Preparation and Evaluation of Sterically Stabilized Liposomes: Colloidal Stability, Serum Stability, Macrophage Uptake, and Toxicity zyxwvu BENGT KRONBERG*, ANNIKA DAHLMAN*, JOHAN CARLFORS', JOHAN KARLSSON*, AND PER ARTURSSON*~ Received June 7, 1989, from the.'lnstitute zyxwvuts for Surface Chemjstty, P.O. zyxwv Box 5607, S-114 86 Stockholm, Sweden, and the *Department zy of 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 incorpo- rating a nonionic surfactant, polysorbate 80 (Tween zyxwvu 80), into the lipid bilayer. The sterically stabilized liposomes exhibited a superior entrap- ment 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 sterically stabilized 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. (OCH,CH, +OH I 0 (a + b + x +y = 20) Nonlonlc Surfactant Polysorbate 80 (Tween 80) Liposomes have been used as experimental drug carriers for more than a decade.1 More recently, they have been intro- duced 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 How- ever, 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 accept- ance. 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 sta- bilization have been thoroughly described by Napper9 and the essential feature is that when two sterically stabilized par- ticles 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 stabi- lized liposomes were investigated with respect to their col- loidal stability and entrapment properties in buffer and serum. The macrophage uptake and toxicity was compared with reference liposomes (containing only lipid and choles- terol) 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 zyxwvu Liposome Preparation-The liposomes were prepared with the fol- lowing materials: DMPC (kz-phosphatidylcholine, dimyristoyl) syn- thetic 99%, DMPA (kz-phosphatidic acid, dimyristoyl sodium salt) 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 prepa- ration of the liposomes: 4.5 mM zyxw 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 an 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 compo- sition 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-phosphati- dyl[N-methyl-3Hlcholine, 1,2-dipalrnitoyl (Amersham, England; spe- cific 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 OO22-3549/90/08OO-0667$0 1 . zyxwvut 0010 0 1990, American Pharmaceutical Association Journal of Pharmaceutical Sciences I 667 Vol. 79, No. 8, August 1990