9450, 11 000, and 21 230)] was a gift from NOF Co., and PEG (Mw 2560, 3410, 5050, and 6250) was purchased from Sowa Science Co. The Mw/Mn of each PEG was within 1.04. 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[monomethoxy poly(ethylene glycol) (5000)] (PEG-DPPE) and 1,2distearoyl-sn-glycero-3-phosphatidylethanolamine-N[monomethoxy poly(ethylene glycol) (2000 or 5000)]

10.1021/bc990135y CCC: $19.00 © 2000 American Chemical Society Published on Web 04/14/2000

PEG-Modification of the Phospholipid Vesicles

Bioconjugate Chem., Vol. 11, No. 3, 2000 373

[PEG-lipid]micelle ) {(It - Ii)/(I0 - Ii)}[PEG-lipid] (1)

Figure 1. Determination of the critical micelle concentration of PEG-lipids by the fluorescence intensity change of DPH incorporated into PEG-lipid micelles. (O) PEG-DPPE and (b) PEG-DSPE.

(PEG-DSPE) were purchased from NOF Co. The mixed lipid powders of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, and 1,2-dipalmitoyl-snglycero-3-phosphatidylglycerol (DPPG) at 5:5:1 in molar ratio (Presome PPG-I) were used for a C16 membrane. 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphatidylglycerol (DSPG) purchased from Nippon Fine Chemical Co. were mixed in the same ratio as for the C18 membrane. Vesicle Preparation. Mixed lipids were dissolved in chloroform and dried in vacuo. to form a film on the wall of a flask. After the film was dispersed into a Tris-HCl buffer solution ([Tris] ) 20 mM, [NaCl] ) 140 mM, pH 7.4) with glass beads, it was extruded through a polycarbonate membrane filter (the final pore size of the filter was 0.2 µm). The diameter of the resulting vesicles was determined to be 250 ( 40 nm with a Coulter submicron analyzer (model N4SD). The total surface area of the vesicle was measured using 6-p-toluidino-2-naphthalenesulfonic acid (Tns) (17), and the number of bilayers of the vesicle was measured to be 2.0 and 1.5 for vesicles with a C16 membrane and a C18 membrane, respectively. Critical Micelle Concentration (CMC). A 1,6-diphenyl-1,3,5-hexatriene (DPH) solution (2 µL, [DPH] ) 30 µM, in THF) was added to various concentrations of PEG-DPPE or PEG-DSPE dispersions (2 mL) and incubated at 37 °C for 2 h. We measured the fluorescence intensity of the dispersions and defined the concentration where the intensity increased abruptly as the CMC (18). These concentrations were determined to be 70 and 9 µM for PEG-DPPE and PEG-DSPE, respectively, as shown in Figure 1. Dissociation of the Micelle. The PEG-lipid dispersion ([PEG-lipid] ) 0.83 mM, [DPH] ) 4.14 µM, 20 µL) was injected into the Tris-HCl buffer (2.7 mL) at 37 °C, and the change in the fluorescent intensity (λex ) 357 nm, λem ) 430 nm) was recorded with a spectrofluorometer (JASCO FP-770). The initial fluorescence intensity (I0) decreased gradually to reach the infinite intensity (Ii), and It is the fluorescent intensity at t seconds. The intensity ratios, It/I0 or Ii/I0, show the dynamic change in the DPH environment or an equilibrated state after dilution of PEG-lipid micelles. The concentration of PEGlipid as micelles at t seconds after diluting them below the CMC is

The rate of demicellization was obtained from the initial change in the [PEG-lipid]micelle. Segmental Motion of Acyl Chains of Micelles and Vesicles. A DPH solution (5 µL, [DPH] ) 1.4 mM, in THF) was added to a PEG-lipid dispersion (3 mL, [PEGlipid] ) 1 mM). A THF solution in the absence of DPH was added to the PEG-lipid dispersion as a blank. These samples were incubated at 37 °C for 2 h. Fluorescence spectroscopy was carried out with a JASCO FP-770 spectrofluorometer equipped with polarizers and connected to a thermo-controller. DPH was excited at 357 nm, and its fluorescence was detected at 430 nm. The anisotropy of the vesicles with a C16 membrane or C18 membrane was measured by the same method. Incorporation Rates. The PEG-lipid dispersion (10 µL, [PEG-lipid] ) 0.83 mM) was injected by a computercontrolled microsyringe into the vesicle dispersion ([lipid] ) 9.9 ≈ 26.0 mM, 1.35 mL) and stirred at 400 rpm, within 2 s. Temperature was strictly controlled to 37 °C. Changes in the calorific values and the total calorific values were measured using an OMEGA titration microcalorimeter (MCS ITC, Microcal, Inc.) (19). Identical injection of the PEG-lipid dispersions into buffer solutions in the absence of vesicles or the injection of buffer into vesicle dispersions were performed as control experiments. The initial rate was analyzed as a second-order reaction, relating to the concentrations of PEG-lipid and the incorporation site from eq 2:

d[PEG-lipid]/dt ) kon[PEG-lipid]t[incorporation site]t (2) where t (seconds) is time passed after injection and [PEGlipid]t and [incorporation site]t are concentrations of PEGlipid and the incorporation site at t (seconds), respectively. The parameter kon was a rate constant for PEGlipid incorporation. The incorporation site was calculated as described below. Equilibrium Constants. The PEG-lipid dispersion (19 mL, [PEG-lipid] ) 17.5 µM) was added to the vesicle dispersion (25 mL, [lipids] ) 17.3 mM) and stirred at 37 °C (final concentration; [PEG-lipid] ) 7.6 µM, [lipids] ) 9.8 mM). The mixture was intermittently sampled and was used to measure the incorporated amount of PEGlipid. The sample was centrifuged (100000g, 60 min) at 37 °C to remove the unincorporated PEG-lipid as a supernatant. The precipitated vesicles were freeze-dried and dissolved in CDCl3. The peak area ratio of the choline methyl proton of DPPC or DSPC (3.39 ppm) to the methylene proton of PEG (3.63 ppm) was measured by 1H NMR spectroscopy (JEOL JNM-LA500) to obtain the incorporated amount of the PEG-lipid (9). The same measurement was carried out for the control sample where unincorporated PEG-lipid was not removed to determine the total amount of PEG-lipid and then free PEG-lipid. The equilibrium constant (K) was calculated from eq 3:

K ) [incorporated PEG-lipid]/([incorporation site] [free PEG-lipid]) (3) where [incorporation site] is the initial concentration of the incorporation site for PEG-lipid in the surface of the mixed lipid vesicles. We estimate it to be the same as the saturated amount of PEG-lipid incorporation, which

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Figure 2. Observation of the PEG-lipid micelle dissociation by dilution below the CMCs at 37 °C from the fluorescence intensity change of DPH. The PEG-lipid dispersions (20 µL, 0.83 mM) were injected into buffer solution (2.7 mL). The final concentration was 6.1 µM (the CMCs of PEG-DPPE and PEGDSPE were 70 and 9 µM, respectively). The concentration of the PEG-lipids forming micelles was calculated from the fluorescence intensity of DPH. (O) PEG-DPPE and (b) PEG-DSPE.

can be obtained from the time course of the PEG-lipid incorporation. Turbidimetry. The PEG solution (500 µL, 0.12 kg/ dm3) was added to the vesicle dispersion (2 mL, 1.39 mM) in a cuvette (l ) 1 cm) at 37 °C. The turbidity change (∆OD) at 600 nm was monitored for 15 min after the addition of PEG by a UV-vis spectrophotometer (Shimadzu, MPS-2000). The minimum molecular weight of added PEG for the aggregation of the vesicles was defined as a critical molecular weight (Mc) (20). Incorporation of the PEG-Lipid into Unmodified Vesicles by Mixing with Modified Vesicles. The dispersion of vesicles having 1.0 mol % of PEG-lipid (15 mL, [lipids] ) 1.39 mM) was mixed with a vesicle dispersion (45 mL, [lipids] ) 1.39 mM). The mixed dispersion was incubated at 37 °C, and the time course of Mc was measured by turbidimetry as described above. The PEG-lipid amount incorporated into the unmodified vesicles was calculated from the relationship between Mc and the incorporation ratio of PEG-lipid, because we can monitor only the Mc change. The incorporation rate of the PEG-lipid into unmodified vesicles was calculated from the slope of the straight line in Figure 7. RESULTS AND DISCUSSION

PEG-Lipid Micelles. As shown in Figure 1, the CMC of the PEG-DSPE is lower than that of PEG-DPPE because of the stronger hydrophobic interaction. However, these values were significantly higher (more than 106 times) than diacyl phospholipids (e.g., DPPC < 10-10 M), indicating the increase in hydrophilicity by PEGconjugation. When the PEG-lipid micelle dispersions are diluted below the CMC, the micelles are dissociated into monomers. The concentration of PEG-lipids assembling as micelles was calculated from the fluorescent intensity of DPH. As shown in Figure 2, the rate constant of dissociation for the PEG-DPPE micelles was estimated to be above 3.0 s-1 at 37 °C. On the other hand, that for the PEG-DSPE micelles was more than 5 × 10-3 s-1 at the same temperature. This also indicates the stronger hydrophobic interaction of PEG-DSPE than that of PEGDPPE. PEG-lipid micelles have a lower anisotropy of DPH compared with 250 nm phospholipid vesicles as shown

Sou et al.

Figure 3. Temperature dependence of the fluorescence anisotropy of the DHP incorporated into micelles or vesicles. (O) PEG-DPPE, (4) C16 vesicle, (b) PEG-DSPE, and (2) C18 vesicle, (0) C16 vesicles with 5 mol % of PEG-DPPE.

in Figure 3. The lower anisotropy indicates a higher segmental motion of the acyl chains (21). The high anisotropy and low temperature dependence of phospholipid vesicles should be due to the cholesterol effect (45 mol % mixture), because cholesterol enhances the membrane packing and alleviates the large change in the segmental motion of acyl chains at the phase transition temperature (22). The PEG-lipid micelles have a larger temperature dependence and a larger effect of acyl chain length, attributed to the lower order of the acyl chain packing in the micelles. The PEG-DPPE micelles have a higher segmental motion of the acyl chains than those of PEG-DSPE because of the lower interaction force. The anisotropy of DPH in the phospholipid bilayer having 5 mol % PEG-DPPE in the outer layer is almost the same as that without PEG-DPPE, indicating that the incorporation of PEG-DPPE does not disturb the acyl chain packing in the bilayer membrane. Therefore, when the PEG-lipid micelles, which are not stable assemblies compared with phospholipid vesicles, are mixed with vesicles, the PEG-lipids should be spontaneously incorporated into the highly packed bilayer membrane. Incorporation of the PEG-lipid into Vesicles. When the PEG-lipid dispersions below the CMC were injected into vesicle dispersions, a heat response could not be detected because of the lower limit of detection (0.2 µcal/s). Therefore, we carried out this experiment above the CMC. When the PEG-lipid dispersion was injected into the vesicle dispersion, an exothermic peak was observed with each addition of titrant as shown in Figure 4, panels a and b. In this figure, the positive and negative values of the y-axis mean the endothermic and exothermic rates, respectively. The first steep increase in the exothermic rate should be caused by the late response of the heat detection after one shot, because the half-response time of this apparatus is around 10 s. If the exothermic rate after baseline compensation with a blank titration were regarded to show the enthalpy change during the incorporation of PEG-lipid, a gradual subsequent return to the baseline would be due to the reduction of the amount of PEG-lipid remaining in the solution. Therefore, we could determine the amount of PEG-lipid in the vesicle from the integration of the exothermic rate and the compensation using molar enthalpy. Figure 4c represents the incorporation profiles of PEGDPPE and -DSPE into a C16 membrane. When the concentration of the vesicles was increased twice, the

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Though the kon values do not show a significant difference between C16 and C18 membranes, the kon values of PEG-DPPE were eight times higher than those of PEG-DSPE. This seems strange because the stronger hydrophobicity of PEG-DSPE should lead to a faster incorporation rate. Two explanations are considered for the incorporation mechanism; one is incorporation as monomers, and the other is incorporation as micelles. The former accompanies the dissociation of micelles, because the PEG-lipids were added in a micelle state, and their concentrations after addition were below the CMC. We consider that the PEG-lipid should incorporate as a monomer after dissociation of the micelles, because the rates of dissociation of the PEG-lipid micelles were faster than the rates of PEG-lipid incorporation. Namely, more than 70% of the PEG-DPPE micelles dissociated within 2 s as shown in Figure 2, and it took more than 2 min for about 70% of the PEG-lipid to incorporate into the C16 membrane. In the case of PEG-DSPE, 70% of the micelles dissociated within 8 min, whereas less than 30% of the PEG-lipid incorporated into the C16 membrane. Therefore, the incorporation of the PEG-lipid would occur as monomer after dissociation of the micelles. As shown in Table 1, the kons of PEG-lipids into the C18 membrane were almost the same as those into the C16 membrane. This supports the conclusion that PEGlipid incorporation was influenced more by the size of the hydrophobic parts of the PEG-lipids than by the nature of the host bilayer membrane. The calorific values (∆H) in Table 1 were calculated from the total peak area, which is seen in Figure 4, panels a and b. The enthalpy changes (∆H) during the incorporation of PEG-lipids into the C16 membrane were small in comparison with those of PEG-lipids into the C18 membrane as depicted in Table 1. The higher ∆H for the C18 membrane would be due to the stronger stabilizing effect of the PEG-lipids after incorporation into the membrane having stronger hydrophobic interaction. Incorporation Stability of PEG-Lipids. A dispersion of C16 membrane vesicles was diluted into an excess amount of PEG-lipid dispersion, and the incorporated amount of the PEG-lipid was measured using 1H NMR after complete separation of the unincorporated PEGlipid. Because PEG-lipid incorporates into only the outer layer of the bilayer membranes, we expressed the molar ratio of the PEG-lipid to the total lipids consisting of the outer layer of the bilayer membrane. This was calculated from the number of bilayer membranes of the vesicles, the average size of the vesicles, and the lipid concentration of the dispersion (17). The average number of the bilayer membrane was calculated to 2.0 for vesicles with the C16 membrane and 1.5 for vesicles with the C18 membrane. The ratio of the lipids in the outer layer to the total lipids was approximated to be 0.25 and 0.33, respectively. As summarized in Table 1, PEG-DPPE and -DSPE were incorporated into the vesicles to around 9 and 5.5 mol %, respectively. There was no significant difference between the C16 and C18 membranes. We estimated that these were maximun values for incorpora-

Figure 4. Detection of the PEG-DPPE and PEG-DSPE incorporated into the preformed C16 vesicles by an isothermal titration calorimeter. (a, b) Raw data from the isothermal titration calorimeter ([lipids] ) 9.9 mM) for PEG-DPPE and PEG-DSPE, respectively, and (c) the incorporation rate calculated from the equation ([PEG-lipid]in ) ∆Ht/∆H[PEG-lipid]0). (O) PEG-DPPE, (b) PEG-DSPE.

incorporation rate of the PEG-lipid also increased twice, indicating the validation of eq 2. The kon values of PEGDPPE and -DSPE being incorporated into the C16 membrane were 41.0 and 5.5 M-1 s-1 (37 °C), respectively, and those for the C18 membrane were 42.8 and 5.5 M-1 s-1 (37 °C), respectively. They are summarized in Table 1.

Table 1. Thermodynamic Parameters of Incorporation and Dissociation of the PEG-Lipid on Vesicles at 37 °C

PEG-DPPE PEG-DSPE a

membrane

max ratio (mol %)

appk on (s-1 M-1)

C16 C18 C16 C18

9.2 8.9 5.7 5.6

41.0 42.8 5.5 5.5

5 off × 10 (s-1)

appk

2.3 1.5 0.24 0.057

incorporation ratioa (%)

K × 10-4 (M-1)

∆Gb (kcal/mol)

∆H (kcal/mol)

72 83 74 93

1.1 1.7 2.0 7.1

-5.7 -6.0 -6.1 -6.9

-16 -21 -13 -23

0.3 mol % of PEG-lipids were added to the vesicle dispersion ([lipids] ) 9.9 mM). b ∆G ) -RT ln K.

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tion because there was no further change with time and we used them for calculation of kon and K. In general, the incorporation of amphiphilic molecules having a micelle-forming property into the bilayer membrane accompanies the transition from vesicles to micelles, if their concentrations were beyond the critical values (23). From the theoretical analysis of the phase behavior of the PEG-lipid and phospholipid mixture in aqueous media, the maximum amount of PEG-lipid being incorporated into the mixed vesicles could be determined from the membrane elasticity, e.g., the cholesterol content in the bilayer and the PEG chain length (6). The maximum concentration of PEG (Mw 5000)-lipid in phospholipid/cholesterol (1/1 mol/mol) was calculated to be ca. 15 mol %. Above this concentration, the thermodynamically favorable structure would be a micelle rather than a vesicle (7). This theoretical speculation was also experimentally confirmed elsewhere (7-9). In those experiments, the PEG-lipids were mixed with phospholipids before preparation of vesicles. In our experimental procedure, the PEG-lipid was incorporated into the vesicles to 9 mol %, not 15 mol %. It is considered that the steric hindrance of the PEG chains extending from the vesicular surface becomes more significant and tends to restrict the further incorporation of the PEG-lipid. Therefore, the steric effect of the PEG chains, which had already been incorporated, and the molecular size of the PEG-lipid should determine the amount of PEG-lipid incorporated into the preformed vesicles. The incorporation behavior in this region (maximum concentration) should not be in an equilibrium state, but in a kinetically limited state. The maximum incorporation does not depend on the acyl chain length of the phospholipids comprising the host membrane but on the acyl chain length of the guest PEGlipids. Theoretically, the entire surface of the vesicle should be covered with 1.5 mol % of PEG (5000) fixed onto the surface (15). The conformation of the PEG chain in this state is considered a mushroom (24). Further PEG incorporation results in a brush conformation, where PEG chains are extended and become compressed with increasing density of the PEG chains. The PEG-lipids have to shove the PEG brush in order to incorporate into the membrane. The size of the PEG-lipids, namely the length of the acyl chains, should influence this process because of the molecular sieving effect of the dense PEG brush. Figure 5a is the time course of PEG-lipid incorporation for all combinations. There are significant differences in the incorporation rates between PEG-DPPE and PEGDSPE. Namely, PEG-DPPE was incorporated very fast in the membrane as predicted from the incorporation rates and reached constant values. The constant values represent equilibrium constants (K) of PEG-lipid incorporation, and the C18 membrane always shows the higher values than the C16 membrane. The saturated incorporation ratios and K values for all combinations are summarized in Table 1. The K value of PEG-DSPE incorporation into the C16 membrane at 37 °C was 2.0 × 104 M-1, which was about twice that (1.1 × 104 M-1) of PEG-DPPE incorporation. However, similar incorporation ratios were obtained for PEG-DPPE and PEG-DSPE. The difference in incorporation sites (maximum incorporation ratio) would explain this result. The incorporation ratio should be dependent on the concentrations of the PEG-lipid and vesicles, in relation to the K values. The constancy of the K values

Sou et al.

Figure 5. Determination of the incorporated PEG-lipids on preformed vesicles by 1H NMR. Incorporated PEG-lipid was determined by the peak area ratio of the choline methyl proton of DPPC or DSPC (3.39 ppm) to the methylene proton of PEG (3.63 ppm). (a) Incorporation into vesicles and (b) dissociation from vesicles by dilution. The combinations of the PEG-lipids and vesicles were (O) PEG-DPPE and C16, (4) PEG-DPPE and C18, (b) PEG-DSPE and C16, and (2) PEG-DSPE and C18.

in different concentrations of PEG-lipids and vesicles confirms the appropriateness of the eq 3 (data not shown here). As depicted in Table 1, despite the smaller kon value of PEG-DSPE than that of PEG-DPPE, the K value of PEG-DSPE was larger than that of PEG-DPPE. This would be explained based on the small dissociation rate (koff) of PEG-DSPE from the C16 membrane. As estimated from the equilibrium constant of PEG-lipid incorporation, the removal of the PEG-lipid from the bilayer membrane should occur after the unincorporated PEGlipid is removed or the mixture dispersion is diluted. The decrease in the incorporation ratio of PEG-lipids by dilution was measured and is shown in Figure 5b. We confirmed the dissociation of PEG-DPPE to be faster than that of PEG-DSPE as predicted from K and kon. The apparent dissociation rate constants (appkoff) of PEG-lipids calculated from the pseudo-first-order of the first stage are summarized in Table 1. The combination of PEGDSPE and the C18 membrane shows the lowest appkoff value of the four combinations. As shown in Table 1, the combination of PEG-DSPE and the C16 membrane is the lowest ∆H of the four combinations, while the combination of PEG-DPPE and the C18 membrane has a relatively high ∆H. It is suggested that the guest PEG-lipid having longer acyl chains than the host phospholipid would not take highly

PEG-Modification of the Phospholipid Vesicles

Figure 6. Relationship between incorporation ratio of PEGlipids into C16 vesicles and Mc. (O) PEG(5,000)-DSPE, (b) PEG(2000)-DSPE at 37 °C. The incorporation ratio was determined by 1H NMR (see Experimental Procedures) and represented against the outer layer of the vesicles.

ordered packing after incorporation, because the guest lipid should oblige to take more number of kink conformation in order to adjust itself to the thickness of the outer layer of the bilayer membrane. The guest PEGlipid having shorter acyl chains than the host phospholipid could order in the bilayer membrane. Stability of the Vesicle Dispersions. To clarify the relationship between the dispersion stability of the PEGmodified vesicles and the incorporation amount or molecular weight of the PEG-lipid, we prepared PEGmodified vesicles incorporating various amounts of the PEG-lipid. The incorporated amount of PEG-lipids was easily controlled by varying the added amount of PEGlipids. Phospholipid vesicles tend to aggregate in the presence of water-soluble polymers by molecular interaction (20). Because the dissociation process of the absorbed polymer from the surface of the vesicle is dominant in this interaction, the molecular weight of the polymers should represent the degree of interaction between the polymer and the surface of the vesicle. We defined the smallest molecular weight of the polymers for aggregation of the vesicles as a critical molecular weight (Mc) (10). The higher Mc of a polymer indicates a smaller degree of interaction. Therefore, it can be influenced by the kinds of polymers, vesicle compositions, and solution composition such as pH and ionic strength. Figure 6 shows the Mc obtained in the vesicles incorporating PEG-DSPE with different PEG lengths. Mc increases with PEG-lipid concentration and reaches a constant value at a certain PEG-lipid concentration. The PEG-lipid concentration was expressed against lipids only in the outer layer of the vesicle. The increase in the Mc indicates the decrease in the interaction force and the restraint of vesicle aggregation by PEG-surface modification. We have already reported that the Mc was decreased after modification with oligosaccharide chains because of the induced interaction between the oligosaccharide chains and PEG (20). Therefore, the PEG chains extending from the surface of the vesicles have the function of excluding the added polymer from the surface. In the case of the PEG(5000)-DSPE, the relationship between the incorporation ratio of the PEG-lipid and Mc could be separated into three regions. The Mc linearly increased below 0.12 mol %, steeply increased from 0.12 to 0.18 mol %, and then became a constant value above 0.18 mol %. Whereas, in the case of PEG(2000)-DSPE, the Mc increased to 0.5 mol % and became a constant value above

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Figure 7. Interbilayer transfer of PEG-lipds from PEGmodified vesicles to unmodified ones at 37 °C. PEG-modified vesicles ([lipids] ) 1.39 mM, 1.0 mol % of PEG-lipid) were added to the unmodified C16 vesicles ([lipids] ) 1.39 mM) in volume ratios of 1 to 3. The combinations of the PEG-lipids and vesicles were (O) PEG-DPPE and C16, (4) PEG-DPPE and C18, (b) PEG-DSPE and C16, and (2) PEG-DSPE and C18. Table 2. Incorporation Rates of the PEG-Lipid into Unmofified Vesicles by Mixing with Modified Ones (PEG-Lipid: 1.0 mol %) at 37 °C PEG-lipid

membrane

d[PEG-lipid]in/dt × 103 (µM/h)

PEG-DPPE

C16 C18 C16 C18

14.9 4.1 0.2 0

PEG-DSPE

this ratio. These critical points should be related to the surface property of the vesicles. To clarify the relationship between Mc and the surface condition, we took a theoretical approach. The average Mws of PEG (2000) and PEG (5000) determined by gel permeation chromatography were 2070 (Mw/Mn ) 1.04) and 5340 (Mw/Mn ) 1.02), respectively. The areas per PEG (2000) and PEG (5000) chain were calculated to be 9.84 and 30.47 nm2, respectively (9). The critical incorporation ratios to reach the constant Mc values were 0.5 and 0.18 mol % for PEG (2000)-DSPE and PEG (5000)-DSPE, respectively. The ratios of surface coverage calculated from the area per PEG chains at these incorporation ratios were 10 and 11% for PEG (2000)-DSPE and PEG (5000)-DSPE, respectively. These values were similar, indicating that the effect of the PEG modification on the prevention of the vesicle aggregation induced by the addition of polymer was sufficient when 10% of the surface was covered with PEG chains. Incorporation of the PEG-Lipid into Unmodified Vesicles by Mixing with Modified Vesicles. When the vesicles modified with 1.0 mol % of PEG-DPPE or PEGDSPE were mixed with unmodified ones, the Mc gradually increased. The Mc of vesicles with 1.0 mol % of incorporated PEG-lipids and unmodified vesicles were 12 200 and 1050, respectively. The Mc of the mixture of vesicles with different Mc corresponded to the lower value. The Mc measured immediately after mixing (t ) 0) was 1050, which was identified as that of unmodified vesicles. Therefore, the Mc increase was caused by the incorporation of PEG-lipid into the unmodified ones. We could convert the Mc into the concentration of incorporated PEG-lipid using the relationship between the incorporation ratio of PEG-lipid and Mc as shown in Figure 6. As shown in Figure 7 and Table 2, the incorporation rate of PEG-DSPE was slower than that

378 Bioconjugate Chem., Vol. 11, No. 3, 2000

of PEG-DPPE. The rate of PEG-DPPE was 70 times higher than that of PEG-DSPE, and no incorporation was observed in the combination of PEG-DSPE and the C18 membrane. As shown in Figure 4, the incorporation rate of the free PEG-lipid was significantly faster than that of incorporated one. Therefore, it is suggested that the incorporation rate of the PEG-lipid into unmodified vesicles would be related to the dissociation step of the PEG-lipid from the vesicles because the incorporation rate decreased with the decrease of dissociation rate from the vesicles. In practice, the incorporation rates of each combination measured by Mc were slower compared with the dissociation rate determined by 1H NMR as shown in Tables 1. This would be caused by the difference in the targeted PEG-lipids. The dissociation rate was calculated against the dissociated total PEG-lipids, while the transfer rate was done against only PEG-lipids incorporated into the unmodified vesicles. All of the dissociated PEG-lipids are not always incorporated into the unmodified vesicles following the equilibrium condition, and some part of the dissociated PEG-lipids should return to the modified vesicles. Therefore, the incorporation rate should reflect the dissociation rate and depend on the molecular weight of PEG and the acyl chain length. The increase in the Mw of PEG chains and the decrease in the acyl chain length resulted in an increase in the incorporation rate. Silvius and Zuckermann also reported that the rate of PEG-lipid transfer between vesicles decreased with the increasing acyl chain length of PEG-lipid (25). CONCLUSION

We studied the PEG-lipid incorporation into the phospholipid vesicles from the kinetics of PEG-lipid micelle dissociation, the incorporation, and dissociation of the PEG-lipid into the bilayer membrane of vesicles for the combinations of PEG-DPPE, PEG-DSPE, vesicles with a C16 membrane and a C18 membrane. The kon values of PEG-DPPE were larger than those of PEG-DSPE; however, the K value of PEG-DPSE was larger than that of PEG-DPPE. This was due to the considerably small koff values of PEG-DSPE. The stability of the incorporation would be determined primarily from the hydrophobicity of the guest molecule and secondarily from the relation of the acyl chains of the host-guest molecules. The increase in the hydrophobic interaction force of the PEG-lipids and the vesicles resulted in a decrease in the dissociation rate. The most stable combination was concluded to be PEG-DSPE and the C18 membrane, though the incorporation rate was small. In the design of the PEG-lipid derivatives with large hydrophilic headgroup, one should consider the hydrophobic interaction not to dissociate from the vesicle and to incorporate the preformed vesicles. The blood circulation time is the most important factor to discuss the effect of PEG-modification to phospholipid vesicles because this is the end to apply this technique for drug delivery systems. If the PEG-lipids were escaped from the surface of the vesicles intravenously injected, this should determine the blood circulation time. We are now studying this effect from blood circulation of PEGmodified vesicles. ACKNOWLEDGMENT

This work was supported in part by a project of the Material Research Laboratory for Bioscience and Photonics in Waseda University and by Health Science Re-

Sou et al.

search Grants (Artificial Blood Project) from the Ministry of Health and Welfare, Japan. We thank to NOF. Co. for the offer of poly (ethylene glycol) (standard). LITERATURE CITED (1) Gregoriadis, G. (1984) Liposome Technology, Vol. I, II, III, CRC Press, Boca Raton. (2) Harris, J. M. (1990) Introduction to Biotechnical and Biomedical Apprications of Poly(Ethylene Glycol). In Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Apprications (J. M. Harris, Ed.) pp 1-14, Plenum Press, New York. (3) Zalipsky, S. (1995) Functionalized Poly(ethylene glycol) for Preparation of Biologically Relevant Conjugates. Bioconjugate Chem. 6, 150-165. (4) Klibanov, A. L., Maruyama, K., Torchilin, V. P., and Huang L. (1990) Amphipatic poly(ethylene glycol)s effectively prolong the circulation time of liposome. FEBS Lett. 268, 235-237. (5) Woodle, M. C., and Lasic, D. D. (1992) Sterically stabilized liposome. Biochim. Biophys. Acta 1113, 171-199. (6) Hristova, K., and Needham, D. (1995) Phase behavior of a lipid/polymer-lipid mixture in aqueous medium. Macromolecules 28, 991-1002. (7) Hristova, K., Kenworthy, A., and McIntosh, T. J. (1995) Effect of bilayer composition on the phase behavior of liposomal suspensions containing poly(ethylene glycol)-lipids. Macromolecules 28, 7693-7699. (8) Kenworthy, A. K., Simon, S. A., and McIntosh, T. J. (1995) Structure and phase behavior of lipid suspensions containing phospholipid with covalently attached poly(ethylene glycol). Biophys. J. 68, 1903-1920. (9) Kenworthy, A. K., Hristova, K., Needham, D., and Mcintosh, T. J. (1995) Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol). Biophys. J. 68, 1921-1936. (10) Warriner, H. E., Keller, Idziak, S. H. J., Slack, N. L., Davidson, P., and Safinya, C. R. (1996) Lamellar biogels: fluid-membrane based hydrogels containing polymer-lipids. Science. 271, 969-973. (11) Polo, D., Haro, I., Alsina, M. A., and Reig, F. (1997) Physicochemical Characterization of Poly(ethylene glycol)Coated liposomes loaded with doxorubicin. Langmuir 13, 3953-3958. (12) Sakai, H., Takeoka, S., Park, S. I., Kose, T., Nishide, H., Izumi, Y., Yoshizu, A., Kobayashi, K., and Tsuchida, E. (1997) Surface Modification of Hemoglobin Vesicles with Poly(ethylene glycol) and Effects on Aggregation, Viscosity, and Blood Flow during 90% Exchange Transfusion in Anesthetized Rats. Bioconjugate Chem. 8, 23-30. (13) Szleifer I., Gerasimov, O. V., and Thompson, D. H. (1998) Spontaneous liposome formation induced by grafted poly(ethylene oxide) layers: Theoretical prediction and experimental verification. Proc. Natl. Acad. Sci. 95, 1032-1037. (14) Senior, J., Delgado, C., Fisher, D., Tilcock, C., and Gregoriadis, G. (1991) Influence of surface hydrophilicity of liposomes on their interaction with plasma protein and clearance from the circulation: studied with poly(ethylene glycol)-coated vesicles. Biochim. Biophys. Acta 1062, 77-82. (15) Yoshioka, H. (1991) Surface modification of hemoglobincontaining liposomes with polyethylene glycol prevents liposome aggregation in blood plasma. Biomaterials 12, 861-864. (16) Uster, P. S., Allen, T. M., Daniel, B. E., Mendez, C. J., Newman, M. S., and Zhu, G. Z. (1996) Insertion of poly(ethylene glycol) derivatized phospholipid into pre-formed liposomes results in prolonged in vivo circulation time. FEBS Lett. 386, 243-246. (17) Takeoka, S., Ohgushi, T., Terase, K., Ohmori, T., and Tsuchida, E. (1996) Layer-controlled hemoglobin vesicles by interaction of hemoglobin with a phospholipid assembly. Langmuir 12, 1775-1759.

PEG-Modification of the Phospholipid Vesicles (18) Chattopadhyay, A., and London, E. (1984) Fluorimetric determination of critical micelle concentration avoiding interference from detergent charge. Anal. Biochem. 139, 408412. (19) Wiseman, T., Williston, S., Brandts, J. F., and Lin, L. N. (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131-137. (20) Takeoka, S., Sou, K., Arase, S., Ohguhsi, T., and Tsuchida, E. (1996) Critical molecular weight effects in the aggregation of phospholipid vesicles triggered by water-soluble polymers and an integrated glycolipid. Macromolecules 29, 8132-8136. (21) Shinitzky, M., and Barenholz, Y. (1974) Dynamics of the hydrocarbon layer in liposomes of lecithin and sphingomyelin containing dicetyl phosphate. J. Biol. Chem. 249, 2652-2657.

Bioconjugate Chem., Vol. 11, No. 3, 2000 379 (22) Ladbrooke, B. D., Williams, R. M., and Chapman, D. (1968) Studies on lecithin-cholesterol-water interactions by differential scanning calorimetry and X-ray diffraction. Biochim. Biophys. Acta 150, 333-340. (23) Fattal, D. R., Andelman, D., and Ben-Shaul, A. (1995) The vesicle-micelle transition in mixed lipid-surfactant system: a molecular model. Langmuir 11, 1154-1161. (24) deGennes, P. G. (1987) Polymer at an interface: a simplified view. Adv. Colloid Interface Sci. 27, 189-209. (25) Silvius, J. R., and Zuckermann, M. J. (1993) Interbilayer transfer of phospholipid-anchored macromolecules via monomer diffusion. Biochemistry 32, 3153-3161.

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