Preparation and Evaluation of Sterically Stabilized Liposomes: Colloidal Stability, Serum Stability, Macrophage Uptake, and Toxicity

Preparation and Evaluation of Sterically Stabilized Liposomes: Colloidal Stability, Serum Stability, Macrophage Uptake, and Toxicity BENGT KRONBERG*, ANNIKADAHLMAN*, JOHAN CARLFORS', JOHAN KARLSSON*, PERARTURSSON*~

AND

Received June 7, 1989, from the.'lnstitute for Surface Chemjstty, P.O. Box 5607, S-114 86 Stockholm, Sweden, and the *Departmentof Pharmaceutics, Uppsala University, P.O. Box 580, S-751 23 Uppsala, Sweden. Accepted for publication October 20, 1989. Abstract 0 Sterically stabilized liposomes were produced by incorporating a nonionic surfactant, polysorbate 80 (Tween 80),into the lipid

bilayer. The sterically stabilized liposomes exhibited a superior entrapment stability compared with surfactant-free liposomes (i.e.,liposomes prepared with lipids and cholesterol).The sterically stabilized liposomes were stable at high calcium ion concentrations,and liposome-entrapped carboxyfluorescein was retained within the stabilized liposomes in the presence of serum for at least 5 h. The macrophage uptake of the sterically stabilized liposomes was comparable to that of liposomes containing lipids and cholesterol.The stericallystabilized liposomes were non-toxic, in concentrations up to 3.0 mM, to macrophages. These results indicate that polysorbate 80 can be used to produce stable liposomes without changing the unique macrophage distribution of this drug delivery system.

Liposomes have been used as experimental drug carriers for more than a decade.1 More recently, they have been introduced in human therapeutics.2 Liposomes fulfill many of the biological requirements of a drug delivery system. They are composed of biodegradable endogenous lipids, are relatively nontoxic, and seem to be well tolerated by humans.2.3 However, some of the common pharmaceutical requirements are not yet fulfilled (e.g., liposomes have a short shelf-life and a poor stability in biological environments such as ser~m).3,4 Several attempts have been made to solve the stability problems,- but so far none of these has gained wide acceptance. Thus, there is a need for a more general method of preparing stable liposomes. In this study, a method for the preparation of sterically stabilized liposomes is presented. The theories of steric stabilization have been thoroughly described by Napper9 and the essential feature is that when two sterically stabilized particles approach there will be a decrease in the chemical potential of the water between the particles, due to the presence of the water soluble chains. This will create osmotic suction of the bulk water into the area between particles and, as a result, the particles will separate. The sterically stabilized liposomes were investigated with respect to their colloidal stability and entrapment properties in buffer and serum. The macrophage uptake and toxicity was compared with reference liposomes (containing only lipid and cholesterol) in cell culture. The results indicate that the sterically stabilized liposomes are nontoxic and have a stability that is superior to that of conventional cholesterol-stabilized, as well as poloxamer-stabilized10 liposomes.

Experimental Section Liposome Preparation-The liposomes were prepared with the following materials: DMPC (kz-phosphatidylcholine,dimyristoyl) synthetic 99%, DMPA (kz-phosphatidic acid, dimyristoyl sodium salt) OO22-3549/90/08OO-0667$01.0010

0 1990, American

Pharmaceutical Association

(OCH,CH, +OH 0

I

( a + b + x + y = 20) Nonlonlc Surfactant Polysorbate 80 (Tween 80)

synthetic 99%, and cholesterol 99% (all obtained from Sigma Chemical Company), as well as the nonionic surfactant polysorbate 80 [Tween-80; Sorbitan monooleate (EO),,; see structure], which was obtained from Atlas Chemicals. The following concentrations were used in the preparation of the liposomes: 4.5 mM DMPC, 0.7 mM DMPA, 5.0 mM cholesterol, and varying amounts of polysorbate 80. The surfactant composition varied from 0 to 4%, counted on the liposome material only. The ingredients were dissolved in a toluene :methanol mixture and then evaporated with nitrogen gas in order to prepare a thin film on the inner wall of the glass vessel." The film was sonicated through a n ultrasonic probe (Ultrasonics, Ltd.) in the presence of PBS (phosphate buffered saline, without magnesium and calcium, pH 7.5; from Flow Laboratories) aqueous solution for a period of 20 min. The dispersion was centrifuged at 3600 x g for 10 min and passed through a Sephadex G-50(Pharmacia AB) column with a PBS-buffered eluant. Fractions at the void volume were collected and used for the subsequent measurements. Note that the surfactant is added before the liposomes are prepared (i.e., before sonication). The surfactant is therefore evenly distributed among the lipid material (i.e., there is surfactant present on the inside as well as on the outside lipid layer of the liposomes). If the liposomes are multilamellar, there is, of course, surfactant present in all lipid layers. The liposome size was determined by dynamic light scattering (at a 90" angle) with a Malvern Autosizer 11. The average size obtained was in the range of 70-200 nm, depending on the liposome composition and the intensity of the ultrasonic probe. Figure 1 shows a typical example of the size distribution of a liposome dispersion. Liposomes containing CF [5(6)-carboxyfluoresceinl were prepared slightly differently. Sonication was performed in a PBS-buffered solution (pH 7.5)containing 1.00 x lo-' M CF (from Eastman Kodak Company). The PBS eluant in the gel filtration step contained 0.100 M NaCl in order to assure that the same osmotic pressure was maintained both inside and outside the liposomes, thus preventing osmotic rupture of the liposomes. The column was thermostated to 10 "C and the collected samples were cooled to 5 "C. Radioactive liposomes were produced by including L-3-phosphatidyl[N-methyl-3Hlcholine,1,2-dipalrnitoyl (Amersham, England; specific radioactivity 103mCi/mg) in the liposome preparation. The specific radioactivity of the liposome preparations were 16-2.1 x lo4 cpml~cg lipid. The radiolabeled lipid marker has previously been used to follow macrophage uptake of liposomes.12 The two radiolabeled liposome

Journal of Pharmaceutical Sciences I 667 Vol. 79,No. 8, August 1990

-9-

200

100

50

Diameter (nm) Figure 1-Size distribution of a typical liposome preparation. The liposome molar composition was 4.4:0.7DMPC:DMPA.

preparations had the following molar compositions: control liposomes, DMPC:DMPA: cholesterol (4.6:0.7 :5.1);sterically stabilized liposomes, DMPC:DMPA:cholesterol:polysorbate80 (4.4:0.7:5.1:0.4). Stability against Salt-The stability of the liposomes was determined by monitoring the change in size of the liposomes with time. The size was determined by dynamic light scattering. Figure 2 shows that the size increases linearly with time, at least in the first 5 min after salt addition. The rate of this size increase is a measure of the colloidal instability. The stability is presented as the relative change in the size after 2 min [i.e., (do- d,)/d,, where do is the particle size before adding salt and d, is the size measured 2 min after the addition of salt]. It should be noted that this method does not discern whether the particles first aggregate and then fuse into larger liposomes, or if they just remain as an aggregate. Nevertheless, the purpose of these measurements is to evaluate the colloidal stability (i.e., whether or not the liposomes aggregate). Fluorescence Measurements-A Pye Unicam SP8-200 UV spectrometer with fluorescence accessory was used in order to evaluate the entrapment efficiency of CF. The excitation beam was set a t 330 nm and a filter, absorbing wavelengths shorter than 475 nm, was placed in front of the detector. The fluorescence of the CF-containing liposome suspensions was determined after 0.25, 0.50,0.75, 1, 2, 3, and 4 h. After 4 h, the total leakage of the liposomes was obtained by

40

1

30 0

P g

h

20-

9 10-

0 0,o

2-0

4,O

6,O

8,O

10,O

Time (minutes) Flgure 2-Relative growth of liposome apparent size as CaCI, is added. Liposomes are without nonionic surfactant in Tris buffer, with CaCI, concentrations of 0.18 and 0.54 rnM, respectively. The liposorne molar composition was 4.4:0.7DMPC: DMPA. 660 I Journal of Pharmaceutical Sciences Vol. 79, No. 8, August 1990

adding a 100 pL of Triton X-100 solution (10%)to the 2-mL sample, thus solubilizing the liposomes. The entrapment studies were performed in a phosphate buffer and in a 20%serum. The serum was obtained from Gibco BRL and heated to 56 “C for 30 min before use. In the experiments involving serum, a small amount of sodium azide was added in order to prevent bacterial growth. Cells-The murine macrophage cell line 5774 was cultivated in Dulbecco’s modified essential medium containing 20% fetal bovine serum, benzylpenicillin (100 U/mL), and streptomycin (10 pg/mL). The cells were mycoplasma negative as determined with the fluorochrome Hoechst 33258. The macrophages were cultivated for 24 h on circular plastic coverslips (Thermanox tissue culture coverslips, 25-mm diameter, Miles Scientific) in tissue culture wells (35-mrn diameter, Costar). The seeding density was lo6 cells/well. Macrophage Uptake-Radiolabeled liposomes corresponding to 47.9-63.4 pg of lipid were added to macrophage monolayers containing 1.6 x 105-2.2 x lo5 cells. After 0.5, 1, 2, and 4 h, the coverslips were removed and washed five times in PBS. The macrophageassociated radioactivity was determined by liquid scintillation counting and expressed as ng lipid/106 cells (n = 3). All incubations were carried out a t 37 “C.The cell culture medium described above was used for experiments performed in the presence of serum. In the serum-free experiments, the serum was exchanged for human serum albumin (0.1%;KABI Diagnostics, Molndal, Sweden). Macrophage Toxicity-The macrophage toxicity was determined with the MTT assay, essentially as described by Mossman.13 The assay is based on the tetrazolium salt MTT [3-(4,5-dimethyIthiazol2-yl)-2,5-dipenthyltetrazolium bromide] that is cleaved by living, but not dead cells, to give a dark blue formazan product. The color development is measured spectrophotometrically using a test wavelength of 570 nm and a reference wavelength of 690 nm. Macrophages were incubated with 0.01-3.0 mM of the different liposomes for 24 or 96 h. At these times, MTT was added and the development of the formazan reaction product was compared with that of the control cultures which were exposed to physiological saline instead of liposomes. The results were expressed as IC,, k e . , the dose required to inhibit the development of the formazan reaction product up to 50% compared with the control cultures). In addition to the liposomes described above, liposomes with the following molar composition were used: DMPC: DMPA:cholesterol:DNP(EO),,(4.4:0.7:5.1:0.4), where DNP(EO),, is a nonionic surfactant [Dinonylphenol polyoxy ethylene(EO),,; from GAF Cooperation]; and DMPC :cholesterol :SA (4.0:5.0:l.O), where SA is stearylamine (from Aldrich Chemicals).

Results and Discussion Polyethylene oxides and their derivatives have a “lower critical solution temperature” in aqueous solutions (i.e,, they precipitate at a certain temperature upon heating). This temperature is termed the cloud point and is normally determined at a 1%by weight surfactant concentration. It has also been shown that sterically stabilized dispersions become unstable and flocculate upon heating if the dispersion is stabilized with polyoxyethylene chains.9 It was therefore important to ascertain that the surfactant system used in our study remained in solution at body temperature. A 1% solution of polysorbate 80 was found to have a cloud point of 79-80 “Cin polyphosphate buffer as well as in serum; that is, far above body temperature. This surfactant should therefore in principle be able to stabilize a liposome dispersion, provided that the surface coverage is sufficiently high. The partition coefficients between the liposome bilayer and the aqueous solutions of surfactants that have a structure similar to that of polysorbate 80 have been shown to be in the order of 106.14 Thus, it can be assumed with good approximation that all the surfactant molecules are absorbed into, or adsorbed onto the liposome lipid bilayer. Most likely, the surfactants absorb with their hydrocarbon tail penetrating into the lipid bilayer. The minimum surfactant concentration producing sterically stabilized liposomes was found to be 4%, calculated on the liposome material only. This value corresponds to 0.12 mol/m2 (assuming a cross-sectional area of 0.65 nm2 per lipid moleculel5), giving a liposome surface area

available per surfactant molecule of 14 nm’. This value of the surface concentration is much lower than the minimum stabilizing surface concentration for similar surfactants on latexes, which has been determined to be in the order of 2 nm2 per molecule.16 In the case of latexes, the surfactants lay down on the surface until the surface is fully covered. Not until then do the ethylene oxide chains of the surfactants start to protrude into the aqueous solution, stabilizing the dispersion. In the case of liposomes, this nonionic surfactant starts to stabilize the dispersion a t a much lower concentration (see below). We therefore tentatively conclude that the ethylene oxide groups have no affinity to the liposome surface and that there is a large liposome surface area that is not covered by the polyethylene oxide units of the surfactant. It should be noted that the calculation above uses only the ratio of surfactant to lipid and thus rests on the fact that the liposomes have been prepared in the presence of the surfactant, giving a n even distribution of the surfactant in the lipid layers. The calculations are thus totally independent of the presence of multilamellar or unilamellar liposomes in the system. If, however, the surfactants were added after the liposomes were made, assumptions on the liposome size and on whether they are uni- or multilamellar have to be made. Since we have prepared the liposomes in the presence of surfactant in this work, we conclude that the deductions above are relevant to our system. Colloidal Stability-A standard test to investigate whether a dispersed system is stericallystabilizedis to expose it to high ionic strengths. This is preferably done by the addition of a noncomplexing salt (e.g., NaCl or CaC1,). This causes the electrical double layer to collapse and the system will coagulate if it is not sterically stabilized. In biological systems, the presence of calcium ions may be a bigger obstacle than high ionic strengths since calcium ions have the tendency to adsorb specifically onto carboxylatedsurfaces,for example.Figure 3showsthat unstabilized liposomes (without nonionic surfactant) are quite susceptible to the presence of calcium ions. At higher calcium ion concentrations, the particle size growth rate decreases,probably as a result of recharging the liposome surface by adsorption of calcium ions on the liposome surface.The affinity constant of Ca2+for the PA group has been determined to be in the order of lo4 M-’.17J* It should be noted that the normal concentrationof free calciumions in serum is -1.3 d . 1 9 Thus, the destabilizing effect of calcium

lo{

0%

ions was observed at physiological calcium concentrations. Figure 3 reveals that the liposomes are not susceptible to calcium ions if cholesterol is incorporated into the liposomes. There are several possible reasons for the enhanced colloidal stability in the presence of cholesterol.The most probable reason is that the liposomes are stabilized through the “condensing,or stiffening, effect” of cholesterol. Thus, upon collision of two liposomes, the bilayers are no longer as deformable as bilayers without cholesterol. In the latter case, the two liposomesform a large area of contact, thus enhancing the probabilityof fusion of the two colliding liposomes.Since this phenomenon is depressed in the presence of cholesterol, there is a larger probability of survival of the identity of two colliding liposomes. Another possible effect ofthe cholesterol is to enhance the strength, or the amount, of the strongly bound water layer (resulting in a repulsive force between liposomes)that is normally found at the surface of lipid mono- or bilayers.20 It is obvious from Figure 3 that calcium ions are not adsorbing onto the surface of liposomes containing cholesterol and the liposomes are stable against coagulation in spite of the depression of the electrical double layer. (This would occur in the concentration range 0.4-1.0 mM studied h e r e P At higher concentrations (>4%) of polysorbate 80, the liposomes are indifferentto the presence of calcium ions, as is shown in Figure 3. Thus, this surfactant is able to prevent coagulation of the liposomes due to the presence of calcium ions. We conclude so far that the presence of cholesterol in the liposomes stabilizes the liposomes, probably against fusion of the liposomes when they interact in a collision, and the polysorbate 80 molecule stabilizes the liposomes against aggregation, provided that the surfactant concentration is sufficiently high. Consequently, the following study was performed with liposomes containing cholesterol, as well as with liposomes containing polysorbate 80. 5(6)-Carboxyfluorescein (CF) Entrapment-Results from the entrapment of CF are shown in Figures 4a and 4b. Here we studied liposomes containing DMPC, DMPA, and cholesterol, with or without polysorbate 80. The entrapment studies were performed in phosphate buffer (Figure 4a) and in 20% serum (Figure 4b). The 20% dilution of serum could be used since -90% of the effects of complete serum on liposomes can be observed a t this lower serum concentration.22 The liposomes were very stable and retained the CF for at least up to 4 h. At this time, Triton X-100was added and the liposomes collapsed, releasing the CF and resulting in a large fluorescence signal. From the Figure we conclude that both types of liposomes, with or without nonionic surfactant, were stable in phosphate buffer. Thus, the polysorbate 80 molecules do not disturb the lipid chain packing such that the CF is released. This is in contrast with the findings of Kellaway et a1.10 who found that nonionic surfactants of the poloxamer series disturb the liposome bilayer to such an extent that the liposomes became leaky. We note in passing that it was impossible to entrap CF in liposomes that did not contain cholesterol. This is hardly surprising since the short myristate chains are highly mobile at room temperature without cholesterol as a “condensing agent”. It is a well known problem that liposomes become leaky in the presence of serum. The most common way to reduce this problem is to include cholesterol into the lipid bilayer,4 thereby decreasing the mobility of the lipid chains. However, as can be seen in Figure 4b, the effect of cholesterol on DMPC :DMPA liposomes is limited, resulting in slowly leaking liposomes in spite of the fact that a very high (50%) cholesterol content was used. In comparison, sterically stabilized liposomes showed a higher stability with respect to entrapment. Thus, the nonionic surfactant polysorbate 80 is able to protect the liposomes from the destructive interaction with the serum components.

.p 4%

I

0,OO

0,20

0,40

0,60

0,80

1,OO

CaCI, concentration (mM) Flgure &Growth rate of liposome apparent size in Tris buffer as a function of CaCI, concentration. Filled symbols represent liposomeswith a molar composition of 4.4:0.7 DMPC:DMPA, and with the indicated levels of polysorbate 80 (calculated with respect to the liposome material only). Open symbols represent liposomes with the molar composition 4.4:0.7:5.2DMPC: DMPA:cholesterol (without any surfactant).

Journal of Pharmaceutical Sciences I 669 Vol. 79, No. 8, August 1990

A

loo\

8o

1

Time (hours)

6o 40

1 Time (hours)

T

'O0I 8o

n

t

1

i4 0

1

2

3

Time (hours)

4

5

t

FlgurerUeakageof CFfromIiposomesinphosphatebuffer(A)andin2W0 serum (B). The liposome molar composition was 4.4:0.7:5.1 DMPC:DMPA:cholesterol,and the indicated levels of polysorbate 80 (calculated with respect to the liposome material only). The arrow indicates the time at w h i i Triton X-100 was added, collapsing the liposomes.

We also studied the size of unstabilized (no polysorbate 80) liposomesin phosphate buffer and in serum (20%)by dynamic light scattering for a period of 5 h in order to verify that no slow flocculation or coagulation occurs. It was found that the size remained unchanged for this time period. Macrophage Uptake-The uptake of liposomes by macrophages can be enhanced by various serum components. Thus, immunoglobulins, complement, and fibronectin are nonspecifically adsorbed to the lipid bilayer.4 This process is known as opsonization. The particle is bound to the specific surface receptors for the adsorbed serum proteins on the macrophages. The binding to these specific 'host-defence' receptors will initiate the internalization of the particles. In the absence of serum, the macrophageuptake (measured as macrophage bound radioactivity) of the polysorbate 80stabilized liposomes was comparable with that of the liposomes containing only the lipids and cholesterol (Figure 5a). In the presence of serum, the uptake of the latter liposomes was reduced compared with the uptake of sterically stabilized l i p some8 (Figure 5b). One explanation for the decreased uptake of the unstabilized liposomes can be found in Figure 4b,which shows that these liposomes are slightly unstable in serum with respect to entrapment. The macrophage uptake of the sterically stabilized liposomes in the presence of serum is comparablewith that in serum-freemedium. The importance of serum opsonins for the macrophage uptake is therefore limited. 670 I Journal of Pharmaceutical Sciences Vol. 79,No. 8, August 1990

-.0.0

2,o

3,O

4,o

Time (hours) Figure M a c r o p h a g e uptake of liposomes, with and without pdysorbate 80,in serum free solution (A) and in serum (6). The liposome molar compositions were 4.6:0.7:5.1 DMPC: DMPA:cholesterol, and 4.4:0.7:5.1:0.4 DMPC:DMPA:cholesterol:polysorbate80 (n = 3 ? SD).

It has been shown that poloxamers, as well as polysorbate 20, a nonionic surfactant of similar structure as polysorbate 80, will reduce the opsonization of latex particles.23.24 A lower uptake of the sterically stabilized liposomes than of the unstabilized liposomes could therefore be expected. However, Illum et al. have shown that the interaction of poloxamercoated latex particles and macrophages is inversely proportional to the length of the stabilizing ethylene oxide chains.25 Their results indicate that the ethylene oxide chain must have a t least 50 monomer units in order to produce a significant inhibition of the macrophage uptake. The ethylene oxide chains of polysorbate 80 have, on the average, only -5 monomer units each (see structure). Thus, although the hydrophilic chains m a y contribute to reduce t h e opsonization,24 they are too short to inhibit the macrophage uptake. Macrophage Toxicity-Polysorbate 80 has a very low toxicity and is therefore widely used as a solubilizer in parenteral pharmaceutical formulations.26 However, liposomes have a body distribution that is different from that of conventional intravenous drug formulations. Shortly after intravenous administration, the liposomes are targeted to the

macrophages of the reticuloendothelial system.' The concentration of the liposomes in the macrophages may introduce new carrier-mediated toxicities of the nonionic detergent. The macrophage toxicity of the sterically stabilized liposomes was therefore compared with some reference liposomes. The sterically stabilized and the corresponding nonstabilized liposomes were nontoxic in the macrophage assay (Figure 6). Liposomes containing stearylamine were included as positive controls and killed 50% of the macrophages at a concentration of 60 pM. Similar IC,, values for liposomes containing stearylamine have been reported previously.27 Liposomes containing DNP(EO),g. were also investigated for macrophage toxicity since stability measurements of liposomes have previously been performed using this surfactant. These liposomeswere less toxic than the liposomes containing stearylamine and had IC,, values of 1900 and 880 @ in the I 24- and 96-h experiments, respectively (see Table I). These results indicate that the M n ' assay was suitable for assessing the toxicity of liposomes containing nonionic detergents. Liposomes containing DNP(EO),, are also sterically stabilized (data not shown). Thus, it is possible to produce stable liposomespreparations with the long ethylene oxide chains that seem to be needed in order to inhibit macrophage interactions.25 Such liposomes would have a prolonged intravascular half-life, which would increase the possibility of targeting the liposomes to other parts of the body than the reticuloendothelial system. However, the results of the MTT assay indicate that this nonionic surfadant may be toxic. The toxicity of new nonionic detergents that are to be included in liposomal drug carriers should therefore always be investigated.

Conclusions This study shows a way of producing sterically stabilized liposomes by the insertion of a well-known and nontoxic nonionic detergent, polysorbate 80 (Tween 801, into the lipid bilayer. The sterically stabilized liposomes have an increased stability in serum compared with the corresponding liposomes without polysorbate 80. The short ethylene oxide chains have no inhibitory effect on the macrophage uptake of the liposomes. The polysorbate 80-stabilized liposomes can therefore be used to target drugs to the macrophages of the reticuloendothelial system. Further, the slow release of the entrapped marker in biological fluids indicates that the

=

1

E! c c

8

h

-5

-4

-3

-2

-1

l

0

.

8

1

.

I

2

slog concentration (mM) Figure &Effect of liposomes on macrophage viability after 24 h of coincubation. The liposomes contained lipids, cholesterol, and the indicated level of surfactants. The highest concentration of liposomes was serially diluted in three-fold steps (n = 3 ? SD).

Table I-Toxic

Effect of Different Liposomes on Macrophages

Liposome Molar Composition DMPC: DMPA:cholesterol (4.5:0.7:5.1) DMPC: DMPA:cholesterol:polysorbate 80 (4.4:0.7:5.1 :0.5) DMPC: DMPA:cholesterol:DNP(E0)49 (4.4:0.7:5.1 :0.5) DMPC:cholesterol:SA (4.0:5.0: 1 .O)

24 h

96 h

>3000

>3000

>3000

~3000

1900

880

60

60

'The dose that causes a 50% reduction in cell viability (see Experimental Section).

liposomes can be used in applications where an extended release of the entrapped material is advantageous.

References and Notes 1. Machy, P.; Leserman, L. Liposomes in Cell Biology and Pharm: John Libbev: London. 1987. 2. LiGomes in the T b m of Infectious Diseases and C a m ? Lo zBerestein, G.; Fidler, I. YE&.; Allan R. Liss: New York, 1989, p g 7 . 3. Kirsh, R.; Bugelski, P. J.; Poste, G. Ann. N.Y. Acad. Sci. 1987, 508,141-154. 4. Bont6, F.; Juliano, R. L. Chem. Phys. Lipids 1986,40, 359-372. 5. Metha, R.; Hsu, M. J.; Juliano, R. L.; Krause, H. J.; Regen, S. L. J . Pharm. Sci. 1986, 75, 579-581. 6. Crowe, J. H., Spargo, B. J.; Crowe, L. M. Proc. Natl. Acad. Sci. U S A . 1987,85, 1537-1540. 7. Payne, N. I.; Timmins, P.; Ambrose, C. V.; Ward, M. D.; Ridgwav. F. J.Pharm. Sci. 1986. 75.3254329. 8. GAizon, A.; Pa ahadjopoulos, D.Proc. Natl. Acad. Sci. U S A . 1988,85,6949-%953. 9. Nap er, D. H. Polymeric Stabilization of Colloidal Dispersions; Acajemic: New York, 1983. 10. Jamshaid M.; Farr, S. J: Kearney, P.; Kellaway, I. W. Int. J. Pharm. 1388,48,125-131. 11. Li osome Technolog :Vol. 1Preparation ofliposomes; Gregoria d s , G., Ed.; CRC: i o c a Raton, FL, 1984. 12. Juliano, R. L.; Hsu, M. J.; Regen, S. L.; Singh, M. Biochim. Biophys. Acta 1984, 770, 109-114. 13. Mosmann, T. J. Immunol. Meth. 1983, 65, 55-63. 14. Kurihara, K., et al., unpublished results. 15. Lis, L. J.; McAlister, M.; Fuller, N.; Rand, R. P.; Parsegian, V. A. Biophys. J. 1982,37,657-666. 16. Kronberg, B., unpublished results. 17. Hendriksson, H. S.; Fullington, J. G. Biochemistry 1965,4,15991605. 18. Abramson, M. B.; Katzman, R.; Gregor, H.; Curci, R. Biochemistry 1966,5. 2207-2213. 19. Lairell, C.-B.; Lundh, B.; Nosslin, B. In Klinisk kemi i praktisk medicin; Student Litteratur: Lund, Sweden, 1976; p 93. 20. Rand, R. P. Ann. Rev. Biophys. Bioeng. 1981,10, 277314. 21. Hiemenz, P. C. Princi les of Colloid and Surface Chemistry; Marcel Dekker: New f o r k , 1977. 22. Lelkes, P.I.; Friedmann, P. Biochim. Biophys. Acfa 1984, 775, 39W1. 23. OMullane, J. E.; Davison, C. J.; Petrak, K.; Tomlinson, E. Biomaterials 1988, 9, 203-205. 24. Rembaum,A.;Ugelstad,J.;Kemshead,J. T.;Chang,M.;Rchards,G.

In Microspheres and Drug Them Pharmaceutical,Immunological and Medical Aspects;Davis, S. S.%lum, L.; McVie, J. G.; Tomlinson, E., Eds.; Elsevier Science: B.V.; 1984, pp 383-391. 25. Illum, L; Jacobsen, L. 0.; Muller, R. H.; Mak, E.; Davis, S. S. Bwmaterials 1987,8, 113-117. 26. Handbook of Pharmaceutical Excipients; American Pharmaceutical Association and The Pharmaceutical Society of Great Britain: Washington, DC, and London, 1986; pp 225-227. 27. Mayhew, E.; Ito, M.; Lazo, R. Exp. Cell Res. 1987,171, 195-202.

Acknowledgments Illuminatin discussions with B. Bergensthhl are gratefully acknowled ed. &e also acknowledge the financial support from the Swedishsoard for Technical Development. Journal of Pharmaceutical Sciences I 671 Vol. 79, No. 8, August 1990