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Characterization of liposomes by scanning electron microscopy and the freeze-fracture technique
Micron and Microscopica .4cta. Vol. 16. No. 2, pp. 109 113. 1985. Printed in Great Britain.
0739 6260/85 53.00+0.00
©
1985 Pergamon Press Ltd.
CHARACTERIZATION OF LIPOSOMES BY SCANNING ELECTRON MICROSCOPY AND THE FREEZE-FRACTURE TECHNIQUE K.
ADLER
and J.
SCHIEMANN
Zentralinstitut für Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, DDR-4325 Gatersieben, Corrensstr. 3, G.D.R. (Received 24 October 1984)
Abstract—This paper describes the application ofboth SEM and freeze-fracture TEMfor the characterization of artificial lipid vesicles (liposomes). Size and shape may be observed in the SEM technique whereas the inner structure could be investigated best in pictures obtained using the freeze-fracture method. Index key words: Liposome, TEM, SEM, freeze-fracture, freeze-drying.
INTRODUCTION MATERIALS AND METHODS The ability of lipid vesicles (liposomes) to Scanning electron microscopy entrap small and large molecules, such as drugs, enzymes, RNA and DNA, and also to deliver The liposomes were pipetted onto the top of them to cells, has attracted great interest in their cleaned specimen mounts. These specimens were use as vehicles (Fraley and Papahadjopoulos, then frozen at —20°C and dried overnight 1981). During recent years a large number of in a freeze-drying unit (LGA 05, VEB papers have been published demonstrating the Zentrifugenbau, Leipzig) at a vacuum of 2.25 Pa. The freeze-dried specimens were sputter-coated delivery of DNA and RNA molecules to a variety with a gold—palladium alloy and observed in an of plant protoplasts (reviewed by Rollo, 1983). In ISI 40 SEM (International Scientific Instruments contrast to other techniques for introducing Inc., Akashi-Seisakusho Ltd., Tokyo, Japan). nucleic acids into cells, liposome-mediated transfer is very efficient (Fukunaga et al., 1981; Fraley, Freeze-fracture electron microscopy 1983). There are few papers presenting electron Suspensions of liposomes were pipetted onto micrographs of liposomes (Deamer and specimen holders made from gold—platinium alloy Bangham, 1976; Harsch et al., 1981; Hauser and and were quick-frozen by immersion in liquid Gains, 1982). Considering the importance of Freon 22. Frozen specimens in gold—platinum liposomes in biochemistry, pharmacy and gen- holders were stored in a cryocontainer in liquid etic engineering, a more frequent control of nitrogen. Freeze-fracturing was conducted in a different liposome preparations should be pro- home-made device based on a B 30-type high vided by electron microscopy. In this paper we vacuum evaporation unit (VEB Hochvakuum, present some results obtained from the appli- Dresden). The specimens were broken in a cation of scanning electron microscopy and vacuum at 2.7 x iO~ Pa at a temperature of freeze-fracture techniques to the analysis and 150°C. Replicas were produced by 45°-angle characterization of liposomes. evaporation of 2 nm platinum strengthened by a vertical evaporated carbon layer. The thickness __________________________________________ of the platinum layer was measured by a quartz —
oscillation thickness monitor which also controlled the evaporation process of the platinum layer. Following cleaning by a sulphuric acid (70%) procedure and rinsing several times with
Abbreviations used: MLV, multilamellar vesicles; LUV, large unilamellar vesicles; SEM, scanning electron microscope; EM, electron microscopy; TEM. transmission electron microscopy. 109
N.
~dIet ,tnd .1 Si.’hten~~tnn
j
I’ tg~. I and 2. Seanntng electron micrograp h~ of L t.~V—I po~omc~ ~ee cxplanatt on~ tIt text magni lication . 1 3 SO• Hg I lb j. magni hcation . 41IS0: F ig 2. magnification I 3~()
.
1- ig I I
Electron Microscop~of Liposomcs
1”
_
II!!! __~MP.
~,, ~iw~j~ Figs. 3 5.1 ransmission electron micrographs offreeze-fracture preparations of LUV- and MLV-liposomes (see explanations in text). Fig. 3. LUV-liposomes (magnification x46.170; Fig. 4, LUV-liposomes (magnification ~43.200); Fig. 5. MLV-liposomes (a magnification x 23,220: h magnilication x 35.550).
Ill
(12
K. Adler and J. Schiemann
aqua bidistil/ata, the specimens were analysed in a TESLA BS 500 transmission electron microscope (Tesla n.p., Brno, CSSR). Chemicals Egg L-c-phosphatidylcholine (type V-E, P5763), bovine brain L-z-phosphatidyl-L-serine (P664 1) and stearylamine (56755)were purchased from Sigma Chemical Co. Preparation of MLV MLV were prepared from phosphatidylcholine and stearylamine according to Lurquin (1979) with some modifications. A chloroform solution of stearylamine (50 ~.tg)and phosphatidylcholine (1 mg) was evaporated in a round-bottomed test tube by applying a stream of nitrogen at room temperature. Plant virus RNA dissolved in 0.5 ml distilled water was vigorously shaken for 1 mm at room temperature with the lipid film. After addition of I ml water the liposomes were underlayed with 0.5 ml D-mannitol solution (9 % in water) and sedimented into the mannitol phase by centrifuging at l0,000g for 10 mm at 10~C. Preparation (?ILUV LUV were prepared from phosphatidylserine according to Fukunaga et al. (1981). After dilution with distilled water the LUV were collected by centrifugation at 20,000 g for 15 mm at 10~Cin 9% D-mannitol solution, RESULTS AND DISCUSSION Figures 1 and 2 show scanning electron micrographs prepared by freeze-drying procedures: besides the egg-shaped liposomes a high proportion of lumps and crusted material is visible which consists mainly of mannitol originating from the preparation medium of the liposomes. The washing procedure is capable of removing this material, but this cleaning process in combination with the necessary additional centrifugation deforms and destroys a part of the liposomes. In these preparations we often observed an aggregation and conglomeration of deformed liposomes. In order to achieve a better structural preservation, we had to accept some difficulties when observing the preparations. The liposomes in our experiments were often seen together (Fig. 2) in small groups or aggregates. Occasionally there appeared to be a connection between the individual spheres (arrow Fig. lb),
which suggested an incomplete separation of the liposome bodies. This may indicate an incomplete separation of the lumen of the liposome from the surrounding medium or from another liposome. As illustrated in the scanning micrographs (Figs. I and 2) the sizes of the liposomes vary over a broad range. The inner structure of both large and small liposomes was very similar, but in the freeze-fracture preparations small fractured partides appeared more frequently. The large LUV-particle in Fig. 3 indicates an incomplete closed membrane (arrow). Moreover, it shows that unilamellar liposomes prepared by the methods described above may contain some smaller particles within their volume (Fig. 3) or they may be filled with a large number of smaller lipid spheres (Fig. 4). In the case of the multilamellar vesicles the images from freeze-fracture specimens (Fig. 5a,b) show a concentric structure of different lipid layers. The MLV-liposomes appear to consist of a flexible system of interlocking spheres in which the spaces between the lipid layers are filled with fluid hydrophilic substances, including any material which was enclosed in the liposomes to be transferred according to the proposal mentioned above. In comparison with other electron microscopic techniques used for liposome investigation, the freeze-drying preparation for SEM as well as the freeze-fracturetechnique for TEM has a number of advantages in that there is no embedding or contact of the liposomes with organic solvents which would dissolve or damage the liposomes. From our results we suggest a combination of the above methods to provide better results in the analysis ofliposomes with the aid of electron microscopy. Acknowledgements We wish to thank W. Panitz for her skilful technical assistance: Dr. H. B. Schmidt and M. MOllet (Institut) fur Phytopathologie der AdL, Aschersleben) for their help with the scanning electron microscope techniques.
REFERENCES Deamer, D. and Bangham, A. D., 1976. Large volume liposomes by an ether vaporization method. Bioehim. hiophy,s. Ada, 443: 629634. Fraley, R. and Papahadjopoulos, D., 1981. New generation Iiposomes: engineering an efficient vehicle for intracellular the delivery ofnucleicofacids. Trends Biochein. Sc,., 6: 77 80 Fraley, R. T., 1983. Liposome-mediated delivery of tobacco
Electron Microscopy of Liposomes mosaic virus RNA into petunia protoplasts. Plant molec. Biol., 2: 5—14. Fukunaga, Y., Nagata, T. and Takebe, J., 1981. Liposome-mediated infection of plant protoplasts with tobacco mosaic virus RNA. Virology, 113: 752—760. Harsch, M., Walter, P. and Weder, H. G., 1981. Targeting of monoclonal antibody-coated liposomesto sheep red blood cells. Biochem. Biophys. Res. Commun., 103: 1069—1076. Hauser, H. and Gains, N., 1982. Spontaneous vesiculation of phospholipids: a simple and quick method of forming
113
unilamellar vesicles. Proc. natn. Acad. Sci. U.S.A., 79: 1683—1687. Lurquin, P. F., 1979. Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts. Nucleic Acids Res. 6: 3773—3784. Rollo, R., 1983. Liposomes as a tool for introducing biologically active viral nucleic acids into plant protoplasts. In: Genetic Engineering in Eukaryotes, Lurquin, P. F. and Kleinhofs, A. (eds.), Plenum Press, New York, 179-185.
0739 6260/85 53.00+0.00
©
1985 Pergamon Press Ltd.
CHARACTERIZATION OF LIPOSOMES BY SCANNING ELECTRON MICROSCOPY AND THE FREEZE-FRACTURE TECHNIQUE K.
ADLER
and J.
SCHIEMANN
Zentralinstitut für Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der DDR, DDR-4325 Gatersieben, Corrensstr. 3, G.D.R. (Received 24 October 1984)
Abstract—This paper describes the application ofboth SEM and freeze-fracture TEMfor the characterization of artificial lipid vesicles (liposomes). Size and shape may be observed in the SEM technique whereas the inner structure could be investigated best in pictures obtained using the freeze-fracture method. Index key words: Liposome, TEM, SEM, freeze-fracture, freeze-drying.
INTRODUCTION MATERIALS AND METHODS The ability of lipid vesicles (liposomes) to Scanning electron microscopy entrap small and large molecules, such as drugs, enzymes, RNA and DNA, and also to deliver The liposomes were pipetted onto the top of them to cells, has attracted great interest in their cleaned specimen mounts. These specimens were use as vehicles (Fraley and Papahadjopoulos, then frozen at —20°C and dried overnight 1981). During recent years a large number of in a freeze-drying unit (LGA 05, VEB papers have been published demonstrating the Zentrifugenbau, Leipzig) at a vacuum of 2.25 Pa. The freeze-dried specimens were sputter-coated delivery of DNA and RNA molecules to a variety with a gold—palladium alloy and observed in an of plant protoplasts (reviewed by Rollo, 1983). In ISI 40 SEM (International Scientific Instruments contrast to other techniques for introducing Inc., Akashi-Seisakusho Ltd., Tokyo, Japan). nucleic acids into cells, liposome-mediated transfer is very efficient (Fukunaga et al., 1981; Fraley, Freeze-fracture electron microscopy 1983). There are few papers presenting electron Suspensions of liposomes were pipetted onto micrographs of liposomes (Deamer and specimen holders made from gold—platinium alloy Bangham, 1976; Harsch et al., 1981; Hauser and and were quick-frozen by immersion in liquid Gains, 1982). Considering the importance of Freon 22. Frozen specimens in gold—platinum liposomes in biochemistry, pharmacy and gen- holders were stored in a cryocontainer in liquid etic engineering, a more frequent control of nitrogen. Freeze-fracturing was conducted in a different liposome preparations should be pro- home-made device based on a B 30-type high vided by electron microscopy. In this paper we vacuum evaporation unit (VEB Hochvakuum, present some results obtained from the appli- Dresden). The specimens were broken in a cation of scanning electron microscopy and vacuum at 2.7 x iO~ Pa at a temperature of freeze-fracture techniques to the analysis and 150°C. Replicas were produced by 45°-angle characterization of liposomes. evaporation of 2 nm platinum strengthened by a vertical evaporated carbon layer. The thickness __________________________________________ of the platinum layer was measured by a quartz —
oscillation thickness monitor which also controlled the evaporation process of the platinum layer. Following cleaning by a sulphuric acid (70%) procedure and rinsing several times with
Abbreviations used: MLV, multilamellar vesicles; LUV, large unilamellar vesicles; SEM, scanning electron microscope; EM, electron microscopy; TEM. transmission electron microscopy. 109
N.
~dIet ,tnd .1 Si.’hten~~tnn
j
I’ tg~. I and 2. Seanntng electron micrograp h~ of L t.~V—I po~omc~ ~ee cxplanatt on~ tIt text magni lication . 1 3 SO• Hg I lb j. magni hcation . 41IS0: F ig 2. magnification I 3~()
.
1- ig I I
Electron Microscop~of Liposomcs
1”
_
II!!! __~MP.
~,, ~iw~j~ Figs. 3 5.1 ransmission electron micrographs offreeze-fracture preparations of LUV- and MLV-liposomes (see explanations in text). Fig. 3. LUV-liposomes (magnification x46.170; Fig. 4, LUV-liposomes (magnification ~43.200); Fig. 5. MLV-liposomes (a magnification x 23,220: h magnilication x 35.550).
Ill
(12
K. Adler and J. Schiemann
aqua bidistil/ata, the specimens were analysed in a TESLA BS 500 transmission electron microscope (Tesla n.p., Brno, CSSR). Chemicals Egg L-c-phosphatidylcholine (type V-E, P5763), bovine brain L-z-phosphatidyl-L-serine (P664 1) and stearylamine (56755)were purchased from Sigma Chemical Co. Preparation of MLV MLV were prepared from phosphatidylcholine and stearylamine according to Lurquin (1979) with some modifications. A chloroform solution of stearylamine (50 ~.tg)and phosphatidylcholine (1 mg) was evaporated in a round-bottomed test tube by applying a stream of nitrogen at room temperature. Plant virus RNA dissolved in 0.5 ml distilled water was vigorously shaken for 1 mm at room temperature with the lipid film. After addition of I ml water the liposomes were underlayed with 0.5 ml D-mannitol solution (9 % in water) and sedimented into the mannitol phase by centrifuging at l0,000g for 10 mm at 10~C. Preparation (?ILUV LUV were prepared from phosphatidylserine according to Fukunaga et al. (1981). After dilution with distilled water the LUV were collected by centrifugation at 20,000 g for 15 mm at 10~Cin 9% D-mannitol solution, RESULTS AND DISCUSSION Figures 1 and 2 show scanning electron micrographs prepared by freeze-drying procedures: besides the egg-shaped liposomes a high proportion of lumps and crusted material is visible which consists mainly of mannitol originating from the preparation medium of the liposomes. The washing procedure is capable of removing this material, but this cleaning process in combination with the necessary additional centrifugation deforms and destroys a part of the liposomes. In these preparations we often observed an aggregation and conglomeration of deformed liposomes. In order to achieve a better structural preservation, we had to accept some difficulties when observing the preparations. The liposomes in our experiments were often seen together (Fig. 2) in small groups or aggregates. Occasionally there appeared to be a connection between the individual spheres (arrow Fig. lb),
which suggested an incomplete separation of the liposome bodies. This may indicate an incomplete separation of the lumen of the liposome from the surrounding medium or from another liposome. As illustrated in the scanning micrographs (Figs. I and 2) the sizes of the liposomes vary over a broad range. The inner structure of both large and small liposomes was very similar, but in the freeze-fracture preparations small fractured partides appeared more frequently. The large LUV-particle in Fig. 3 indicates an incomplete closed membrane (arrow). Moreover, it shows that unilamellar liposomes prepared by the methods described above may contain some smaller particles within their volume (Fig. 3) or they may be filled with a large number of smaller lipid spheres (Fig. 4). In the case of the multilamellar vesicles the images from freeze-fracture specimens (Fig. 5a,b) show a concentric structure of different lipid layers. The MLV-liposomes appear to consist of a flexible system of interlocking spheres in which the spaces between the lipid layers are filled with fluid hydrophilic substances, including any material which was enclosed in the liposomes to be transferred according to the proposal mentioned above. In comparison with other electron microscopic techniques used for liposome investigation, the freeze-drying preparation for SEM as well as the freeze-fracturetechnique for TEM has a number of advantages in that there is no embedding or contact of the liposomes with organic solvents which would dissolve or damage the liposomes. From our results we suggest a combination of the above methods to provide better results in the analysis ofliposomes with the aid of electron microscopy. Acknowledgements We wish to thank W. Panitz for her skilful technical assistance: Dr. H. B. Schmidt and M. MOllet (Institut) fur Phytopathologie der AdL, Aschersleben) for their help with the scanning electron microscope techniques.
REFERENCES Deamer, D. and Bangham, A. D., 1976. Large volume liposomes by an ether vaporization method. Bioehim. hiophy,s. Ada, 443: 629634. Fraley, R. and Papahadjopoulos, D., 1981. New generation Iiposomes: engineering an efficient vehicle for intracellular the delivery ofnucleicofacids. Trends Biochein. Sc,., 6: 77 80 Fraley, R. T., 1983. Liposome-mediated delivery of tobacco
Electron Microscopy of Liposomes mosaic virus RNA into petunia protoplasts. Plant molec. Biol., 2: 5—14. Fukunaga, Y., Nagata, T. and Takebe, J., 1981. Liposome-mediated infection of plant protoplasts with tobacco mosaic virus RNA. Virology, 113: 752—760. Harsch, M., Walter, P. and Weder, H. G., 1981. Targeting of monoclonal antibody-coated liposomesto sheep red blood cells. Biochem. Biophys. Res. Commun., 103: 1069—1076. Hauser, H. and Gains, N., 1982. Spontaneous vesiculation of phospholipids: a simple and quick method of forming
113
unilamellar vesicles. Proc. natn. Acad. Sci. U.S.A., 79: 1683—1687. Lurquin, P. F., 1979. Entrapment of plasmid DNA by liposomes and their interactions with plant protoplasts. Nucleic Acids Res. 6: 3773—3784. Rollo, R., 1983. Liposomes as a tool for introducing biologically active viral nucleic acids into plant protoplasts. In: Genetic Engineering in Eukaryotes, Lurquin, P. F. and Kleinhofs, A. (eds.), Plenum Press, New York, 179-185.