Effect of Hydrophilic Polymers on Physical Stability of Liposome

2374

J. Phys. Chem. B 2001, 105, 2374-2385

Effect of Hydrophilic Polymers on Physical Stability of Liposome Dispersions Kohsaku Kawakami,* Yoshitaka Nishihara, and Koichiro Hirano Formulation R & D Laboratories, Shionogi & Co., Ltd., 12-4 Sagisu 5-chome, Fukushima-ku, Osaka 553-0002, Japan ReceiVed: January 6, 2000; In Final Form: NoVember 20, 2000

Interactions between various hydrophilic polymers and liposomes composed of hydrogenated soybean lecithin were investigated by means of isothermal titration calorimetry (ITC), FT-IR, and dynamic light scattering (DLS) measurements. Their effect on the dispersity of liposomes was investigated by turbidity measurement. Sodium alginate was found to bind to lipid bilayers via electrostatic interaction and to stabilize dispersity over a wide concentration range despite its high hydrophilicity. This was elucidated in terms of the irreversible adsorption of sodium alginate onto the liposome surfaces. Carboxymethyl cellulose sodium salt and poly(vinyl alcohol) gave similar results, but the stabilization effect was reduced. Other polymers, which were adsorbed onto the liposome surfaces very weakly or not adsorbed at all, destabilized the dispersity significantly, just below the overlap concentration of polymers. This was interpreted in terms of depletion flocculation. The adsorption of the polymers onto the liposome surfaces was detected by ITC and DLS measurements, although the adsorbed fraction seemed to be very small. Notably, ITC enabled us to quantitatively estimate the heat flow accompanied by the adsorption. The sign of the heat flow due to the adsorption enabled us to detect the perturbation of the lipid membrane, which was also proved by FT-IR measurements. The relationship between the interaction strength and the stabilization effect is discussed.

Introduction The stability of colloidal dispersion can be modified by adding polymers. The polymers are, in many cases, adsorbed onto the colloidal particles. When their concentration is very low, it is often the case that the polymers bridge the particles and the dispersion is destabilized. However, the dispersion is usually stabilized by increasing the polymer concentration to the order of 1%. This is because most of the surfaces of the particles are covered by the adsorbed polymers. The effect of nonadsorbing polymers on the physical stability of colloidal particles has also attracted much attention. In general, colloidal particles become aggregated due to an imbalance of osmotic pressure between the bulk and gaps between colloidal particles. Such attractive interaction between colloidal particles is termed the depletion force.1-17 Asakura and Oosawa were the first to claim that there can be attractive forces between particles suspended in a nonadsorbing macromolecule solution, which would be strong enough to flocculate particles.1,2 More than 20 years later, Vincent et al.3 and Feigin et al.4 showed that the depletion force could stabilize particles from flocculation at higher concentrations due to unfavorable overlap of polymer coils between colloidal particles. A theoretically acceptable explanation for such depletion stabilization was derived by Fleer et al.5 by showing that the polymer-depleted layer around the particles was thinner at higher polymer concentrations. This reduced the depletion energy and restabilized the particles, because the flocculation entropy loss was greater in that concentration range. In the 1990s, on the other hand, Walz et al.10 showed the existence of repulsive barriers at larger separations by calculating in a virial expansion to the second order of the particle volume fraction. * To whom correspondence should be addressed. Fax: +81-6-64580987, E-mail: [email protected].

Mao et al.11 extended the calculation to the third order and found a secondary minimum in the interaction potential between particles. A recent theory proposed by Walz and Sharma10,14-16 and their experiments have shown that the depletion energy could be enhanced by charges on the particles. Direct experimental observation of the depletion force remains a difficult issue. To our knowledge, the most elegant method for this purpose has been to use the optical technique of total internal reflection microscopy,14-16 although its application is limited to the wall-particle interaction. Surface force apparatus can also be a very powerful tool for investigating the forcedistance relationship.12 However, some parameters such as the concentration of the larger particles cannot be altered. Further development of theory and experimental technique is still required. The interaction between cells (or liposomes) and polymers, notably poly(ethylene glycol) (PEG) has been an attractive issue also in the field of biochemistry. This is because PEG has been frequently utilized as cell fusogen,18-22 although its mechanism has not been clarified yet. Biologists have explained this phenomenon by supposing that the hydration state of lipid membranes was altered due to the strong binding affinity between PEG and water.18-20 Their interpretation may be partially true. However, additional experiments with the diluted system are needed to prove the nature of the interaction, because a few tens of percent of PEG is usually present in experiments on cell fusion. It should be very helpful to clarify the mechanism of the interaction between PEG and lipid membranes from the viewpoint of colloidal chemistry. The effect of adding hydrophilic polymers into a colloidal system is also discussed frequently among food scientists.23-26 This is because some polymers play important roles in controlling the stability and rheology of food colloids such as emulsions. The general observation is that small amounts of

10.1021/jp000087a CCC: $20.00 © 2001 American Chemical Society Published on Web 03/02/2001

Hydrophilic Polymer/Liposome Interactions

J. Phys. Chem. B, Vol. 105, No. 12, 2001 2375

TABLE 1: Polymers Used in This Study

a

polymer

abbrev

mw

radius (nm)

supplier

poly(ethylene glycol) methyl cellulose poly(vinyl alcohol) dextran pullulan sodium alginate carboxymethyl cellulose sodium salt

PEG MC PVA DEX PUL Alg-Na CMC-Na

20 000 65 000 150 000 60 000 75 000 90 000 200 000

5.5a 7.0d 14.2b 6.8c 7.5d 8.1d 11.4d

Nacalai Tesque Shin-Etsu Kagaku Wako Pure Chemical Nacalai Tesque Wako Pure Chemical Kanto Kagaku Wako Pure Chemical

Reference 3. b Reference 47. c Calculated according to ref 48. d Calculated by the same equation for dextran.

polysaccharides increase the creaming rate, whereas large amounts lead to stabilization. The detailed mechanism is under discussion. Liposomes have been, needless to say, hopefully regarded for 30 years as possible new carriers of active agents in pharmaceutical and cosmetic fields.27-33 Some products, such as skincare products,27,28 injectable formulations,29-31 and topical formulations,32,33 have already been launched and others are under development. Various other applications have also been suggested in the literature, such as their use for ocular delivery,34,35 inhalation,36,37 oral delivery,38,39 and nonvirus vector for gene therapy.40,41 When liposomes are utilized as drug carriers, the formulation often contains polymers for two purposes: (1) to modify the surface characteristics29-31,42,43 and (2) to increase the formulation viscosity. In the former case, lipid-conjugated poly(ethylene glycol) is the most frequently used material and the stabilized liposomes are called stealth liposomes.43 The polymers employed for this purpose contain hydrophobic parts, because they must bind strongly enough to liposome surfaces. The effect of the decoration by such amphiphilic polymers has been frequently studied, and the decorated liposomes were found to prevent serum protein binding onto their surfaces, resulting in long circulation time after injection. On the other hand, highly hydrophilic polymers are used in the latter case.44-46 There are relatively few studies on this topic, but recent progress in studies of the depletion force clearly indicates the importance of such work. This paper discusses the interaction between liposomes and polymers and the effect of addition of hydrophilic polymers on liposome dispersity. Experimental Section Materials. Hydrogenated soybean lecithin (HSL) was purchased from Nippon Oil & Fats (Tokyo, Japan). HSL consisted of ca. 87% of distearoyl phosphatidylcholine and ca. 13% of dipalmitoyl phosphatidylcholine. Polymers together with their abbreviation, molecular weight, radius of gyration, and supplier are listed in Table 1. All polymers were used as supplied but stored in a desiccator before use. Preparation of Liposome Dispersions. Liposome dispersions were prepared by the freeze-dry method.49,50 Briefly, weighed lipid powder was dissolved in tert-butyl alcohol above the phase transition temperature (at ca. 65 °C), and then the alcohol solution was freeze-dried. The powder obtained was dispersed in distilled water also above that temperature with vigorous vortexing. In some experiments, phosphate buffer (1 mM, 10 mM, or 200 mM, pH 6.5) was used as the dispersion medium. The diameter of the liposomes was ca. 2 µm, and there were two lamellar layers on the average.50 Polymer-containing dispersions were prepared by mixing liposome dispersions with polymer solutions. The size of the liposome for the turbidity measurement was reduced by Extruder (Lipex Biomembranes, Vancouver, Canada) using polycarbonate membranes. The pore

size of the membrane was 600 nm, and the extrusion was repeated for 5 times. The diameter of the obtained liposome was confirmed to be ca. 600 nm by He-Ne laser light scattering measurements on Coulter N4 Plus (Coulter, FL). Preparation of Polymer Solutions. The well-dehydrated polymers were weighed and dissolved in distilled water or the phosphate buffer, and the solution was degassed under vacuum. These solutions were hydrated at least for 1 day at room temperature. Isothermal Titration Calorimetry. The heat flow due to the adsorption of polymers onto liposome surfaces was measured using a Thermometric 2277 thermal activity monitor (Thermometric BA, Sweden). The stainless steel sample cell was filled with 3 mL of water or a liposome dispersion, into which 20 µL of a polymer solution was injected at 15-min intervals. The concentrations of both the liposome dispersion in the cell and the polymer solution in the syringe were 10 mg/mL except for the Alg-Na solution, which was reduced to 5 mg/mL because of its high viscosity. The sample cell and the reference cell filled with 3 mL of water were immediately placed to the equilibrium position of the calorimeter. After that, they were slowly immersed into the measuring position and left for at least 3 h. After confirming that the baseline was stable, the injection was repeated twelve times. The heat flow was monitored and integrated. The first injection was excluded from the analysis because it contained large experimental errors. All measurements were taken at 25 ( 0.0001 °C and repeated for at least 3 times. The heat flow accompanied by the interaction between PEG molecules and salt was measured in the same manner. The sample cell was filled with 3 mL of water or a PEG solution, into which 20 µL of water or a phosphate buffer solution was injected. The concentrations of PEG and buffer were 10 mg/ mL and 200 mM, respectively. Fourier Transform Infrared Spectroscopy. The physical effect of polymers on lipid membranes was evaluated by Fourier transform infrared spectroscopy (FT-IR). ATR spectra were acquired using a Magna-IR 560 spectrometer (Nicolet, WI), by coaddition of 128 interferograms collected at 1 cm-1 resolution. To exclude the effect of the osmotic pressure on the FT-IR spectra due to the polymers outside, liposome dispersions for this experiment were prepared using polymer solutions. Therefore, polymers were located both inside and outside the liposomes at nearly the same concentration. HSL concentration was 50 mg/mL and the measurement was done at room temperature. Surface Tension Measurements. Surface tension measurements of the polymers were performed by the Wilhelmy plate method using Kyowa CBVP surface tensiometer A3 (Kyowa Kagaku, Tokyo, Japan). The sample room was maintained at 25 ( 0.2 °C by circulating temperature-controlled water, and the polymer solutions for the measurements were immersed in a thermobath controlled at 25 ( 0.2 °C before use. Before every measurement, the platinum plate was heated with a burner,

2376 J. Phys. Chem. B, Vol. 105, No. 12, 2001

Figure 1. Surface tension of polymers. Polymer type: PEG (O), MC ([), PVA (2), DEX (9), PUL (4), Alg-Na (]), and CMC-Na (0).

followed by rinsing with distilled water and acetone. All the data were read at 3 min after the creation of new surfaces. The measurement accuracy was ca. (0.1 mN/m, except for the case in which the time dependence was very strong. Bulk Viscosity Measurements. The viscosity of polymer solutions was obtained using a Cannon-Fenske type viscometer immersed in a thermobath controlled at 25 ( 0.2 °C. Measurements were done at least twice and experimental errors were confirmed to be within 1%. The values obtained from these measurements were adopted as the bulk viscosity of polymerliposome mixtures. Microviscosity Measurements. Microviscosity, which can be defined as the local viscosity around the liposome surfaces, was evaluated from the laser light scattering measurements on Coulter N4 Plus. The scattering angle was 90° and the obtained data was analyzed by the cumulant method. Each measurement was repeated 3 times, and the averaged self-diffusion coefficients were converted to viscosity values via the Einstein-Stokes relationship. The concept of this experiment is that polymer adsorption onto liposome surfaces should enrich the polymer fraction around liposomes in comparison to the bulk and therefore the local viscosity would increase, which should depress the Brownian motion of liposome particles, and vice versa. The change in the apparent diameter of liposome particles due to the polymer adsorption is negligible, since liposomes are much larger than polymers. The osmotic shrinkage can also be ignored, because the polymer concentration for this experiment is very low. Turbidity Measurements. To evaluate the stability of liposome dispersions, the turbidity of dispersions was measured at 600 nm after mild centrifugation (1500 rpm × 10 min, 25 °C), which precipitated most of the liposomes. The size distribution of the supernatant was the same as that of the whole dispersion, suggesting that only the weakly flocculated liposome particles were precipitated by the centrifugation. Therefore, the increase in the turbidity could be attributed to the stabilization of dispersions and vice versa. All measurements were done 24 h after mixing of liposomes and polymers. Results Surface-Active Properties of Polymers. Although all of the employed polymers were highly hydrophilic, they had different surface-active properties depending on whether their hydrophobic parts could be separated from the hydrophilic parts at the water/air interfaces.51 Figure 1 shows the surface tension of the polymers at 3 min after the creation of new surfaces. As can be seen, the employed polymers can be separated into two groups.

Kawakami et al. PEG, MC, and PVA were relatively surface-active, while the other polymers hardly reduced surface tension, even at very high concentrations. Three minutes is too short to attain the equilibrium state, because more than several hours are required for most hydrophilic polymers.51-53 However, it was enough for the qualitative evaluation. If the surface-active property of polymer plays a dominant role in the polymer-liposome attractive interaction, PEG, MC, and PVA should be adsorbed onto lipid membranes. Isothermal Titration Calorimetry. Isothermal titration calorimetry (ITC) is a very powerful tool to investigate weak physical interactions between different molecules due to its high sensitivity.54-57 Figure 2 shows examples of titration experiments using Alg-Na as a hydrophilic polymer. When 20 µL of water was periodically injected into 3 mL of water (Figure 2a) or liposome dispersion (Figure 2b), no heat flow was observed. Therefore, the heat flow due to the mixing of solvent can be neglected in this experiment. However, when a small amount of polymer solution was injected into a large amount of water (Figure 2c), constant heat flow was obtained. This was also the case when polymer solutions were added to liposome dispersions (Figure 2d). The heat flow was not polymerconcentration dependent, because no significant decrease with injection number was observed. This was because the liposome concentration was very high. The decrease of the heat flow was investigated when the liposome concentration was lowered. However, it seemed to be desirable to perform the titration experiments under the liposome-rich condition, because the tendency at low liposome concentration was not very clear, perhaps due to the very weak interaction. In normal cases, the existence of the liposome in the cell changed the heat flow significantly compared to the liposomefree referential experiments, as in the case of Alg-Na. The difference in the heat flow can be explained by the liposomepolymer interaction. Table 2 lists the averaged heat flow accompanied by addition of polymer solutions. The heat flow of the dilution was very large for the charged polymers (AlgNa, CMC-Na), but almost negligible for highly hydrophilic polymers (DEX, PUL). The large heat flow of the charged polymers can be explained in terms of the electrostatic charging energy. In the presence of liposome, the significant change in the heat flow compared to the liposome-free solution was investigated for PEG, MC, PVA, Alg-Na, and CMC-Na according to Student’s t-test. The change was exothermic for MC and Alg-Na, but endothermic for PEG, PVA, and CMCNa. It can be assumed that there are direct interactions between these polymer molecules and the liposomes. However, the interaction seemed to be very weak and the adsorbed fraction to be very small, as discussed later. To obtain further information to prove the interaction mechanism, we performed the same experiment in the presence of salt for PEG and Alg-Na. Table 3 shows that the heat flow of the dilution of the polymer itself was almost the same for PEG, but decreased significantly for Alg-Na in the presence of the salt. This is most likely a screening effect. The presence of the liposome led to no significant change in the heat flow for both polymers, suggesting that salt inhibited the interaction between liposomes and polymers. Although the hindrance of the adsorption of Alg-Na can be easily elucidated by the screening effect, that of PEG is difficult to explain. To overcome this problem, we also observed the PEG-salt interaction by ITC. Figure 3 shows that the injection of water into PEG solution yielded a small exothermic heat flow and that of the phosphate buffer into water a large endothermic heat



Figure 2. Examples of the heat flow observed in the ITC measurement: (a) periodic injection of water into a large volume of water, (b) periodic injection of water into a large volume of liposome dispersion, (c) periodic injection of Alg-Na solution into a large volume of water, (d) periodic injection of Alg-Na solution into a large volume of liposome dispersion.

TABLE 2: Heat Flow Accompanied by Addition of Polymer Solution

a

polymer

w/o liposome (kcal/mol)

w/ liposome (kcal/mol)


1.53 ( 0.30 5.66 ( 1.62 13.9 ( 2.7 1.89 ( 0.49 0.565 ( 0.878 50.1 ( 7.0 150 ( 39

1.11 ( 0.31b 6.94 ( 0.53b 11.2 ( 1.8a 2.33 ( 0.60 0.798 ( 1.192 68.4 ( 10.6b 125 ( 16b

p < 0.01. b p < 0.001

TABLE 3: Heat Flow Accompanied by Addition of Polymer in Presence of Salt polymer

w/o liposome (kcal/mol)

w/ liposome (kcal/mol)

PEG Alg-Na

1.40 ( 0.50 25.0 ( 2.9

1.43 ( 0.56 26.4 ( 6.1

flow. However, that of the phosphate buffer into PEG solution produced much a larger endothermic heat flow than into water, suggesting that there was an endothermic interaction between PEG and salt. The heat flow due to the PEG-salt interaction was estimated to be ca. -70 cal/mol. Although the details of the interaction are under investigation, it is very likely that the PEG-salt interaction inhibited the PEG-liposome interaction. FT-IR Measurements. Fourier transform infrared spectroscopy (FT-IR) has been widely used to evaluate the conforma-

tional disorder of the lipid molecules.58-60 Figure 4 shows the examples of FT-IR spectra in the presence and absence of polymers. The strongest bands from the symmetric and antisymmetric CH2 stretching vibrations were found around 2900 cm-1, as reported in the literature. The conformational disorder of acyl chains is usually evaluated by the shifting of these bands to higher frequencies. In the same manner, the interfacial and the hydrophilic region can be investigated from the band at ca. 1700 and 1250 cm-1, respectively. The addition of DEX hardly affected the membrane structure, suggesting that the interaction between the lipid membrane and polymer was very weak. This was also the case when PUL was added. On the other hand, the addition of MC significantly affected the membrane structure. Because its influence was observed in all the regions, that is, hydrophilic, interfacial, and hydrophobic regions, MC molecules strongly perturbed the lipid membranes. Alg-Na was also observed to affect the IR spectra, suggesting that a strong interaction also exists between the Alg-Na and lipid membrane. However, the lower concentration was found to be more effective than the higher concentration. This may be due to a change in the adsorption manner, that is, the conversion from train-type adsorption to tail-type adsorption accompanied by the increase of the Alg-Na concentration. The other polymers, PEG, PVA, and CMC-Na, were found to slightly affect the spectra, suggesting that they interact weakly to the membrane and hardly disturb the membrane structure. The additional experiments using the phosphate buffer as the medium were also performed for PEG and Alg-Na (data not


Kawakami et al.

Figure 3. Heat flow accompanied by the PEG-salt interaction: (a) periodic injection of water into a large volume of 10 mg/mL PEG solution, (b) periodic injection of 200 mM phosphate buffer into a large volume of water, (c) periodic injection of 200 mM phosphate buffer into a large volume of 10 mg/mL PEG solution.

shown). The interaction strength was shown to be slightly reduced by the presence of the salt. Viscosity Measurements. If adsorption layers or depletion layers are formed around liposome particles, the bulk viscosity and the microviscosity should not coincide.9,13,61 In the case of the formation of adsorption layers, the microviscosity should be higher than the bulk viscosity, and vice versa for the formation of depletion layers. Figure 5 shows the relationship between the bulk viscosity and the microviscosity investigated. Clearly, the polymers can be separated into two groups. The microviscosity of PEG, MC, PVA, Alg-Na, and CMC-Na showed higher values than the bulk in the low polymer concentration region, whereas at higher polymer concentrations, the bulk viscosity became higher. On the other hand, the microviscosity of DEX and PUL always showed values lower than those of the bulk. Therefore, polymers of the former group seemed to adsorb onto liposome surfaces, although the amount was not very large. The reason of this interpretation is as follows. At the low polymer concentration, the microviscosity becomes higher than the bulk if there are adsorbed polymers on the liposome surfaces regardless of the amount. However, at the high polymer concentration, the microviscosity can be lower if the amount of adsorbed molecules is small. This is because of the more significant contribution of the depletion layers around the adsorbed layers. Polymers of the latter groups, DEX and PUL, are most likely to be depleted from liposome surfaces without significant adsorption.

Figure 4. Examples of FT-IR spectra of HSL in the absence and the presence of polymers. Polymer concentrations were 0 mg/mL, 0.05 mg/mL, and 5 mg/mL (top to bottom). The lipid concentration was 50 mg/mL.

As mentioned in the Experimental Section, the change in liposome size due to osmotic shrinkage should affect the diffusion coefficient and reduce the calculated value of microviscosity to a certain degree. However, the change in the diffusion coefficient was too large, as it reached values that are (at most) 5 times larger than the case when no polymer was added (see Discussion). Therefore, it seemed to have a minor effect on this experiment. Figure 6 shows the effect of the addition of salt. The adsorption of PEG seemed to be reduced slightly and that of Alg-Na significantly. Turbidity Measurements. From the various observations mentioned above, each polymer was found to interact with lipid membranes in a different manner, although all the polymers employed are well-known as being highly hydrophilic. We next investigated the effect of the addition of the polymers on the physical stability of liposome dispersions with turbidity measurements. Figure 7 shows the observed turbidity in the presence of polymers. The data are shown as relative turbidity, which was derived by dividing the turbidity of the polymer-liposome mixture by that of the polymer-free dispersion. The stabilization



Figure 6. Effect of salt on microviscosity around liposome surfaces. Open symbols represent dispersions prepared with distilled water, and closed symbols those prepared with 10 mM phosphate buffer. Polymer type: PEG (0, 9), Alg-Na (4, 2). The lipid concentration was 0.4 mg/mL.

free liposome dispersion becomes more unstable as the amount of salt increases. These results clearly show the importance of the electrostatic interaction for the stabilization of liposome dispersions. Discussion

Figure 5. Microviscosity around liposome surfaces as a function of bulk viscosity. The lipid concentration was 0.4 mg/mL.

of dispersions resulted in an increase in the relative turbidity, since the aggregated liposome particles should precipitate. As can be clearly seen, Alg-Na was the most effective polymer to stabilize the dispersion, since increase in turbidity was observed over the entire concentration range investigated. PVA and CMC-Na showed the similar results, although the effectiveness was reduced. However, the other polymers destabilized the dispersity significantly, notably in the middleconcentration region (0.1-10 mg/mL). No bridging flocculation seemed to occur, because the dispersity at the low polymer concentration range was not affected for all adsorbing polymers. Addition of salt to the PEG/liposome mixture did not have a significant effect (data not shown), but was very effective in the case of the Alg-Na/liposome mixture as shown in Figure 8. When the salt was absent from the medium, the stabilization effect of Alg-Na was investigated over the whole concentration range of Alg-Na as shown earlier. However, in the presence of the salt, there was a critical concentration to stabilize the liposome dispersity: ca. 0.15 mg/mL for 1 mM buffer, 0.5 mg/ mL for 10 mM buffer, and 1 mg/mL for 200 mM buffer. This finding clearly indicates that a higher Alg-Na concentration is needed if the salt concentration becomes higher. We also observed that the relative turbidity becomes greater with an increase of the salt concentration. This was because the polymer-

Interactions between Liposomes and Polymers. As mentioned in Results, each polymer was found to interact with lipid membranes in a different manner. Below we discuss in detail the interactions for each polymer investigated. ITC was found to be a very powerful tool to investigate the interaction between liposomes and polymers. The results obtained agreed with those from light scattering measurements; DEX and PUL showed no direct interactions with the membrane, whereas the other polymers were adsorbed. However, the observed heat flow is so small that the adsorbed fraction seems to be very small for all the polymers. The binding free energy of polymers to the lipid bilayers has often been estimated with peptide-bilayer interactions. Engelman et al. calculated the free energy of insertion of polyalanine due to the hydrophobic effect to be -30 kcal/mol.62 The enthalpy change of hydrophilic polymers seems to be much smaller than this. It is likely to be comparable to the entropic contribution, because the observed sign of the heat flow change depends on the polymer type. The exothermic heat flow change was observed for MC and AlgNa. In these cases, we can assume that the entropy change, which seems to originate from the configuration change of polymer molecules or the motional restriction of lipid molecules, plays an important role in the heat flow change. It is very interesting to note that these polymers also affected the FT-IR spectra significantly. Therefore, the structural change of the lipid bilayer may occur when these polymers are added. Table 4 summarizes the experimental results of ITC, DLS, and FT-IR, and the supposed action of polymers on liposomes. As can be clearly seen, the surface-activity or the charged moieties seem to be essential for the adsorption onto liposome surfaces. The results from ITC and DLS measurements agreed very well, suggesting that these methods were adequate for


Figure 7. Relative turbidity of liposome dispersions after centrifugation. The lipid concentration before centrifugation was 1 mg/mL.

Figure 8. Relative turbidity of liposome dispersions prepared by the phosphate buffer after centrifugation. Alg-Na was employed as a hydrophilic polymer. The buffer concentration was 200 mM (9), 10 mM (O), 1 mM (2), and 0 mM (×). The lipid concentration before centrifugation was 1 mg/mL.

detecting such a weak interaction. On the other hand, the results of FT-IR measurement revealed a different aspect. It was likely to detect the entropic contribution, that is, membrane perturba-

Kawakami et al. tion by polymer molecules, which could also be judged from ITC measurements by the sign of the heat flow difference. As indicated in earlier literature,44,45 Alg-Na seemed to have relatively strong interactions with lipid membranes, since 0.05 mg/mL was enough to change the FT-IR spectra. It is most likely to be explained in terms of the electrostatic interaction between negative charges of Alg-Na and the local positive charges of phospholipids. The interaction strength of CMC-Na is weaker than that of Alg-Na. It must be the reason for the reduced stabilization effect of the dispersity. PEG, MC, and PVA were also observed to interact with lipid membranes. PEG was adsorbed onto membranes according to microviscosity measurements, but FT-IR measurement revealed that it hardly disturbed the membrane structure. This was consistent with the earlier observation made by Ariga et al.63 They employed a lipid Langmuir-Blodgett film and found that PEG could not be adsorbed onto the air/water interface when the surface pressure was high enough. In addition to this, some NMR experiments have proven the adsorption of PEG onto lipid bilayers. For example, Kuhl et al.12 employed 31P NMR to investigate the microviscosity around liposome surfaces and concluded that PEG20000 became adsorbed onto lipid bilayers. Ohno et al.64 also investigated that the motion of the choline methyl group was suppressed by the addition of PEG7500 by using 1H NMR, although the possibility of change in the osmotic pressure was not discussed. Therefore, it is very likely that PEG can become adsorbed onto lipid bilayers. The interaction strength seemed to be weakened by the addition of salt according to the ITC measurement. Because the PEG-salt interaction yielded a large endothermic heat flow, it seemed to be an enthalpy-driven interaction. We also titrated PEG solution with sodium chloride and disodium hydrogenphosphate solution and found that only the latter solution produced the endothermic heat flow due to the polymer-salt interaction (data not shown). Therefore, phosphate ions seem to interact with PEG molecules. The results of PVA are consistent with those of an earlier study. Takeuchi et al.65 also assumed that PVA was adsorbed onto lipid membranes and the mechanism was elucidated in terms of the surface adsorption. DEX and PUL were observed to have almost no direct interaction with lipid bilayers according to all of the employed measurements. In early literature, both of them were believed to interact directly with lipid bilayers, because they enhanced the aggregation.66,67 It was also reported that the osmotic shrinkage of liposomes was not significant in the presence of DEX unlike the case of PEG.68,69 This was explained by supposing a direct interaction between DEX and the headgroups of lipids. These assumptions are certainly possible but each of them was based on indirect observation. In addition, the enhanced aggregation can be explained by the depletion force as discussed later. Polymer Concentration Profile at Liposome Surface. We concluded that Alg-Na, CMC-Na, PEG, MC, and PVA directly interacted with lipid membranes, but not DEX and PUL. In the case that the polymer adsorbs onto lipid membranes, the self-similar grid model70 is useful for expressing the decay profile of the polymer fraction φp, that is,

φp(r) )

(mr)

4/3

(1)

where m is the monomer size and r is the distance from the liposome surface. This equation means that the local mesh size is equal to the distance from the surface. If we suppose the existence of an adsorption layer of thickness a, the polymer



TABLE 4: Summary of the Liposome-Polymer Interaction characteristics

exptl results

supposed action

polymer

surface activitya

chargesb

heat flowc

DLSd

FT-IRe

adsorptionf

perturbationg

stabilizationh


O O O -

O O

negative positive negative positive negative

O O O O O

O O -

O O O O O

O O -

× × 4 × × O O

a Surface Activity: Ability to lower surface tension. b Charges: Existence of charged moieties. c Heat Flow: Heat flow due to liposome-polymer interaction d DLS: Existence of polymer-adsorbed layer on liposome surfaces detected by DLS measurements. e FT-IR: Change in the FT-IR spectra of lipids due to polymers. f Adsorption: Polymer adsorption on liposome surfaces. g Perturbation: Membrane perturbation due to existence of polymer. h Stabilization: Physical stabilization of liposome dispersion by addition of polymer.

was obtained from the light scattering experiments, we must know the distance range which influences the Brownian motion of particles. Defining the distance range as L, we obtain the ratio of the microconcentration and the bulk concentration Rc from the following equation:

Rc )

Figure 9. Polymer concentration profile calculated from eqs 2-5. This profile can be divided into four regions as described in the text.

fraction profile can be divided into four regions as shown in Figure 9. The first region is the adsorption layer, which can be written as

φp(r) ) 1

(0 < r < a)

(2)

The second region can be expressed by the self-similar grid model, that is,

φp(r) )

(r + mm - a)

4/3

(a < r < r*)

(3)

where r* is the distance at which the polymer fraction shows the minimum value. The equation for the third region can be given in a similar manner:

φp(r) ) φb

(d + mm - r)

4/3

(r* < r < d)

(4)

where φb is the polymer fraction in bulk and d is the depletion layer thickness, which is equal to the polymer size when the polymer concentration is low enough. At a long distance from the liposome surface, we obtain

φp(r) ) φb

(r > d)

(5)

If no adsorption of polymers onto liposome surfaces occurs, the concentration profile is expressed by eqs 4 and 5. From these four equations, we can calculate the polymer “microconcentration” around liposome particles. To explain the difference in the bulk concentration and the microconcentration, which

∫0Lφp(r)dr Lφb

(6)

If no adsorption occurs, Rc becomes constant according to this model. Figure 10 shows the Rc values of all the polymers as a function of φb. As can be seen, the results investigated for DEX and PUL display a trend that is apparently different from those of the other polymers, with Rc being a decreasing function of φb. It seems that the Rc values of DEX and PUL do not depend on φb and can be approximated as constant. We can estimate L as 40 nm from the plot of DEX and PUL, because it is the only fitting parameter. The same figure also shows the profiles calculated with eq 6, except in the case of PVA. By fixing L as 40 nm, a becomes the only fitting parameter. As can be seen, the experimental plot and the calculated line showed very good agreement, suggesting that this model succeeded in assuming the polymer profiles. The calculation also enabled us to estimate the adsorption layer thickness a roughly for all of the polymers. The fitting for PVA failed, suggesting that PVA may form a loose-packed adsorption layer. The employed parameters and the calculated adsorption layer thickness are summarized in Table 5. Calculation of the Depletion Energy. Next, let us consider how the physical stability of liposome dispersions is affected by the presence of polymer. If polymers do not adsorb on the liposome surfaces at all, the depletion force can be an important factor that affects the dispersity. On the other hand, the adsorption of polymers often sterically stabilizes the dispersion, although the depletion interaction also exists. Below we try to calculate the depletion energy Fdep. (1) Case of ReVersible Adsorption. Fdep is given by using the Derjaguin approximation,71 that is,

∫0d∆Π dh

Fdep ) -πrL

(7)

where rL is the radius of liposomes and h is the shortest distance between the two liposome surfaces. ∆Π is the difference in the osmotic pressure between the bulk and the gap of liposome particles, which can be calculated from72

∆Π 1 ) (1 - 2χpw)(φ2b - 〈φp〉2) kT 2

(8)


Kawakami et al.

Figure 10. Ratio of the microconcentration and the bulk concentration as a function of polymer fraction. Open and closed symbols represent the experimental data derived from the light scattering measurement, and the solid and dotted lines were obtained using eq 6.

TABLE 5: Parameters of Polymers polymer PEG MC PVA DEX PUL Alg-Na CMC-Na a

a (nm) d (nm) m (nm) L (nm) 2.4 0.8 -a 0.0 0.0 1.4 0.4

11.0 13.9 14.8 13.6 15.0 16.2 22.8

0.3

40.0

φ/p

C* (mg/mL)

0.0355 0.0559 0.0155 0.0563 0.0525 0.0500 0.0398

1.69 4.20 0.32 4.26 3.70 3.36 2.13

{(

d ) 2rp 1-

)}

φ/wφp - φ/pφw φ/wφp

1/2

(10)

where

Could not be determined (see text).

φ/w ) 1 - φL - φ

where χpw is the χ parameter between polymer and water. The variables k and T have their usual meanings. The averaged polymer fraction between the gap, 〈φp〉, can be regarded as zero if polymers between the gap can freely diffuse to the bulk in the case that liposome particles come close. However, when the bulk polymer concentration exceeds its overlap fraction φp*, the diffusion becomes restricted and the polymers between the gap become condensed during the approach of two liposome particles. The degree of the condensation seems to be difficult to estimate, because it depends on the velocity and angle of two approaching particles. Assuming that the difference in the osmotic pressure is unchanged during the approach, eq 8 can be rewritten using C as a constant. 2

∆Π Cφb ) (1 - 2χpw) kT 2

of the occupied volume per polymer molecules (although polymer molecules do not keep the spherical shape), we obtain

(9)

The depletion thickness d is also a function of φb above φp*.5,6,9 It is calculated using a simple geometric consideration. Assuming that the depletion thickness above φp* is equal to the size

/ p

(11)

The terms rp and φL stands for the radius of a polymer molecule and the fraction of liposome, respectively. The calculated depletion thickness is shown in Figure 11, which is similar to the results of earlier literature.5 Figure 12 shows the calculated depletion energy. It can be observed that significant destabilization of the dispersion occurs below φp*, whereas the restabilization is investigated above that. The same results could be found in older literature.5 However, the segment density profile of polymers between larger particles was taken into account and was essential in the theory. Although such an approach also seems to be possible, there is no need for the smaller particles to be polymer molecules according to the depletion theory. This is the first time that the unstable concentration region was calculated without taking into account the polymer configuration. (2) Case of IrreVersible Adsorption. If polymer molecules are tightly bound to the liposome surfaces, the adsorbed layer can be regarded as the part of the liposome particles. Therefore, Fdep is expressed by


J. Phys. Chem. B, Vol. 105, No. 12, 2001 2383 The result calculated from eq 12 is also shown in Figure 12. This clearly indicates that the stability of the dispersity can be significantly improved by supposing the irreversible adsorption of polymer molecules. However, it should be noted that we do not assume the bridging of two surfaces by polymer molecules here. Thermodynamic Interpretation on Liposome Dispersity. Turbidity measurements revealed that (1) Alg-Na stabilizes the dispersity over the wide polymer concentration range, (2) CMC-Na and PVA also stabilize but the effectiveness is reduced, and (3) the other polymers destabilized significantly, notably in the middle-concentration range. Below we try to explain these behaviors from the viewpoint of thermodynamics. The difference in the free energy before and after the aggregation of particles, F, can be separated into three terms:

F ) Fmix + Fpair + Fdep Figure 11. Depletion thickness calculated from eq 10.

where Fmix, and Fpair, are the free energy change due to the mixing entropy and the pair-interaction energy, respectively. According to the Flory-Huggins mean-field theory, by defining the volume and the fraction of component i as ni and φi, the change in the mixing entropy and the pair-interaction energy is expressed as

1

(Fmix + Fpair) ) -

kT

Figure 12. Calculated depletion energy per unit liposome surfaces by supposing the reversible adsorption of polymers (solid line) and the irreversible adsorption (dotted line). Parameters employed: χpw ) 0.1, φ/p ) 0.05, φL ) 0.02, d ) 10 nm, rL ) 1000 nm, C ) -0.01, m ) 0.3 nm, a ) 4 nm.

Fdep ) -πrL

∫rd+2a∆Πdh

(12)

+

Here r+ is the shortest distance between liposome surfaces to which they can approach each other. ∆Π can be calculated from eq 8 and 〈φp〉 from the following equation:

2

d+2a 2 r+ 2

∫

〈φp〉 )

(r + mm - a)

4/3

dr (13)

h-2a

In this case, r+ satisfies the following equation:

r+ -a) 2

∫a∞(r + mm - a)

4/3

dr

(15)

(14)

∑i

φi ln φi

+

ni

1

∑i ∑j χijφiφj

2

(16)

where χij is a χ parameter between components i and j. The first mixing entropy term can be approximated as a constant, because we do not take into account the entropy change due to the demixing of both water and polymer molecules. The entropy change due to the aggregation of liposome particles can be negligible, because they occupy only a small fraction. According to the dispersity data, the simplest result was obtained when Alg-Na was added to the dispersion. In this case, the dispersity was improved over the entire concentration range of Alg-Na, which can be successfully elucidated by assuming that Alg-Na adsorbed onto liposome surfaces irreversibly. In other words, steric repulsion between the adsorbed Alg-Na chains played a significant role on the dispersity as calculated in Figure 12. Because a strong interaction between lipid membranes and Alg-Na has often been suggested in early literature,44,45 this is the most likely explanation. Of course, the electrostatic repulsion between liposome particles due to the adsorption of Alg-Na must be another reason for the stabilization. On the other hand, the effect of adding PEG and MC can be interpreted by supposing their reversible adsorption onto the surfaces. These two polymers affected the dispersity in a similar manner, that is, they have two unstable concentration regions. One unstable region was located just below the overlap concentration and the other above that. The overlap fraction φ/p of each polymer can be calculated by assuming the hexagonal close packing using the next equation.3

φ/p )

8M 5.63d3 NAF

(17)

Here, M, NA, and F are the molecular weight of polymer, Avogadro’s constant, and the density of polymer, respectively. The term F is simplified to 1 g/cm3. The calculated results for all polymers are shown in Table 5. These values can be converted into the overlap concentration C* via the following


Kawakami et al.

equation:

Conclusion

C* )

6Mφ/p πd3 NA

(18)

The obtained values are also presented in the same table. As can be seen, all C* values fall within 1.5-4.5 mg/mL except PVA. Therefore, according to the results shown in Figure 12, the dispersions should be unstable below this polymer concentration because of the depletion interaction. The results of the turbidity measurement agreed very well with this calculation, that is, the most unstable region was located just below C*. Therefore, the lower unstable region was likely to have originated due to the depletion interaction. The upper region seemed to appear to be due to unfavorable pair-interaction energy, that is, direct contact between liposome surfaces and polymer molecules seemed to be thermodynamically unfavorable. Because polymer chains cannot escape from liposome surfaces above the overlap concentration, it is natural to assume that the pair-interaction energy plays an important role at such a high concentration range. CMC-Na and PVA are likely to have stronger interaction than PEG and MC, although MC can perturb the membrane structure. Their adsorption may be partially irreversible and contribute to the improved stability. The effect of adding DEX and PUL can be interpreted by only the depletion interaction. The unstable region was located only below C*, and its effect on the stability is better than that of PEG or MC above their overlap concentration. This may be elucidated in terms of a better miscibility with liposome surfaces compared to PEG and MC, and the total free energy difference in that region may be near zero. One may ask why DEX and PUL are more miscible with liposome surfaces than PEG or MC despite their not adsorbing onto surfaces. The ability of the adsorption depends on whether there are attractive parts to surfaces in the polymer structure, that is, the total structure has a lesser effect. However, the pair-interaction energy is governed by the total structure. Practical Application. Because all of the observed polymers are well-known as being highly hydrophilic, their interaction with lipid membranes has been frequently ignored. However, we have shown that a slight difference in the strength of the interaction can lead to significant differences in the stabilization of liposome dispersions. Alg-Na, which carries negative charges and therefore interacts with membranes strongly, was proved to stabilize the dispersity significantly. This could be explained by its irreversible adsorption onto liposome surfaces. Therefore, we conclude that Alg-Na is a very useful polymer for stabilizing liposome dispersions. However, we must also keep in mind that it can destroy the lipid membranes due to the interaction. The next choice for the stabilization is CMC-Na or PVA. These findings revealed that a certain degree of interaction between liposomes and polymers is needed to avoid destabilization of the dispersity. Charged or surface-active polymers are favorable from this viewpoint. Other hydrophilic polymers, which interact with membranes very weakly or do not interact at all, destabilize dispersions especially below the overlap concentration. Therefore, when such polymers are added to liposome dispersions, the concentration should be selected carefully. These findings provide important information for controlling the stability of liposome dispersions by adding hydrophilic polymers.

Interactions between various hydrophilic polymers and liposomes were investigated. To stabilize liposome dispersions, some extent of the interaction between polymers and liposome membranes is needed, because lack of such interaction can lead to depletion flocculation. Alg-Na was proved to have such a favorable ability due to its charged moieties, which interact with membranes electrostatically. When there is no interaction or very weak interactions between polymers and membranes, the concentration must be determined carefully so as not to destabilize the dispersity. These observations provide suggestions for how to select polymers to stabilize liposome dispersions. The weak interaction between hydrophilic polymers and liposomes has been difficult to investigate. Therefore, the origin of the aggregation of liposomes, that is, the depletion flocculation or the bridging aggregation, has been difficult to differentiate. We have shown that ITC and DLS could be powerful tools to observe such interaction. Acknowledgment. The authors thank Dr. Mark Phipps, Thermometric AB, for the technical advice and useful discussions on the ITC experiments. References and Notes (1) Oosawa, F.; Asakura, S. J. Chem. Phys. 1954, 22, 1255. (2) Asakura, S.; Oosawa, F. J. Polym. Sci. 1958, 33, 183. (3) Vincent, B.; Luckham, P. F.; Waite, F. A. J. Colloid Interface Sci. 1980, 73, 508. (4) Feigin, R. I.; Napper, D. H. J. Colloid Interface Sci. 1980, 75, 525. (5) Fleer, G. J.; Scheutjens, J. M. H. M. Croat. Chem. Acta 1987, 60, 477. (6) Vincent, B.; Edwards, J.; Emmett, S.; Croot, R. Colloids Surf. 1988, 31, 267. (7) Lekkerkerker, H. N. W. Colloids Surf. 1990, 51, 419. (8) Calderon, F. L.; Bibette, J.; Biais, J. Europhys. Lett. 1993, 23, 653. (9) Krabi, A.; Donath, E. Colloids Surf. A 1994, 92, 175. (10) Walz, J. Y.; Sharma, A. J. Colloid Interface Sci. 1994, 168, 485. (11) Mao, Y.; Cates, M. E.; Lekkerkerker, H. N. W. Physica A 1995, 222, 10. (12) Kuhl, T.; Guo, Y.; Alderfer, J. L.; Berman, A. D.; Leckband, D.; Israelachvili, J.; Hui, S. W. Langmuir 1996, 12, 3003. (13) Donath, E.; Krabi, A.; Allan, G.; Vincent, B. Langmuir 1996, 12, 3425. (14) Sharma, A.; Walz, J. Y. J. Chem. Soc., Faraday Trans. 1996, 92, 4997. (15) Sharma, A.; Tan, S. N.; Walz, J. Y. J. Colloid Interface Sci. 1997, 190, 392. (16) Sharma, A.; Tan, S. N.; Walz, J. Y. J. Colloid Interface Sci. 1997, 191, 236. (17) Ye, X.; Tong, P.; Fetters, L. J. Macromolecules 1997, 30, 4103. (18) Maggio, B.; Lucy, J. A. FEBS Lett. 1978, 94, 301. (19) Blow, A. M. J.; Bothan, G. M.; Fisher, D.; Goodall, A. H.; Tilcock, C. P. S.; Lucy, J. A. FEBS Lett. 1978, 94, 305. (20) Saéz, R.; Alonso, A.; Villena, A.; Gon˜i, F. M. FEBS Lett. 1982, 137, 323. (21) Roots, D. S.; Davidson, R. L.; Choppin, P. W. Cell Fusion; Plenum Press: 1987. (22) Lentz, B. R. Chem. Phys. Lipids 1994, 73, 91. (23) Hibberd, D. J.; Howe, A. M.; Robins, M. M. Colloids Surf. 1988, 31, 347. (24) Cao, Y.; Dickinson, E.; Wedlock, D. J. Food Hydrocolloids 1990, 4, 185. (25) Dickinson, E.; Goller, M. I.; Wedlock, D. J. Colloids Surf. A 1993, 75, 195. (26) Tuinier, R.; de Kruif, C. G. J. Colloid Interface Sci. 1999, 218, 201. (27) Perrier, P.; Redziniak, G. Skincare 1989, Feb, 29. (28) Gregoriadis, G. Biochemist 1994, Feb/Mar, 8. (29) Lasic, D. D.; Papahadjopoulos, D. Science 1995, 267, 1275. (30) Sharma, A.; Sharma, U. S. Int. J. Pharm. 1997, 154, 123. (31) Storm, G.; Crommelin, D. J. A. Pharm. Sci. Technol. Today 1998, 1, 19. (32) Naeff, R. AdV. Drug DeliVery ReV. 1996, 18, 343.

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Effect of Hydrophilic Polymers on Physical Stability of Liposome

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