Journal of Microencapsulation Micro and Nano Carriers
ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: http://www.tandfonline.com/loi/imnc20
Incorporation of the reovirus M cell attachment protein into small unilamellar vesicles: incorporation efficiency and binding capability to L929 cells in vitro W. Rubas, A. C. Banerjea, H. Gallati, P. P. Speiser & W. K. Joklik To cite this article: W. Rubas, A. C. Banerjea, H. Gallati, P. P. Speiser & W. K. Joklik (1990) Incorporation of the reovirus M cell attachment protein into small unilamellar vesicles: incorporation efficiency and binding capability to L929 cells in vitro, Journal of Microencapsulation, 7:3, 385-395 To link to this article: http://dx.doi.org/10.3109/02652049009021848
Published online: 27 Sep 2008.
Submit your article to this journal
Article views: 9
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=imnc20 Download by: [Deakin University Library]
Date: 05 November 2015, At: 20:09
J. MICROENCAPSULATION,
1990, VOL. 7,
NO.
3, 385-395
Incorporation of the reovirus M cell attachment protein into small unilamellar vesicles: incorporation efficiency and binding capability to L929 cells in vitro
Downloaded by [Deakin University Library] at 20:09 05 November 2015
W. RUBAS?$, A. C. BANERJEA5, H. GALLATIJI, P. P. SPEISERt and W. K. JOKLIK5 Department of Pharmacy, Swiss Federal Institute of Technology ETH, 8092 Zurich, Switzerland 9 Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710, USA. 1) F. Hoffmann-La Roche & Co. Ltd, 4002 Basel, Switzerland (Received 6 November 1989; accepted 16 November 1989)
Neutral phosphatidylcholine/cholesterol(10 :3) liposomes with the reovirus M cell attachment protein a1 were made by means of detergent dialysis method. An immunological evaluation method revealed an incorporation efficiency (pg proteinlpg lipid) of 314.Binding studies with mouse fibroblasts (L929 cells) yielded a 10 fold improvement of uptake of coated liposomes compared to uncoated liposomes. Competition studies with reovirus serotype 3 demonstrated that coated liposomes were capable of binding to the reovirus receptor. In vitro incubation of rat Peyer’s patches with either coated or uncoated liposomes resulted in a 10-20 fold higher uptake of the coated liposomes. These results suggest that selective adherence of a carrier system to M cells may facilitate delivery of carrier contents to mucosal underlying lymphoid tissue, thereby enhancing immune response.
Introduction Administration of macromolecules such as antigens and lymphokines to man via the oral route must overcome the serious problem of poor absorption due to the environmental and absorptive barriers of the intestine. I t is possible to improve the absorption rate by using absorption promoters such as medium chain glycerides (Higaki et al. 1987), fatty acids, chelators, nonsteroidal anti-inflammatory agents, surfactants (Fix 1987), enamines (Nishihata et al. 1985)and trypsin inhibitors (Fujii et al. 1985). Promoters act by increasing membrane fluidity and calcium ion sequestration in the membrane (Muranishi 1985, Sakai et al. 1986). These mechanisms are not selective to drug absorption and therefore not suitable for a long term treatment. I n the gastrointestinal tract a specific mechanism exists for uptake of macromolecules. Embedded in Peyer’s patches membranous epithelial (M) cells endocytose and transport a variety of macromolecules such as horseradish peroxidase, ferritin, carbon particles, latex, ricin, reovirus, TGE-virus, rotavirus, astrovirus, Bredavirus, Chlamydia and Vibrio cholera from the lumen to underlying lymphoid tissue (Egberts et al. 1985). Reovirus adheres to M cells through the minor outer
$ Present Address: c/o Syntex Research Institute of Pharmaceutical Sciences, 3401 Hillview Ave, Palo Alto, CA 94303, USA. Please use this address for correspondence. 0265-2048/90 $ 3 4 0
0 1990 Taylor & Francis Ltd.
Downloaded by [Deakin University Library] at 20:09 05 November 2015
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W. Rubas et al.
capsid (al) protein (Lee et al. 1981). More recently several authors have expressed the a1 protein in procaryotic or eucaryotic cells (Masri et al. 1986, Pelletier et al. 1987, Banerjea et al. 1988). Recently, gastric resistant liposomes have become available (Rowland and Woodley 1980, European Patent Application 249-561 -lA 1986). Therefore liposomes could be a potential vehicle for protecting macromolecules, that would not normally survive in the gastrointestinal tract from premature digestion. Incubation of insulin-liposomes with a proteolytic enzyme led only to minor degradation of the protein (Axt et al. 1983). Liposomes enter absorptive cells of the intestine but are digested by lysosomal enzymes (Pate1et al. 1985). In contrast M cells largely, if not completely, fail to sequester and digest luminal macromolecules (Owen et al. 1986). T o investigate whether the protein 01 is suitable for targeting liposomes efficiently to the reovirus receptor, we have coated them with the attachment protein. In this work we describe an immunological approach to measuring the incorporation efficiency of protein a1 into liposomes and present results of binding studies to mouse L929 fibroblasts and rat Peyer’s patches in vitro.
Materials and methods Chemicals Sojaphosphatidylcholine (SPC) was obtained from Lucas Meyer, Hamburg, FRG (Epikuron 200); the sodium salt of deoxycholic acid (SDOC), Tween@20 and anti-mouse IgG fluorescein isothiocyanate (FITC) conjugates absorbed with Human serum proteins were purchased from Sigma, St Louis, MO, USA; cholesterol (Chol) was obtained from Fluka AG, Buchs, Switzerland. [3H] Inulin (163 GBqlmmol) and [1251]protein A (1.11 GBq/mg) were from Amersham Int., Amersham, UK. Tris(hydroxymethy1)aminomethane (Tris) was from Boehringer Mannheim Biochemicals, Indianapolis, USA. The organic solvents, sucrose and sodium chloride (NaC1) were purchased from Mallinckrodt Inc., Paris, Kentucky, USA. Sepharose CL-4B was obtained from Pharmacia LKB, Uppsala, Sweden. As liquid scintillation fluid Safety-Solve from Research Products Int. Corp., Mount Prospect, IL, USA was used. Foetal bovine serum (FBS) was supplied by HyClone Laboratories Inc., Logan, UK. Triton X-100 was purchased from Beckman Instruments Inc., Fullerton, CA, USA. Male Sprague-Dawley rats (subline CD) were purchased from Charles River Labs. Inc, Wilmington, MA, USA. Preparation of a1 protein Protein a1 was routinely prepared according to the method described by Banerjea et al. (1988). The concentration of the protein in the final solution was determined by the Bio-Rad protein assay (Bio Rad, South Richmond, CA, USA). Solutions were diluted to a final concentration of 0.1 pg/pl. Liposome preparation Liposomes were routinely prepared according to the detergent dialysis method described elsewhere (Milsmann et al. 1978, Rubas et al. 1986). Briefly, SPC, Chol and SDOC at a molar ratio of 10: 3 :22 (referred to SPC) were dissolved in chloroform/methanol (1 :1 v/v per cent). The mixture was evaporated to dryness at room temperature. The lipid film was then resuspended in 10mM Tris-HC1 (pH 7.4) containing 150 mM NaCl (TBS) including appropriate amounts of protein
Downloaded by [Deakin University Library] at 20:09 05 November 2015
Incorporation of protein a1 into liposomes
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01 and [3H] inulin to yield a lipid concentration of 2mg SPC/ml. T h e micelle solution was dialysed for 20 h at 4°C against 1 1 T B S using a cellulose membrane with a molecular weight cut off of 10000 D in a Lipoprep apparatus (Diachema, Langnau, Switzerland) to yield a liposome dispersion with incorporated and unincorporated protein. [3H] Inulin containing micelles were first dialysed at 4°C overnight against 1 1 T B S containing equimolar amounts unlabelled inulin. T h e liposomes were then dialysed for another 20 h at 4°C to remove unincorporated inulin. T h e liposomes were purged with nitrogen gas and stored at 4°C in sterile tubes until use. T o avoid leakage of r3H] inulin during storage, these liposomes were used immediately. Liposomes were separated from unincorporated protein a1 on Sepharose CL-4B columns (25 x 0.8 cm). T h e flow rate was 0.2 ml/min and the elution was monitored at 254nm using an Isco UA-5 absorbance/fluorescence detector (Isco Inc., Lincoln, Nebraska, USA). T h e concentration of liposomes in each fraction (size = 0.25 ml) was determined by liquid scintillation counting of the incorporated [3H] Inulin in a Beckman LS-233 counter. Amount of protein a1 incorporated per mg lipid was measured as follows. Increasing amounts of protein a1 were added either to the micelles or incubated with pre-formed liposomes (3 h, 4°C). Unincorporated protein was separated from liposomes by column chromatography (see above). Aliquots of protein coated liposomes were disrupted with detergent and spotted onto a nitrocellulose membrane with a pore size of 0 4 5 pm (Schleicher & Schuell, Keene, N H , USA) using a MilliBlot-D transfer apparatus (Millipore Corp., Bedford, MA, USA). After drying at 37°C the membrane was washed for 30 min at R T with 0.05%Tween/phosphate buffered saline (TPBS) to avoid nonspecific binding of antiserum to the membrane. T h e membrane was then incubated for 2 h with a polyclonal antiserum against reovirus serotype 3 and after incubation was rinsed with TPBS for 30min with 3 buffer changes. T h e washed membrane was then incubated for 30 min at R T with [1251]labelled Protein A (370 Bq) and after washing for 30 min with PBS at R T with 3 buffer changes, the dried membrane was placed overnight next to X-ray film (Eastman Kodak Comp., Rochester, NY, USA). Visible spots were marked on the membrane, cut out and radioactivity was counted in a Beckmann Biogamma I1 counter. T h e standard curve for pure protein a1 was obtained by the method described above with the exception of the chromatography, which was not done. From this curve the amount of incorporated protein into liposomes could be estimated. T h e ratio of protein a1 per mg lipid was then calculated on the basis of the measured protein and the liposome recovery using r3H] inulin liposomes.
Cell binding studies A monolayer of L929 cells (2.5 x lo5 cells/well) was grown overnight in Joklik’s modified Minimal Essential Medium (MEM) (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 5 per cent fetal calf serum. Twenty-four well, flat bottom tissue culture plates (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) were used. T h e medium was then removed and replaced with cold PBS (4°C) for several minutes. In uptake studies column purified [3H] inulin liposomes either uncoated or coated with the protein 01 (protein/SPC ratio 0 . 0 5 4 1 ) were incubated at increasing liposome/cell ratios with all monolayers for 90 min at 4°C with shaking (100 rpm) on a Gyrotory Shaker (New Brunswick Scientific Co. Inc., New Brunswick, NJ, USA). T h e thoroughly washed monolayers were then harvested in PBS and transferred to
388
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Eppendorf tubes and centrifuged for 3 min (Fisher Microcentrifuge Model 235 A, Fisher Scientific Company) in order to pellet the cells. T h e pellets were transferred to scintillation cocktail and counted for radioactivity. Competition experiments were carried out as follows. Either 1OOpl of purified [3H] inulin protein a1 coated liposome and appropriate amounts of reovirus serotype 3 were mixed and incubated as described above, or the cell monolayers were preincubated with appropriate amounts of reovirus serotype 3 for 30min at 4"C, washed and then incubated with loop1 purified [3H] inulin protein a1 coated liposomes as described above.
Downloaded by [Deakin University Library] at 20:09 05 November 2015
Tissue binding studies Nonfasted male Sprague-Dawley rats (subline CD) weighing 450-500 g were anaesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal Sodium, 50 mg/ml, Abbot Laboratories, North Chicago, IL, USA) ( 0 1 m l = 5.0mg per lOOg body weight). T h e abdomen was entered and a loop of distal ileum containing several Peyer's patches was opened by incision on both ends. T h e loop was gently rinsed with MEM. Segments with Peyer's patches were carefully removed and opened by longitudinal incision. Small mucosal samples ( 5 x 5 mm) were excised with fine scissors, placed in a 24 well culture plate and immerged in 2 0 0 ~ 1MEM. T h e tissue was incubated for 1 h at 4°C with loop1 of [3H] inulin liposomes either coated with protein a1 or uncoated. After exposure the samples were washed 3 times with MEM and transferred into a scintillation cocktail for radioactivity counting.
Results Stable neutral SPC :Chol (10 :3) liposomes containing protein a1 could be prepared at 4°C either by detergent dialysis or incubation of pre-formed liposomes with the protein. No visible aggregates of protein containing liposomes could be detected after 60 days of storage. In order to characterize the quantity of protein a1 associated with the phospholipid bilayers, the liposome dispersions were separated from unincorporated protein by means of column chromatography. Figure 1 illustrates the elution profile obtained from a typical liposome preparation with a molar protein/phospholipid ratio of 3.7 x lo-'. T h e recovery of liposomes was 1 0 f 5 per cent ( n = 5 ) as determined with r3H] inulin liposomes. Small amounts of protein cannot be detected by commonly used protein assays. To overcome this limitation we immobilized protein 01 on a nitrocellulose membrane. Since it retains all of its antibody binding capacity it can be quantified using [1251] labelled Protein A as an amplifier (Banerjea, pers. communication). Figure 2 represents a standard curve for protein a1 (y= 1 . 4 6 9 6+ ~ 2.7461, r =0.991). T o investigate the incorporation efficiency of protein a1 into the bilayer, we prepared micelles with increasing protein/SPC ratios or incubated pre-formed liposomes with various protein/SPC ratios. T h e addition of the protein to micelles led to saturation in the investigated range (314 pg protein/pg sojaphosphatidylcholine). However, the incubation of pre-formed liposomes with increasing amounts of protein did not result in saturation of the bilayer. T h e uptake efficiency was 100 times higher if the protein was added to the micelles compared to incubation of preformed liposomes with the protein (figure 3).
Incorporation of protein a1 into liposomes
389
100-
908070w 0
60-
u
Downloaded by [Deakin University Library] at 20:09 05 November 2015
v,
3
50-
LL
,\" 40-
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ml Figure 1 . Elution profile of a liposome preparation with a molar protein/phospholipid ratio of 3.7 x lo-' from a Sepharose C1-4B column (25 x 08cm). Liposome recovery was 10f 5% (n= 5) as determined with [3H] inulin liposomes.
00 80 --
O 60--
0
10
20
30
40
50
u, -PROTEIN [ng] Figure 2.
Correlation between blotted protein a1 onto nitrocellulose membrane and gamma counts (y=1*4696x + 2.7461; r=0991).
390
W . Rubas et al.
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0 Added to Liposomes
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Downloaded by [Deakin University Library] at 20:09 05 November 2015
EI
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0.100
CL
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0.010 0,010 Figure 3.
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0.100 1 .ooo pg a; -PROTEINIpg SPC A D D E D
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Effect on incorporation efficiencyof adding protein crl either to micelles or to preformed liposomes.
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Figure 4. Effect of incubating a monolayer (2.5 x lo5 cells/well) of L929 fibroblasts either with coated (01-liposomes) or uncoated liposomes on their uptake. The data were combined from different experiments (n= 3).
Incorporation of protein crl into liposomes
I
Downloaded by [Deakin University Library] at 20:09 05 November 2015
A
391
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I
A -Added as mixture A - Pre-incubated with Reovi ru s
3 4 5 6 7 8 9 10 11 RAT I0 V I RU S PA RTI CLE / L I PO S 0 M E Figure 5. Effect of competing ol-coated liposomes with reovirus, added to a monolayer (2.5 x lo5cells/well) of L929 fibroblasts either as a liposome-virus mixture or after preincubation of the monolayer with reovirus and subsequent incubation with liposomes (12.75 x lo5 liposomes/well), on their uptake. 0
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Figure 6. Effect of in witro incubation of rat Peyer’s patches with either a1 coated or uncoated liposomes on their uptake. Level of significance: P
ISSN: 0265-2048 (Print) 1464-5246 (Online) Journal homepage: http://www.tandfonline.com/loi/imnc20
Incorporation of the reovirus M cell attachment protein into small unilamellar vesicles: incorporation efficiency and binding capability to L929 cells in vitro W. Rubas, A. C. Banerjea, H. Gallati, P. P. Speiser & W. K. Joklik To cite this article: W. Rubas, A. C. Banerjea, H. Gallati, P. P. Speiser & W. K. Joklik (1990) Incorporation of the reovirus M cell attachment protein into small unilamellar vesicles: incorporation efficiency and binding capability to L929 cells in vitro, Journal of Microencapsulation, 7:3, 385-395 To link to this article: http://dx.doi.org/10.3109/02652049009021848
Published online: 27 Sep 2008.
Submit your article to this journal
Article views: 9
View related articles
Citing articles: 4 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=imnc20 Download by: [Deakin University Library]
Date: 05 November 2015, At: 20:09
J. MICROENCAPSULATION,
1990, VOL. 7,
NO.
3, 385-395
Incorporation of the reovirus M cell attachment protein into small unilamellar vesicles: incorporation efficiency and binding capability to L929 cells in vitro
Downloaded by [Deakin University Library] at 20:09 05 November 2015
W. RUBAS?$, A. C. BANERJEA5, H. GALLATIJI, P. P. SPEISERt and W. K. JOKLIK5 Department of Pharmacy, Swiss Federal Institute of Technology ETH, 8092 Zurich, Switzerland 9 Department of Microbiology and Immunology, Duke University Medical Center, Durham, NC 27710, USA. 1) F. Hoffmann-La Roche & Co. Ltd, 4002 Basel, Switzerland (Received 6 November 1989; accepted 16 November 1989)
Neutral phosphatidylcholine/cholesterol(10 :3) liposomes with the reovirus M cell attachment protein a1 were made by means of detergent dialysis method. An immunological evaluation method revealed an incorporation efficiency (pg proteinlpg lipid) of 314.Binding studies with mouse fibroblasts (L929 cells) yielded a 10 fold improvement of uptake of coated liposomes compared to uncoated liposomes. Competition studies with reovirus serotype 3 demonstrated that coated liposomes were capable of binding to the reovirus receptor. In vitro incubation of rat Peyer’s patches with either coated or uncoated liposomes resulted in a 10-20 fold higher uptake of the coated liposomes. These results suggest that selective adherence of a carrier system to M cells may facilitate delivery of carrier contents to mucosal underlying lymphoid tissue, thereby enhancing immune response.
Introduction Administration of macromolecules such as antigens and lymphokines to man via the oral route must overcome the serious problem of poor absorption due to the environmental and absorptive barriers of the intestine. I t is possible to improve the absorption rate by using absorption promoters such as medium chain glycerides (Higaki et al. 1987), fatty acids, chelators, nonsteroidal anti-inflammatory agents, surfactants (Fix 1987), enamines (Nishihata et al. 1985)and trypsin inhibitors (Fujii et al. 1985). Promoters act by increasing membrane fluidity and calcium ion sequestration in the membrane (Muranishi 1985, Sakai et al. 1986). These mechanisms are not selective to drug absorption and therefore not suitable for a long term treatment. I n the gastrointestinal tract a specific mechanism exists for uptake of macromolecules. Embedded in Peyer’s patches membranous epithelial (M) cells endocytose and transport a variety of macromolecules such as horseradish peroxidase, ferritin, carbon particles, latex, ricin, reovirus, TGE-virus, rotavirus, astrovirus, Bredavirus, Chlamydia and Vibrio cholera from the lumen to underlying lymphoid tissue (Egberts et al. 1985). Reovirus adheres to M cells through the minor outer
$ Present Address: c/o Syntex Research Institute of Pharmaceutical Sciences, 3401 Hillview Ave, Palo Alto, CA 94303, USA. Please use this address for correspondence. 0265-2048/90 $ 3 4 0
0 1990 Taylor & Francis Ltd.
Downloaded by [Deakin University Library] at 20:09 05 November 2015
386
W. Rubas et al.
capsid (al) protein (Lee et al. 1981). More recently several authors have expressed the a1 protein in procaryotic or eucaryotic cells (Masri et al. 1986, Pelletier et al. 1987, Banerjea et al. 1988). Recently, gastric resistant liposomes have become available (Rowland and Woodley 1980, European Patent Application 249-561 -lA 1986). Therefore liposomes could be a potential vehicle for protecting macromolecules, that would not normally survive in the gastrointestinal tract from premature digestion. Incubation of insulin-liposomes with a proteolytic enzyme led only to minor degradation of the protein (Axt et al. 1983). Liposomes enter absorptive cells of the intestine but are digested by lysosomal enzymes (Pate1et al. 1985). In contrast M cells largely, if not completely, fail to sequester and digest luminal macromolecules (Owen et al. 1986). T o investigate whether the protein 01 is suitable for targeting liposomes efficiently to the reovirus receptor, we have coated them with the attachment protein. In this work we describe an immunological approach to measuring the incorporation efficiency of protein a1 into liposomes and present results of binding studies to mouse L929 fibroblasts and rat Peyer’s patches in vitro.
Materials and methods Chemicals Sojaphosphatidylcholine (SPC) was obtained from Lucas Meyer, Hamburg, FRG (Epikuron 200); the sodium salt of deoxycholic acid (SDOC), Tween@20 and anti-mouse IgG fluorescein isothiocyanate (FITC) conjugates absorbed with Human serum proteins were purchased from Sigma, St Louis, MO, USA; cholesterol (Chol) was obtained from Fluka AG, Buchs, Switzerland. [3H] Inulin (163 GBqlmmol) and [1251]protein A (1.11 GBq/mg) were from Amersham Int., Amersham, UK. Tris(hydroxymethy1)aminomethane (Tris) was from Boehringer Mannheim Biochemicals, Indianapolis, USA. The organic solvents, sucrose and sodium chloride (NaC1) were purchased from Mallinckrodt Inc., Paris, Kentucky, USA. Sepharose CL-4B was obtained from Pharmacia LKB, Uppsala, Sweden. As liquid scintillation fluid Safety-Solve from Research Products Int. Corp., Mount Prospect, IL, USA was used. Foetal bovine serum (FBS) was supplied by HyClone Laboratories Inc., Logan, UK. Triton X-100 was purchased from Beckman Instruments Inc., Fullerton, CA, USA. Male Sprague-Dawley rats (subline CD) were purchased from Charles River Labs. Inc, Wilmington, MA, USA. Preparation of a1 protein Protein a1 was routinely prepared according to the method described by Banerjea et al. (1988). The concentration of the protein in the final solution was determined by the Bio-Rad protein assay (Bio Rad, South Richmond, CA, USA). Solutions were diluted to a final concentration of 0.1 pg/pl. Liposome preparation Liposomes were routinely prepared according to the detergent dialysis method described elsewhere (Milsmann et al. 1978, Rubas et al. 1986). Briefly, SPC, Chol and SDOC at a molar ratio of 10: 3 :22 (referred to SPC) were dissolved in chloroform/methanol (1 :1 v/v per cent). The mixture was evaporated to dryness at room temperature. The lipid film was then resuspended in 10mM Tris-HC1 (pH 7.4) containing 150 mM NaCl (TBS) including appropriate amounts of protein
Downloaded by [Deakin University Library] at 20:09 05 November 2015
Incorporation of protein a1 into liposomes
387
01 and [3H] inulin to yield a lipid concentration of 2mg SPC/ml. T h e micelle solution was dialysed for 20 h at 4°C against 1 1 T B S using a cellulose membrane with a molecular weight cut off of 10000 D in a Lipoprep apparatus (Diachema, Langnau, Switzerland) to yield a liposome dispersion with incorporated and unincorporated protein. [3H] Inulin containing micelles were first dialysed at 4°C overnight against 1 1 T B S containing equimolar amounts unlabelled inulin. T h e liposomes were then dialysed for another 20 h at 4°C to remove unincorporated inulin. T h e liposomes were purged with nitrogen gas and stored at 4°C in sterile tubes until use. T o avoid leakage of r3H] inulin during storage, these liposomes were used immediately. Liposomes were separated from unincorporated protein a1 on Sepharose CL-4B columns (25 x 0.8 cm). T h e flow rate was 0.2 ml/min and the elution was monitored at 254nm using an Isco UA-5 absorbance/fluorescence detector (Isco Inc., Lincoln, Nebraska, USA). T h e concentration of liposomes in each fraction (size = 0.25 ml) was determined by liquid scintillation counting of the incorporated [3H] Inulin in a Beckman LS-233 counter. Amount of protein a1 incorporated per mg lipid was measured as follows. Increasing amounts of protein a1 were added either to the micelles or incubated with pre-formed liposomes (3 h, 4°C). Unincorporated protein was separated from liposomes by column chromatography (see above). Aliquots of protein coated liposomes were disrupted with detergent and spotted onto a nitrocellulose membrane with a pore size of 0 4 5 pm (Schleicher & Schuell, Keene, N H , USA) using a MilliBlot-D transfer apparatus (Millipore Corp., Bedford, MA, USA). After drying at 37°C the membrane was washed for 30 min at R T with 0.05%Tween/phosphate buffered saline (TPBS) to avoid nonspecific binding of antiserum to the membrane. T h e membrane was then incubated for 2 h with a polyclonal antiserum against reovirus serotype 3 and after incubation was rinsed with TPBS for 30min with 3 buffer changes. T h e washed membrane was then incubated for 30 min at R T with [1251]labelled Protein A (370 Bq) and after washing for 30 min with PBS at R T with 3 buffer changes, the dried membrane was placed overnight next to X-ray film (Eastman Kodak Comp., Rochester, NY, USA). Visible spots were marked on the membrane, cut out and radioactivity was counted in a Beckmann Biogamma I1 counter. T h e standard curve for pure protein a1 was obtained by the method described above with the exception of the chromatography, which was not done. From this curve the amount of incorporated protein into liposomes could be estimated. T h e ratio of protein a1 per mg lipid was then calculated on the basis of the measured protein and the liposome recovery using r3H] inulin liposomes.
Cell binding studies A monolayer of L929 cells (2.5 x lo5 cells/well) was grown overnight in Joklik’s modified Minimal Essential Medium (MEM) (GIBCO Laboratories, Grand Island, NY, USA) supplemented with 5 per cent fetal calf serum. Twenty-four well, flat bottom tissue culture plates (Falcon, Becton Dickinson, Lincoln Park, NJ, USA) were used. T h e medium was then removed and replaced with cold PBS (4°C) for several minutes. In uptake studies column purified [3H] inulin liposomes either uncoated or coated with the protein 01 (protein/SPC ratio 0 . 0 5 4 1 ) were incubated at increasing liposome/cell ratios with all monolayers for 90 min at 4°C with shaking (100 rpm) on a Gyrotory Shaker (New Brunswick Scientific Co. Inc., New Brunswick, NJ, USA). T h e thoroughly washed monolayers were then harvested in PBS and transferred to
388
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Eppendorf tubes and centrifuged for 3 min (Fisher Microcentrifuge Model 235 A, Fisher Scientific Company) in order to pellet the cells. T h e pellets were transferred to scintillation cocktail and counted for radioactivity. Competition experiments were carried out as follows. Either 1OOpl of purified [3H] inulin protein a1 coated liposome and appropriate amounts of reovirus serotype 3 were mixed and incubated as described above, or the cell monolayers were preincubated with appropriate amounts of reovirus serotype 3 for 30min at 4"C, washed and then incubated with loop1 purified [3H] inulin protein a1 coated liposomes as described above.
Downloaded by [Deakin University Library] at 20:09 05 November 2015
Tissue binding studies Nonfasted male Sprague-Dawley rats (subline CD) weighing 450-500 g were anaesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal Sodium, 50 mg/ml, Abbot Laboratories, North Chicago, IL, USA) ( 0 1 m l = 5.0mg per lOOg body weight). T h e abdomen was entered and a loop of distal ileum containing several Peyer's patches was opened by incision on both ends. T h e loop was gently rinsed with MEM. Segments with Peyer's patches were carefully removed and opened by longitudinal incision. Small mucosal samples ( 5 x 5 mm) were excised with fine scissors, placed in a 24 well culture plate and immerged in 2 0 0 ~ 1MEM. T h e tissue was incubated for 1 h at 4°C with loop1 of [3H] inulin liposomes either coated with protein a1 or uncoated. After exposure the samples were washed 3 times with MEM and transferred into a scintillation cocktail for radioactivity counting.
Results Stable neutral SPC :Chol (10 :3) liposomes containing protein a1 could be prepared at 4°C either by detergent dialysis or incubation of pre-formed liposomes with the protein. No visible aggregates of protein containing liposomes could be detected after 60 days of storage. In order to characterize the quantity of protein a1 associated with the phospholipid bilayers, the liposome dispersions were separated from unincorporated protein by means of column chromatography. Figure 1 illustrates the elution profile obtained from a typical liposome preparation with a molar protein/phospholipid ratio of 3.7 x lo-'. T h e recovery of liposomes was 1 0 f 5 per cent ( n = 5 ) as determined with r3H] inulin liposomes. Small amounts of protein cannot be detected by commonly used protein assays. To overcome this limitation we immobilized protein 01 on a nitrocellulose membrane. Since it retains all of its antibody binding capacity it can be quantified using [1251] labelled Protein A as an amplifier (Banerjea, pers. communication). Figure 2 represents a standard curve for protein a1 (y= 1 . 4 6 9 6+ ~ 2.7461, r =0.991). T o investigate the incorporation efficiency of protein a1 into the bilayer, we prepared micelles with increasing protein/SPC ratios or incubated pre-formed liposomes with various protein/SPC ratios. T h e addition of the protein to micelles led to saturation in the investigated range (314 pg protein/pg sojaphosphatidylcholine). However, the incubation of pre-formed liposomes with increasing amounts of protein did not result in saturation of the bilayer. T h e uptake efficiency was 100 times higher if the protein was added to the micelles compared to incubation of preformed liposomes with the protein (figure 3).
Incorporation of protein a1 into liposomes
389
100-
908070w 0
60-
u
Downloaded by [Deakin University Library] at 20:09 05 November 2015
v,
3
50-
LL
,\" 40-
3020100-
5.0 100 15.0 20.0
ml Figure 1 . Elution profile of a liposome preparation with a molar protein/phospholipid ratio of 3.7 x lo-' from a Sepharose C1-4B column (25 x 08cm). Liposome recovery was 10f 5% (n= 5) as determined with [3H] inulin liposomes.
00 80 --
O 60--
0
10
20
30
40
50
u, -PROTEIN [ng] Figure 2.
Correlation between blotted protein a1 onto nitrocellulose membrane and gamma counts (y=1*4696x + 2.7461; r=0991).
390
W . Rubas et al.
n
looo'ooo
2
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Effect on incorporation efficiencyof adding protein crl either to micelles or to preformed liposomes.
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Figure 4. Effect of incubating a monolayer (2.5 x lo5 cells/well) of L929 fibroblasts either with coated (01-liposomes) or uncoated liposomes on their uptake. The data were combined from different experiments (n= 3).
Incorporation of protein crl into liposomes
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3 4 5 6 7 8 9 10 11 RAT I0 V I RU S PA RTI CLE / L I PO S 0 M E Figure 5. Effect of competing ol-coated liposomes with reovirus, added to a monolayer (2.5 x lo5cells/well) of L929 fibroblasts either as a liposome-virus mixture or after preincubation of the monolayer with reovirus and subsequent incubation with liposomes (12.75 x lo5 liposomes/well), on their uptake. 0
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Figure 6. Effect of in witro incubation of rat Peyer’s patches with either a1 coated or uncoated liposomes on their uptake. Level of significance: P
